BORRELIATESTIT

Asiantuntijana Soile Juvonen TTT

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BORRELIATESTIT

ViestiKirjoittaja soijuv » To Tammi 22, 2009 21:24

Uusi DNA-testi sekä B.burgdorferin että B.miyamotoin testaamiseen. Testi maksaa n. 120€.

Testiä voi tilata osoitteesta: http://www.dnalymetest.com/home.html
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Borrelioosin laboratoriodiagnostiikka

Soile Juvonen TTT

Borrelioosin laboratoriodiagnostiikka perustuu ensisijaisesti serologiaan. Viljely ja PCR ovat erityistutkimuksia, jotka eivät sovellu seulontaan. Punkin/verta imevän hyönteisen puremakohdan ympärille saattaa kehittyä nopeasti laajeneva rengasihottuma, erythema migrans (EM) - se kehittyy kuitenkin vain noin 50 %:lle ihmisistä. EM on ehdoton hoitoindikaatio ilman muuta taudin varmistusta. Taudin myöhäisoireet voivat ilmaantua viikkojen, kuukausien tai jopa vuosienkin kuluttua tartunnasta, yleisimmin hermoston, nivelten, ihon tai sydämen taholta.

Borreliabakteerin kuten muidenkin mikrobien aiheuttamat infektiot käynnistävät elimistössä ns. immuunivasteen, joka ilmenee vasta-aineiden muodostumisena. Vasta-aineiden esiintymistä tutkitaan borrelioosissa useimmiten ELISA (EIA) ja/tai Western Blot (immunoblottaus)-menetelmillä. Yleensä sairauden alkuvaiheessa IgM -luokan vasta-aineet lisääntyvät ja IgG -luokan vasta-aineet ilmaantuvat vasta myöhemmin. Useimmiten infektiotautien kohdalla IgM -luokan vasta-aineiden osoittaminen riittää akuutin infektion toteamiseen, mutta IgG -vasta-ainepitoisuus on harvoin riittävä osoitus tuoreesta infektiosta. Joissakin taudeissa (esim. vatsan helikobakteeri) pelkkä vasta-aineiden esiintyminen tarkoittaa kroonista infektiota. Näin ei ole ainakaan vielä tällä hetkellä borrelioosin kohdalla.

Vasta-ainereaktioon perustuvat menetelmät eivät ole osoittautuneet borrelioosin diagnostiikassa kovinkaan luotettaviksi. Siihen on useita syitä. Borreliabakteeri hakeutuu nopeasti jo varhaisvaiheessa sellaisiin paikkoihin, esim ihon tai sydämen kollageeni, limakalvot, aivot jne, joissa sen kontakti immuunijärjestelmään on vähäistä ja näin ollen myös vasta-ainereaktio on heikko. Borreliabakteerin on myös havaittu muuntuvan sellaisiin muotoihin joissa immuunijärjestelmä ei tunnista sitä. Vasta-ainetestit eivät ole myöskään herkkiä toteamaan erilaisia borreliabakteerin alalajeja, joten vääriä negatiivisia tuloksia voi esiintyä.

"Borreliosis is difficult to diagnose by serologic evaluation and Western blot interpretation. In our patient, no intrathecal antibodies were produced to support clinical suspicion of disease. The low antibody titers could be attributed to antigenic variation between /B. valaisiana/ and /B. burgdorferisensustricto/, which was used as antigen because no commercial kit is specific for /B. valaisiana/. Differences between the strain causing infection and the antigen may play a role in the false-negative results.
http://wwwnc.cdc.gov/eid/article/10/9/0 ... rticle.htm

Slovakia 2010.
32 potilasta. Kaikilla ELISA negatiiivinen mutta Western blot positiivinen. Kaikilta kroonisia oireita sairastavista tulee ottaa Wb vaikka ELISA on olut negatiivinen. Negatiivinen vasta-ainetulos on tutkijoiden mukaan todennäköisesti immuunipuutoksesta johtuvaa.

Suomessa tehdään ensin vain ELISA. Mikäli se on negatiivinen jatkotutkimusta ei tehdä. JAtkotutkimus tehdään yleensä vain mikäli ELISA on ollut positiivinen.
Useimmilla Suomessa negatiivisen testituloksen saaneista, Saksan, BCA klinikan testit ovat kuitenkin olleet positiiviset ja sen lisäksi immuunipuolustuksesta kertova CD 57 erittäin alhainen. Se saatttaa osaltaan selittää Suomen testien virheellisen negatiivisuuden.


Bratisl Lek Listy. 2010;111(3):153-5.

Our experience with examination of antibodies against antigens of Borrelia burgdorferi in patients with suspected lyme disease.
Durovska J, Bazovska S, Ondrisova M, Pancak J.



Bakteeri voi levitä jo varhaisvaiheessa esim. ihomuutosvaiheessa (erythema migrans) eri puolille elimistöä:

http://www.terveysportti.fi/xmedia/duo/duo94555.pdf

Borrelia burgdorferin DNA:ta erythema migrans -potilaiden ihossa, veressä ja virtsassa
Italialaisessa tutkimuksessa selvitettiin geenimonistustekniikalla Borrelia burgdorferin DNA: n esiintymistä iho-, veri - ja virtsanäytteissä.

Kaikilla 30 potilaalla DNA:ta löytyi ainakin yhdestä näytteestä. Seitsemällä potilaalla sitä löytyi kaikista, 16 potilaalla iho- ja verinäytteestä, kahdella ihosta ja virtsasta ja viidellä vain ihosta.
Tulos viittaa siihen, että Lymen borrelioosissa, joka on monen elimen sairaus, taudinaiheuttaja voi levitä jo varhain paikallisen ihoinfektiokohdan ulkopuolelle. (Acta Derm Venereol 2004;84:106)



Seuraavan tutkimuksen mukaan (WienKlinWochenschr (2002) 114/13-14: 601-605) sellaisilla potilailla joilla oli eläviä spirokeettoja elimistön nesteissä (esim. nivelissä, selkäytimessä), esiintyi alhaisia tai negatiivisia veren vasta-ainepitoisuuksia: "There is no correlation between the level of antibodies (ELISA), the number of protein bands (Western blot) and the presence of spirochetes in body fluids (culture and PCR?Lymeborreliosis patients who have live spirochetes in body fluids have low or negative levels of borrelial antibodies in their sera"

http://www.springer.at/periodicals/full ... 13_601.pdf

Vasta-aineisiin perustuvien testien heikkoutena on myös se, että ne eivät välttämättä kykene kertomaan infektion todellista aiheuttajaa esim. ristireaktioiden vuoksi (infektio on aiheuttanut aiemmin sairastettujen mikrobitautien vasta-aineiden muodostumisen). Borrelioosissa ristireaktioita voivat aiheuttaa mykoplasma, Epstein-Barr -virus jne. Todellisen infektion aiheuttajaa ei sen vuoksi ole aina helppo määrittää ? toisaalta kyseessä voi olla myös useiden mikrobien aiheuttama sekainfektio kuten borrelioosia sairastavien kohdalla on usein esitetty.

Negatiivinen vasta-ainetulos voi johtua myös siitä että henkilön vasta-aineiden tuotanto on heikentynyt esim. jonkin muun perustaudin, kroonisen infektion, lääkityksen (esim. antibiootit, immunosuppressiiviset lääkkeet kuten kortisoni jne) vuoksi.  Eri laboratorioista saadut vasta-ainemääritykset saattavat myös poiketa toisistaan hyvin paljon, sillä testit eivät ole standardoituja. Lisäksi laboratoriot määrittelevät itse mikä on diagnostisesti merkitsevä vasta-ainepitoisuuden nousu.

Testien epäluotettavuudesta on ilmestynyt vuosien aikana kymmeniä julkaisuja. Serologisia testejä tulisikin pitää ainoastaan muita diagnostisia menetelmiä täydentävänä, eikä pääasiallisena diagnostisena menetelmänä.

Kansanterveyslaitoksen mukaan: "Borrelioosin diagnoosi on kliininen, anamneesi on tärkeä. Tueksi voidaan käyttää joitakin epäspesifisiä laboratoriolöydöksiä ja borreliavasta-aineiden mittausta. Taudin varhaisvaiheessa vasta-ainetuotanto on heikkoa ja vasta-ainepitoisuuksien muutokset hitaita. Seronegatiivisia tapauksia voi esiintyä taudin kaikissa vaiheissa. Matalan tiitterin vääriä positiivisia tuloksia esiintyy mononukleoosia, LEDiä tai reumaa sairastavilla. Kuppa voi aiheuttaa ristireaktion."

(http://www.ktl.fi/portal/suomi/julkaisu ... loydokset/ )

Huslabin: mukaan "Negatiivinen vasta-ainemäärityksen tulos ei siten sulje pois Borrelia burgdorferi -infektiota. Neuroborrelioosin diagnosoinnissa intratekaalisen vasta-ainetuotannon toteamisen herkkyys flagella- antigeenilla on noin 60 %. Arviolta 10 % tapauksista tulee esille seulonta-antigeenillä, vaikka intratekaalista vasta-ainetuotantoa ei päästä osoittamaan. Kokonaisherkkyys on siten noin 70 %."

http://www.huslab.fi/ohjekirja/3975.html
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Lymen borrelioosin diagnoosi on kliininen, jota laboratoriotutkimukset tukevat. Serologia on edelleenkin borrelioosin laboratoriodiagnostiikan kulmakivi. Siinä ei kuitenkaan saisi rajoittua vain yhden menetelmän käyttöön. Flagelliinia käyttävät menetelmät ovat kaupallisista testeistä luotettavimpia. Varhaisborrelioosi on diagnostinen ongelma, johon uudet antigeenit eivät ole tuoneet ratkaisua. Vasta-aineet voivat jäädä kehittymättä myös myöhäisborrelioosissa, ja taudilla on taipumus olla sitä vaikeampi mitä heikompi immuunivaste on. Jos diagnoosi perustuu pelkkään serologiaan, voivat juuri eniten hoitoa tarvitsisivat potilaat jäädä hoidotta.

http://www.ktl.fi/portal/suomi/julkaisu ... ko_maassa/

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http://www.ktl.fi/portal/suomi/julkaisu ... loydokset/

Borrelioosin diagnoosi on kliininen, anamneesi on tärkeä. Tueksi voidaan käyttää joitakin epäspesifisiä laboratoriolöydöksiä ja borreliavasta-aineiden mittausta. Taudin varhaisvaiheessa vasta-ainetuotanto on heikkoa ja vasta-ainepitoisuuksien muutokset hitaita. Seronegatiivisia tapauksia voi esiintyä taudin kaikissa vaiheissa.



http://www.punkki.net/artikkelit/lymenb ... tml#diagno

LB:n diagnoosi on aina perustaltaan kliininen. Perusteellinen anamneesi eli keskustelu potilaan kanssa oireiston alkamistavasta sekä siihen liittyvistä asioista jne. on borrelioosidiagnostiikan kulmakivi.



http://www.therapiafennica.fi/wiki/inde ... n_kannalta

Lymen taudin diagnostiikassa ovat anamnestiset tiedot mahdollisesta borreliatartunnasta ensiarvoisen tärkeitä:

Erythema migransissa ei tarvita lainkaan lisätutkimuksia vaan ihottuma hoidetaan toteamisen jälkeen. Myöhäisvaiheissa ovat sekä erotusdiagnostiset tutkimukset, tautiprosessin kuvantaminen sekä aktiivisen infektion osoittaminen paikallaan ennen hoitopäätöksen tekoa.

Serologia on siis edelleen tärkein diagnostinen keino, aiheuttaen samalla hyvin suuria tulkintaongelmia. Negatiivinen tulos ei sulje pois Lymen artriittia.

Lymen tauti on seronegatiivinen ainoastaan aikaisessa erythema migrans-vaiheessa; myöhemmin taudin kuluessa tapahtuu käytännössä aina serokonversio, joten käytännössä [b]seronegatiivista myöhäisborrelioosia ei ole olemassa, vaan "Lymen tauti on seronegatiivinen vain koska serologinen tekniikka on puutteellinen."



http://oyslab.fi/ohjekirja/3552.html

Negatiivinen vasta-ainetulos ei poissulje tartuntamahdollisuutta.



http://notes.helsinki.fi/halvi/tiedotus ... enDocument

LB:n diagnoosi on kliininen, mutta etenkin muissa kuin iho-oireisissa taudeissa vasta-aineiden mittaukseen perustuvia serologisia menetelmiä käytetään varmistamaan diagnoosi. Nykyiset vasta-aine testit käyttävät antigeeneina flagella- ja ns. kokosoluvalmisteita. Nämä antigeenit eivät kuitenkaan ole diagnostiikassa eivätkä taudin hoitovasteen seurannassa riittävän herkkiä ja tarkkoja. Alkuvaiheen EM-ihottuman aikana jopa 60%:lta potilaista ei nykymenetelmin voida todeta borrelia-vasta-aineita. Myöhemmin
infektion aikana vasta-aineita voi olla vähän, niiden ilmaantuminen verenkiertoon voi viivästyä tai jopa puuttua kokonaan.
Tutkimuksen tarkoituksena olikin tuottaa ja testata uusia antigeeneja Lymen borrelioosin serologiassa.

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Huslab. Seppälä

-"Kun EM on kliinisesti tyypillinen, ei tarvita vasta-
aineiden mittausta.

- Negatiivinen vasta-ainemäärityksen tulos ei silti sulje pois Borrelia burgdorferi -infektiota.

-Voi olla uskallettua luottaa, että yksi VlsE-antigeeni
toteaisi kaikkien infektioiden kehittämät vasta-aineet

-Kaupallisilla firmoilla voi olla kolmen Euroopassa
esiintyvän Borrelian proteiinit mukana"

[/b]
Tutkimus

S ?BorrAbG
S ?BorrAbM
S ?VlsEAbG
S ?VlsEAbM

Borrelia burgdorferi, IgG vasta-aineet
Borrelia burgdorferi, IgM vasta-aineet
Borrelia burgdorferin VlsE- proteiini, vasta-aineet IgG
Borrelia burgdorferin VlsE ja OspC proteiinit, vasta-aineet IgM

IgG- ja IgM-vasta-aineiden seulontavaiheen mittaus entsyymi-immunomenetelmällä (EIA), antigeenina Borrelia afzelii-kanta, johon on IgG-vasta-ainemittauksessa lisätty myös Borrelia burgdorferi ryhmän bakteerien VlsE-antigeenia. Seulontavaiheessa positiivinen tulos varmennetaan mittaamalla vasta-aineet myös toisella antigeenillä (IgG-vasta-aineet spesifisesti VlsE yksinään, IgM-vasta-aineet VlsE + OspC-proteiiniyhdistelmällä).

Noin 60% varhaisvaiheen infektioista (erythema migrans) tulee esille. Myöhempien taudin vaiheiden toteaminen noin 80-95% herkkyydellä.

Viitearvot:

S ?BorrAbG: <9, harmaa alue 9-11, positiivinen >11.
Yksikkö VE/ml

S -BorrAbM: <9, harmaa alue 9-11, positiivinen >11.
Yksikkö VE/ml

S ?VlsEAbG: <10, harmaa alue 10-15, positiivinen >15.
Yksikkö AU/ml

S ?VlsEAbM: <18, harmaa alue 18-22, positiivinen >22.
Yksikkö AU/ml

Seulontavaiheen testit S ?BorrAbG ja S ?BorrAbM ovat herkkiä, mutta ei täysin spesifisiä osoittamaan Borrelia burgdorferi-infektiota.
Varmistuksessa käytetyt S ?VlsEAbG ja S ?VlsEAbM tutkimukset suoritetaan vain seulonnassa positiivisille näytteille. Viimemainittujen tulos on huomattavan spesifinen Borrelia burgdorferi ryhmän bakteereiden aiheuttamille infektioille.

Jos vain seulontavaiheen testi osoitti positiivisuutta, tulee tulkinnassa ottaa huomioon ristireaktiivisen immuniteetin mahdollisuus muun immuunireaktion aiheuttamana. Tyypillisiä ristireaktion aiheuttajia IgG-vasta-ainemäärityksessä ovat syfilis ja lievemmin tuberkuloosi, IgM-vasta-ainemäärityksessä erityisesti infektiöösi mononukleoosi ja primaarinen sytomegalo- tai herpes simplex-virusinfektio.

Jos reaktion spesifisyyden selvittäminen on kliiniseltä kannalta tärkeää, suosittelemme Borrelia burgdorferi-vasta-aineiden jatkotutkimusta S ?BorAbJt (no 8529), missä vasta-aineet mitataan rekombinanttiantigeeneja käyttävällä EIA-menetelmällä sekä Line Blot-tyyppisellä immunoblottimäärityksellä. Lisäksi voi olla hyödyksi poissulkea syfilis S -TrpaAb (KL 4942) testillä.

IgG-vasta-aineita Borrelia burgdorferi-bakteeria kohtaan syntyy puutiaisen pureman välityksellä saadun infektion jälkeen yleensä kolmen kuukauden kuluessa. Erythema migransin alkuvaiheessa testi on yleensä vielä negatiivinen.

Myöhäisoireiden yhteydessä (meningiitti, artriitti, acrodermatitis chronica atrophicans) testi on useimmiten positiivinen, jos Borrelia burgdorferi on taudin aiheuttajana. Negatiivinen vasta-ainemäärityksen tulos ei silti sulje pois Borrelia burgdorferi -infektiota.

Vasta-aineet häviävät hoidon jälkeen yleensä niin hitaasti, että vasta-ainetuloksia ei voida käyttää luotettavana osoituksena Borrelia burgdorferi ryhmän bakteerien häätymisestä.
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Borrelia burgdorferi ?infektion diagnostiikka

Ilkka Seppälä
HUSLAB
Kliininen mikrobiologia
Immunologian osasto
24.11.2009

Borreliaviljelyn positiivinen saalis on edelleen niin
satunnaista, että menetelmää ei kannata käyttää
rutiinidiagnostiikkaan

Borrelian osoitus PRC-menetelmällä on sinänsä
toimivaa ihobiopsiasta, nivelnesteestä ja
selkäydinnesteestä.
Vain ihossa bakteeri viihtyy pidempään ja osuu
säännöllisesti nukleiinihappodiagnostiikan
näytteeseen.


Serologinen osoitus on siten edelleen tärkeää ja
herkin infektion osoitusmenetelmä

B. burgdorferi -bakteerin kasvattaminen on
hyvin rajoitettua
Saalis on minimaalisen pieni verrattuna saman
elatusainevolyymin kykyyn kasvattaa esim
koliformeja bakteereita

Rekombinanttiantigeeneilla saadaan
massatuotanto aikaiseksi

Genomin sekvensointi antaa avun
rekombinanttien etsintään

Western blot-määritykset
kokobakteeriantigeeneilla ovat olleet
ohjaamassa kohteiden valintaa.

Hyödyllisiä antigeeneja

VlsE

Viljelty bakteeri ei ekspressoi VlsE-antigeenia!

Variable Lipoprotein Surface Expressed

Proteiinilla on vakiorunko, johon kuuteen
kohtaan otetaan geeninkonversiolla
varastokaseteista geeninpätkiä.

Osin myös sallitaan tuntemattomalla
mekanismilla yksittäisten nukleotidien
mutaatioita ilman tunnistettavan templaatin
käyttöä

Ct-peptidi = B. burgdorferi sensu stricto kannan VlsE-proteiinin C-
terminaalisen vakio-osan peptidi
Ei toimi Euroopan borreliainfektioissa

IR6 = kasettivarastosta otettu 26-28 aminohapon mittainen
peptidisekvenssi
Vaihtelee kannan sisällä varaston sisällöstä otettuna.
Vaihtelee hieman lajien välillä, mutta kasettivarasto on varsin
samanlainen eri B. burgdorferi ryhmän lajeilla. Proteiinin runko sen
sijaan vaihtelee enemmänkin.

C6 = kaupallinen IR6-peptidiin perustuva vasta-ainemääritys
Käyttää B. garinii -peräistä sekvenssiä

Muiden kaupallisten kittien VlsE

Osin vaikeaa saada selville, mitä kitti sisältää
Voi olla VlsE-peptidi tai melkein kokonainen
proteiini, liikesalaisuuksia

Kaupallisilla firmoilla voi olla kolmen Euroopassa
esiintyvän Borrelian proteiinit mukana

Voi olla uskallettua luottaa, että yksi VlsE-antigeeni
toteaisi kaikkien infektioiden kehittämät vasta-aineet


Jotensakin varmasti infektoineen kannan VlsE-
ekspressio on ainakin osittain erilainen kuin firman
myymä antigeeni

Tietyn näytteen vasta-aineiden vahvuusaste voi
poiketa eri valmistajien testiä käytettäessä
Raja-arvotapauksissa tulos voi vaihdella reagenssista
riippuen


Alkuperäinen Immunetics C6-testi mittaa
kaikkien vasta-aineluokkien vasta-aineet

Myöhemmin markkinoille tulleet kitit erottelevat
IgG- ja IgM-vasta-aineet

Useissa vasta-ainekiteissä on yhdistetty
bakteeriekstrakti rekombinantti-VlsE-
antigeeniin.
HUSLAB käyttää tällaista seulontaan

Erythema migrans-vaiheen infektio (EM) on ollut
vasta-ainetesteissä n. 40% positiivinen
bakteeriekstraktiantigeeeneilla.

VlsE-antigeenilla n. 60-70% herkkyys EM-vaiheessa

Kun EM on kliinisesti tyypillinen, ei tarvita vasta-
aineiden mittausta

Puolet EM-tapauksista voi olla epätyypillisiä tai jäädä
havaitsematta


Sekundaarivaiheen taudissa VlsE-vasta-ainemittaus
on hyödyllistä, yli 90 % herkkyys.
Spesifisyys oireisilla potilailla noin 96% (→100% ??)

OspC-antigeeni

Antigeenin ekspressio alkaa, kun puutiainen syö veriaterian,
samalla OspA-antigeenin ekspressio vähenee

OspC ohjaa borreliaa puutiaisen sylkirauhaseen

OspC vaihtelee, noin 14 ryhmää ja vielä lisää tyyppejä

C-terminus on vakiorakenteinen;
immunogeeninen ihmisellä IgM-vasta-aineiden tuotantoon, mutta
ei immunogeeninen hiirille

Runko-osalle voi tulla IgG-luokan vasta-aineita, jotka ovat uutta
infektiota estäviä hiirimalleissa.

Tyyppien runsauden vuoksi OspC:n IgG-luokan vasta-aineita
päästään harvoin mittaamaan

OspC on IgM-luokan vasta-aineiden pääkohde
ihmisellä
(flagella toinen merkittävä kohde)

OspC-IgM-vasta-ainetuotanto voi jäädä päälle infektion
jälkeen

Ei tiedetä miksi ja miten?

Ristireaktiivinen epitooppi muiden infektioiden
agenssien kanssa?

HUSLAB:in käyttämä VlsEAbM ?nimike sisältää sekä
VlsE-IgM:n että OspC-IgM:n kaupallisena paketointina

Oireettomien henkilöiden VlsE-vasta-
ainepositiivisuuden spesifisyys on epävarmaa pätevän
standardin puutteen vuoksi

Infektion insidenssistä voidaan arvioida, että
oireettomien VlsE-vasta-ainepositiivisuuksien taustalla
voi usein olla B. burgdorferi ?kontakti

Yleensä nähdään, että hoidon jälkeen IgM-luokan
vasta-aineet VlsE-antigeenille laskevat ensin ja sitten
parin vuoden sisällä IgG-luokan vasta-aineet laskevat
myös huomattavasti, usein viitealueellekin
Samalla bakteeriekstraktin vasta-aineet jäävät
positiivisiksi

Immunoblottitutkimukset

Luotettavaan serologiseen diagnostiikkaan tarvitaan vasta-
aineiden osoitus ainakin kahta mikrobin komponenttia
kohtaan. Yhden komponentin vasta-aineet voisivat olla
satunnaisen ristireaktion seurausta

Kaupallisesti on saatavilla liuskoja sekä Western blot-
tutkimuksiin bakteeriekstraktiantigeeneilla että Line Blot-
määrityksiin rekombinanttiantigeeneilla.
Testissä voi olla 7-15 antigeenikohdetta joko yhdestä
borrelialajista tai useammasta

Borrelialla on tapahtunut geenien siirtoa lajista toiseen. Siten
esim. OspC-proteiinin vasta-aineet mitattuna B. afzelii -
peräiselle antigeenille ei tarvitse merkitä sitä, että B. afzelii
olisi ollut aiheuttamassa tämän potilaan infektion.

Selkäydinnesteen vasta-ainemääritys

Intratekaalinen vasta-ainetuotanto voidaan määrittää,
kun tunnetaan seerumin ja likvorin
immunoglobuliinipitoisuudet ja mitataan borreliavasta-
ainepitoisuudet samalla skaalalla

Sitten lasketaan indeksi (>1.5 positiivinen)

Positiivinen indeksi kertoo paikallisen vasta-
ainetuotannon

Se on merkki siitä, että bakteeri on käynyt paikalla ja
jättänyt antigeenia; ei sitä, että bakteeri olisi paikalla nyt

Tulevaisuutta??

Galaktolipidiantigeenit

Kaksi immunogeenista Borrelian glykolipidiä

ACG = Acylated Cholesteryl Galaktoside
MgalD= monogalactosyl diacylglycerol

ACG on nyt onnistuttu syntetisoimaan

Myöhäisen infektiovaiheen merkkivasta-aine
(Jones ym. Clinical Immunology 132:93-102, 2009;
Stübs ym J Biol Chem 284:13326-1334, 2009)
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Tri Jon Sterngold:
"Borreliabakteeri on ainutlaatuinen bakteerien joukossa esim DNA rakenteensa vuoksi. Lukuisat DNA:t mahdollistavat bakteerin selviämisen elimistössä ja pakenemisen immuunipuolustuksen tuhoamisyrityksiltä. Bakteeri kykenee esim. muuntamaan pintaproteiinejaan. Bakteeri kykenee muuntamaan myös muotoaan korkkiruuvimaisetsa muodosta kystamuotoon. Kystamuodossa bakteeri on erittäin vastustuskykyinen.

Antibioottihoidon teho riippuu bakteerin aktiivisuustasosta; miten nopeasti bakteeri kasvaa ja lisääntyy. Useimmat bakteerit (useimmat virtsatieinfektiot, keuhkokuume jne) lisääntyvät alle 24 tunnissa. Tällaisissa infektioissa ihminen saa yleensä nopeasti avun antibiooteista. Borreliabakteerin lisääntymissykli on jopa yhdeksän kuukauden mittainen. Passiivisessa tilassa olevaa bakteeria on erittäin vaikea saada tuhotuksi hoidoilla.

Sen lisäksi, joidenkin tutkimusten mukaan, borreliabakteeri muodostaa muiden taudinaiheuttajien kanssa myös ns. biofilmi-yhteisöjä.
Mikroskoopissa tämä nähdään bakteerien kerääntymisenä geelimäisiin "kasoihin". Tällöin ne ovat suojassa immuunipuolustukselta ja antibiooteilta. Tässä "bunkkerissa" niistä kuitenkin vapautuu (aineenvaihdunnallisen kierron aikana) esim. hermostolle myrkyllisiä aineita. Kyseessä on taistelu elintilasta. Bakteerit voivat tarpeidensa mukaisesti esim. heikentää ihmisen puolustujärjestelmiä tai muuttaa muita elintomintoja. Niiden tarkoituksena ei ole tuhota isäntäelimistöä vaikka sitäkin joskus tapahtuu.

Aivo-, hermo-, sydän-, verisuoni-, tukikudosten-, nivelten jne tulehdusten lisäksi borreliabakteeri pystyy aiheuttamaan ns. autoimmuunisairauksia kuten ALS, lupus, MS jne. Kyseisiä oireita pidetään parantumattomina mutta monissa tapauksissa pitkillä, korkea-annoksisilla antibioottihoidoilla taudit on saatu jopa parannettua.

Borrelioositestit ovat ongelmallisia. Yleensä testeissä mitataan elimistön kykyä muodostaa vasta-aineita borreliabakteeria kohtaan. MUTTA bakteeri kykenee piiloutumaan, muuntumaan, heikentämään elimistön kykyä muodostaa vasta-aineita. SIKSi testitulokset voivat olla täysin negatiiviset vaikkka henkilö sairastaa Borrelioosia. Näemme usein, että esim. vuoden antibioottihoitojen jälkeen immmuunipuolustus viimein kykenee havaitsemaan joitakin bakteerin jäänteitä ja testitulokset muuttuvat positiivisiksi. Potilaalla on kuitenkin ongelmia mikäli häntä hoitava lääkäri luottaa ainoastaan negatiivisiin testituloksiin. Tällöin hän ei saa asianmukaista hoitoa ja kärsimykset jatkuvat.

Tässä vaiheessa erottuvat sellaiset lääkärit, jotka ymmärtävät testeihin liittyvät ongelmat ja hoitoihin liittyvät haasteet niistä lääkäreistä, jotka eivät osaa tai eivät halua ymmärtää bakteeriin/tautiin liittyviä ongemia. Ongelman laajuus ja syvyys on erittäin suuri tänä päivänä."

http://www.willitsnews.com/ci_13453460


--------------------------------------------------------------------------------

"Pohjois Floridan yliopiston professorin, Kerry Clarkin, mukaan borreliatestit kykenevät osoittamaan korkeintaan n. 40% positiivisista Borrelioositapauksista sillä testit osoittavat immuunipuolustuksen kykyä tunnistaa bakteeri eivätkä sitä onko elimistössä borreliabakteereita esim. piiloutuneena jänteisiin, keskushermostossa jne.

Tri Clark on nyt kehittänyt testin joka perustuu itse borreliabakteerin DNA:n löytämiseen vasta-aineiden muodostumisen sijaan. Hän on tutkimuksissaan jo huomannut, että Borrelioosia esiintyy esim. Floridassa huomattavsti aiempia arviointeja enemmän."


"Tick-borne Disease Research Area - Please Do Not Enter," the sign says on the front door of Kerry Clark's University of North Florida office.

If that's not enough of a deterrence, there are always the photographs of Florida's three most common tick species blown up to larger-than-life proportions.

But it's worth poking inside the seemingly menacing door if only to meet Clark and listen to his story.

"It's like a great mystery," Clark said.

The villain of his story is Lyme disease, a poorly understood illness that's spread by tick bites to tens of thousands of Americans each year. After a decade of paltry funding and suffering countless tick bites himself, the 40-year-old epidemiology professor has reached a scientific breakthrough that stands to revolutionize the way doctors diagnose and treat Lyme.

In addition, his toil has revealed an unsettling message for the people of Florida and other parts of the South: Lyme-carrying ticks are spreading the illness here at vastly higher rates than what public health statistics and experts have suggested.

Disease's spread

Lyme disease follows a perplexing arc that begins with a bull's eye-shaped rash and vague, flu-like symptoms. Without treatment, Lyme digs in deep, progressing to potentially disabling effects, like severe arthritis, fatigue, numbness in the hands or feet and neurological problems.

The vast majority of the more than 265,000 cases of Lyme disease reported since 1993 have come from the Northeast and upper Midwest.

That's a conservative number. Scientists think there are seven to 12 cases for each one that is reported. And even that dire-sounding estimate may be too low. Only about 40 percent of positive cases are getting detected by traditional diagnostic tools, which test the body's reaction to the Lyme bacteria, Clark said.

Clark thinks that his test, which involves looking for Lyme's DNA in the victim's blood, is a more accurate way of detecting the disease.

For many, an inaccurate test is a life-changer.

Caught early, the Lyme bacteria usually can be wiped out with antibiotics. But many cases go undetected for years because people, though sick, often don't know they've been bitten by a tick or don't develop the tell-tale rash.

Not safe in the South

People like Dane Boggs. For a decade, Boggs, a home builder, felt tired all the time and his joints hurt. But his symptoms were mild, so he figured they were merely the side effects of getting older.

Things got worse, though, after he was bitten by a tick on a job site in Atlantic Beach five years ago. He now thinks that his previous decade of troubles were caused by a tick bite that went unnoticed.

The double whammy of bites nearly crippled him, he said.

"My immune system was kind of fighting it off for 10 years, but when I got bit [the second time], that's when I got super-sick," Boggs said. "I just wanted to go to bed all the time. It was like an 18-wheeler ran over my body."

The Ponte Vedra Beach man retired early to devote all his time to fighting the illness. He took powerful antibiotics for two years with little improvement. So he turned to an alternative therapy that uses electrical frequencies to zap microscopic invaders like Lyme disease.

Today, the 55-year-old is healthy, though he cautions his results from the alternative treatment probably aren't the norm. After his battle, Boggs co-founded a research and support organization called the Northeast Florida Lyme Association.

"Nobody even believes Lyme disease is in Florida. But it does exist, and a lot of people are sick," said Boggs, who has found a sympathetic ear and a NEFLA board member in Kerry Clark.

Finding new strains

Clark's research has revealed that Lyme disease is much more common in Florida than previously known.

State disease-surveillance efforts confirmed 88 cases last year, 11 of which are believed to have originated in the state. But Clark has found the Lyme bacteria in virtually every corner of the state, including hordes on the First Coast.

The perception that the South doesn't have a Lyme problem has biological roots.

In the Northeast, mice are the primary reservoir of the Lyme bacteria, known among scientists as Borrelia burgdorferi sensu lato. But in the South, lizards are ticks' prime target. And since studies in California showed that reptiles were poor reservoirs, many scientists concluded that the South was relatively safe.

But Clark's studies of lizards in South Carolina and Florida revealed that 54 percent were positive for Lyme disease. That research petered out because of a lack of funding - a frequent complaint of Clark's - but it led him to perfect what he believes to be the most sensitive testing method yet for the disease.

Lyme disease is hard to detect in lizards because their blood is highly concentrated with their own DNA, overwhelming the genetic tidbits of any other organisms that might be in their systems. By applying the same amplifying methods he developed for lizard samples, Clark started getting positive readings in human samples that had previously tested negative.

Clark put his theory to the test on 150 blood and skin samples collected from patients suspected of having Lyme disease.

Forty-four percent came back positive, including 20 of the 49 samples from Florida.

What's more, for the first time anywhere in the United States, he found two additional strains of Lyme disease in humans: Borrelia andersonii and another that has not yet been named.

At least five strains of Lyme are known to infect animals and ticks, but researchers had never seen more than one in humans, Clark said. Most diagnostic tests were only developed to detect one Lyme strain. So if more are infecting humans, Clark thinks, that may explain why they have such a high error rate.

A paper detailing his findings is in review with the Journal of Clinical Microbiology.

Andrea Varela-Stokes, a parasitologist at Mississippi State University, said she is intrigued by Clark's research. She called the understanding of Lyme in the South a "tricky situation" because scientists have been unable to grow the Lyme bacteria in laboratory cultures from sick patients.

Although Clark ran into the same problem, he thinks he's had a breakthrough.

"I think the paper is a really big deal," he said. "One of two things is going to happen: They're going to say, 'This is that weirdo who did all that lizard stuff.' Or they're going to say, 'Why didn't we do that?' "

jeremy.cox@jacksonville.com, (904) 359-4083

http://www.jacksonville.com/lifestyles/ ... _mysteries
------------------------------------------------------------------
CD 57 -testi:

Krooninen borreliabakteerin aiheuttama infektio heikentää immuunipuolustusta. HIV-viruksen tavoin borreliabakteeri vaikuttaa kaikkiin immuunipuolustuksesta huolehtiviin soluihin mutta erityisesti tappajasoluihin, kuten CD 57.

CD 57-testi osoittaa kuinka aktiivinen infektio on tutkimushetkellä. Aktiivisessa vaiheessa CD 57 -arvo on yleensä alhainen (usein jopa alle 60, >200 on normaali).

http://www.healthcentersofamerica.com/i ... cfm?id=144

Lyme CD57 Test
THE CD-57 Striker Panel Test

Our ability to measure CD-57 counts represents a breakthrough in Chronic Lyme Disease treatment. It can be used to help determine how active the infection is, how well the treatment is working, and whether, after treatment ends, a relapse is likely to occur!

This is how it works: Chronic Lyme infections are known to suppress the immune system. The Lyme spirochete can affect all major cell types of the immune system, but it most clearly can impact a specific subset of the natural killer cells. This is called the CD-57 subset. Just as in HIV infection, which suppresses T-cell counts, Lyme suppresses Natural killer cell count such as CD57. As in HIV infection, where abnormally low T-cell counts are routinely used as a marker of how active the infection is, in Lyme we can use the CD-57 count to indicate how active the Lyme infection is. When Lyme is active, the CD-57 count is suppressed. We currently are having our tests run by LabCorp because published research on this test was based on their methods. At this lab, the expected range for the CD 57 count is above 60. However, in the chronic Lyme patient, CD-57 counts are usually well below 60 and may be at risk with levels of 60-100.


This test can be run at the start of therapy, then every several months to document the effectiveness of treatment. One hopes to see a stable number or a rising trend over time. When antibiotic therapy is finally at an end, if the CD-57 count is not above 60, then a Lyme relapse is more likely to occur.

Test interpretation: Low CD57 occurs in chronic Lyme or when the disease has been active for over 1 year. A review of the affects of other infections, only Lyme spirochetes lowers the CD57. Following is the criteria established by research.

Test interpretation: Low CD57 occurs in chronic Lyme or when the disease has been active for over 1 year. The count reflects the degree of infection. It is not a diagnostic test but is used as a marker for Lyme being active. Test done by LabCorp.
# >200 is normal
# < 20 severe illness
# 0-60 is seen in chronic Lyme disease
# > 60 Lyme activity indicates improvement

http://www.anapsid.org/lyme/strickerpanel.html

Another test gaining popularity amongst physicians treating Lyme patients is one of the antigen tests, CD-57. For information on the use of the test Lyme patients, please read Long-Term Decrease in CD-57 Lymphocyte Subset in Patients with Chronic Lyme by Stricker, Burrascano and Winger. Your physician can order the Stricker NK Panel CD-57 from Labcorp (who recently purchased IDL, the lab that developed this particular test). If your physician or lab has questions about this test, they can call LabCorp at 510-635-4555. As of this writing, this test was not yet on Labcorp's website.

KAIKKI MITÄ OLET IKINÄ HALUNNUT TIETÄÄ CD-57 - TESTISTÄ:

EVERYTHING YOU ALWAYS WANTED TO KNOW ABOUT THE CD57 TEST

By: GINGER SAVELY, RN, FNP-C

From coast to coast, frustrations abound among patients and
clinicians regarding the diagnosis of chronic Lyme disease.
Misinformed health care providers in Texas and surrounding states
consider the infection rare and non-endemic. They are inclined to
rule out Lyme disease based on the negative result of a laboratory
test that, unbeknownst to them, is highly insensitive. In the absence
of a reliable laboratory test or adequate experience in the
recognition of the varied and complex presentations of the illness,
most clinicians are ill-equipped to diagnose chronic Lyme disease.
Many patients suffer needlessly for years, hopelessly lost in the
maze of the health care system, looking for answers and enduring the
skepticism of practitioners inexperienced with the disease?s signs
and symptoms.

What is needed is a better Lyme test or some other objective measure
to persuade the practitioner to consider the diagnosis of chronic
Lyme disease. Enter the CD57 test! You may have heard the term ?CD57″
tossed around on chat groups, or your Lyme-literate health care
provider may have even explained the test to you in one of your
moments of brain-fogged stupor. What is this number that sounds more
like a type of Heinz steak sauce than a lab test, and what in the
world does it have to do with Lyme disease?

Let?s start by going back to basic high school biology. You may
remember that white blood cells (a.k.a. leukocytes) are the
components of blood that help the body fight infections and other
diseases. White blood cells can be categorized as either granulocytes
or mononuclear leukocytes. Mononuclear leukocytes are further sub-
grouped into monocytes and lymphocytes.

Lymphocytes, found in the blood, tissues and lymphoid organs, attack
antigens (foreign proteins) in different ways. The main lymphocyte
sub-types are B-cells, T-cells and natural killer (NK) cells. B-cells
make antibodies that are stimulated by infection or vaccination. T-
cells and NK cells, on the other hand, are the cellular aggressors in
the immune system and are our main focus in the discussion that
follows.

Let?s pause a moment and introduce something you probably never
learned about in high school biology class: CD markers. CD, which
stands for ?cluster designation?, is a glycoprotein molecule on the
cell surface that acts as an identifying marker. Think of comparing
cells as comparing people. Humans are made up of innumerable
superficial identifying characteristics (such as hair color, eye
color, etc.) and so are cells. Cells probably have thousands of
different identifying markers, or CDs, expressed on their surfaces,
but 200 or so have been recognized and named so far.

Each different marker (or CD) on a cell is named with a number, which
signifies nothing more than the order in which the CD was discovered.
On any given cell there are many different cluster designation
markers (CDs), giving each cell its unique appearance and function
but also linking certain cells by their similarities (like grouping
all people with brown hair or all people with blue eyes). Cells that
have a certain kind of CD present on their surface are denoted as +
for that CD type (e.g., a cell with CD57 markers on its surface is
CD57+).

NK cells have their own specific surface markers. The predominant
marker is CD56. The percentage of CD56+ NK cells is often measured in
patients with chronic diseases as a marker of immune status: the
lower the CD56 level, the weaker the immune system. You may have
heard Chronic Fatigue Syndrome patients talk about their CD56 counts.
A smaller population of NK cells are CD57+.

A below-normal count has been associated with chronic Lyme disease by
the work of Drs. Raphael Stricker and Edward Winger. No one knows for
sure why CD57+ NK cells are low in Lyme disease patients, but it is
important to note that many disease states that are often confused
with chronic Lyme (MS, systemic lupus, rheumatoid arthritis) are not
associated with low CD57+ NK counts. The good news is that for most
Lyme patients the CD57+ NK level increases as treatment progresses
and health is regained.

CD57 markers can also be expressed on other kinds of cells, including
T-cells, so it is important to distinguish between CD57+ T-cells and
CD57+ NK cells. Clinicians need to be aware that many testing
laboratories claiming to perform the CD57 test are actually looking
at CD57+ T-cells rather than CD57+ NK cells, which are the cells of
interest in chronic Lyme disease.

In order for a testing laboratory to measure the CD57+ NK level, it
first measures the percentage of lymphocytes that are CD57+ NK cells.
Then an absolute count is calculated by multiplying that percentage
by the patient?s total lymphocyte count. The standard normal range
for the absolute CD57 NK count is 60 to 360 cells per microliter of
blood. This wide range was established based upon test results of
hundreds of healthy patients. By these laboratory standards, a test
result below 60 cells per microliter would be considered below normal
and therefore associated with chronic Lyme disease. However, a recent
study of my Austin patients has led me to believe that 100 cells per
microliter is a more reliable threshold separating Lyme patients and
healthy controls.

When Drs Stricker and Winger discovered that CD57+ NK cells are low
in chronic Lyme patients and tend to increase with patients? clinical
improvement, an opportunity arose for Lyme-literate practitioners to
utilize a handy tool to aid in the diagnosis of chronic Lyme disease,
to follow treatment progress, and to determine treatment endpoint.
Just as AIDS patients have always held great store in their CD4 T-
cell count, Lyme patients now have a fairly reliable marker of the
status of their illness.

It is important to remember that the CD57 result is just a number;
far more important is the patient?s clinical status. An old professor
of mine used to say, ?treat the patient, not the lab test!? There is
still much we do not know about the CD57 marker and what other
factors may lower or raise it. However, overall, the CD57+ NK count
is a useful tool in diagnosing and treating chronic Lyme disease in
most patients. As a measure of immune status, it provides an indirect
measure of bacterial load and severity of illness. Furthermore, in a
patient who has a negative or indeterminate Lyme test but is highly
suspect for the disease, the clinician may utilize the CD57+ NK count
as one more piece in the complex puzzle of a Lyme disease diagnosis.

Postscript: If you would like your health care provider to order the
CD57 NK test for you, your blood sample needs to be drawn into an
EDTA tube (lavender top) on Monday through Thursday and sent
immediately to either LabCorp in Burlington, NC, or Clinical
Pathology Laboratories (CPL) in Austin, TX. LabCorp and CPL are the
only two labs that perform this test properly. Quest does NOT. The
LabCorp test code is #505026 and is named HNK1 (CD57) Panel. The CPL
test code is #4886, CD57 for Lyme disease. The test is time-sensitive
and must be performed within 12 hours of collection, so blood should
not be drawn on a Friday or results may be inaccurate.

....................................................................................


KOTITESTI

Artikkelin mukaan testsi on tarkoitettu tuoreen tartunnan toteamiseen. Toisaalta nimenomaan myöhäis-/kroonistuneen borrelioosin diagostiikkaan kaivataan luotettavaa testiä.


Luotettavuus? Mielenkiintoinen lisä joka tapauksessa.


http://www.apteekkishop.fi/TikAlert-Bor ... eoen-1-kpl

Tik´Alert on uusi pikatesti tuoreen borrelioosin tunnistamiseen. Tuote on immunikromatografinen pikatesti, joka mittaa IgM vasta-aineita ihmisen sormenpäänäytteestä.

Testi tulee suorittaa 3 - 4 viikon kuluttua punkin puremasta. Tuote on CE hyväksytty.


http://www.diagnostica.fi/fileadmin/use ... 1_2011.pdf




------------------------------------------------------------------------
Mahdollisen borreliatartunnan voi yrittää selvittää myös punkista itsestään. Punkki irrotetaan ihosta ja lähetetään allaolevien ohjeiden mukaisesti Saksaan laboratorioon tutkittavaksi. Tartunnan riski on sitä suurempi mitä pidempään punkki on ehtinyt imeä verta.

http://www.wallrich.fi/index.htm
Punkin tutkiminen nopeuttaa ja täsmentää tartuntariskin arviointia
Diagnoosin teko on lääkärille infektion alkuvaiheessa hyvin vaikeaa, koska tartunnan mahdolliset oireet ovat sangen moninaisia ja epäspesifisiä. Oireet ilmaantuvat - jos yleensä ilmaantuvat - vasta infektion jo pitkälle edetessä. Osa oireista jää myös huomaamatta.

Ajoissa infektion alkuvaiheessa tehty diagnoosi onkin tärkeää, koska tartunta on vielä silloin suhteellisen helposti hoidettavissa.

Punkissa borrelioita - tartuntariski 10-kertainen
Vaikka Lymen borrelioosiin sairastuu vuosittain tuhansia, on infektioriskiä aliarvioitu. Heidelbergin yliopiston tutkimuksen mukaan tartuntariski on 10-kertainen, jos punkissa on borrelioita
Universität Heidelberg

Etukäteen varmuuden vuoksi määrätyistä antibioottihoidosta kiistellään, koska punkin puremista tartuntaan johtaa vain noin 1 - 3 %. Jos punkki sattuu olemaan borrelioosin kantaja, kasvaa tartuntariski kymmenkertaiseksi eli aina 30 %:iin. Tässä tapauksessa antibioottihoito voi olla perusteltua. Joka tapauksessa tulisi seurata hyvin valppaasti mahdollisen infektion mahdollisia oireita.


BorrelioseCheckin tilaus:
Toimi näin:
Paketoi punkki kirjekuoressa lähetettäväksi. Esimerkkipaketointi: Punkki kääritään pienen paperipalan sisään, käärö laitetaan muovipussiin ("MiniGrip"), ja pussi kirjekuoreen
Soita numeroon 044 - 93 77 643. Saat viitenumeron.
Kirjoita kuoreen osoitteeksi:
JenaGen GmbH
Löbstedter Str. 80
D-07749 Jena / Thüringen

BORRE-FIN #VIITE
Kirjoita kuoren päälle vielä "BORRE-FIN" ja saamasi viite esim. "2888"
Maksa analyysimaksu 38,60 euroa tilille:
SAMPOPANKKI WALLRICH KY/DNA,ANALYYSIMAKSUT
800016 - 71016476 muista viite !
Tuloksen saat 4 - 6 arkipäivässä haluamallasi tavalla

Jos tulos on positiivinen (punkissa on borrelioita), voidaan tutkia borrelioiden genotyypit(alalajit). Tämä analyysi maksaa 16 euroa.
Yhteystiedot

:: wallrich, dna24
Vesa Vallivaara
Merisotilaantori 1 C 26
00160 Helsinki

Puhelin: 044 - 93 77 643

Sähköposti: analysen@wallrich.fi

"TUTKIMUSTULOS yhdestä laitokseen lÄhetetystä punkista:

Pvm 09.06.2008
Toimeksiannon suorittaja
JenaGen GmbH
Toimeksianto
Borrelia burdorferi sensu lato -spesifisen DNA:n tutkiminen punkista

Näytteen laji ja kuvaus:
1 punkki
Näytteenotto toimeksiantajan toimesta
Tutkimusaika 04.06. bis 09.06.2008
Näytteen käsittely:
Punkki pienennetty skalpellilla. Solukkolyseeraus (hajoittaminen) ja geneettisen DNA:n

Näytteitä säilytetään 3 kuukautta laboratoriossa. Tutkimustulokset kohdistuvat vain tutkittuihin näytteisiin

Tutkimusmenetelmä:
Analyysi suoritettiin Gooskensin Gel-PCR:llä. Evaluation of an internally controlled
real-time PCR targeting the ospA gene for detection of Borrelia burgdorferi sensu lato DNA in
cerebrospina fluid. Clinical Microbiology and Infection, 12: 894-900

Tutkimusmetodi:
A-Pintaproteiini-Geenin (137 Bp) monentaminen (40 reaktisykliä)osp A-Pintaproteiini-Geenin (137
Bp) osalta ja erityisesti Lyme-Borreliosiin yhteydessä esiintyviin Borrelia burgdorferi (sensu lato)
ja sen muotoihin B. burgdorferi sensu stricto, B. afzelii, B. garinii, B.valaisiana .

Tulos:
ospA Geenilöytö negatiivinen
Kontrollireaktiot:
Negatiivisyyskontrolli negatiivinen
Positiivisuuskontrolli (B. afzelii) positiivinen

Yhteenveto:
Tutkitusta punkista ei löytynyt Borrelia burdorferi sensu lato ? DNA:ta

Huomautamme kuitenkin, että infektiota ei voi sulkea pois 100 %:lla varmuudella."

................................................................................................

2. Borrelioosin diagnostiikassa on käytössä useita erilaisia laboratoriodiagnostiikkaan perustuvia testejä. Yksi niistä on MELISA- testi.

Viiden suomalaisen verinäytteet tutkittiin MELISA- laboratoriossa. Tutkittavien aiemmat, Suomessa suoritetut, borreliatestit (vasta-ainetesti ELISA) olivat olleet negatiiviset. Viidestä tapauksesta kolmessa MELISA osoitti henkilöillä olevan aktiivivaiheessa olevan Borrelioosin. Kahden henkilön kohdalla suoritettiin tämän jälkeen Suomessa Western blot ? testi ja kummassakin tapauksessa vastauksessa mainittiin: "Tulos viittaa vanhaan immuniteettiin." Eroa saattaakin esiintyä nimenomaan tulosten tulkinnassa. Western blotin kohdalla vasta-aineiden nousun saatetaan tulkita viittaavan vanhaan immuniteettiin kun taas samanaikaisesti tehdyn MELISA- testin mukaan henkilöllä on aktiivi infektio.

Suomea lähinnä olevat MELISA-laboratoriot löytyvät Ruotsista, Norjasta ja Saksasta.

MELISA-testejä on erilaisia. Laboratorion sivuilla mainitaan, että esim. kroonista väsymysoireyhtymää sekä MS-tautia sairastavilta on testeissä löydetty korkeita raskasmetallipitoisuuksia.

Testistä löytyy tietoa ja tutkimustuloksia sivulta
http://www.melisa.org/index.php

"Minkälaisia borrelioositestejä käytetään normaalisti?
Perinteisesti käytetään laboratoriotestejä nimeltään ELISA, jonka spesifisyys on alhainen ja Western blot (immunoblotti) joka on jonkin verran ELISAa luotettavampi. Kummatkin testit mittaavat elimistössä muodostuneiden borreliavasta-aineiden määrää. Ne eivät siis pysty osoittamaan onko elimistössä borreliabakteereita vai ei. Vasta-aineiden muodostumiseen perustuvat testit eivät ole kovin luotettavia. Virheellisiä negatiivisia tuloksia esiintyy usein, erityisesti taudin varhaisvaiheessa. Tästä syystä virhediagnoosien mahdollisuss on suuri. Borrelioosi diagnosoidaan virheellisesti esim. reumaksi, MS-taudiksi, krooniseksi väsymysoireyhtymäksi, fibromyalgiaksi ja moneksi muuksi autoimmunni- tai neurologiseksi sairaudeksi. Tämän seurauksena bakteeri pääsee vapaasti leviämään eri puolille elimistöä aiheuttamaan vakaviakin terveysongelmia."

Miten MELISA-testi eroaa vasta-ainetesteistä?

MELISA-testi ei testaa vasta-aineiden muodostumista vaan borreliabakteerille ominaista solujen immunoreaktiivisuutta joka kertoo taudin aktiivisuudesta. Testi parantaa diagnostiikkaa vahvistamalla sen, onko bakteeri aktiivinen oireisilla henkilöillä. Testistä on hyötyä myös antibioottihoidon tuloksellisuutta arvioitaessa. Vasta-aineita sen sijaan voi esiintyä elimistössä vuosien ajan sellaisissakin tapauksissa joissa hoito on ollut tuloksellista."

Testin hinta on tällä hetkellä (2009): 160 ?.
http://www.melisa.org/borrelia-lyme-disease.php

MELISA - testiä suorittavista laboratorioista löytyy lista sivulta
http://www.melisa.org/melisa-laboratories.php

[b]Näytteitä otetaan myös MELISA - klinikoilla joita on laboratorioita enemmän
http://www.melisa.org/melisa-clinics.php .


Lyme disease is a bacterial infection caused by a spirochete, a type of bacteria, called Borrelia burgdorferi, which is passed to the patient by a tick bite. Lyme disease, also called active lyme borreliosis, has many symptoms, but skin symptoms, arthritis and various neurological symptoms are usually present. Examples include a reddish skin rash, head aches, neck pain, chronic fatigue, fibromyalgia, joint pains, emotional instability and mental confusion.

Unfortunately, standard laboratory testing is often unable to give clear results of whether a patient is infected or not. The standard serological test ELISA has the broadest detection rate but low specificity. Specificity can be improved with the Western Blot. The test with the highest specificity but with a fairly low detection rate is the PCR test. Borrelia infections are usually treated with antibiotics.

Lyme disease is often misdiagnosed as chronic fatigue syndrome, multiple sclerosis, fibromyalgia, rheumatoid arthritis, and many other autoimmune and neurological diseases, which leaves the infection untreated and allows it to further disseminate the organism. If the neurologic form of borreliosis is left untreated for years, it may lead to severe debility of the patient.

The MELISA® technology can now be applied to diagnose active Lyme disease, especially in serologically and clinically unclear cases. To date, hundreds of patients have been tested and successfully treated. Laboratory Center Bremen in Germany does this testing routinely, so please contact Dr Elizabeth Valentine-Thon (evt@laborzentrum-bremen.de) at +49 421 4307 -305/206 if you want to be tested.

Dr Elizabeth Valentine-Thon's article "A novel lymphocyte transformation test (LTT-MELISA®) for Lyme borreliosis" is published in Diagnostic Microbiology and Infectious Disease (scroll to the bottom of the page for abstract and to download article).

Frequently Asked Questions about Lyme disease (Lyme Borreliosis)

What is Lyme disease? Lyme disease, also known as Lyme Borreliosis, is caused by the bacterium Borrelia burgdorferi, which is a spirochete.

How is Lyme disease transmitted to humans? Lyme disease is a vector borne disease and transmitted to humans primarily through the bite of a tick that is infected with this bacterium. An infection with the bacterium is a prerequisite for developing Lyme disease. At the same time, not every infected person will develop the disease.

What are the symptoms of Lyme disease? After an incubation period of two to thirty days, the typical symptoms include fever, headache, fatigue, and a characteristic skin rash called erthema migrans, which often takes a bull?s-eye appearance and is seen in 50-80% of Lyme disease patients. This represents ?Stage I? of the disease. If untreated, infection can spread to the joints, the heart, and the nervous system, causing a large variety of symptoms which may persist over months or years (chronic Lyme).

How is Lyme disease diagnosed? Lyme disease diagnosis is based on symptoms and the possibility of exposure to infected ticks. Laboratory testing on the blood can then be preformed to confirm the infection (presence of antibodies).

How is Lyme disease treated? Patients treated in the early stages of the disease usually recover rapidly and completely with a few weeks of oral antibiotics. Patients treated during the late stages of the disease may require longer antibiotic therapy and may have recurrent symptoms.

What types of standard tests are available for Lyme disease? Conventional laboratory testing includes the serological test ELISA, which has a low specificity, and the Western blot has a higher specificity. Both tests look for Borrelia specific antibodies. These tests can give false negative results, especially in patients with early disease (Stage I) and therefore a misdiagnosis can occur. Lyme disease is often misdiagnosed as chronic fatigue syndrome, multiple sclerosis, fibromyalgia, rheumatoid arthritis, and many other autoimmune and neurological diseases, which leaves the infection untreated and allows it to further disseminate in the organism. If the neurological form of Lyme disease is left untreated for years it may lead to severe debility of the patient.


What is MELISA® and how is it different? MELISA® is a lymphocyte transformation test, which detects not antibodies but cellular immunoreactivity characteristic of active infections of Borrelia burgdorferi. The test improves laboratory diagnosis by confirming active disease in patients with clinical symptoms of Lyme. In addition, this test provides an early marker for successful antibiotic therapy, while antibodies may persist for years in successfully treated patients.

What is needed to use the MELISA®? If you believe you may have Lyme disease, you should immediately contact your general practitioner. In order to perform the MELISA® test for Lyme disease, 20-30 ml of blood along with a special Request Form is needed.

How much does the MELISA® cost and does insurance cover the test? The MELISA® currently costs 160 euro. Many insurance providers will cover the cost of the test along with one round of oral antibiotic treatment; however, you should contact your insurance provider regarding your policy and benefits.

How common is Lyme disease in the UK? There are 500 confirmed cases of Lyme disease in the UK each year according to the Health Protection Agency (HPA). However, this does not necessarily reflect all the cases of the disease. HPA estimated that there could be up to 2,000 new cases occurring every year in the UK.

Who is at risk for Lyme disease? In the UK, Lyme disease is carried by the sheep tick, Ixodes ricinus, which can also feed on deer, birds, and other wild and domestic mammals. The tick is commonly found in woods, heath, and moorland, although it does not live exclusively in these habitats. People who live in the parts of the country where the tick is prevalent are at greater risk.

How can Lyme disease be prevented? Reducing exposure to ticks is the best defense against Lyme disease. If in a wooded area, always wear long pants and a long-sleeved shirt and use insect repellent with 20-30% DEET. Also, remove ticks promptly from clothes and perform a tick check before going indoors. If bitten, remove the tick as soon as possible to reduce the risk if infection.

A novel lymphocyte transformation test (LTT-MELISA®) for Lyme borreliosis
Valentine-Thon E, Ilsemann K, Sandkamp M. Diagn Microbiol Infect Dis. 2006 Jul 27

Diagnosis of active Lyme borreliosis (LB) remains a challenge in clinically ambiguous, serologically indeterminant, and polymerase chain reaction-negative patients. Lymphocyte transformation tests (LTTs) have been applied to detect specific cellular immune reactivity, but their clinical application has been severely hampered by the poorly defined Borrelia antigens and nonstandardized LTT formats used. In this study, we describe the development and clinical relevance of a novel LTT using a validated format (MELISA®) together with well-defined recombinant Borrelia-specific antigens. From an initial screening of 244 patients with suspected Borrelia infection or disease, 4 informative recombinant antigens were selected: OspC (Borrelia afzelii), p41-1 (Borrelia garinii), p41-2 (B. afzelii), and p100 (B. afzelii). Thereafter, 30 seronegative healthy controls were tested in LTT-MELISA® to determine specificity, 68 patients were tested in parallel to determine reproducibility, and 54 lymphocyte-reactive symptomatic patients were tested before and after antibiotic therapy to assess clinical relevance. Most (86.2%) of the 36.9% (90/244) LTT-MELISA® positive patients were seropositive and showed symptoms of active LB. Specificity was 96.7% and reproducibility 92.6%. After therapy, most patients (90.7%) showed negative or markedly reduced lymphocyte reactivity correlating with clinical improvement. This novel LTT-MELISA® assay appears to correlate with active LB and may have diagnostic relevance in confirming LB in clinically and serologically ambiguous cases.

Download article as pdf
Viimeksi muokannut soijuv päivämäärä Ke Huhti 18, 2012 20:19, muokattu yhteensä 17 kertaa
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » To Tammi 22, 2009 21:25

VlsEprotein IR(6) peptidi ja synteettinen C6 peptidi eivät olleet riittävän luotettavia euroopplaisten potilaiden ihomuutosvaiheen diagnostiikassa. (Suomi, Ruotsi)

Int J Med Microbiol. 2009 Jan 8; [Epub ahead of print]

Antibody responses to borrelia IR(6) peptide variants and the C6 peptide in Swedish patients with erythema migrans.

Tjernberg I, Sillanpaa H, Seppala I, Eliasson I, Forsberg P, Lahdenne P. Department of Clinical Chemistry, Kalmar County Hospital, S-391 85 Kalmar, Sweden; Division of Infectious Diseases, Department of Clinical and ExperimentalMedicine, University of Linkoping, Linkoping, Sweden.

The aim of this study was to evaluate the antibody responses to different VlsEprotein IR(6) peptide variants and the synthetic C6 peptide in acute andconvalescent (2-3 and 6 months) serum samples from Swedish patients withclinical erythema migrans (EM). Serum samples were prospectively collected from148 patients with EM and compared to serum samples obtained from 200 healthyblood donors. The IgG responses to 3 IR(6) peptide variants originating fromBorrelia burgdorferi (B. burgdorferi) sensu stricto, B. garinii, and B. afzeliiwere measured by enzyme-linked immunosorbent assays (ELISAs) and compared to acommercial C6 peptide ELISA. Seropositivity rate in the IR(6) or C6 peptideELISAs ranged from 32% to 58% at presentation, 30-52% after 2-3 months, and20-36% after 6 months. At presentation, positive antibodies in any of the 4ELISAs were found in 66%. In 7/52 (13%), C6-negative EM cases, serologicalreaction was found to the B. burgdorferi sensu stricto-derived IR(6) peptide. Inpatients reporting previous LB compared to those without previous LB,significantly higher seropositivity rates were noted for all IR(6) peptides, butnot for the C6 peptide.

In the serology of EM in Europe, C6 ELISA does not seem to cover all cases. An ELISA using a mixture of B. burgdorferi sensu strictoIR(6) peptide and the C6 peptide could be of value in the serodiagnosis of LB inEurope. Further studies on combinations of variant IR(6) peptides and the C6peptide in other manifestations of LB are needed to address this issue. PMID: 19138558 [PubMed - as supplied by publisher]

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Artikkelissa esitetään uusi borrelioositesti - LightCycler TaqMan analyysi. PCR tyyppinen testi sopii eri näytteiden testaukseen (seerumi, kudokset, selkäydinneste jne). Testi on artikkelin mukaan erittäin herkkä ja erityisen spesifi Bb sl:n tutkimiseen.

http://www.ncbi.nlm.nih.gov/entrez/quer ... &DB=pubmed

Diagn Microbiol Infect Dis. 2006 Sep 19; [Epub ahead of print]

A LightCycler TaqMan assay for detection of Borrelia burgdorferi sensu lato in clinical samples.

* Ivacic L,
* Reed KD,
* Mitchell PD,
* Ghebranious N.

Molecular Diagnostics Genotyping Laboratory, Marshfield Clinic, Marshfield, Wisconsin, WI 54449, USA.

Lyme disease (LD) is an infection caused by an ixodid tick-borne spirochete, Borrelia burgdorferi sensu lato. LD manifests itself as a multisystem inflammatory disease that affects the skin in its early localized stage and spreads to the joints, nervous system, heart, and, to a lesser extent, other organ systems in its later disseminated stages. If diagnosed and treated early with appropriate antibiotics, LD is almost always readily cured. Developing a highly sensitive and specific real-time polymerase chain reaction assay could be very useful in improving the diagnostic accuracy and decreasing turnaround time for results. We report the development of a LightCycler TaqMan assay targeting the OspA gene for clinical detection of B. burgdorferi sensu lato in various types of biologic samples. This assay was validated by testing a variety of clinical samples including cerebrospinal fluid, synovial fluid, skin biopsies, and blood and culture isolates from skin biopsies. The TaqMan testing results were 100% concordant with previously reported results. Reference strains representing isolates from other geographic regions were also successfully amplified. The developed assay is robust, is highly sensitive and specific for B. burgdorferi sensu lato, and is suitable for clinical detection of the bacterium in biologic samples.

PMID: 16989975 [PubMed - as supplied by publisher]
Viimeksi muokannut soijuv päivämäärä Ke Helmi 04, 2009 10:06, muokattu yhteensä 1 kerran
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » To Tammi 22, 2009 21:26


Borreliatesteistä ja niiden luotettavuudesta esiintyy erilaisia näkemyksiä.
Artikkelin lopussa on esitetty Saksassa esim. BCA klinikalla käytössä olevia borreliatestejä. Monien kohdalla Suomessa suoritetut borreliatestit ovat olleet negatiiviset ja esim. Saksan testit positiiviset. Allalolevan artikkelin mukaan Suomen testit ovat hyvin kehittyneitä. http://www.mtv3.fi/uutiset/kotimaa.shtm ... linikoilla
".....Saksassa joillakin yksityisklinikoilla käytössä olevista testeistä. ...nimenomaan nämä testit ovat huonoja, koska ne antavat vääriä positiivisia tuloksia".


Useiden näkemysten mukaan kuitenkin nimenomaan perinteiset vasta-ainetestit ovat taudin kaikissa vaiheissa varsin epäluotettavia ja siksi tarvitaan uudenlaisia testejä. Vuonna 2011, FDA; Amerikan Elintarvike- ja Lääkevirasto ja CDC; Yhdysvaltojen tartuntatautien valvonta- ja ehkäisykeskus, hyväksyivät ELISPOT LTT tekniikan käytön. tuberkuloosiin. Nyt tätä tekniikkaa käytetään useiden eri taudinaiheuttajien tutkimiseen.
http://www.infectolab.com.au/Pages/LTT.aspx

Tekniikka ei ole käytössä Suomessa Borrelioosin diagnostiikassa ./size]

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http://www.infectolab.de/index.php?id=51&L=1

Borrelia Elispot-LTT (LymphocyteTransformationsTest)

Actual news:

The Elispot-LTT method has been approved by the FDA in May 2011 for M. tuberculosis (Not ITT or MELISA)

The FDA argues in this paper:

"... A positive result (in the Elispot-LTT) suggests that an infection is likely, a negative result that an infection is unlikely..."

"... Results (of the Elispot-LTT) can be available within 24 hours (ITT or MELISA not)..."

A Borrelia infection does not only activate the humoral immune response, but also activates T-lymphocytes at the same time. Once Borrelia bacteria are not active anymore, the T-cellular immune response is not present.

It is not possible to test the treatment success by Borrelia antibodies, because the 'titer" or antibodies can be measured in the blood over years. Furthermore, Lyme infections in Stage I (e.g. 'bulls-eye rash' or 'summer flu') only show antibodies in the blood after weeks and sometimes do not show them at all.

The Borrelia Elispot-LTT eliminates these problems. The test reflects the actual, current Borrelia burgdorferi activity of chronic and also acute Lyme infections. The Elispot-LTT is highly sensitive and can detect even one single Borrelia-reactive T-cell in the blood. The Elispot-LTT is very helpful when monitoring a chronic or acute Lyme therapy. The Elispot-LTT should usually become negative about 6 to 8 weeks after completion of an effective therapy.

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Artkkeli LTT-tekniikastta eri mikrobeja tutkittaessa:
Elispot®-LTT: FDA and CDC approved LTT technique in U.S.

Actual T-cellular activity in the blood against Borrelia burgdorferi, Chlamydia pneumoniae, Chlamydia trachomatis, Ehrlichia/Anaplasma, Epstein-Barr-Virus, Yersinia

In May 2011 the U.S. Food and Drug Administration (FDA) and the Centers for Disease Control and Prevention (CDC) have approved the Elispot®-LTT (T-Spot) technique beneath the QuantiFERON® TB Gold In-Tube test. Both tests represent Interferon-Gamma Release Assays (IGRAs) in form of Lymphocyte Transformation Tests (LTT). No other laboratory T-cell tests have been approved (ie: MELISA® or ITT® techniques) in the field of all Lymphocyte Transformation Tests (LTT) by the FDA/CDC yet. In the paper of the CDC regarding Interferon-Gamma Release Assays (IGRAs) from May 2011 the CDC says: "... A positive result suggests that an infection is likely, a negative result that an infection is unlikely...", and "...results can be available within 24 hours..."


The Elispot®-LTT is available for the following infections:
- Borrelia burgdorferi
- Chlamydia pneumoniae
- Chlamydia trachomatis
- Ehrlichia/Anaplasma
- Epstein-Barr-Virus
- Yersinia species


LTT in Borrelia Testing

A Borrelia infection leads to a vitalisation of T-lymphocytes apart from the humoral immune answer. The T-cellular immune response vanishes as soon as the Lyme disease is not active anymore.

A therapy success control of a Lyme infection is not possible by the Borrelia antibodies, as the “titer” of antibodies in the blood can be found for years after an infection. Additionally in stage I (e.g. “bull´s eye rash” or “summer flue” after a tick bite) antibodies can be found after several weeks or in stage III they cannot be found in every case (weak immune system). These diagnostic gaps are closed by the Elispot-LTT for Borrelia, which detects the actual cellular activity against Borrelia burgdorferi in chronic and also acute Lyme disease. The Elispot is so sensitive, that even a single Borrelia can reactivate T-cells in the blood. The Elispot is 20- to 200-fold more sensitive than an ELISA-test on Borrelia and will already find 1 reactive T-cell in 100.000 lymphocytes. The Elispot for Borrelia is very important for controlling a therapy of a chronic or acute Lyme infection. In general the Elispot-LTT is going to be negative app. 6 to 8 weeks after the end of a successful therapy.

Advantages of the Elispot-LTT for Borrelia as performed by Infectolab in contrast to other lymphocyte transformation or proliferation tests from other laboratories are:
· The result is available within 5 days (Other similar tests: 2 – 3 weeks)
· The use of cell stabilising CPDA-tubes means a stability of 3 days for the measured cells after taking the blood (others use Heparin for a stability of only 24 hours)
· Better reliability than the different "unspecific" tests using the proliferation of T-cells (e.g. ITT®)

This offers a significant improvement of the stability of the T-cells and a very quick decision possibility for Lyme treating physicians to extend a therapy or to switch to a new treatment option for Lyme disease!

Elispot-LTT for Borrelia:
Material: 3 x 9 ml CPDA-tubes (yellow cap, kept at room temperature, do not cool or centrifuge)
Time required for analysis: 2 days (Results in 5 days)
Indications: - Diagnosis of chronic Lyme disease
- Diagnosis of acute Lyme disease
- Decision for the length of Lyme disease therapies
- Success control of therapies after Lyme treatment

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http://www.aacc.org/events/Annual_Meeti ... 9-B150.pdf

Chenggang Jin, MD, PhD/Bradley Bush, ND

[size=150]Development and Validation of a Novel Interferon-γ ELISPOT Assay for Sensitive and Specific Detection of Antigen-Specific T cell Response to Borrelia burgdorferi


The enzyme-linked immunospot (ELISPOT) technology has proven to be extremely sensitive in detecting antigen specific reactive T cells and has been applied in laboratory diagnostic for Tuberculosis approved by FDA. A novel T-cell based assay for diagnosis of Lyme disease -Lyme ELISPOT was successfully developed and validated.

ABSTRACT
Learning Objectives: After the presentation, an individual will be able to:
⦁ Describe ELISPOT assay technology
⦁ Contrast ELISPOT assays to conventional antibody detection methods utilized for the diagnosis of Lyme disease
⦁ Evaluate the potential application of ELISPOT as a diagnostic tool for Lyme disease
Presentation Outline:
Background: Lyme disease, caused by infection with the spirochete Borrelia burgdorferi, is an emerging infectious disease in the United States that has become an important public health problem. Both B-cell immunity and T-cell immunity develop in natural infection with Borrelia burgdorferi. Detection of specific antibody response mediated by B cells against Borrelia burgdorferi is utilized conventionally in aiding the clinical diagnosis of Lyme disease. However, the limitation of these antibody-based immunoassays is that they have low sensitivity and specificity, causing significant false negative and false positive results. Furthermore, Borrelia specific antibodies cannot be detected at the early stage of infection and in a fraction of seronegative Lyme patients who lack Borrelia specific antibody responses. In contrast, Borrelia specific T-cell based immune assays have not yet been well developed. Thus, highly sensitive and specific T-cell based clinical laboratory assays are needed to help in diagnosing Lyme disease in conjunction with antibody-based immunoassays. The enzyme-linked immunospot (ELISPOT) technology has proven to be extremely sensitive in detecting antigen specific reactive T cells and has been applied in laboratory diagnostic for Tuberculosis approved by FDA. We here explore the potential application of ELISPOT as diagnostic tool for Lyme disease.
Objective: The aim of this study is to develop and validate a novel T-cell based assay for diagnosis of Lyme disease using newly developed digitalized ELISPOT technology.
Methods: To develop the novel T-cell based diagnostic assay for Lyme disease, we detected the Borrelia antigen-specific memory T cells that were activated ex vivo by recombinant Borrelia specific antigens, using Th1 cytokine Interferon-γ ELISPOT at the single cell level. The human peripheral blood mononuclear cells (PBMC) were stimulated with single or a combination of recombinant Borrelia specific antigens, DbpA, OspC, p100 and VlsE. In addition, we added costimulatory cytokine IL-7 into the cell culture to increase the detection of T memory cells. The results of ELISPOT were analyzed using CTL S6 Ultimate-V Analyzer/BioSpot 5.0 Software and reported as IFN- γ Spot Forming Units (SFU). To validate the Lyme ELISPOT assay, a cohort of 21 clinically diagnosed Lyme patients and 45 healthy control subjects were further studied and compared with Western Blot test. The performance of the Lyme ELISPOT assay, including clinical sensitivity, clinical specificity, accuracy and precision, is also evaluated.
Results: The frequency of Borrelia specific T memory cells can be detected by Interferon- γ ELISPOT and therefore can be used as a biomarker for Borrelia infection. The detection of antigen specific T cells was significantly increased by a combination of recombinant Borrelia antigens and addition of constimulatory cytokine IL-7. The signal enhancing effect of IL-7 was observed even at saturating antigen concentration in terms of frequency, but IL-7 did not increase the amount of IFN- γ secreted by individual cells. A strong correlation was observed between ELISPOT and IFN- γ concentration measured by Bio-plex suspension system (R=0.8, P<0.0001). The Lyme ELISPOT assay cut-off value was determined using Receiver Operating Characteristic (ROC) curve analysis and it was found that a cut-off value of >25 PFU maximized assay sensitivity and specificity. It has a significantly higher specificity (96%) and sensitivity (76%) compared with Western blot (Sensitivity 24%). The results also demonstrated that there was dissociation between B cell response and T cell response during Borrelia infection, suggesting a comprehensive immunological diagnostic panel should include both B cell and T cell diagnostics. Further studies will include more Lyme patients, other related diseases and independent studies by other laboratories.
Conclusion: A novel T-cell based assay for diagnosis of Lyme disease -Lyme ELISPOT was successfully developed and validated. This newly developed Lyme ELISPOT assay may be a helpful laboratory diagnostic test for Lyme disease, especially for seronegative Lyme patients. A comprehensive evaluation of both antibody response and T cell response to Borrelia infection will provide new insights into the pathogenesis and diagnosis of Lyme disease.





Lymfosyyttitransformaatiotestistä/Borrelioosista lisää esim.

http://www.cdc.gov/lyme/stats/chartstab ... yyear.html
Yound, J.D. Underreporting of Lyme disease. N Engl J Med. 1998. 338(22):1629-1629.
http://www.cdc.gov/hiv/topics/surveillance/basic.htm
http://www.cdc.gov/ncidod/dvbid/westnil ... tailed.htm
http://www.who.int/influenza/human_anim ... 1cases.pdf
Maloney, E.L. The Need For Clinical Judgment in the Diagnosis and Treatment of Lyme Disease. Journal of American Physicians and Surgeons. 2009. 14(3):82-89.
Lehmann, P.V., Zhang, W. Unique Strengths of ELISPOT for T Cell Diagnostics. In: Alexander Kalyuzhny, E. Handbook of ELISPOT: Methods and Protocols, Methods in Molecular Biology, vol. 792. 2nd Ed. New York, NY: Spriner Science+Business Media, LLC; 2012: 3-23.
T-Spot.TB 96 [Package Insert]. Oxfordshire, UK: Oxford Immunotec Limited; 2009.
Tary-Lehmann M., Hamm, C.D., Lehmann, P.V. Validating reference samples for comparison in a regulated ELISPOT assay. In: Uma Orabhakar and Marian Kelley Eds. Validation of Cell-Based Assays in the GLP Setting: A Practical Guide. 1st Ed. West Sussex, England: John Wiley & Sons. Ltd; 2008: 127-146.
Forsberg, P., Ernerudh, J., Ekerfelt, C., et al. The outer surface of Lyme disease borrelia spirochetes stimulate T cells to secrete interferon-gamma (IFN-γ): diagnostic and pathogenic implications. Clin Exp Immunol. 1995; 101:453-460.
Ekerfelt C., Forsberg P., Svenvik, M., et al. Asymptomatic Borrelia-seropositive individuals display the same incidence of Borrelia-specific interferon-gamma (IFN-γ)-secreting cells in blood as patients with clinical Borrelia infection. Clin Exp Immunol. 1999. 115: 498-502.
Dattwyler, R.J., Volkman, D.J., Luft, B.J., et al. Seronegative Lyme Disease- Dissociation of Specific T- and B-Lymphocyte Responses to Borrelia burgdorferi. New England Journal of Med. 1988. 319(22): 1441-1446.
Dressler, F., Yoshinari, N.H., Steere, A.C. The T-Cell Proliferative Assay in the Diagnosis of Lyme Disease. Annals of Internal Medicine. 1991. 115:533-539.
Martinuzzi, E., Scotto, M., Enee, E., et al. Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISPOT detection. Journal of Immunol Meth. 2008. 333: 61-70.
Seriburi, V., Ndukwe, N., Chang, Z., et al. High frequency of false positive IgM immunoblots for Borrelia burgdorferi in clinical practice.Clin Microbiol Infect. 2012 Dec;18(12):1236-40.
Kuerten, S., Batoulis, H., Recks, M.S., et al. Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays. Cells 2012, 1(3), 409-427


*Patent Pending (USPTO Application No: 61779064)

Conculsion

Lyme disease is an increasingly common condition that can have debilitating effects if not diagnosed and treated appropriately. A comprehensive approach to diagnosis will lead to the most positive outcomes and healthcare savings; however, testing options thus far have the potential for false negative results, making diagnosis difficult. NeuroScience’s testing options (iSpot Lyme™ including Western Blot analysis) can aid in the diagnosis of B. burgdorferi infection and allow for early detection leading to earlier treatment. iSpot Lyme™ is a highly sensitive T cell-based enzyme-linked immunospot (ELISPOT) method which enumerates the B. burgdorferi-specific activated effector/memory T cells. This test represents a breakthrough in diagnostic accuracy, and can increase the speed of diagnosis and treatment leading to improved clinical outcomes.
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http://www.mtv3.fi/uutiset/kotimaa.shtm ... linikoilla

Professori: Suomessa paremmat borrelioositestit kuin Saksan klinikoilla

Punkkitauti borrelioosista vuonna 1996 väitellyt professori kehuu Suomessa käytössä olevia borrelioositestejä.

Suomen testejä on kritisoitu julkisessa keskustelussa, ja ja ihmisten on jopa kerrottu matkustavan testeihin ulkomaille.

Turun yliopistollisen keskussairaalan infektiotautien ylilääkäri, infektiotautien professori Jarmo Oksi sanoo, että Suomessa käytössä olevat vasta-ainetestit borrelioosin diagnosoimiseksi ovat hyvin kehittyneitä.

– Väittäisin, että meillä käytetään todennäköisesti parempia testejä kuin suurimmassa osassa muuta Eurooppaa. Uskoisin, että juuri missään ei ole saatavilla parempia testejä, Oksi sanoo.


"Toinen testi antaa melkein kenelle tahansa positiivisen"

Oksi haluaa sanoa sanansa myös Saksassa joillakin yksityisklinikoilla käytössä olevista testeistä. Oksin mukaan nimenomaan nämä testit ovat huonoja, koska ne antavat vääriä positiivisia tuloksia.

– Joissakin Saksassa olevista yksityisklinikoista näytetään käytettävän niin sanottua lymfosyyttitransformaatiotestiä, joka on vähän eri asia kuin vasta-ainetesti ja selvästi ongelmallisempi. Tämä testi näyttää positiivista, jos on koskaan vähänkään törmännyt borreliabakteeriin, vaikkei bakteeria nykyään kehossa olisikaan, Oksi tyrmää.

Lymfosyytit kuuluvat ihmisen valkosoluihin, ja Oksin mukaan lymfosyytit voivat siis reagoida borreliabakteeriin, vaikka bakteeria ei ole elimistössä.

Oksi myöntää, että borrelioosin diagnosoiminen voi olla vaikeaa ja että Suomessa käytössä olevat vasta-ainetestitkään eivät ole täydellisiä.

– Ensimmäisen kahden kuukauden aikana punkin puremasta vasta-aineet eivät ole ehtineet vielä nousta. Toisaalta voi olla täysin terve, vaikka vasta-ainetesti olisi positiivinen. Vasta-aineet nousevat todennäköisesti kuitenkin hitaammin kuin lymfosyyttitunnistus, joka saattaa antaa melkein kenelle tahansa positiivisen tuloksen.

-----------------------------------------------------------------

Saksan Borrelioosiklinikan testit:

http://www.b-c-a.de/index.php?id=97\\\\\\\%27&L=1

http://www.b-c-a.de/fileadmin/img/bca/6 ... .07.11.pdf


Ihr kompetenter Laborpartner
http://www.infectolab.de
BCA-clinic
Betriebs
GmbH & Co. KG
Dr. med. Armin Schwarzbach
Facharzt für Labormedizin
Infecto
lab
, ein Geschäftsbereich der BCA-clinic Betriebs GmbH
& Co. KG
Geschäftsführer: Dr. med. Carsten Nicolaus, Dr. med
. Armin Schwarzbach
Amtsgericht Augsburg HRA 15697
·
Morellstraße 33
·
86159 Augsburg
·
Tel. +49/ 0821/ 455 074-0
·
Fax +49/ 0821/ 455 074-1
http://www.infectolab.de
·
e-mail: service@infectolab.de
·
Umsatzsteuer-Ident.-Nr. DE250941023
·
Bankverbindung:
Kreissparkasse Augsburg (BLZ 720 501 01)
·
Konto-Nr. 19885
·
IBAN: DE48 7205 0101 0000 0198 85
·
BIC: BYLANDEM1AUG
Patient information:
Laboratory services and diagnosis for Lyme disease
and possible
co-infections
- The detection of tick-borne infections makes high
demands on laboratory analysis in
combination with the diagnostic findings (anamnesis
) -
It is important to detect Borrelia infections at an
early stage. The earlier the detection the simpler
the treatment measures (usually antibiotics) and th
e shorter the ordeal of the infected patients.
This is how you can diagnose Lyme Disease

a Lyme disease infection progresses in 3 stages dep
ending on
symptoms and ailments
(time specification after the tick bite):
1.
Stage I
(after days up to weeks): “bull’s eye rash“ (“Eryth
ema chronicum
migrans“, only in 40-70% of all cases), Borrelia l
ymphocytoma,
headache, fever, sweating,
“Summer flue“ (app. 20% of all
cases), exhaustion and fatigue, facial palsy (espec
ially with
children/pupils).
2.
Stage II
(after weeks up to months): inflammation of the bra
in; meninges;
spinal marrow; any nerve in the human body, inflamm
ations of
the joints (“arthritis”), joint and muscle pain, in
flammation of the
eye, liver and kidneys, myocarditis, pericarditis,
Cardiac
arrhythmia
.
Borrelia burgdorferi – a
spirochete bacteria with a range
of over 300 proven species world
wide.
3.
Stage III
(after months up to years): Thinning of the skin at
the back of the hand, (“Acrodermatitis chronica
atrophicans“), Borrelia lymphocytoma (ear, nose, sc
rotum), lethargy, fatigue, paraesthesia, cognitive
dysfunction, muscle inflammations, joint inflammati
ons and swelling, tendon inflammations,
inflammation of the bursa, vasculitis, myocardinal
diseases, depression.
Symptoms of Lyme disease occur in thrusts with alte
rnating intensity and appearance in contrast
to a classic organic illness. Many patients also su
ffer from slightly higher temperature during those
thrusts. Co-infections with other bacteria and viru
ses have been increases over the past years and
often lead to a complicated course of the disease.
Often the tick bite is not detected early enough
or the acute treatment by the attending physician i
s not sufficient. The chronic Lyme disease
patients often go through a true “martyrdom” as the
y do not only suffer from actual physical and
mental ailments, but also from not receiving a reli
able diagnosis and the fact that their illness is n
ot
taken seriously.
There are three main reasons why a Lyme infection i
s not immediately detected in daily practice,
so that a treatment at an early stage is inhibited:
1. There is no
bull’s eye rash
(“Erythema chronicum migrans“)! Research has prov
en that
this classic symptom of Lyme disease only appears i
n 40% to a maximum of 70% of all
cases.
2.
No tick bite detected
! Such a bite can be induced even by very small tic
ks (larvae). Or it
was not detected because there was no specific skin
reaction. In addition, the scientific
community now assumes that Borrelia bacteria can al
so be transmitted by infected insects.
3.
Only conventional blood tests
have been performed. This was either too early
(antibodies can be tested positive only after a per
iod of up to six weeks after the tick bite)
or there have been absolutely no antibody productio
n in the body or the cellular stage was
not or not sufficiently tested (necessary tests are
: Elispot
®
-LTT and CD3-/CD57+ cells).

The BCA-clinic Augsburg (
BCA
) is specialised in the diagnosis and therapy of ti
ck-borne
diseases. In case of a suspected tick-borne disease
the diagnosis is performed by experienced
physicians of the “Medical Partnership” cooperating
with the BCA-clinic. The diagnosis is based on
an extensive anamnesis in which precise ailments an
d the antecedent are recorded and reviewed
along with a physical examination (=
clinical findings
). Additionally, the physician will initiate
specific laboratory tests of your blood, which will
be undertaken and analysed in the specialised
laboratory of the BCA-clinic.
First the
laboratory diagnosis
is crucial for the detection
of Borrelia. There is a difference between the test
s of the
humoral
(antibodies) and
cellular
level (lymphocytes,
NK-cells). Both levels have to be examined at the s
ame
time when a Lyme disease or an apparent illness is
suspected.
1. Laboratory testing of the humoral level (antibo-
dies):
Borrelia IgM- and IgG-EIA (Enzymimmuno-assay)
as well as Borrelia IgM- and IgG-Immunoblot
2. Laboratory-testing of the cellular level:
Elispot
®
-LTT (Borrelia Elispot Lymphocyte Transformation
Test), CD3-/CD57+ cells (NK-cells)
Explanations of these tests:
1. Antibodies – humoral level: In case of the Borre
lia antibodies
examinations up to 19% of the antibodies results in
the EIA are
falsely negative due to the minimal sensitivity (re
sponsiveness)
of the EIA in contrast to the Immunoblot. Therefore
it is
essential to check the Borrelia IgM- and IgG-Immuno
blot along
with the Borrelia IgM- and IgG-EIA (even with a neg
ative EIA)!
Important: The laboratory has to always tests for V
lsE (Variable
major protein-like sequence Expressed) in EIA and
Immunoblot. VlsE describes the characteristics of
the Borrelia
as a “chameleon“, which permanently changes the sur
face
structure VlsE in vivo to resist the detection via
the immune
system. VlsE has the highest sensibility for the an
tibody search!
Beware of positive antibodies constellations!
Armin Schwarzbach M.D.,
medical specialist for laboratory medicine,
head of the BCA laboratory and
specialist for Lyme and co-infections.
Borrelia IgM- as well as IgG-antibodies can remain
in the body for
months or years even without an active Lyme disease
infection.
Hence a positive Borrelia antibodies test does not
give any evidence
of the activity of a Lyme infection, but gives only
one conclusion: There must have been a tick bite o
r tick
bites in the past during which Borrelia bacteria ha
ve been transmitted – no more or less!
2. Cellular level:
a) The
Borrelia Elispot
®
-LTT
provides information about the current activity of
the Borrelia bacteria and is
20 to 200 fold more sensitive than a EIA-antibodies
test.
b) The
CD57+ cells
indicate the extent of immune suppression during a
chronic Lyme disease and are the
prognostic factor during and after the antibiotic t
reatment.
The complete laboratory diagnosis is very complex a
nd - considering the co-infections - has to be
put together like a mosaic. This demands experience
d Lyme disease analysts who can diagnose
and evaluate all other infections as well.
Armin Schwarzbach, M.D., PhD, is head of laboratory
medicine and diagnostics in the BCA.-clinic.
Dr. Schwarzbach is very experienced in laboratory m
edicine and has been a specialist in Lyme
disease and co-infections for years.
The medical laboratory diagnosis is primarily orien
tated on the guidelines of the German Society
for Hygiene and Medical Microbiology (MiQ12 Lyme-Bo
rreliose). However, the complexity of a
Lyme disease infection is not sufficiently covered
by this and needs additional laboratory testing.

herefore further internationally accepted methods
are applied which are of considerable
importance for the patient before and after the the
rapy (e.g. for the cellular level: Elispot
®
-LTT and
the CD3-/CD57+ cells).
The laboratory integrated in the BCA for the analys
is of vital parameters ensures, especially in the
case of the medical treatment (e.g. infusion therap
y), that the physicians of the medical
partnership make further therapy decision as soon
as possible according to the blood results.
In practice, the following laboratory constellation
s indicate a Borrelia infection:
(1) Positive antibody detection and positive cellul
ar results
(Elispot
®
-LTT and/or CD57+) – often active infection
(2) Positive antibody detection without positive ce
llular test findings – What kind of indications
are shown by the clinical findings (symptoms)?
(3) Negative antibodies detection, but positive cel
lular test results
- Indication for an active Lyme disease at an early
stage, but also chronic stage – what
kind of indications are shown by the clinical findi
ngs (symptoms)?
Attention: A negative antibody detection in the EI
A and/or Immunoblot does not give evidence of
a Lyme infection! Because: the antibodies productio
n of a Lyme disease in stage I needs several
weeks, 10 to 14 days at a minimum. Thus the immedia
te measurement of cellular activity in
Elispot
®
-LTT is a compulsory necessity, as normally the cel
lular activity precedes the humoral
activity in stage I of a Lyme disease infection.
In practice the following combinations of blood tes
ts have proven useful:
Laboratory diagnosis stage I
(Costs: 393,44 €
plus extra charges.
)
1. Borrelia IgG- and IgM-EIA incl. VlsE
2. Borrelia IgG- and IgM-Immunoblot
incl. VlsE
3. Borrelia Elispot
®
-LTT
Laboratory diagnosis stage II and
stage III
(Costs app.: 544 €
plus extra charges.
)
1. Borrelia IgG- and IgM-EIA incl. VlsE
2. Borrelia IgG- and IgM-Immunoblot
incl. VlsE
3. Borrelia Elispot
®
-LTT
4. CD3-/CD57+ cells
Necessary “
Staging
“ (evaluation of progression) of Lyme disease durin
g and after an antibiotic or
holistic approach to therapy: The above mentioned p
arameters also have to be checked during
Lyme therapy.
Recommended laboratory diagnosis during the therapy
:
Stage I
Performance of above mentioned 3 tests
4 weeks after beginning of therapy and 8
weeks after the end of therapy.
Stage II and III
Performance of above mentioned 4 tests after
beginning of therapy every 8 weeks as well as
8 weeks after end of therapy.
For explanation:
Borrelia antibodies or titer cannot be “eliminated”
, but they can exist in the
blood for months or even years. After a successful
antibiotic therapy Elispot
®
-LTT as well as
CD57+ cells should have come to a “normal” level 8
weeks after the end of therapy. But
: Inspite
of a symptom free status after treatment there migh
t be still positive findings of Elispot
®
-LTT
and/or CD57+ cells, so the patient should be consid
ered for a “monitoring” of the activity tests and
possible future symptoms. Otherwise there will be t
he risk of a relapse, a new infection or a co-
infection.
It should be noted that laboratory parameters alone
do not give a definite proof of Lyme disease,
but very important evidence and details about a pos
sible infection. The parameters are also very
important to make decisions about the duration and
success of therapy.

Adjoining is an overview
of relevant diagnostic
parameters of tick-borne
diseases.
These may be extended
or limited individually
during an extensive
consultation meeting with
your attending physician
(e.g. in case of pre-
findings).
Based on the
co-
infections questionnaire
indications are
established if such
additional laboratory tests
are necessary.
An overview of prices can
be found in the
attachment “Order for
laboratory tests with
declaration of consent“ for
patients.
Note: The laboratory analysis is only one part
of a comprehensive diagnosis. The
clinical
report
based on an extensive
anamnesis
and
a physical examination is the crucial part. The
experienced physicians of the cooperating
Medical Partnership know the broad range of
possible disease patterns, which can also
become noticeable as „
unspecific general
symptoms
“ (note adjoining chart).
Generally, the laboratory diagnosis causes the foll
owing costs:
Laboratory costs
1. for Borrelia IgM- and IgG- antibodies including
VLSE,
Borrelia Elispot
®
-LTT and CD3-/CD 57+ T-lymphocytes
with blood sampling and special tubes
app.
544 €
2. Basic laboratory (blood count, liver-kidney-clot
ting) app.
80 €
3.
Possible necessary additional examination when susp
icion of co-
infections (Each test app. 61 – 97 €):
up to app. 945 €
Note:

Elispot- and CD3-/CD57+ are principally not covered
by health insurances!

The unit prices for different laboratory tests resu
lt from the laboratory order form in the attachment
:
blood samplings and special tubes will be billed se
parately

Laboratory tests of possible co-infections
Laboratory tests of possible co-infections have an
increasing importance when adequate evidence
exists (BCA co-infections sheet). Especially Ehrlic
hia/Anaplasma, Babesia, Bartonella, Rickettsia,
Chlamydia and Mycoplasma are to be named.
Because: numerous symptoms of the co-pathogens over
lap the same symptoms of Lyme disease
patients. Without an exact knowledge of a patient’s
possible co-infections the therapist cannot
make a exact decision about the antibiotic therapy.
Not all co-infections can be treated by the
generally used antibiotics for Lyme disease. Howev
er, the testing for co-infections can induce
extensive additional laboratory costs (up to 945€).
But these are justified by the additional securit
y
in the diagnosis and especially by the correct anti
biotic decision, i.e. more success in antibiotic
therapies as well as generally less expensive costs
for medication during the antibiotic therapy
(vs. the “classical” antibiotic therapy of chronic
Lyme disease).
Apart from the LTT for Borrelia further cellular ac
tivity tests for Ehrlichia/Anaplasma, Chlamydia
pneunomiae and Chlamydia trachomatis have been deve
loped in the meantime (Babesia cellular
activity testing is coming soon) and can therefore
be determined with Elispot-LTT-technique. With
the help of this new Elispot
®
-LTT many activities of Chlamydia and Ehrlichia hav
e been detected
in the BCA! A serum examination concerning co-infec
tions is perfomed at the same time. There
are now well standardised antibody tests for Chlamy
dia, Mycoplasma, Ehrlichia, Bartonella,
Rickettsia, Babesia, Yersinia etc. The same applie
s here as well as with the Borrelia-LTT: the
antibodies alone do not have any significance towar
ds the activity of an infection – but the
Elispot
®
-LTT is significant as it attests the high-specific
interferon release against the respective
co-pathogen in the blood.
It should be noted that in some cases it is the co-
pathogen itself which is responsible for the
symptoms and not the Borrelia infection: e.g. Chla
mydia cause diseases patterns such as Morbus
Alzheimer, Multiple Sclerosis, fibromyalgia, Chroni
c Fatigue Syndrome (CFS), myocardinal
infarcts, strokes, vasculitides, visual disturbance
s.
The Lyme infection may well have been treated succe
ssfully with antibiotics, but the co-infection
might not have been destroyed by it. Therefore, an
exact anamnestic documentation of the
symptoms during the therapy is necessary before, du
ring and after a Borrelia infection.
Further information regarding the importance of co-
infections can be found in the separate
information for patients: „Increasing importance of
co-infections for Lyme disease patients” (article
by Armin Schwarzbach, M.D., PhD).
Laboratory transmittals
In case of a suspected tick-borne disease physician
s have the opportunity to execute specific
laboratory analyses in cooperation with the BCA. T
he blood kits contain special anamnestic
questionnaires by the BCA and a questionnaire to ch
eck for co-infections. The patient should fill
out these questionnaires and send them to the BCA t
ogether with the blood kits.
We are delighted to provide interested physicians w
ith further information about possibilities in
terms of collaboration and co-operation to provide
a successful treatment for patients suffering
from tick-borne diseases. Especially for the diagn
osis and therapy of chronically ill patients, who
are not living in the Augsburg area we can coordina
te the procedure of the treatment with the
attending physician at home if wanted. However, in
case of complicated symptoms and severe
illness patterns we recommend the physicians of the
Medical Partnership which cooperates with
the BCA for the first diagnosis and therapy plan pr
eparation. We also recommend the Lyme

Disease “Intensive Treatment and Rehabilitation” Pr
ogram of the BCA, which is a special Compact
Treatment clinic for chronically ill patients here
in Augsburg.
The BCA regularly offers seminars and workshops, as
well as exchange and sharing of
experiences to interested physicians and cooperatio
n partners.
Below you will find as
attachments
further information regarding the laboratory tests
on the
cellular level (Borrelia Elispot
®
-LTT, CD3-/CD57+ cells and the Elispots for Chlamyd
ia and
Ehrlichia) as well as the order form for laboratory
tests with declaration of consent for the BCA.
Attachments

Further information regarding the laboratory tests
on cellular levels: Borrelia Elispot
®
-LTT,
CD3-/CD57+ cells, Chlamydia Elispot
®
-LTT and Ehrlichia Elispot
®
-LTT

Order form for laboratory tests with declaration o
f consent
Borrelia Elispot
®
-LTT
-
Determination of actual activity in the blood regar
ding Borrelia burgdorferi
A Borrelia infection simultaneously leads to an act
ivation of T-lymphocytes lateral to the humoral
immune response. The T-cellular immune answer vanis
hes as soon as the Lyme disease infection
is not active anymore.
A control of success of a Lyme infection therapy is
not possible via Borrelia antibodies, as the
“titer” or the antibodies can still be found in the
blood for years after a cured infection. Addition
ally,
in the first stage of Lyme disease (e.g. “bull ́s ey
e rash” or “summer flu” after a tick bite) antibodi
es
can only be detected and measured after several wee
ks or not at all.
The diagnostic gaps are suggested to be Borrelia ac
cording to the Elispot, which measures the
actual activity regarding Borrelia burgdorferi with
chronic and also acute Lyme disease infections.
The Elispot is so sensitive, that it can even detec
t a single Borrelia reactive T-cell in the blood. T
he
Elispot is 20- to 200-fold more sensitive than an E
LISA-test on Borrelia and is able to find 1
reactive cell under 100.000 lymphocytes.
The Elispot of Borrelia is very important for contr
olling a therapy of a chronic or acute Lyme
infection. In general the Elispot is negative appr
oximately 6 to 8 weeks after the end of a
successful therapy.

Advantages of the Borrelia Elispot-LTT
(- as performed in the BCA -) in contrast to tradi
tional
lymphocyte transformation tests:

The result is obtainable within 2 days (LTT: 1 – 2
weeks) !

The use of cell stabilising CPDA tubes means a sta
bility of 3 days for the measured cells
(LTT: Heparin blood only 24 hours) !
This offers a significant improvement of the stabil
ity of the examined cells and a fast decision-
making possibility for Lyme disease therapists to e
xtend the duration of a therapy or start a new
treatment approach!
Borrelia Elispot:
Materials:
2 x 8.5 ml CPDA-tubes (kept at room temperature, d
o not
refrigerate)
Time required for analysis:
2 days
Billing according to GOÄ:
Number 3694 (3 different antigen approaches) factor
1.5 (3x
49.84 €) + number 4003 (Lymphocytes isolation) Fact
or 1.5 (34.97 €)
- total sum: 184.49 €
Indications:
- Diagnosis of a chronic Lyme disease
- Diagnosis of a acute Lyme disease
- Decision-making regarding the length of therapy
- Control of therapy after a Lyme disease therapy
Borrelia CD 57+ cells
-
Determination of chronic activity in the blood rega
rding Borrelia burgdorferi
A chronic progression (stage III) of a Lyme infecti
on leads to a weakening of the immune system.
This is reflected by the decrease of the CD3-/CD57+
NK-cells in case of chronic Lyme disease.
The CD3-/CD57+ cells are a subpopulation of the Nat
ural-Killer-Cells (NK-cells).
A decrease of the CD57+ cells indicates (an untreat
ed) chronic or not sufficiently treated chronic
Lyme disease and does not appear in cases of a acut
e Lyme infection (e.g. “bull’s eye rash“ or
“summer flu“ after a tick bite).
The CD57+ cells reflect the degree of activity of a
chronic Lyme disease and decrease to a normal
level after a successful Lyme disease therapy (afte
r the end of treatment).
In contrast there is no decrease of CD57+ cells wit
h clinical similar diseases, such as Multiple
Sclerosis (MS), Systemic Lupus Erythematosus (SLE)
or an Amyotrophic Lateral Sclerosis (ALS).
In addition there is no significant fluctuation of
CD57+ cells throughout the day.
CD57+ cells are appropriate laboratory parameters i
n cases where chronic Lyme disease is
suspected and for therapy monitoring. These should
be measured parallel to the Borrelia Elispot,
which reflects the actual T-cellular activity.
Advantages of the CD57+ cells determination of Borr
elia
:
1. The result is available within 2 days !
2. The use of cell-stabilising Heparin-tubes assure
s a stability of the measured NK-cells for 2
days!
In combination with the Borrelia Elispot this resul
ts in a significant improvement of the stability of
the examined cells and a fast decision-making possi
bility for Lyme disease therapists to extend
the duration of a therapy or start a new treatment
approach!
CD3-/CD57+ NK-cells:
Material:
1 x 10 ml Heparin-tube + 1 x EDTA-blood/blood coun
t-tube (kept
at room temperature, do not
refrigerate)
Time required for analysis:
2 days

Seite 9
Ihr kompetenter Laborpartner
Ehrlichia/Anaplasma Elispot
®
-LTT
-
Determination of actual activity in the blood regar
ding Ehrlichia/Anaplasma
Ehrlichia or Anaplasma are intracellular pathogens,
which can be found obligatory within the white
blood cells. Ehrlichia are transmitted to the human
by ticks contaminated with Ehrlichia
(approximately 6% of all ticks are contaminated wit
h the pathogen).
The symptoms of an Ehrlichia infection begin with f
lu like problems and express themselves with
strong headache, which are very often located “behi
nd the eyes”. Furthermore muscle aches and
numerous neurologic symptoms may be caused by Ehrli
chia. Rarely skin rashes on various parts
of the skin can be found, also on the palm of the h
and and soles of the feet.
An immune suppression is often the risk factor for
an Ehrlichia infection amongst others
depending on age, but also on chronic infections li
ke Lyme disease.
In the case that Ehrlichiosis (illness pattern of a
n infection with Ehrlichia) existing parallel to a
Lyme infection it is called co-infection. Accordin
g to the latest scientific literature additional co
-
infections with Ehrlichia should be taken into acco
unt in ca. 30% of all chronic Lyme disease
patients.
An infection with Ehrlichia leads to an activation
of the T-lymphocytes parallel to the humoral
immune answer (Ehrlichia-IgM- and Ehrlichia-IgG-ant
ibodies). The T-cellular immune answer
vanishes as soon as the Ehrlichia infection shows n
o activity.
The Elispot on Ehrlichia/Anaplasma measures the act
ual activity of this disease.
The Ehrlichia-Elispot helps with the diagnosis of t
his infection and also is a means of a successful
control during the course of the therapy.
Advantages of the Ehrlichia Elispot
:

The result is available within 2 days !

The use of cell stabilising CPDA tubes means a sta
bility of up to 3 days for the measured
T-cells !

The right choice of a specific antibiotic therapy
!
Ehrlichia Elispot:
Material:
2 x 8.5 ml CPDA-tubes (kept at room temperature, d
o not
refrigerate)
Time required for analysis:
2 days
Billing according to GOÄ:
Number 3694, factor 1.5 (49.84 €) + number 4003 (ly
mphocytes
isolation), factor 1.5 (34.97 €), total sum 84.81 €
Indications:
- Diagnosis of an infection with Ehrlichia
- Decision-making regarding the length of therapy
- Control of therapy success after an Ehrlichia-spe
cific therapy
Attachment: Order form for laboratory tests with de
claration of consent
-
This order is to be filled out by your physician a
nd has to be signed and dated by you in case of
an order to the infecto
lab
laboratory. The costs of the respective laboratory
order result from the
following individual prices (GOÄ), plus possible si
de costs for blood sampling, blood tubes,
shipment.

BCA klinikan testejä: http://www.b-c-a.de/index.php?id=97\\\\\\\%27&L=1
----------------------------------------------------------------------------------------------------------------------------
Warthin-Starry -värjäys on yksi menetelmä jolla borreliabakteereita kyetään etsimään kudosnäytteistä.

http://www.ncbi.nlm.nih.gov/entrez/quer ... &DB=pubmed

Pol Merkuriusz Lek. 2006 Jun;20(120):731-4.

Clinico-pathological collations in borreliosis
[Article in Polish]

* Wagner T,
* Legatowicz-Koprowska M,
* Prochorec-Sobieszek M.

Zaklad Anatomii Patologicznej, Instytutu Reumatologii w Warszawie. twagner@vp.pl

Borreliosis is an infectious disease caused by spirochetal microorganisms Borrelia burgdorferi transmitted by ticks. Due to versatile clinical symptomatology and many pathogenetic aspects not explained yet, diagnosis of this disease is often very difficult. Borreliosis may affect various human organs and systems such as movement system, nervous system (central and peripheral), skin, heart and vessels. One of the diagnostic methods for detection of microbial organisms in tissues is the histochemical staining by Warthin-Starry. Very important problem connected with this illness is the relationship between borrelial antigens and autoimmunity.

PMID: 17007281 [PubMed - in process]
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Pe Tammi 23, 2009 14:37

Mark Stroud kertoo miten hän on itse tutkinut ostamallaan mikroskoopilla omaa verinäytettään ja nähnyt siinä borreliabakteereita. Hän neuvoo sivullaan miten muutkin voivat toteuttaa tutkimuksen.

Mark Stroud: "Olen viimeisen 14 vuoden aikana olen etsinyt syitä sairasteluuni. Minut on lähetetty lääkäriltä toiselle, mutta diagnoosi ei ole selvinnyt tai sitten oireiden on sanottu johtuvan psyykkisistä tekijöistä. Kun viimein löysin syyn oireilleni, olin hämmästynyt miten epäluotettavia borrelioositestit ovat. Siitä lähtien olen käyttänyt aikaani opettaakseni ihmisille, miten he voivat itse tehdä borrelioositestin kotona ottamalla verinäytteen sormenpäästään ja tutkimalla sitä mikroskoopilla. Menetelmä on erittäin yksinkertainen, ja silti kuulemme toistuvasti miten vaikeaa borreliabakteereita on löytää verestä. Siksi olen käyttänyt paljon aikaa löytääkseni yksinkertaisen tavan tutkia bakteereita. En yritä sanoa, että menetelmää käytettäisiin diagnoosin tekemiseen - se olisi väärin.. Mutta siitä saa mielenkiintoisen harrastuksen, joka auttaa lisäksi selvittämään mitä elimistössäsi tapahtuu. Näyttää siltä, että verinäytteessä ei näkyisi ainoastaan borreliabakteerin spirokeettamuotoja, vaan sen lisäksi myös bakteerin L-muotoja."

Koko artikkeli ja yksityiskohtaiset ohjeet mikroskoopin käyttöön löytyvät sivulta

http://www.lyme-diagnosis.org.uk/ Sivulla olevissa videolinkeissä esitetään Markin verinäytteessä näkyvät löydökset.

Suomenkieliset ohjeet mikroskoopin rakenteesta: http://physics.oulu.fi/fysiikka/oj/7661 ... _s2006.pdf


(Suom. huom. en tiedä vielä tällä hetkellä, miten luotettava sivun teksti ja siinä esitetty menetelmä on. Mielenkiintoinen sivu joka tapauksessa. Tällaista aktiivisuutta tarvitaan, varsinkin kun Borrelioosin laboratoriodiagnostiikka on vielä tällä hetkellä näinkin epäluotettavaa. Löytyykö yhdistyksestämme ketään joka pystyy vahvistamaan/kumoamaan Markin ohjeet tai toteuttamaan testin ohjeiden pohjalta? )

How YOU can see Borrelia Burgdorferi using common, low cost equipment.

Click below for movies taken down the microscope
Video1

Video2

Video3

From: Mark Stroud
Sunday, 10:14 a.m.

For the last 14 years, I have been searching for a reason for my sickness. I have been thrown around from doc to doc without a diagnosis, or if they have tried to diagnose me then it has been "all in my head".

When I finally found the cause, I was amazed at just how unreliable the testing for this pathogen is. That's why I have pulled out all the stops and I am trying to teach people how to implement the SAME strategies that I use to see Borrelia Burgdorferi in a tiny drop of blood from my finger.

The technique described below is so astoundingly easy to do yet we still hear that Bbsl is not very easily found in the blood leaving 1,000s of potential lyme patients with a non diagnosis due to the testing set out by the CDC.

So I've spent a lot of time looking for a solution that's easy to use...

Now, I am not saying that this procedure is to be used as a diagnosis tool for your own illness. That would be wrong. It is more of an interesting hobby to prove to yourself what is really going on. It appears that you can not only see the spirochetes, you can see the l-forms as well (see the videos at the top of this page)



That said, let's get into the procedure...

I look at my own blood under a normal microscope. My microscope was bought from Digilens in the far east but there is nothing special about this scope, so perhaps you can find an equivalent locally or on ebay. These people ship world wide and the URL is http://www.digilens.com.tw/06/product_d ... =25&ID=544

As I said, there is nothing special about this microscope, but I will say that you should get a "BIO" microscope and one that follows the DIN standard. The DIN standard means that the objective lenses are interchangeable with other DIN standard scopes. (don't worry if you don't understand what that means at the moment - all will be clear later)

About the microscope.

You will notice from the picture below that the objective is quite big. This is a full size microscope with the DIN standard of lens. It is this lens that looks directly at the slide.



We are going to modify the scope and put a different lens on it. More on that later.

Below, and just under my finger, is what you place the slide on and that is called the "stage". There is adjustment underneath the scope so that you can move the slide around without touching it yourself.
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Pe Tammi 23, 2009 23:23

Borreliabakteeri leviää elimistössä verenkierron välityksellä eri puolille elimistöä. Tämä selittää sen miksi sellaiset henkilöt, joilla on ihomuutos ja jotka eivät ole saaneet antibioottihoitoja, saattavat saada oireita kaukana ihomuutoksesta. Tutkimuksessa huomattiin myös, että plasma soveltuu bakteerin viljelyyn paremmin kuin kokoveri tai seerumi.


Wien Klin Wochenschr. 2006 Nov;118(21-22):634-7.

Hematogenous dissemination in early Lyme disease.
Wormser GP.
Division of Infectious Diseases, Department of Medicine of New York Medical College, Valhalla, NY, USA, gary_wormser@nymc.edu.

Hematogenous dissemination has long been considered an important pathogenetic event in Lyme borreliosis but has been hard to document and characterize due to insensitive blood culture methods. In a series of recent investigations involving adult patients from the United States with erythema migrans, it was concluded that plasma was a better source of culture material than serum or whole blood, and yield correlated directly with the volume of material cultured.

The rate of recovery of Borrelia burgdorferi from 9 mL of plasma exceeded 40%. Both the genotype of the strain of B. burgdorferi introduced by the tick and host factors, such as having a first episode of Lyme borreliosis and being more than 55 years of age, appear to affect the risk of hematogenous dissemination. Although the majority of spirochetemic patients are symptomatic, spirochetemia can be a subclinical event.

In summary, hematogenous dissemination is a common and important feature of early Lyme borreliosis in the United States. The high rate, early onset and prolonged duration of risk of spirochetemia probably explain why untreated patients with erythema migrans may develop objective clinical manifestations at a distant anatomic site.

PMID: 17160600 [PubMed - in process]
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » La Tammi 24, 2009 18:21

"Laboratoriotestit voivat olla hyödyllisiä kliinisen diagnoosin tukena. Kuitenkaan tällä hetkellä käytössä olevista testimenetelmistä yksikään ei ole optimaalinen borrelioosin diagnosointiin."

http://www.pubmedcentral.nih.gov/articl ... 1448627#r1
Two-year evaluation of Borrelia burgdorferi culture and supplemental tests for definitive diagnosis of Lyme disease.
Coulter P,
Lema C,
Flayhart D,
Linhardt AS,
Aucott JN,
Auwaerter PG,
Dumler JS.
Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Lyme disease is usually diagnosed and treated based on clinical manifestations. However, laboratory testing is useful for patients with confusing presentations and for validation of disease in clinical studies. Although cultivation of Borrelia burgdorferi is definitive, prior investigations have shown that no single test is optimal for Lyme disease diagnosis. We applied high-volume blood culture, skin biopsy culture, PCR, and serodiagnosis to a cohort of patients with suspected Lyme disease acquired in Maryland and southern Pennsylvania. The study was performed to confirm the relative utility of culture and to identify laboratory testing algorithms that will supplement clinical diagnosis. Overall, 30 of 86 patients (35%) were culture positive, whereas an additional 15 of 84 (18%) were seropositive only (51% total sero- and culture positive), and PCR on skin biopsy identified 4 additional patients who were neither culture nor seropositive. Among 49 laboratory test-positive patients, the highest sensitivity (100%) for diagnosis was obtained when culture, skin PCR, and serologic tests were used, although serologic testing with skin PCR was almost as sensitive (92%). Plasma PCR was infrequently positive and provided no additional diagnostic value. Although culture is definitive and has a relatively high sensitivity, the results required a mean of 3.5 weeks to recovery. The combination of acute-phase serology and skin PCR was 75% sensitive, offering a practical and relatively rapid alternative for confirming clinical impression. The full battery of tests could be useful for patients with confusing clinical signs or for providing strong laboratory support for clinical studies of Lyme disease. PMID: 16207966 [PubMed - indexed for MEDLINE]

Related Links

Diagnosis of lyme borreliosis. [Clin Microbiol Rev. 2005] PMID: 16020686
Polymerase chain reaction in diagnosis of Borrelia burgdorferi infections and studies on taxonomic classification. [APMIS Suppl. 2002] PMID: 11985118
Laboratory testing for suspected Lyme disease. [Med Clin North Am. 2002] PMID: 11982304
Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions. [J Clin Microbiol. 1992] PMID: 1452688
Improvement in the laboratory recognition of lyme borreliosis with the combination of culture and PCR methods. [Mol Diagn. 2003] PMID: 15068385
See all Related Articles...
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » La Tammi 24, 2009 18:26

Uusi nykyistä immunoblottausta tarkempi menetelmä on saanut FDA:n hyväksynnän.

http://centralfloridaresearch.com/lab/


Information
Dear Physician:
Central Florida Research, Inc. is replacing the laboratory operations of Bowen Research and Training Institute, Inc. Bowen Research and Training Institute, Inc. will continue providing Bowen Therapy as a separate and distinct corporation.

The new Borrelia burgdorferi antigen test Central Florida Research, Inc. will be offering is a much more definitive test than the Western Blot. A Borrelia burgdorferi fluorescent antibody is used to detect the antigen in whole blood. The test is set up manually and read by Flow Cytometry.

The Flow Cytometer can count the number of organisms in 100,000 events in 2 minutes and 50,000 in 1 minute. To visually count the organisms in 100,000 events or 50,000 events using a microscope would be almost an impossibility. The Flow Cytometer counts the number of all events passing through the aperture and enumerates the organisms that react with the antibody. The test result will be reported as a percent of the counted events.
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » La Tammi 24, 2009 18:38

Central Florida Research -tutkimuslaitos on saanut Medicaren hyväksynnän uudelle borrelioositestilleen nimeltään The RAIBb Flow Cytometry test. Testi on antigeeniin perustuva testi eikä elimistön vasta-aineisiin kuten Elisa ja immunoblottaus jotka ovat normaalit Suomessa tehdyt testit. Artikkelissa mainitaan borrelioosidiagnoosin testistä huolimatta olevan edelleenkin kliininen. Testitulos voi olla negatiivinen esim. siitä syystä että verenkierrossa ei ole paljon borreliabakteereita eikä niitä osunut näytteeseen.

Artikkelissa mainitaan sairaalla elimistöllä olevan vaikeuksia muodostaa vasta-aineita. Siinä on yksi syy nykyisten vasta-aineiden muodostumiseen perustuvien testien epäluotettavuuteen. Laboratorion mukaan borreliabakteeri on soluseinätön bakteeri joka kykenee peittämään itsensä elimistön omalla DNA:lla. Immuunipuolustus ei tunnista sitä vaan tunnistaa bakteerin osana elimistöä. Tällöin ei elimistö muodosta vasta-aineita. Laboratorion mukaan spirokeettoja löydetään vain harvoin verestä, mutta sen sijaan soluseinättömiä (CWD) ja kystamuotoja löydetään.


"Understanding the microbiology behind the cyst or cell wall deficient form of Bb and its ability to hide from our immune system helps us understand the Lyme disease controversies and the complications for testing for the bacteria. If the antibodies a person's body normally produces against an invading pathogen aren't always present then we must assume the accepted antibody test are flawed. Testing for the spirochetes presents a problem because spirochetes are rarely found in the blood. However, the antigen Bb in its cysts, or cell wall deficient (CWD) form can be found in the blood."

The Central Florida Research is dedicated to the research of Lyme Disease and other CSID ( chronic, systemic, infectious diseases). Besides the bacteria that causes Lyme disease Borrelia burgdorferi (Bb), recent research supports the fact that other pathogenic bacteria and viruses contribute to the symptoms found in Lyme and CSID patients.

www.centralfloridaresearch.com

The bacteria Bb is transmitted by ticks and other vectors including mosquitoes, fleas and mites. A recent study at the University of New Haven, in Connecticut revealed that ticks tested carried not only Bb, but Babesia, Ehrlichia, Bartonella as well as other organism that have previously been found such as HHV6-A. These three organisms Babesia, Ehrlichia and HHV6-A require different treatment protocols than Bb.

The life-cycle of Bb is related to the life cycle of the tick (usually Ixodes scapularis). The tick has four stages in its two year life cycle: egg, larva, nymph and adult. The spirochete is usually acquired during the tick's larval state. It is in this stage that the tick then becomes the host for the spirochete.

Willy Burgdorferi, Ph.D, who discovered the cause of Lyme disease in 1981, states that the complexity of the arthropod-borne spirochetes of Bb are well known. Lyme diagnosis is complicated because Lyme disease mimics many other diseases. Like Syphilis, another disease caused by a spirochete, Lyme is called 'The Great Imitator'. Two of the most common misdiagnosis are Chronic Fatigue and Fibromyalgia. The symptoms of other disease like RA, MS, Lupus and ALS often overlap Lyme disease symptoms. In diagnosing Lyme disease most physicians look for the accepted telltale sign of a Lyme infection which is the EM (bull's eye) rash seen at the bite's site. However, only about 50% of those infected exhibit this rash. More often other symptoms appear after initial infection. These may include joint/muscle pain that migrates, stiffness of joints, flu like symptoms, headaches, neck pain and TMJ. Neurological, Neuropsychiatric, Cardiac/Pulmonary and Gastrointestinal problems appear to develop if the disease goes untreated and the infection is allowed to progress. To better understand the complicated issues in diagnosing Lyme disease one should read the essay 'When to Suspect Lyme Disease' by John D. Bleiweiss, M.D.

The nature of the spirochete complicates the treatment of Lyme disease. Antibiotics are the chosen mainstream treatment, but success is difficult if the patient goes untreated and the disease becomes chronic. Prompt treatment within six weeks of antibiotics for a bite is recommended by the International Lyme and Associated Disease Society ( ILADS ) in order to prevent relapses and chronic illness. If not caught early extended treatment is usually necessary.

Upon entering the body, the spirochete cloaks itself with a coating of the person's own DNA. The immune system which is normally in Cellular Immunity recognizes that there is a foreign invader and goes into Humeral Immunity, the state of attack. The invader due to its ability to cloak itself and hide from the immune system evades detection. Hence, the referral to the spirochete as being a stealth pathogen. The white blood cells, the Neutrophils and Macrophages, whose job it is to engulf foreign pathogenic invasion allows the cloaked spirochetes to go in and out of these cells as they don't recognize it as the enemy. They see it as self (part of the person's own DNA).

Under these circumstances the immune system stays in Humeral Immunity overdrive due to the fact that it can't locate the invader and destroy it. Normally when an enemy is destroyed the immune system returns to normal Cellular Immunity. When an immune system is left in Humeral Immunity overdrive, the results are negative. Autoimmune issues occur and the immune system starts to attack many systems in the body. It is not unusual to see people with Borrelia or Mycoplasma infections to exhibit autoimmune issues such as diagnosed in Lupus, Parkinson's, ALS, Lupus, RA, and MS. Chronic Fatigue Immune Dysfunction (CFIDS) and/or Fibromyalgia are other common misdiagnosis. Specific diagnosis is the result of a combination of factors present in the individual. These include the general health of the individual's immune system, their genetics, and the infectious agent. There are over 300 strains of Bb, and many species, therefore combinations of symptoms vary in each individual.

At the beginning of the last century, bacteriologist entered into an intense debate over pleomorphism, which is the ability of a bacteria to change from one form to another and as in the case of Bb back again into its original form. The debate split microbiologist into two camps: the monomorphists and the pleomorphists. The monomorphists believe that each bacterial cell by binary fission divides transversely to produce two new cells which evolve to replicate the original cell in size and shape. The pleomorphists believe that even common bacteria show complex life cycles, and have the ability to shed their cell wall. In the Spring of 2006, a research study was published on 'A Life Cycle for Borrelia Spirochetes,' by Dr. Alan MacDonald, Ph.D., What is a Bb life cycle? According to Dr. MacDonald, study the "....life cycles are diverse arrays of life forms which emerge in an ordered sequence, which are 'connected' to one another across primary and secondary hosts, and constitute a cycle with 'circular' relationship between hosts."

In 'The Complexity of Arthropod-borne Spirochetes' by Dr. Burgdorferi, he states ."...the most recent findings confirm the development of membrane-derived cysts, blebs, spherules, vesicles and potential transformation to motile, helical spirochetes. These are not a part of a complex developmental cycle but rather as a 'survival mechanism' of spirochetes to overcome and escape unfavorable conditions. Such conditions prevail during early phases of infection when spirochetes ingested into the midgut of ticks or lice become exposed to the vectors' digestive enzymes and tissue barriers. As a result, most detectable spirochetes produce numerous cysts often filled with granular material."

Understanding the microbiology behind the cyst or cell wall deficient form of Bb and its ability to hide from our immune system helps us understand the Lyme disease controversies and the complications for testing for the bacteria. If the antibodies a person's body normally produces against an invading pathogen aren't always present then we must assume the accepted antibody test are flawed. Testing for the spirochetes presents a problem because spirochetes are rarely found in the blood. However, the antigen Bb in its cysts, or cell wall deficient (CWD) form can be found in the blood.

There are only a couple of laboratories that do antigen testing for Bb. Antigen tests check for the organism itself and aren't dependent upon a sick immune system to produce antibodies. The Central Florida Research Laboratory in South Florida is a laboratory doing antigen testing. CFR's antigen test is a one of a kind. It is an immune fluorescence test using a special kind of machine called Flow Cytometry. Flow Cytometry is a specific instrument designed to identify the various stages of Borrelia burgdorferi. Cells acting with fluorescent antibodies are counted. The Flow Cytometer can count the number of organisms in 100,000 cells in three minutes and 50,000 cells in one minute. It counts all the cells passing through the aperture and enumerates the organisms that react with the fluorescent antibody. The test result will be reported as a percent of the counted cells.

The Flow Cytometer prints out in a percentage ratio of how many cells are enumerated and fluorescing - .02 negative - .03 borderline -.04 positive. This test named the ' RAIBb' is more definitive than the Bowen Q-RIBb test. However, it must be stated the final diagnosis for Lyme disease is a clinical diagnosis.

Since Borrelia burgdorferi is a CWD and a stealth pathogen, an antigen test result is more accurate in detecting its presents in the blood than a test looking for antibodies. Our blood supply is protected by an antigen test to detect for HIV, thereby making the blood supply much safer for the detection of HIV. The RAIBb Flow Cytometry test from Central Florida Research is most beneficial in picking up Borrelia Burgdorferi the causative agent for Lyme Disease.

Mycoplasma organisms run the course from non-pathogenic to very pathogenic. Many patients suffering from chronic systemic infectious diseases are also found to have some of the pathogenic species of Mycoplasma present. Mycoplasma Fermentans (especially the Incognitos Strain have been found in many CSID. Another pathogenic Mycoplasma that appears to be present in many CSID is Mycoplasma Pneumonia. These organisms are very good at evading detection by the immune system and are also good at keeping the immune system in Humural (overdrive). These organisms are Cell Wall Deficient from their inception, and can go into little DNA blebs making it difficult not only for the immune system to detect but also difficult for laboratory testing.

Presently the best test for Mycoplasma is the PCR. It is important to use a laboratory that specializes in this type of testing such as MDL in New York. According to some of the scientist who study these organism, patients that have been on antibiotics of the Tetracycline family need to be off these antibiotics for at least 3 months in order to detect the Mycoplasmas.

Central Florida Research, Inc.
245 N. Seminole Avenue
Lake Alfred, FL 33850
Phone: (863) 956-3538
Fax: (863) 956-0839


"This test detects the antigen or spirochete in the blood. The antigen may not be present in the sample tested if there are very few spirochetes within the blood or if they are in some other organ of the body. Therefore, a negative test does not mean the patient does not have Lyme disease. It means that we were unable to detect Borrelia burgdorferi in the specimen."
Viimeksi muokannut soijuv päivämäärä Su Touko 30, 2010 11:57, muokattu yhteensä 1 kerran
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » La Tammi 24, 2009 18:49

USEITA TUTKIMUKSIA TESTEISTÄ:

1. Tämän tutkimuksen mukaan vasta-ainetesteissä IgG -luokan vasta-aineet ovat merkittävin laboratoriodiagnostinen osoitus borrelioosista.


http://www.ncbi.nlm.nih.gov/entrez/quer ... &DB=pubmed

Ann Agric Environ Med. 2006;13(2):307-11.

Correlation of tests for detection of Borrelia burgdorferi sensu lato infection in patients with diagnosed borreliosis.

* Chmielewska-Badora J,
* Cisak E,
* Wojcik-Fatla A,
* Zwolinski J,
* Buczek A,
* Dutkiewicz J.

Department of Occupational Biohazards, Institute of Agricultural Medicine, Jaczewskiego 2, 20-090 Lublin, Poland. jcb@galen.imw.lublin.pl

A group of 180 patients with diagnosed Lyme borreliosis were examined for the presence of infection with Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) by serologic tests with B. burgdorferi s.l. antigens (IgM-ELISA, IgG-ELISA, IgM-immunoblot, IgG-immunoblot) and by polymerase chain reaction (PCR, nested-PCR) for detection of B. burgdorferi s.l. DNA in peripheral blood. A total of 61.7 %, 53.9 %, 62.2 %, and 59.4 % of the examined patients' sera showed positive or borderline results in the serologic tests IgM-ELISA, IgG-ELISA, IgM-immunoblot, and IgG immunoblot, respectively.

The results of the tests IgM-ELISA and IgM-immunoblot were significantly correlated (p < 0.001). A higher degree of the correlation (p < 0.000001) was found at the comparison of results obtained with IgG-ELISA and IgG-immunoblot. The correlation between the positive findings in the IgM-ELISA and detection with IgM-immunoblot the diagnostically important B. burgdorferi s.l. OspC surface protein was relatively low but statistically significant (0.01 < p < 0.05). Much higher correlation was found between the positive findings in the IgG-ELISA and detection with IgG-immunoblot other diagnostically important B. burgdorferi s.l. antigen, the VlsE protein (p < 0.000001). The presence of B. burgdorferi s.l. DNA was found by PCR in 20 out 180 examined blood samples (11.1 %). No correlation was found to exist between the PCR results and the results of any of the serologic tests for detection of anti B. burgdorferi s.l. antibodies of IgM class. PCR results correlated significantly at a relatively low level (0.01 < p < 0.05) with the results of IgG-ELISA, but not with the results of IgG-immunoblot with regard to total reactions (0.2 < p < 0.1). By contrast, a distinctly significant correlation was found between the PCR results and detection of the VlsE protein with IgG-immunoblot (0.001 < p < 0.01).

In conclusion, the results of the present study suggest that antibodies of IgG class are the most reliable marker in laboratory diagnostics of Lyme borreliosis, in particular those directed against VlsE surface protein of Borrelia burgdorferi sensu lato. PMID: 17196006 [PubMed - in process]

Related Links

* Enzyme-linked immunosorbent assay, immunofluorescent assay, and recombinant immunoblotting in the serodiagnosis of early Lyme borreliosis. [Int J Immunopathol Pharmacol. 2003] PMID: 14611730
* Prevalence of DNA and antibodies to Borrelia burgdorferi sensu lato in dogs suspected of borreliosis. [Ann Agric Environ Med. 2005] PMID: 16457474
* Immunoblot using recombinant antigens derived from different genospecies of Borrelia burgdorferi sensu lato. [Med Microbiol Immunol (Berl). 1994] PMID: 8202030
* Diagnosis of lyme borreliosis. [Clin Microbiol Rev. 2005] PMID: 16020686
* Polymerase chain reaction in diagnosis of Borrelia burgdorferi infections and studies on taxonomic classification. [APMIS Suppl. 2002] PMID: 11985118
* See all Related Articles...

FREE FULL text can be read here :- http://www.aaem.pl/pdf/13307.htm



2. Tutkimuksessa vertailtiin kahta borrelioosin laboratoriodiagnostiikassa käytettyä vasta-ainetestiä; IFA ja LIAISON. LIAISON havaitsi vasta-aineet IFA:aa useammin, lukuunottamatta kroonista borrelioosia sairastavien kohdalla. Kummankin testin spesifisyys oli samaa luokkaa.


Wien Klin Wochenschr. 2006 Nov;118(21-22):686-90.
Comparison of immunofluorescence assay (IFA) and LIAISON((R)) in patients with different clinical manifestations of Lyme borreliosis.

Cerar T, Ruzic-Sabljic E, Cimperman J, Strle F.

Institute of Microbiology and Immunology, Medical Faculty Ljubljana, University of Ljubljana, Slovenia, tjasa.cerar@mf.uni-lj.si.

Serological tests for detection of borrelial antibodies are frequently used in laboratory diagnostics of Lyme borreliosis. Unfortunately these tests are not standardized and the results obtained with different assays may not be concordant. The aim of the present study was to compare two different serological tests, IFA and LIAISON((R)), for detection of Borrelia burgdorferi sensu lato IgM and IgG antibody. We analyzed the serological immune response in 383 patients with different clinical manifestations of Lyme borreliosis and in 49 healthy blood donors.

LIAISON((R)) detected IgM and IgG antibodies more often than IFA in all groups of patients except those with chronic Lyme borreliosis. The differences were significant for IgM and IgG antibodies in patients with solitary erythema migrans and in those with early disseminated Lyme borreliosis. There was no significant difference in the specificity of the two tests.

PMID: 17160608 [PubMed - in process]


3. IDSA:n suositusten takana oleva IDSAn näkemystä borrelioosin hoidosta edustava A. Steere tekee yhteistyötä helsinkiläisten Haartman-instituutin tutkijoiden kanssa, mm. I. Seppälä, Peltomaa jne. IDSA ei mm. tunnusta borrelioosin kroonistumista.

Tutkimuksen mukaan vasta-ainetesti C6 ELISA (IR 6) antoi vaihtelevia tuloksia riippuen bakteerin alalajista. Testi oli epäluotettavin nimenomaan Euroopassa esiintyviä alalajeja, B.garinii ja B.afzelii, kohtaan.


Int J Med Microbiol. 2007 Jan 16; [Epub ahead of print]

Immune responses to borrelial VlsE IR(6) peptide variants.
Sillanpaa H, Lahdenne P, Sarvas H, Arnez M, Steere A, Peltomaa M, Seppala I.
Department of Bacteriology and Immunology, Haartman Institute, Helsinki, Finland.

Laboratory confirmation of Lyme borreliosis (LB) relies mainly on the demonstration of anti-borrelial antibodies. In recent studies, a novel VlsE protein IR(6) peptide-based assay has been introduced. Our aim was to evaluate the IR(6) peptides from three Borrelia burgdorferi sensu lato genospecies in the serodiagnosis of European and North American patients. Five VlsE protein IR(6) peptide variants representing sequences from B. burgdorferi sensu stricto, B. garinii, and B. afzelii were used as antigens in both IgG and IgM enzyme- inked immunosorbent assays (ELISA). Serum antibodies of 187 patients at different stages of LB from Europe and the United States were evaluated for serodiagnosis. For comparison samples were tested with one of the commercial IR(6) ELISAs.

Three B. afzelii IR(6) variant peptides revealed antibodies that were concordant with each other. B. burgdorferi sensu stricto peptide antibodies mostly paralleled B. afzelii peptide antibodies, and positive values were also obtainedin the majority of European sera. For several sera, B. garinii IR(6) peptide antibodies were discordant to B. afzelii peptide antibodies. The commercial IR(6) peptide antibody assay (C6 ELISA) results correlated better with B. burgdorferi sensu stricto IR(6) than with B. garinii IR(6) peptide IgG results, especially in sera from patients with facial palsy. Thus, antibody specificity to IR(6) peptides may vary according to the infecting Borrelia species. In some manifestations of the disease, C6 ELISA may not cover all LB cases. Evidently, the methodological aspects in ELISA design for peptide antibody measurements are important as well as the amino acids sequence of the antigen.

PMID: 17234451 (PubMed - as supplied by publisher)

4. Varhaisvaiheen borrelioosin diagnosointi PCR-tekniikalla on luotettavampi virtsasta kuin verestä. Bakteerin DNA:ta löydettiin 8 %:lta virtsasta vielä vuoden kuluttua. Sairastuneilla ei ollut kliinisiä oireita. (Suom.huom. Bb:a löydettiin virtsateistä siis jo sairauden varhaisvaiheessa.)

Acta Derm Venereol. 2007;87(1):39-42.
Course of Borrelia burgdorferi DNA Shedding in Urine after Treatment.
Aberer E, Bergmann AR, Derler AM, Schmidt B.
Department of Dermatology, Medical University of Graz, Auenbruggerplatz 8.

Diagnosis of Lyme borreliosis by urine polymerase chain reaction (PCR) has been recognized as having better diagnostic sensitivity in patients with erythema migrans than serological methods. We made serial tests with 192 urine specimens from 70 patients with erythema migrans and 60 urine specimens from 21 patients with acrodermatitis chronica atrophicans to evaluate the course of positive urine PCR after antibiotic treatment. Before treatment, urine samples from patients with erythema migrans showed a positive PCR in 27/34 samples (79%), and those from patients with acrodermatitis chronica atrophicans in 7/11 (63%).

The specificity of bands was proven by hybridization with GEN-ETI- TM-DEIA kit in 40/41 samples. Borrelia DNA in urine decreased gradually within the observation period of one year in both patients with erythema migrans and acrodermatitis chronica atrophicans, and persisted without clinical symptoms in 4/45 patients with erythema migrans (8%) after 12 months. Urine PCR can serve as a diagnostic method in early Lyme borreliosis and also in seropositive patients
with unclear clinical symptoms.

PMID: 17225014 [PubMed - in process]


5. Bb on vaikeasti löydettävissä normaaleilla ihobiopsia testeillä. Itävaltalaiset tutkijat ovat kehittäneet uuden menetelmän borreliabakteerien löytämiseksi ihonäytteistä. Testistä käytetään nimeä FFM (focus floating microscopy). FFM osoittautui PCR -testiä herkemmäksi menetelmäksi.

Am J Clin Pathol. 2007 Feb;127(2):213-22.

Focus floating microscopy: "gold standard" for cutaneous borreliosis? Eisendle K, Grabner T, Zelger B.

Department of Dermatology and Venerology, Innsbruck Medical University, Innsbruck, Austria

Borrelia burgdorferi is difficult to detect in routine biopsy material from patients with skin lesions of borreliosis. In this study, a new immunohistochemical method, focus floating microscopy (FFM), was developed to detect B burgdorferi in tissue sections and was compared with polymerase chain reaction (PCR). By using standard histologic equipment, tissue sections stained with a polyclonal B burgdorferi antibody were simultaneously scanned through 2 planes: horizontally in serpentines and vertically by focusing through the thickness of the section.Borrelia were detected in 47 of 71 ticks, 34 of 66 tick bites, 30 of 32 erythema chronicum migrans cases, 41 of 43 borrelial lymphocytomas, and 50 of 51 acrodermatitis chronica atrophicans cases. FFM proved to be more sensitive than PCR (96.0% vs 45.2%) and nearly equally specific (99.4% vs 100%). All 169 control cases, except 1 false-positive case of secondary syphilis, were negative with FFM. FFM is an easy, quick, and inexpensive method to reliably detect Borrelia in cutaneous tissue sections.
PMID: 17210530 [PubMed - in process]


6. Muutama ote kroonista fatiikkia käsittelevän konferenssin luennoista: Tri Nicholson kertoo punkkien välittävän useita sairauksia. Kroonista fatiikkia sairastavilta on löydetty kohonneita vasta-ainepitoisuuksia borreliabakteeria ja useita muita taudinaiheuttajia esim. mykoplasmaa kohtaan.

Tri Vojdani on kehittänyt uuden antigeeniin perustuvan borrelioositestin nimeltä IVAT.



http://phoenix-cfs.orgIACFS%202007%20Co ... une,%20Gut,%2Pain%20and%20Sleep.htm

LYME INFECTION RATES IN CFS PART I: Garth Nicolson ? Chronic bacterial co-infections in Chronic Fatigue Syndrome and Chronic Fatigue Syndrome patients subsequently diagnosed with Lyme disease.

Dr. Nicolson was another impressive speaker. He has been involved in elucidating pathogen prevalence in CFS for many years now. Some researchers lost interest in pathogens when they failed to find the pathogen that caused CFS. It?s clear now that no one pathogen causes CFS. We know that CFS patients are more susceptible to viral reactivation and bacterial and viral infection than are healthy people but we didn?t really know how much more susceptible until now. Dr. Nicolson apparently added up all the numbers and found that CFS 18x?s more likely to harbor a pathogen than expected!

Dr. Nicolson also presented the first data on Lyme prevalence in CFS. Dr. Nicolson noted that ticks carry a number of different diseases, then indicated that he found that that 9% of Western U.S and a higher percentage of Eastern U.S. CFS patients tested positive on a Western Blot test for Borrelia burgdorfii antigens. These patients appeared susceptible to multiple infections as about 2/3rds of them also tested positive for a mycoplasma infection.

LYME INFECTION RATES IN CFS PART II: Aristo Vojdani, Bernard Raxlen. In vivo induced antigen technology for detection of antibodies against Borrelia burgdorferi and its cross-reactive antigens in patients with Chronic Fatigue and Fibromyalgia (poster)

As with some other pathogens associated with CFS the diagnostic capability of the different Lyme tests has been in question. Here Dr. Vodjani introduces a new test for Lyme disease called In Vivo-Induced Antigen Technology (IVAT) that identifies antigens or immune reactive proteins produced by Borrelia infections. An antigen is something that provokes the immune system. Many pathogenic tests look not for the pathogen itself but for indications that the immune system has been activated by an infection. The test Dr. Vodjani is describing appears to be looking for specific proteins that Borrelia produces.

Dr. Vodjani subjected 206 samples from CFS and Fibromyalgia patients to two Lyme tests; the ELISA and Western Blot (WB) tests and the new IVIAT multiple peptide-based ELISA (IVIAT-MPE) test. He found a much higher rate of positivity ? about 45% (92/206 samples) using WB than Dr. Nicolson did. Hence the questions regarding the efficacy of these tests! Were Dr. Vodjani?s patients from a Lyme hotspot? Or are the tests just unreliable? The IVIAT-MPE was positive in almost all of these as well (88/92).

The IVIAT-MPE test was also positive in a high percentage (32/42) of the WB tests with equivocal results AND it was positive in a good number of the samples the WB test found were negative (26/44). All told the IVIAT-MPE test was positive in almost 60% (146/206) of the CFS/FM samples.

Does this mean that 60% of CFS patients have Lyme disease? Not necessarily so. Dr. Vodjani indicates that the IVIAT-MPE test "detects antibodies against unrelated peptides and proteins of different infectious agents". He further notes that because of this physicians must make sure that other spirochetes such as Yersinia entercolitica, Brucella, Chlamydiae pneumoninae, Ricketssia ricketsii and even the glutathione-S-transferase protein need to be excluded before a physician begins on a protocol of long term antibiotic treatment to attack the Lyme pathogen. He suggests that this test should be used in combination with the Western blot test.


7. "CD-57 -pitoisuuden mittaaminen on merkittävä edistysaskel borrelioosin diagnostiikassa ja hoidossa. Tunnetusti krooninen borrelioosi heikentää immuunijärjestelmää ja saattaa alentaa CD-57 osuutta (tappajasolut). HIV-infektiossa alhaisia T-solupitoisuuksia pidetään merkkinä taudin aktiivisuudesta. CD-57 -pitoisuudella voidaan seurata vastaavasti taudin aktiivisuutta borrelioosissa ja sitä onko antibioottihoidon jälkeen odotettavissa taudin aktivoituminen uudelleen.

Testiä voidaan käyttää myös joukkotutkimusmenetelmänä sillä se on halpa ja yksinkertainen suorittaa. Mikäli henkilön CD-57 on kovin korkea, johtuvat hänen oireensa todennäköisesti jostakin muusta kuin borreliabakteerista (esim. lisäinfektiosta). CD-57:n normaaliarvo on yli 200. Mikäli CD-57:n pitoisuus ei ole antibioottihoidon loputtua normaaliarvon rajoissa, on erittäin todennäköistä, että potilaan oireet palaavat. "


..."Our ability to measure CD-57 counts represents a breakthrough in LB diagnosis and treatment. Chronic LB infections are known to suppress the immune system and can decrease the quantity of the CD-57 subset of the natural killer cells. As in HIV infection, where abnormally low T-cell counts are routinely used as a marker of how active that infection is, in LB we can use the degree of decrease of the CD-57 count to indicate how active the Lyme infection is and whether, after treatment ends, a relapse is likely to occur. It can even be used as a simple, inexpensive screening test, because at this point we believe that only Borrelia will depress the CD-57. Thus, a sick patient with a high CD-57 is probably ill with something other than Lyme, such as a co-infection.

When this test is run by LabCorp (the currently preferred lab, as published studies were based on their assays), we want our Lyme patients to measure above 60; a normal count is above 200. There generally is some degree of fluctuation of this count over time, and the number does not progressively increase as treatment proceeds. Instead, it remains low until the LB infection is controlled, and then it will jump. If the CD-57 count is not in the normal range when a course of antibiotics is ended, then a relapse will almost certainly occur.

http://www.ilads.org/files/burrascano_0905.pdf

---------------------------------------------------

Immunol Lett. 2001 Feb 1;76(1):43-8

Decreased CD57 lymphocyte subset in patients with chronic Lyme disease.

Stricker RB,

Winger EE.
Department of Medicine, California Pacific Medical Center, 450 Sutter Street, Suite 1504, San Francisco, CA 94108, USA. rstricker@usmamed.com

BACKGROUND: Chronic Lyme disease (LD) is a debilitating illness caused by tickborne infection with the spirochete Borrelia burgdorferi. Although immunologic abnormalities appear to play a role in this disease, specific immunologic markers of chronic LD have not been identified.

METHODS: We evaluated 73 patients with chronic LD for lymphocyte subset abnormalities using flow cytometry. Of these, 53 patients had predominant musculoskeletal symptoms, while 20 patients had predominant neurologic symptoms. The estimated duration of infection ranged from 3 months to 15 years, and all patients had positive serologic tests for B. burgdorferi. Ten patients with acute LD (infection less than 1 month) and 22 patients with acquired immunodeficiency syndrome (AIDS) served as disease controls. RESULTS: All 31 chronic LD patients who were tested prior to antibiotic treatment had significantly decreased CD57 lymphocyte counts (mean, 30+/-16 cells per microl; normal, 60-360 cells per microl, P<0.001). Nineteen of 37 patients (51%) who were tested after initiating antibiotic therapy had decreased CD57 levels (mean, 66+/-39 cells per microl), and all five patients tested after completing antibiotic treatment had normal CD57 counts (mean, 173+/-98 cells per microl). In contrast, all 10 patients with acute LD and 82% of AIDS patients had normal CD57 levels, and the difference between these groups and the pre-treatment patients with chronic LD was significant (P<0.001). Patients with chronic LD and predominant neurologic symptoms had significantly lower mean CD57 levels than patients with predominant musculoskeletal symptoms (30+/-21 vs. 58+/-37 cells per microl, P=0.002). CD57 levels increased in chronic LD patients whose symptoms improved, while patients with refractory disease had persistently low CD57 counts.

CONCLUSIONS: A decrease in the CD57 lymphocyte subset may be an important marker of chronic LD. Changes in the CD57 subset may be useful to monitor the response to therapy in this disease.

PMID: 11222912 [PubMed - indexed for MEDLINE]

7. Turkulainen infektiolääkäri Oksi mainitsee sivuillaan borrelioosin diagnoosin olevan aina kliininen, lab.testeissä löydetään mm. tumavasta-aineita, kiertäviä immuunikomplekseja ja kohonneita maksa-arvoja.

Norjalainen tutkija O. Brorson kertoi tänään, että he käyttävät immuunikompleksien testausta taudin aktiivisuuden seurannassa.


Oksi:
"LB:n diagnoosi on aina perustaltaan kliininen. Perusteellinen anamneesi eli keskustelu potilaan kanssa oireiston alkamistavasta sekä siihen liittyvistä asioista jne. on borrelioosidiagnostiikan kulmakivi. Punkinpureman ja EM:n puuttuminen ei kuitenkaan poissulje LB:a. Borrelia-vasta-ainetutkimusta ei ole syytä ottaa oireettomalta ihmiseltä esim. pelkän potilaan uteliaisuuden vuoksi.
Epäspesifisistä laboratoriolöydöksistä laskon ja CRP:n kohoaminen on suhteellisen harvinaista. Verikokeissa voidaan todeta ns. kiertäviä immuunikomplekseja, lievästi koholla olevia tumavasta-aine- ja "maksaentsyymi"tasoja todetaan melko usein.

Hermojuuritulehdukselle ja aivokalvontulehdukselle on tyypillistä selkäydinnesteen tulehdussolulödös ja kohonnut proteiinipitoisuus.
Spesifinen diagnoosi perustuu yleisimmin vasta-aineiden osoittamiseen verestä. IgM- luokan vasta-aineet kehittyvät yleensä 1 kk:n kuluessa ja häviävät normaalisti 6 kk:n kuluessa tartunnasta. IgG vasta-aineet kohoavat 1.5 - 2 kk:ssa ja ne ovat huipussaan 6 kk:n kuluttua. Korkealla pysyvä IgM tai IgG vasta-aineiden taso viittaa pitkittyneeseen infektioon. Varhaisvaiheessa vasta-aineiden tuotanto on hyvin heikkoa ja diagnoosiin päästään menetelmästä riippuen vain 30 - 70 %:ssa tapauksista..." http://www.punkki.net/artikkelit/art2_lb.html#diagno


8. "Borreliabakteeria on vaikea löytää nykyisillä käytössä olevilla rutiinitesteillä. Serologiset testit (Elisa, immunoblottaus) eivät myöskään ole luotettavia. Ne antavat 20 - 80 %:ssa tapauksista väärän negatiivisen ja joskus väärän positiivisen tuloksen esim. ristireaktio syfiliksen aiheuttajan kanssa tai positiivisen endeemisen taustan vuoksi eri puolilla Eurooppaa." Tutkimuksessa esitetään uusi immunohistokemiallinen menetelmä, FFM, borreliabakteerin löytämiseksi kudoksista.


Focus Floating Microscopy: "Gold Standard" for Cutaneous Borreliosis?
http://www.medscape.com/viewarticle/551675

Posted 02/27/2007

Klaus Eisendle, MD, PhD; Tanja Grabner, MD; Bernhard Zelger, MD, MSc
Author Information

http://adv.medscape.com/js.ngord%3D1174 ... 26mpg%3D43"

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Abstract and Introduction

Abstract
Borrelia burgdorferi is difficult to detect in routine biopsy material from patients with skin lesions of borreliosis. In this study, a new immunohistochemical method, focus floating microscopy (FFM), was developed to detect B burgdorferi in tissue sections and was compared with polymerase chain reaction (PCR). By using standard histologic equipment, tissue sections stained with a polyclonal B burgdorferi antibody were simultaneously scanned through 2 planes: horizontally in serpentines and vertically by focusing through the thickness of the section.

Borrelia were detected in 47 of 71 ticks, 34 of 66 tick bites, 30 of 32 erythema chronicum migrans cases, 41 of 43 borrelial lymphocytomas, and 50 of 51 acrodermatitis chronica atrophicans cases. FFM proved to be more sensitive than PCR (96.0% vs 45.2%) and nearly equally specific (99.4% vs 100%). All 169 control cases, except 1 false-positive case of secondary syphilis, were negative with FFM. FFM is an easy, quick, and inexpensive method to reliably detect Borrelia in cutaneous tissue sections.

Introduction
The spirochetal origin of borreliosis (Lyme disease) was first demonstrated by Burgdorfer et al[1] in 1982. The microorganisms (Borrelia burgdorferi) are transfected by ticks, in Europe usually Ixodes ricinus, and cause characteristic disease. Similar to syphilis, borreliosis has been separated into 3 stages. In stage 1, erythema chronicum migrans (ECM) (Figure 1) and borrelial lymphocytoma (BL) (Figure 2) appear weeks after the tick bite; in stage 2, borrelial arthritis, lymphocytic meningoradiculoneuritis (Bannwarth syndrome), and borrelial carditis appear after months; and in stage 3, acrodermatitis chronica atrophicans (ACA) (Figure 3) appears after years.[2-4] Stages may overlap or be skipped. Many other tissues such as muscle, soft tissues, and internal organs may be involved, and unusual clinical manifestations may be seen.[4,5]


Figure 1. (click image to zoom)
A, Characteristic clinical manifestations of erythema chronicum migrans. B, Histologic examination (H&E, ×20) reveals superficial and middermal dense perivascular infiltrate of (C) lymphocytes, some plasma cells, and an increase of fibroblasts and mucin between collagen bundles (H&E, ×200).

Figure 2. (click image to zoom)
A, Borrelial lymphocytoma with characteristic histologic features of dense infiltrates of lymphocytes (B, H&E, ×10) with follicle formation (C, H&E, ×200).

Figure 3. (click image to zoom)
A, Acrodermatitis chronica atrophicans of left leg characterized by ill-defined, hyperpigmented, and atrophic patch (note prominent veins). B, Histologic examination (H&E, ×10) reveals a dense lichenoid and middermal perivascular infiltrate with hints of follicle formation (C, H&E, ×100) composed of lymphocytes, some plasma cells, and an increase of fibroblasts between fibrosclerotic collagen bundles (D, H&E, ×200).

Histologic examination of borrelial infection reveals an infiltrate of lymphocytes, macrophages, plasma cells, and fibroblasts with variable fibrosclerosis.[2] After initial enthusiasm,[6] detection of microorganisms has turned out to be difficult, frequently unreliable, and almost always extremely time-consuming by different procedures, including histochemical stains (Gram, Wright, Wright-Giemsa, and polychromes), fluorochromes (thioflavine-T, acridine orange, and rhodamine), silver impregnation techniques (Warthin-Starry, modified Dieterle, modified microwave-Dieterle, and Bosma-Steiner) in the 1980s,[7-11] and immunohistochemical analysis in the 1990s.[7,8,12-14] In our opinion and that of others, there is need for a reliable and easy method of direct detection of Borrelia in tissue.[12] To our knowledge, no company supplies positive control sections, making assessment of methods difficult.

Serologic techniques (immunofluorescence, enzyme-linked immunosorbent assay [ELISA], and immunoblot) are similarly unsatisfying, with false-negative (20%-80%) and false-positive results occasionally due to cross-reactions with Treponema pallidum or, more commonly, to a positive endemic background of 20% to 50% in many parts of Europe.[8,15,16] Cultures with specified media such as modified Pettenkofer-Kelly or Barbour-Stoenner-Kelly can detect Borrelia in all clinical forms, but these techniques are not generally available and are unreliable, with less than 50% sensitivity.[8,17] Molecular techniques initially seemed to solve the riddle,[18-20] but in due course, it became clear that sensitivity varies (30%-90%) according to the Borrelia strains, the material (fresh frozen tissue or paraffin material), and the applied primers.[8,21-24] Borreliosis remains a diagnosis based on circumstantial evidence combining clinicopathologic and laboratory information and clinical response to therapy.

To the best of our knowledge, this article describes for the first time a new immunohistochemical detection method that is highly sensitive and reliable, works on routinely formalin-fixed, paraffin-embedded tissue samples, and, according to performance, is called focus floating microscopy (FFM).

Section 1 of 4
Next Page: Material and Methods

9. Borrelioosia epäiltäessä tutkitaan aina vasta-aineiden esiintymistä veressä (Borrelia IgM ja IgG). Italialaisessa tutkimuksessa vertailtiin 3:a kaupallista ELISA -testiä. Tämäkin uusi tutkimus vahvisti lukuisia aiempia tutkimuksia ja kannanottoja siitä että ELISA testimenetelmä on epäluotettava. Tutkimuksessa käytettyjen testien herkkyys vaihteli 36,8 % - 70 %:n välillä - eli borrelioosidiagnoosi on edelleen kliininen eikä yksinomaan verikokeisiin yms perustuva:


Comparative evaluation of three different ELISA methods for the diagnosis of early culture-confirmed *Lyme* disease in Italy.

http://www.ncbi.nlm.nih.gov:80/entrez/q ... s=15770021

AUTHORS: Antonella Marangoni, Monica Sparacino, Francesca Cavrini, Elisa Storni, Valeria Mondardini, Vittorio Sambri, Roberto Cevenini

AFFILIATION: Sezione di Microbiologia - DMCSS, University of Bologna, St Orsola Hospital, via Massarenti 9, Bologna, Italy.

REFERENCE: J Med Microbiol 2005 Apr 54(Pt 4):361-7

In this study the raising and development of the immune response to *Borrelia burgdorferi* infection in 45 Italian patients suffering from culture-confirmed *Lyme* borreliosis erythema migrans was investigated. A total of 95 serially collected serum samples were tested by using three different commercial ELISAs: recomWell Borrelia (Mikrogen), Enzygnost Borreliosis (DADE Behring) and Quick ELISA C6 Borrelia (Immunetics).

The sensitivities of the ELISAs were as follows: Enzygnost Borreliosis IgM , 70.5 %; Quick ELISA C6 Borrelia, 62.1 %; recomWell Borrelia IgM, 55.7 %; recomWell Borrelia IgG, 57.9 %; and Enzygnost Borreliosis IgG, 36.8 %. In order to compare the specificity values of the three ELISAs, a panel of sera obtained from blood donors (210 samples coming from a non- endemic area and 24 samples from an endemic area) was tested, as well as sera from patients suffering from some of the most common biological conditions that could result in false-positive reactivity in *Lyme* disease serology (n = 40). RecomWell Borrelia IgG and recomWell Borrelia IgM were the most specific (97.1 % and 98.9 %, respectively), followed by Quick ELISA C6 Borrelia (96.7 %). Enzygnost Borreliosis IgG and IgM achieved 90.1 % and 92.3 % specificity, respectively. Sera that gave discrepant results when tested by the three ELISAs were further analysed by Western blotting.

PMID: 15770021 http://refscout.com/cgi-bin/exportAbstr ... d=15770021


10. "Immunoglobuliini E saattaa antaa immuniteetin borreliabakteerin aiheuttamaa infektiota kohtaan. Immuniteetti voi kestää koko elämän ajan. Tutkimuksessa seurattiin lapsia jotka olivat jossakin vaiheessa saaneet elämäänsä saaneet borreliatartunnan."


Scand J Immunol. 2007 Apr;65(4):376-82.Click here to read Links
IgE Anti-Borrelia burgdorferi Components (p18, p31, p34, p41, p45, p60) and Increased Blood CD8(+)CD60(+) T Cells in Children with Lyme Disease.

* Bluth MH,
* Robin J,
* Ruditsky M,
* Norowitz KB,
* Chice S,
* Pytlak E,
* Nowakowski M,
* Durkin HG,
* Smith-Norowitz TA.

Department of Surgery, S.U.N.Y. Downstate Medical Center, Brooklyn, NY, USA.

Immunoglobulin (Ig) E may provide immunity against Borrelia burgdorferi infection (Lyme disease) in children which lasts throughout adulthood. We investigated the presence and persistence of IgE anti-B. burgdorferi antibodies (Abs) in paediatric patients infected with Lyme disease over time. Serum immunoglobulin levels, presence of IgG and IgE anti-B. burgdorferi components, and distributions of blood T, B and natural killer lymphocyte subsets were studied in B. burgdorferi-infected and -uninfected children (nephelometry, UniCAP Total IgE Fluoroenzymeimmunoassay, Western blot, flow cytometry). Total serum IgM, IgG, IgE and IgA levels, and distributions of blood lymphocytes (CD4(+), CD8(+), CD19(+)) of both groups, excluding CD8(+)CD60(+) T cells, were within normal ranges. However, infected, but not uninfected children made IgG anti-B. burgdorferi proteins p18, p31, p34, p41, p45, but not IgG anti-p60, and IgE anti-B. burgdorferi proteins p31, p34, p41, p45, p60, but not IgE anti-p18. These proteins were also detected in an infected child 1 year post-infection. Interestingly, CD8(+)CD60(+) T-cell numbers were significantly increased (fourfold) in infected, compared with uninfected, patients (P = 0.001). These results demonstrate that specific IgE anti-B. burgdorferi Abs are generated and persist in children with Lyme disease and that CD8(+)CD60(+) T cells may play an important role in these responses.

PMID: 17386029 [PubMed - in process]
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ViestiKirjoittaja soijuv » La Tammi 24, 2009 18:52

Artikkelin mukaan borreliavasta-ainetestit voivat olla virheellisesti negatiiviset esim. sellaisilla henkilöillä joiden immuunipuolustus on heikentynyt.

Infection. 2007 Apr;35(2):110-113.
Seronegative Lyme Neuroborreliosis in a Patient on Treatment for Chronic Lymphatic Leukemia.

* Harrer T,
* Geissdorfer W,
* Schoerner C,
* Lang E,
* Helm G.

Dept. of Medicine III, University Hospital Erlangen, Krankenhausstr. 12, 91054, Erlangen, Germany, Thomas.Harrer@med3.imed.uni-erlangen.de.

We report on a patient who developed seronegative Lyme neuroborreliosis complicating chemotherapy for chronic lymphatic leukemia. After the fifth cycle of chemotherapy (FCR: fludarabine, cyclophosphamide, rituximab and prednisone) the 63-year-old patient developed night sweat, arthralgia in elbows, wrists, proximal interphalangeal joints (PIPs) and strong neuropathic pain in both legs, followed by paresthesia and hypesthesia in the feet, arms and face. Laboratory analysis revealed an elevated C-reactive protein (CRP), a slight elevation of liver enzymes and decreased IgG levels. Cerebrospinal fluid (CSF) analysis showed a lymphomononuclear pleocytosis and an elevation of protein. A broad diagnostic work-up was negative including a negative Borrelia IgG and IgM ELISA. The patient did not remember recent tick bites, but after specific questioning he recollected a transient erythema on his leg developing just before the start of the last cycle of chemotherapy. As the combination of neuropathic pain and arthralgia, the transient erythema and the lymphomononuclear pleocytosis raised the suspicion of Lyme neuroborreliosis, the patient was treated for 3 weeks with ceftriaxone. On therapy all symptoms resolved and CRP normalized. Retrospective PCR analysis of a CSF sample confirmed the clinical diagnosis by detecting Borrelia garinii DNA.

This case demonstrates that in immunosuppressed patients borrelial serology may be negative and that additional diagnostic approaches (including tests for direct Borrelia detection) may be needed to demonstrate borrelial infection.

PMID: 17401717 [PubMed - as supplied by publisher]
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ViestiKirjoittaja soijuv » La Tammi 24, 2009 20:55

Miten borrelioosi diagnosoidaan?

Borrelioosin diagnoosi on ensisijassa kliininen. Testejä käytetään tukemaan diagnoosia.


http://www.lymediseaseaction.org.uk/diagnose.htm

How is Lyme Disease Diagnosed?
The LDA believes Lyme Disease should be diagnosed clinically. This is view is supported by Clinical Answers, the NLH'S Primary Care Question Answering Service* for the NHS.

The following Q&A are dated 7th September 2005.

http://www.clinicalanswers.nhs.uk/index ... stion=1076

What is a recognised means of diagnosing Lyme Disease?
A search of the TRIP Database located a number of guidelines and eTextbook articles that discuss diagnosis. A recent American guideline reports on the diagnosis of Lyme Disease [1]. It has a section ?Diagnostic Concerns? which discusses various aspects of diagnosis. This is summarised in the key recommendations as:

?Since there is currently no definitive test for Lyme disease, laboratory results should not be used to exclude an individual from treatment.

Lyme disease is a clinical diagnosis and tests should be used to support rather than supersede the physician?s judgment.?

However, the guideline discusses many other aspects and we recommend you read these at http://www.guidelines.gov/summary/summa ... oc_id=4836

eMedicine, an American online textbook, has a chapter on Lyme Disease [2]. This also has an extensive discussion of diagnosis which, due to length, cannot be reproduced here. Therefore, we recommend you read the relevant section at http://www.emedicine.com/med/topic1346.htm

Finally, GP Notebook, has a section on the diagnosis of Lyme Disease [3] which states:

?It is worth assessing the risks by considering the pathogenesis and clinical features of Lyme disease - negative serology does not exclude the diagnosis.

In more than half of people affected by this condition there is no history of tick bite.

The diagnosis is confirmed by serologic testing by indirect immunofluorescence or enzyme-linked immunosorbent assays. IgM peaks at 3-6 weeks; IgG appears more slowly and may take months or years.

The diagnosis is unreliable early. Check Treponema pallidum haemoglutination is negative before accepting a positive result.

The antibody is not affected by treatment. ESR is elevated.

Use of the polymerase chain reaction to detect the presence of Borrelia burgdorferi DNA in specimens from patients may become the most reliable means of determining who has been infected with this organism and when infection has been eliminate.
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Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » La Tammi 24, 2009 20:56

Unkarilainen borrelioosilääkäri B. Bozsik on sitä mieltä että borrelioosia esiintyy epidemioina eri puolilla maailmaa. Hänen mukaansa borrelioosin kykenee diagnosoimaan luotettavasti yhdellä laboratoriotutkimusmenetelmällä, pimeäkenttämikroskooppisella tutkimuksella. Tutkimus on tunnettu jo vuodesta 1909.

(Suom.huom. Tiedossani ei ole henkilöitä joille olisi tehty Suomessa kyseistä tutkimusta borrelioosin selvittämiseksi.)

Bozsikin mukaan borrelioositapauksia on huomattavasti enemmän kuin yleensä annetaan ymmärtää. Useimmat tautitapaukset kuitenkin "häviävät" sairastuneille annettujen erilaisten diagnoosien viidakkoon.

Bozsikin power point -esitys asiasta löytyy sivulta :

http://lymerick.net/Sheffield2005/Bozsi ... ,1,Telling The Truth ? my experiences with Lyme borreliosis ?
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » La Tammi 24, 2009 21:08

Suomalainen tutkimus v. 2007: Selkäydinnestenäyte saattaa olla hyödyllinen selvitettäessä neuroborrelioosin aktiivisuutta (Elisa)

Antibodies to recombinant decorin-binding proteins A and B in the cerebrospinal fluid of patients with Lyme neuroborreliosis
Panelius, J., et al. - Cerebrospinal fluid (CSF) and serum samples from 34 patients with proven neuroborreliosis (NB) and 22 patients with suspected neuroborreliosis (SNB) from Finland were analysed for antibodies to decorin-binding proteins A (DbpA) and B (DbpB). Antibodies to recombinant protein antigens originating from Borrelia burgdorferi sensu stricto, B. afzelii, or B. garinii species were studied by enzyme-linked immunosorbent assay (ELISA)...The results suggest that CSF antibodies to DbpB might be useful as a marker of active infection whereas antibodies to DbpA seem to persist a long time after acute phases of NB [more...]
Scandinavian Journal of Infectious Diseases, 08/20/07
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » La Tammi 24, 2009 21:09

Muutamat amerikkalaiset tutkijat lähettivät v. 2004 72 borrelioosia sairastavan henkilön, joiden sairaus oli jo aiemmin vahvistettu borrelioosiksi, näytteet tutkittavaksi eri laboratorioihin eri puolilla Amerikkaa (julkaisematon tutkimus). Tuloksissa esiintyi suuria eroja eri laboratorioiden välillä. Huonoimman laboratorion testi tunnisti vain 20 % borrelioositapauksista. Parhaimman laboratorion testi havaitsi yli 85 % tapauksista.


Results of blood tests for evidence of exposure to Lyme disease


June 2004

72 total samples were sent to labs from Lyme disease patients in 9 different states (CA to IN to NY to FL).

44 samples were sent to IGeneX, MDL, or Bowen Lab.

39 were accurate and indicated positive results- 5 were false negative.

28 samples were sent to Stoney Brook, Quest, or Labcorp.

8 were accurate and indicated positive results- 20 were false negative.

Individual lab results-

In patients with confirmed cases of Lyme disease and/or tick borne coinfections:

IGeneX- More than 85 percent of the samples tested were positive

MDL- Approximately 65 percent of the samples tested were positive

Stoney Brook- Approximately 30 percent of the samples tested were positive

Labcorp- Only 25 percent of the samples tested were positive

Quest- Only 20 percent of the samples tested were positive
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » La Tammi 24, 2009 21:55

Mikroskoopisella testillä (focus floating microscopy) pystyttiin havaitsemaan ihonäytteistä borreliabakteereita.

(Suom.huom. En tiedä onko kyseinen testi käytössä Suomessa)


Journal of the American Academy of Dermatology
Volume 57, Issue 6, December 2007, Pages 1026-1030

Dermatopathology
Detection of spirochetal micro-organisms by focus-floating microscopy in necrobiotic xanthogranuloma.

Bernhard Zelger MD, MSc, Klaus Eisendle MD, PhD,Christian Mensing MDand Bettina Zelger MD


Background Necrobiotic xanthogranuloma (NXG) is a rare histiocytic disorder of unknown origin.
Objective
We conducted an investigation of skin biopsy specimens from 7 patients with NXG for the presence of Borrelia by focus-floating microscopy.

Methods
Focus-floating microscopy is a recently described, modified immunhistochemical technique in which the sections of a slide are simultaneously scanned both horizontally and vertically. Focus-floating microscopy is more sensitive for the detection of micro-organisms than polymerase chain reaction.

Results
Borrelia could be detected as single, paired, or clusters of spirochetes in 6 cases of NXG whereas two cases investigated with a Borrelia-specific polymerase chain reaction (23s-RNA) remained negative.

Limitations
Limited biopsy material in each patient prohibited a more detailed study of the life history of cutaneous lesions in NXG.

Conclusions
The detection of this micro-organism in NXG points to a specific involvement of B burgdorferi or other similar strains in the development of or as a trigger of this disease.



Abbreviations: FFM, focus-floating microscopy; NXG, necrobiotic xanthogranuloma; PCR, polymerase chain reaction
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 10:35

Borrelioosi vaikuttaa eri puolille elimistöä, esim. niveliin. Ultraäänitutkimus saattaa olla käyttökelpoinen tutkimusmenetelmä esim. polvinivelen tulehdustapauksia tutkittaessa.

http://www.ncbi.nlm.nih.gov/entrez/quer ... &DB=pubmed

Przegl Epidemiol. 2006;60 Suppl 1:34-8.

[Ultrasonography in diagnosing Lyme arthritis of the knee joints in correlation with anti-CCP antibodies]
[Article in Polish]

* Czeczuga A,
* Targonski A,
* Zajkowska J,
* Hermanowska-Szpakowicz T,
* Swierzbinska R.

Zaklad Diagnostyki Obrazowej Wojewodzkiego Szpitala Specjalistycznego im. K. Dluskiego w Bialymstoku.

Lyme disease, a multi-system disorder may be associated with arthritis. Lyme arthritis most commonly affects the knee joints. Ultrasonography can show the inflammation changes of the knee joint and can be a usefull method in diagnosis of Lyme arthritis. The most freguent ultrasonographic finding was knee joint effusion. Because of Lyme arthritis similarities to rheumatoid arthritis, a serologic test antibodies against cyclic cytrulinated peptid (anty CCP) can be helpfull in distinguishing of these two diseases.

PMID: 16909773 [PubMed - in process]
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 10:58

Yleisesti tunnustetaan, että nykyiset borrelioositestit ovat epäluotettavia. Merkittävä osa positiivisista tapauksista jää huomaamatta - joidenkin arvioiden mukaan jopa yli puolet tapauksista. Viime elokuussa on alkanut tutkimus jossa selvitetään olemassaolevien ja uusien testien diagnostista luotettavuiutta. Tutkimus päättyy kesällä 2008.

Grant Number: 1R43AI069564-01
Project Title: Improving Lyme disease diagnosis through bioinformatics
PI Information: Name Email Title

PORWANCHER, RICHARD B. porwancher@aol.com PRESIDENT

Abstract: DESCRIPTION (provided by applicant): Due to limited physician knowledge of diagnostic criteria for Lyme disease (LD) and excessive utilization of laboratory tests, over-diagnosis and over-treatment of LD has become a significant public health problem. This grant is the first phase of a 2-part process that will develop a bioinformatic framework to maximize the predictive power of new and existing laboratory tests.


The current 2-step serologic approach detects antibody to Borrelia burgdorferi by whole-cell EIA, followed by Western blot confirmation of positive or equivocal EIA results; while this approach is highly specific, it lacks sensitivity for early LD. We will evaluate a new, patented algorithm based on Bayes' theorem that combines the pretest risk of LD with multi-antibody serology to generate a posterior probability for each patient. This new algorithm has demonstrated sensitivity superior to the 2-step method in a pilot study. To provide comparators to the algorithm, multivariate models will be developed concurrently using partial ROC regression and logistic-linear regression, assisted by penalized likelihood functions.


Data from 280 LD patients and 559 controls, already tested using the 2-step method and VIsE, C6, and pepdO ElAs, will be used to develop these models. The new algorithm will utilize those variables selected by partial ROC regression that contribute significantly to the predictive model. The pretest risk of LD will be estimated for each patient and control based on available clinical data. A partial ROC curve (at least 80% specific) will be generated for each predictive method and the 2-step approach by varying their respective posterior probability cutoffs. The diagnostic power of each method will be determined by the area under its partial ROC curve (AUC), and compared to that of the 2-step approach using a bootstrap technique.


The primary study end-point is to identify at least 1 new predictive method with performance equivalent or superior to the 2-step approach, thereby justifying a Phase II study. Phase II will consist of a prospective multi-centered study to collect both serum and clinical data from a diverse set of LD patients and controls. Using the biostatistical techniques developed in Phase I, new clinical and serologic predictive models will be developed in Phase II. This bioinformatic approach has the potential for integrating clinical and serologic diagnostic approaches using a standardized serum bank.

Thesaurus Terms:


There are no thesaurus terms on file for this project.
Institution: INFECTIOUS DISEASE CONSULTANTS, PC

Suite 411

Trenton, NJ 086193831
Fiscal Year: 2006
Department:

Project Start: 15-AUG-2006
Project End: 31-JUL-2008
ICD: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
IRG: BCHI
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 11:18

"Henkilöllä voi olla aktiivinen keskushermostotulehdus negatiivisesta selkäydinnestentestistä huolimatta. Negatiiviset tulokset ovat yleisiä eikä sen perusteella voida tehdä päätelmää että henkilöllä ei ole neuroborrelioosia.."

Alla aihetta käsitteleviä tutkimuksia:



Seronegative Spinal Taps

A patient may have active central nervous system (CNS) infection despite negative spinal fluid tests for B. burgdorferi [1, 2] Negative results are often obtained on cerebrospinal fluid (CSF) of known Lyme patients, including normal cell count and chemistry evaluations and absent Lyme antibody titers.[1, 2] Consequently the absence of antibodies against B. burgdorferi in CSF cannot be relied on to rule out CNS infection with this organism. Fallon explained the issue:



CSF antibody studies in chronic Lyme encephalopathy may be misleadingly normal. In a study of 35 patients with late neurological Lyme disease who had B. burgdorferi OspA antigen in the CSF n experimental testing, 40% had normal CSF antibody study results and 20% had no evidence of either Lyme antibodies or other typical CSF abnormalities. Normal CSF results therefore cannot be used to rule out neuroborreliosis.[3]

Similar findings were made by Dr. Coyle at Stonybrook University:

CSF changes were not marked in either group of adult patients. In adults with acute disease, 45% had reactive or borderline CSF serology, 7% had intrathecal antibody production (a common test for CNS infection), 30% had an elevated white blood cell count (WBC), 23% had elevated protein, only 14% had evidence of oligoclonal bands, and only 7% had an elevated IgG index. Similarly, in the adult chronic disease group, 35% had reactive or borderline CSF serology, 6% had intrathecal antibody production, 15% had elevated WBC, 15% had elevated protein, 6% had evidence of oligoclonal band testing, and 3% had an elevated IgG index.[4]

Given the foregoing, the diagnosis of B. burgdorferi infection should be made primarily on clinical grounds, with current serologies playing only supportive roles.

1. Coyle, P.K., et al., Detection of Borrelia burgdorferi-specific antigen in antibody-negative cerebrospinal fluid in neurologic Lyme disease. Neurology, 1995. 45(11): p. 2010-5.

http://www.ncbi.nlm.nih.gov/entrez/quer ... ds=7501150

2. Pfister, H.W., et al., Latent Lyme neuroborreliosis: presence of Borrelia burgdorferi in the cerebrospinal fluid without concurrent inflammatory signs. Neurology, 1989. 39(8 ): p. 1118-20.

http://www.ncbi.nlm.nih.gov/entrez/quer ... ds=2668788

3. Fallon, B.A., et al., Functional brain imaging and neuropsychological testing in Lyme disease. Clin Infect Dis, 1997. 25 Suppl 1: p. S57-63. http://www.journals.uchicago.edu/CID/jo ... 57.web.pdf

4. Coyle, P.K. Neurologic Lyme disease. in 14th International Scientific Conference on Lyme Disease & Other Tick-Borne Disorders. 2001. Hartford, Connecticutt.

http://www.medscape.com/viewarticle/418441.

Also see http://www.freewebs.com/teenswithlyme/f ... etests.htm
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 12:18

1. Arvostetun lääketieteellisen lehden British Medical Journalin artikkelin mukaan borrelioositestit ovat erittäin epäluotettavia. "Ne jättävät havaitsematta puolet borrelioositapauksista. Niiden luotettavuus on samaa luokkaa kuin mitä saavutettaisiin mikäli taudin diagnostiikassa käytettäisiin kolikon heittoa."

http://www.wddty.com/033638003692252840 ... -coin.html

2. Myös tuoreessa slovakialaisessa tutkimuksessa todettiin borrelioositestien epäluotettavuus. Ongelmana on testien standardoinnin puute. Asiaa vaikeuttaa borreliabakteerien heterogeenisyys eri puolilla Eurooppaa. Testeissä tulisi testata vasta-aineiden muodostusta eri antigeenejä kohtaan.

Bratisl Lek Listy. 2007;108(9):399-402.

3. Borrelioositestit ovat osoittautuneet epäluotettaviksi. Joidenkin tutkimusten mukaan ne pystyvät osoittamaan tartunnan vain noin puolessa tapauksista - ensimmäisen artikkelin mukaan yhtä hyvän tuoksen saa kolikkoa heittämällä.

Vääriä negatiivisia testituloksia siis esiintyy merkittävässä määrin. Myös vääriä positiivisia voi esiintyä joissakin tapauksissa. Tällainen tilanne voi tulla kyseeseen vasta-ainetestien (esim. ELISA, EIA) kohdalla silloin kun ainoastaan IgM on koholla. Henkilöllä saattaa olla ainoastaan tai sekainfektio samanaikaisesti jonkin muun taudinaiheuttajan kanssa, esim. Epstein Barr -virus (EBV), syfilis, toisintokuume, jokin muu spirokeettojen aiheuttama sairaus, reuma, lupus, sytomegalovirus. Siksi oireiden tarkka selvittäminen ja lisätutkimukset ovat tarpeen: esim. immunoblottaus (Western blot) jne.



Lyme disease: The tests are only as good as the toss of a coin

22 November 2007

Tests for Lyme disease are so poor that they can miss up to half of all cases. Even test kits that have been approved by drug regulators such as the Centers for Disease Control in the US miss an average of 88 cases out of every 200.

Current testing kits are no better than tossing a coin, says the Lyme and Associated Diseases Society, and, as a result, many people with the disease are walking around undiagnosed.

(Source: British Medical Journal, 2007; 335: 1008).
----------------------------------------------------------------------------

Antibodies to Borrelia burgdorferi in erythema migrans patients.

Trnovcova M, Bazovska S, Svecova D.

Department of Epidemiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia. mariana.trnovcova@fmed.uniba.sk

Determination of antibodies against Borrelia burgdorferi has supporting value in the diagnose of Lyme disease. The purpose of this study was to determine the production of antibodies in a defined group of patients.

MATERIAL AND METHODS:

The study analysed antibodies in the group of 25 patients with erythema migrans. For the detection of antibodies Immunofluorescence methods, ELISA and Western blot were used.

RESULTS:

The detection of antibodies by Imunoflorescence methods proved positivity of the titre 1:256 in 14 patients and 9 patients were borderline, with the titre 1:128. Majority of the antibodies detected were of IgM class. The ELISA IgM test found positive reaction in 12 patients and 2 borderline results. IgG antibodies were found significantly more often by ELISA test than by Immunofluorescence. The Western blotting results were IgM positive in 9 patients, 6 were borderline. Only 5 patients have positive IgM results in all tests (Immunofluorescence, ELISA and at least one positive result out of three IgM Western blots).

DISCUSSION AND CONCLUSION:

The tests which detect Borrelia burgdorferi antibodies are not standardized. They have variable sensitivity and specificity and their standardization is complicated with respect to great heterogenicity of Borrelia burgdorferi strains circulating in individual regions of Europe. The high specificity of antibodies to individual borrelia antigens are presently pointing towards the need to use, when in diagnostic confusion, more tests, which could detect antibodies also to other borrelia antigens (Tab. 3, Ref. 14).

** Full Text (Free, PDF) www.bmj.sk. **

Publication Types:
Research Support, Non-U.S. Gov't

PMID: 18225477 [PubMed - in process]

................................................................

Cross reactivety of serologic tests is often the rationale but note that cross-reaction is discribed only for ELISA IgM. Co- infection of Bb and EBV is also possible.

False positive EIA results can occur in patients with syphilis, relapsing fever, Rocky Mountain Spotted Fever, other spirochetal diseases, autoimmune disease, rheumatoid arthritis, systemic lupus erythematosus, EBV or CMV infection (4). Clinical symptoms, epidemiology, and other laboratory tests such as the more specific Western blot should allow these conditions to be distinguished from Lyme disease.

H. A. T. Goossens1, A. E. van den Bogaard1 and M. K. E. Nohlmans Patients with Epstein-Barr virus or cytomegalovirus infection who had a positive reaction in the IgM EIA could not be discriminated from patients with early Lyme borreliosis with the help of Western blotting.


Hulinska D
National Reference Laboratory on Borreliosis, Electron Microscopy, National Institute of Public Health, Prague, Czech Republic.


Since the possibility of interruption of latent EBV infection has been suggested by the induction of the lytic virus cycle with chemical substances, other viruses, and by immunosuppression, we hypothesized that the same effect might happen in B. burgdorferi sensu lato infection as happens in Lyme disease patients with positive serology for both a00000gents. We have observed EBV replication in lymphoblastoid cells after superinfection with B. garinii and B. afzelii strains after 1 and 4 h of their interaction. We found that viral and borrelial antigens persisted in the lymphoblasts for 3 and 4 days. Morphological and functional transformation of both agents facilitate their transfer to daughter cells. Association with lymphoblasts and internalization of B. garinii by tube phagocytosis increased replication of viruses more successfully than B. afzelii and chemical inductors. Demonstration of such findings must be interpreted cautiously, but may prove a mixed borrelial and viral cause of severe neurological disease.


PMID: 12630667 [PubMed - indexed for MEDLINE]
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 13:14

http://www.hi.helsinki.fi/content.asp?r ... .2824.3452

Ilkka Seppälän ja Heikki Sarvaksen tutkimusryhmä

Lymen borrelioosin immunologia ja diagnostiikka

Vastaava tutkija
Ilkka Seppälä, LKT, dosentti
Bakteriologia ja immunologian osasto
Huone: EK142
Puhelin: (09) 191 26394
GSM: 040 838 4011
ilkka.j.seppala(at)helsinki.fi

Vanhemmat tutkijat
Heikki Sarvas, LKT, dosentti
Pekka Lahdenne, LKT, dosentti (HUS, Lasten ja nuorten sairaala)

Jatko-opiskelija
Heidi Sillanpää, FM

Avustava henkilökunta
Pirkko Kokkonen, laboratoriomestari

Laboratorio: B311
Toimisto: B326
Puh. (09) 191 26395 (laboratorio)
Puh. (09) 191 26615 (toimisto)


Tutkimuskohteet

Lymen borrelioosi on punkkien välityksellä leviävä bakteeri-infektio. Tautia aiheuttavia Borrelia-bakteereita on useita lajeja, joista ainakin kolmea lajia (B. burgdorferi, B. afzelii ja B. garinii) tavataan Suomesta. Lymen borrelioosia esiintyy Euroopassa, Aasiassa ja Yhdysvalloissa; Suomessa erityisesti rannikkoalueilla ja Ahvenanmaalla. Taudille tyypillinen oire alkuvaiheessa on rengasmainen ihottuma puutiaisen puremakohdan ympärillä. Hoitamattomana tauti voi edetä, jolloin tauti ilmenee mm. sydän-, nivel- ja hermosto-oireina.

Tutkimuksemme on painottunut uusien antigeenien etsimiseen kolmesta Suomessa tavattavasta Borrelia-lajista. Nykyisin käytössä olevien immunologisten testien ongelmana on esimerkiksi se, että ne eivät kerro taudin aktiivisuudesta mitään. Uusia rekombinanttiantigeeneja (proteiineja ja peptidejä) hyödynnetään immunologisissa testeissä kuten esimerkiksi ELISA- ja immunoblottaustesteissä.


Keskeiset julkaisut

Panelius, J. et al. Antibodies to recombinant decorin-binding proteins A and B in the cerebrospinal fluid of patients with Lyme neuroborreliosis. Scand J Infect Dis 2007; 39: 775-780.

Sillanpää, H. et al. Immune responses to borrelial VlsE IR6 peptide variants. Int J Med Microbiol 2007; 297:45-52.

Lahdenne, P. et al. Antigenicity of borrelial protein BBK32 fragments in early Lyme borreliosis. J Med Microbiol 2006; 55:1499-1504.

Heikkilä, T. et al. New antigens for serologic diagnosis of neuroborreliosis in children. Pediatr Infect Dis J 2005; 24:709-12.

Panelius, J. et al. Diagnosis of Lyme neuroborreliosis with antibodies to recombinant proteins DbpA, BBK32, and OspC, and VlsE IR6 peptide. J Neurol 2003;250:1318-1327.
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 13:21

1. Tutkimusten mukaan borrelioositestit ovat erittäin epäluotettavia. Johns Hopkinsin kaksivuotisen tutkimuksen mukaan verikokeet anatavat virheellisen negatiivisen tuloksen 75 %:ssa tapauksia. ILADS:in mukaan standarditestit antavat virheellisen negatiivisen tuloksen jopa 90 %:ssa borrelioositapauksia. Ihokokeista ja selkäydinnesteestä ei ole myöskään useissa tapauksissa diagnostista apua.


Äskettäin julkaistun tutkimuksen mukaan nykyiset borrelioosistestit eivät ole riittävän herkkiä ja aiheuttavat sen vuoksi jatkuvan painajaisen sellaisille henkilöille joille kerrotaan että heillä ei ole borrelioosia koska testitulos oli negatiivinen. Sen seurauksena sairastuneelta evätään hoidot. Noin puolet sairastuneista ei myöskään muista nähneensä itsessään puutiaista.

Borreliabakteeri aiheuttaa elimistössä mitä erilaisimpia oireita ja siksi sairastuneet diagnosoidaan usein virheellisesti (MS, Parkinson, masennus, skleroderma, paniikkikohtaukset, niveltulehdus, lisääntyvät kivut erityisesti lapsilla jne.)

2. Kaikkien vasta-aineisiin perustuvien testien ongelmana on havaitsevatko ne riittävän lajasti taudinaiheuttajan kaikki alalajit. Ongelma on merkityksellinen C6 -testin kohdalla sillä testi on reaktiivinen ainoastaan yhtä peptidiä kohtaan.

Wormserin ym. (2008) tutkimuksen mukaan allaolevassa tutkimuksessa käytetty C6 testi on aiempaa testiä luotettavampi. Uudella testillä testattiin varhaisvaiheen borrelioosia sairastavia (ihomuutosvaihe). Testin ongelmana on mm. riittämätön herkkyys.

Erilaisten laboratoriotestien, myös borrelioositestien, ongelmana on testiin itseensä liittyvien tekijöiden lisäksi testien kehittelijöihin liittyvät puolueettomuusongelmat. Testin saaminen markkinoille on tuottavaa bisnestä. Monet testien kehittelijöistä ovat tavalla tai toisella sidoksissa esim. lääketeollisuuteen. Esim. Wormser on saanut palkkioita kahdelta C6 -testiä markkinoivalta yritykseltä (joista toisen omistaa Dattwyler, molempien näkemys edustaa IDSA:n näkemyksiä borrelioosin diagnostiikasta ja hoidosta). Amerikkalaisten tutkijoiden näkemyksiä asiasta on esitetty tutkimuksen jälkeen.



1. Lyme Tests- A Lemon?

As Maryland climbed the ladder to become the 4th highest in the country for new cases of Lyme disease, Johns Hopkins released the results of a two year study indicating blood tests missed 75 percent of the people with Lyme. Hopkin?s latest results confirmed what the International Lyme and Associated Disease Society (ILADS) has been stating for years- "up to 90 percent" of people with Lyme were being missed using the standard testing methods.

The recently released study admits the sensitivity required for Lyme testing is still "not obtainable", causing a potential nightmare for those who were told they didn?t have Lyme due to a negative test and therefore, were denied treatment for a serious and potentially chronic infectious disease.

The study determined skin biopsies of the rash and cultures can be inconclusive. In addition, waiting for results, which may take a month or more, may delay necessary treatment. Without prompt and adequate treatment, the Lyme infection can become more entrenched and more difficult and costly to treat. Testing spinal fluid, an invasive procedure with risks to the patient, has also been determined to be of little diagnostic value.

According to Hopkin?s, months after becoming infected, untreated patients can develop chronic major manifestations and can experience serious neurologic, cardiovascular, or musculoskeletal disorders.

To compound the diagnostic problems, less than 50 percent of those with Lyme disease recall a tick bite or remember having a rash. Tick borne infections, such as Lyme, Babesiosis, Bartonella, Ehrlichiosis, STARI, and Rocky Mountain Spotted Fever, which are being detected in Maryland patients by physicians experienced in treating Lyme, can have devastating consequences and can increase the severity of the illness, increase treatment time and associated costs, and can cause permanent disability. An increasing number of deaths have also been associated with Lyme and tick borne infections.

Lyme disease, often referred to as the great imitator, has been misdiagnosed as Fibromyalgia, Multiple Sclerosis, Parkinson?s, Lou Gehrigs, depression, panic attacks, arthritis, Alzheimer?s, heart related disorders, scleroderma, ADD and growing pains (especially in children), as well as other conditions.

Blood and tissue specimens for the Hopkin?s study were collected from 86 Maryland and Pennsylvania residents who were evaluated at the Johns Hopkins Medical Institutions in 2001 and 2002. The results were not published until October, 2005.

Anyone who suspects they may have Lyme disease should contact a knowledgeable physician who follows ILADS guidelines and is familiar with sensitive, up-to-date testing methods which can be utilized to aid in a proper clinical diagnosis.

For more information, please contact the Lyme Disease Association?s toll free number, 1-888-366-6611 or visit the following web sites: www.lymediseaseassociation.org AND www.ilads.org


2. Clin Infect Dis. 2008 Aug 25; [Epub ahead of print]

Effect of Borrelia burgdorferi Genotype on the Sensitivity of C6 and 2-Tier Testing in North American Patients with Culture-Confirmed Lyme Disease.

Wormser GP, Liveris D, Hanincová K, Brisson D, Ludin S, Stracuzzi VJ, Embers ME, Philipp MT, Levin A, Aguero-Rosenfeld M, Schwartz I

1Division of Infectious Diseases, Department of Medicine, and Departments of 2Microbiology and Immunology and 3Pathology, New York Medical College, Valhalla; 4Department of Biology, University of
Pennsylvania, Philadelphia; 5Division of Bacteriology and Parasitology, Tulane National Primate Research Center, Tulane University Health Sciences Center, New Orleans, Louisiana; and 6Immunetics, Boston, Massachussetts.

Background. A potential concern with any serologic test to detect antibodies to Borrelia burgdorferi is whether the epitopes incorporated in the test provide sufficient cross-reactivity to detect infection with all of the pathogenic strains of the species.

This is a particular concern for the C6 test, which is based on reactivity to a single peptide.

Methods. C6 testing and 2-tier testing were performed on acute-phase serum samples obtained from >158 patients with erythema migrans for whom the genotype of the borrelial isolate was defined on the basis of an analysis of the 16S-23S ribosomal DNA spacer region and/or on the genetic variation of the outer surface protein C gene (ospC).

The sonicated whole cell-based enzyme-linked immunosorbent assay, the immunoblots used in the 2-tier testing, and the C6 assay all used antigens from B. burgdorferi sensu stricto strain B31.

Results. The sensitivity of C6 testing (69.5%) was greater than that of 2-tier testing (38.9%) ([Formula: see text]); the difference in sensitivity, however, was statistically significant only for patients infected with 2 of the 3 ribosomal spacer type-defined genotypes.

The lower sensitivity of 2-tier testing was attributable to the low sensitivity of the immunoblot tests, rather than the first-tier enzyme-linked immunosorbent assay. There was also a trend for the sensitivity of 2-tier testing to vary according to the ospC genotype for the 14 genotypes represented in the study ([Formula: see text]); this relationship was not observed with C6 testing.

Conclusions. Lack of sensitivity of the C6 test because of strain diversity seems less likely to be a limitation of this serologic test, compared with 2-tier testing in North American patients with early Lyme disease.

PMID: 18724824 [PubMed - as supplied by publisher]

--------------------------------------------------------------------------------------------------------
Dr. Wormser has received consulting fees from two companies that market C6 Lyme tests -- Immunetics and Biopeptides (owned by Dattwyler and Gomes-Solecki of NYMC).


Barbara Johnson of the CDC is listed as an inventor on C6 test processes, so she is eligible to receive up to $150,000 a year in patent royalties, which would double her government salary of $140,000 a year. Of course, there is no way to tell if she is earning royalties, because her salary is below the threshold where these payments have to be disclosed. The C6 technology is currently used in tests marketed by Biomerieux, IDEXX, Biopeptides, and Immunetics.


This is old market data, and the market for Lyme tests is significantly larger now that testing is mandatory for non-EM rash cases, but this gives you an idea of the dollars involved:
Income Potential of Lyme Vaccines/Tests1
Vaccine: 2.8M shots per year, at $100 per shot = $280M revenue per year.
WB Assays: 2M units per year
ELISAs: 5M units per year
Babesia tests $30M-$50M per year
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 16:40

Tutkimusten mukaan varhaisvaiheen borrelioosissa C3 ja C4 ovat usein
koholla. Allaolevan tutkimuksen mukaan kroonisessa borrelioosissa
sen sijaan esiintyy C4:n kohoamista.


Ps. Onko keneltäkään koskaan otettu C4 testiä? Kertokaa mitä testitulokset osoittivat.

Complement Split Products C3a and C4a in Chronic Lyme Disease

Authors: Stricker, R. B.; Savely, V. R.; Motanya, N. C.1; Giclas, P. C.2
Source: Scandinavian Journal of Immunology, Volume 69, Number 1, January
2009 , pp. 64-69(6)


Abstract:
Complement split products C3a and C4a are reportedly elevated in patients with
acute Lyme disease. We have now examined these immunologic markers in
patients with chronic Lyme disease compared to appropriate disease controls.
The study population consisted of 29 healthy controls, 445 patients with chronic
Lyme disease, 11 patients with systemic lupus erythematosus (SLE) and six
patients with AIDS. The Lyme disease patients were divided according to
predominant musculoskeletal symptoms (324 patients) or predominant
neurologic symptoms (121 patients). C3a and C4a levels were measured by
radioimmunoassay. All patients with chronic Lyme disease and AIDS had normal
C3a levels compared to controls, whereas patients with SLE had significantly
increased levels of this marker. Patients with predominant musculoskeletal
symptoms of Lyme disease and AIDS patients had significantly increased levels
of C4a compared to either controls, patients with predominant neurologic
symptoms of Lyme disease or SLE patients. Response to antibiotic therapy in
chronic Lyme disease was associated with a significant decrease in the C4a
level, whereas lack of response was associated with a significant increase in this
marker. In contrast, AIDS patients had persistently increased C4a levels despite
antiretroviral therapy. Lyme patients with positive single-photon emission
computed tomographic (SPECT) scans had significantly lower C4a levels
compared to Lyme patients with normal SPECT scan results. Patients with
predominant musculoskeletal symptoms of Lyme disease have normal C3a and
increased C4a levels. This pattern differs from the increase in both markers seen
in acute Lyme disease, and C4a changes correlate with the response to therapy
in chronic Lyme disease. C4a appears to be a valuable immunologic marker in
patients with persistent symptoms of Lyme disease.

Document Type: Research article
DOI: 10.1111/j.1365-3083.2008.02191.x
Affiliations: 1: Union Square Medical Associates, San Francisco, CA, USA 2:
Allergy and Immunology Division, Department of Pediatrics, National Jewish
Medical and Research Center, Denver, CO, USA
The full text electronic article is available for purchase. You will be
able to download the full text electronic article after payment.
$43.08 plus tax
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 16:48

Kaikki suositukset perustuvat yleensä ohjeiden kirjoittajien valitsemaan näkemykseen asiasta ja ohjeiden laatijoiden valitsemiin tutkimuksiin. Ne eivät siksi välttämättä edusta koko kuvaa/oikeaa kuvaa asiasta. Allaolevissa lähteissä esiintyy toistuvasti useille tuttuja nimiä kuten A. Steere, Wormser, Klempner, Barbour, Dattwyler jne. joiden näkemys borrelioosista ei suinkaan edusta kaikkien asiantuntijoiden näkemystä taudista ja sen diagnostiikasta. Yhtään esim. Brorsonien tai muutamaa suomalaistakaan tutkimusta bakteerin kyvystä piiloutua esim immuunisolujen sisälle tai muuntua ei lähdeluettelossa ja siten suositusten laatimisessa ole käytetty.

Kirjallisuudessa on esitetty lukuisia syitä siihen miksi borrelioositestit voivat näyttää virheellisiä negatiivisia tuloksia. Esim. bakteerin piiloutuminen/muuntuminen vaikuttaa ratkaisevasti siihen tunnistavatko immuunisolut vihollisen vai eivät ja sillä on suora vaikutus vasta-aineiden muodostumiseen. Esim. tämän seikan vaikutusta testituloksiin ei ole artikkelissa pohdittu. Artikkelissa pyritään paremminkin painottamaan väärien positiivisten kuin väärien negatiivisten mahdollisuutta.

Pääperiaate tulee kuitenkin esille eli borrelioositestit eivät ole luotettavia. Niiden spesifisyys ja herkkyys ovat heikkoja.

Ps. Testit ovat samoja joita Suomessakin käytetään.

Pps. Ajanpuutteen vuoksi en nyt ehdi kääntämään artikkelia. Jos jollakulla on aikaa, sen saisi mielihyvin kääntää.

KOKO artikkeli: lähteineen http://www.pubmedcentral.nih.gov/articl ... d=18923770


DIAGNOSIS OF LYME DISEASE
The diagnosis of Lyme disease can be made clinically or in conjunction with laboratory test results. When the skin rash (EM) is typical and when a patient has been exposed to an environment where blacklegged ticks are known to be established, the diagnosis can be made on clinical grounds alone. In parts of Canada where adventitious, unestablished populations of blacklegged or western blacklegged ticks have been noted, a clinical diagnosis is more challenging. When the rash is atypical or occurs in circumstances in which exposure to the appropriate vector tick species was unlikely, diagnosis is based on the demonstration of a serological response to B burgdorferi. Immunoglobulin (Ig) M antibodies are usually detectable within weeks of the onset of symptoms; however, a significant proportion of patients with EM may not have detectable antibody at the time of initial presentation (11). Furthermore, when patients are treated very early in the course of illness, antibodies may not develop. When an initial antibody determination is negative, it is suggested that a second serum specimen be collected four weeks later.

When patients have a credible possibility of exposure to infected ticks and objective findings are suggestive of any of the known neurological manifestations of Lyme disease or Lyme arthritis, then serological testing is recommended. Because available serological screening tests have limitations to their specificity, screening of patients with nonspecific subjective symptoms is strongly discouraged (12).

Current evidence suggests that commercially available enzyme immunoassays (EIAs) used for the purpose of screening are sufficiently sensitive (beyond the first month of infection).

Used alone, EIA tests presently in use lack the specificity necessary to base a diagnosis of Lyme disease on an unconfirmed result. Although newer EIAs offer the promise of improved performance characteristics, neither peptide-based nor recombinant antigen-based EIAs have been approved for use in Canada at this time. Consequently, it is the recommended practice to confirm initial EIA screening test results with a Western blot test (the two-step approach) (13,14). To assure optimal performance characteristics, guidelines for the interpretation of Western blot testing have been developed (15). Within the first four weeks of illness, both IgM and IgG Western blot tests should be performed for confirmation of initially positive EIA screening test results. Beyond the first four weeks of illness, the IgG Western blot alone should be used for confirmation. The MarDx Western blot (Marblot, MarDx Diagnostics Inc, USA) commonly used in Canada adheres to the United States Centers for Disease Control and Prevention (CDC) criteria for a positive Western blot (eg, of the 10 possible designated bands detectable by the IgG test, at least five should be positive). When an IgM Marblot is performed, it is recommended that two of the three designated bands be present. Lyme disease incidence is still low in Canada compared with the eastern United States and very low or zero in most parts of central Canada and in certain parts in western Canada; in this situation, considering the pretest likelihood of disease, the possibility of false-positive Western blot results should not be ignored (16).

B burgdorferi has been recovered from EM lesions, synovial biopsies, and blood and cerebrospinal fluid. Although the culture method is highly specific, the isolation of B burgdorferi is expensive, lacks clinical sensitivity, and requires special media, an invasive procedure and possibly up to six weeks of incubation (11,17). The vast majority of positive cultures have been from patients in the early stage of Lyme disease, especially from patients with EM lesions (11). Culture is unavailable in most Canadian centres.

Nucleic acid amplification testing (NAT) has been applied in many of the same settings as cultures (11,18?20). As with cultures, positive tests are most frequently seen in the early stage of the disease. A variety of targets, both chromosomal and plasmid, have been used for polymerase chain reaction (PCR) testing, and protocols vary greatly among laboratories. Positivity rates depend greatly on the epidemiological circumstances, the clinical features, the nature of the specimen submitted, the primer sets and testing conditions, and the experience of the testing laboratory. Although PCR has been used to detect B burgdorferi in urine, this procedure lacks both sensitivity and specificity, and is not recommended (11). At this time, NAT should be considered for research purposes only.

Antigen detection has also been used in both spinal fluid and urine. As with NAT, antigen tests cannot be recommended unless their sensitivity and specificity significantly improve (21).

Serological testing for Lyme disease should not be undertaken without a thorough appreciation of the geographic and seasonal setting in which the diagnosis is being considered, as well as an assessment of the likelihood that a specific symptom or symptoms complex is due to Lyme disease. For example, a patient residing in an area where blacklegged ticks are established who presents with a typical bull's eye rash in July should be considered to have Lyme disease until proven otherwise. A negative serological test should not dissuade the clinician from treating empirically and notifying public health officials. On the other hand, a patient with typical findings of multiple sclerosis or chronic fatigue without objective findings is highly unlikely to have Lyme disease, and both the physician and patient should be dissuaded from serological testing. In such settings, the low pretest likelihood of Lyme disease greatly increases the chance of a false-positive result; such false-positive results are often difficult to discount by either the ordering physician or the patient, and these results often lead to unnecessary treatment. Subsequent escalations of treatment often follow treatment failure (16).

Such situations are further complicated when an initial screening test is negative and subsequent Western blot testing is performed. When the initial likelihood of Lyme disease is small and when subsequent initial serological testing is negative, the pre-Western blot probability of Lyme disease is even more remote. Because Western blot testing for Lyme borreliosis is itself associated with false-positive test results, a positive result in the setting described above is usually a false positive (22).

For this reason, the Canadian Public Health Laboratory Network continues to support a two-step approach for the serodiagnosis of Lyme borreliosis. The use of the initial EIA is recommended. If a positive or borderline result is obtained, then it is recommended that a second-step Western blot be performed. When the epidemiological setting is appropriate and when patients have findings suggestive of Lyme disease, this two-step approach should assure that the vast majority of cases of Lyme borreliosis are recognized. If the patient is seen shortly after the onset of infection, then repeated serological testing may be recommended. In patients with suspect neuroborreliosis, additional testing, such as PCR testing of spinal fluid or joint fluid, may be indicated.

HEALTH LABORATORY NETWORK RECOMMENDATIONS
The appearance of a typical EM rash occurring in season and with a history of exposure to ticks should be considered an indication for antibiotic treatment, irrespective of the results of serological testing.
An EM-like rash occurring out of season and/or after exposition in a Lyme nonendemic area where ticks are not known to be established should be investigated with antibody testing.

Initial negative serological tests in patients with skin lesions suggestive of EM should have testing repeated after four weeks.
Patients with symptoms and signs suggestive of early disseminated or late Lyme disease should be tested for antibodies to B burgdorferi.

Initial testing should include an EIA commercially available and approved for use in Canada.

Sera that are positive or indeterminate by an EIA should be subjected to Western blot confirmatory testing.
Sera that are screened negative for antibodies using an EIA should not be subjected to Western blot testing.

Western blot tests should be interpreted using criteria set forth by the CDC Working Group.
Western blot tests that fail to meet all of the criteria set out by the CDC Working Group should be reported as negative; testing may be repeated when it is appropriate to do so.

The specific banding patterns seen on Western blots should not be reported.

When serological testing is requested for Lyme borreliosis, and when the initial screening test is positive and the subsequent Western blot confirmatory test is negative, specimens should be reported as ?negative for antibodies to B burgdorferi?.

Culturing for B burgdorferi is a low-yield procedure and is not encouraged; if performed, it should be done only on biopsies from EM lesions and synovial or spinal fluid.

There is inadequate evidence to support the use of B burgdorferi antigen testing as an adjunct to the diagnosis of Lyme borreliosis.
The role of NAT (eg, PCR) is limited, and its use should be restricted to patients with objective evidence of joint or central nervous system infection.

There is inadequate evidence to recommend PCR testing of blood and urine for the diagnosis of Lyme disease.

Patients without objective findings suggestive of B burgdorferi infection should not be ?screened? for B burgdorferi antibodies.
The diagnosis of Lyme borreliosis should not be based on positive serological tests in the absence of objective findings of infection and a credible epidemiological link to infected ticks.

Bypass of laboratories that apply the two-step testing procedure (initial EIA followed by Western blot testing) is strongly discouraged.
Patients should be made aware that antibody testing is subject to false-positive results, and that a positive test in the absence of objective findings and credible exposure histories usually represent false-positive results.

The role of antibody testing to monitor the results of therapy has not been established and is therefore not recommended.
The role of the microbiology laboratory in the assessment of patients with the persistence of symptoms following antibiotic treatment has not yet been established.

In patients in whom tick exposure occur outside of North America, physicians should seek diagnostic advice on testing from a Canadian laboratory with expertise in the diagnosis of Lyme disease.

Testing patients suspected of Lyme disease for other tick-associated diseases should not be routinely performed; instead, testing should be based on risk exposure and clinical symptoms.
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 16:49

Dan Kinderlehrer MD kertoo kirjassaan borrelioositestien epäluotettavuudesta ja miten niissä esiintyviä heikkouksia ei huomioida käytännön hoitotilanteissa.

Lyme Testing: The Problems Rarely Appreciated
Many good and sincere physicians have been trained to perceive Lyme testing falsely, and some are even infectious disease consultants.

Lyme is a very sophisticated bug. It is partially related to the bug that causes syphilis. There are literally well over a dozen reasons for missing the diagnosis.

First, that bulls eye rash is a good sign you have it. But many other "bite" patterns or rash patterns can also be Lyme. In fact only 1/2 get any kind of mark or rash. And only 25-50% have the popular bulls eye rash. Sometimes a bulls eye rash is not on a part of you body you easily see and so is missed.

Lyme can hide by a number of ways from your immune system.
It your immune system is not tuned up and working very well you can be found fully negative on multiple lab tests.

Most tests for Lyme are antibody tests. Antibodies, also known as immunoglobulins, are proteins that recognize something foreign in the bodyÑlike infecting bacteria and help remove it. The first and most common test your doctor usually orders is an ELISA antibody test. Again, if the Lyme is hiding well or your immune system is fair, you will come up normal. Specifically, the ELISA test missed 56% of confirmed Lyme patients (Archives of Internal Medicine 15:761-0763, 1992).

In another study, it was in some ways worse. In this one the ELISA test missed over 70% of people with early Lyme disease, and 46% with late manifestations of Lyme. (Laboratory Medicine 21:299-304, 1990). Meaning, it missed 70 out of 100 people with the early disease. But it was still negative after the bug was in the body for a long time -- still missing 46 of 100 seriously infected people.
For some, the Lab is a place of perfect science. A place which has purely objective fact. In Lyme this is not valid. In one study, 55% of the labs could not accurately identify blood samples with Lyme, which led to the conclusion in a prestigious infection journal, that: screening tests for Lyme disease are not adequate (Journal of Clinical Microbiology 35:537-543, 1997).

What About the Western Blot? Is That Definitive?
The Western Blot is merely another antibody test. However, it is more specific than the ELISA. The test can test for 25 possible "bands" that relate to parts of Lyme or other infections.

But the routine Western Blot typically done has massive errors. In one serious test of the Lyme Western Blot testers, there was a stunning finding. They used nine clearly infected patients and sent their blood to 18 labs. Of the IgG type of antibody, some labs were wrong. They missed 10 of 18 samples. For the IgM type of antibody, the labs were occasionally so bad they falsely reported Lyme as absent in 16 of 18 samples (Arch Intern Med 150:761-763, 1990).

Most physicians are taught to do the ELISA first. If that is positive then "confirm" with the Western Blot. The big confusion is that this is not a way to diagnose. It is the CDC's way of generally tracking the movement of Lyme in locations and states. It is not a way to determine whether you, personally, have Lyme!

If you use the Elisa first method with the confirmation Western Blot you miss massive numbers of individuals with Lyme (Journal of Clinical Microbiology 34: 10-9, 1996). From this two-stage approach, you may have a sense that Lyme is entering your state at an increased rate, but that does not address your individual concern.

The CDC guidelines seem to express clearly to me that these two lab tests were never intended to be the final measure of whether you have Lyme. They report the main diagnostic criteria are what you report to your doctor and what they find on a physical, i.e., "clinical findings." (http://www.cdc.gov/ncidod/dvbid/lyme/diagnosis.htm)

Another government agency, the conservative FDA, has issued a bulletin explaining that a person may have active Lyme disease and yet may have a negative lab result. Meaning, diagnosis should be based on the history of what happened to you, symptoms, exposure to the tick and physical findings (http://www.fda.gov/medbull/summer99/lyme.html).

Congress and the President have felt that negative labs have been used to keep people from needed treatment. United States Congress Public Law 107-116 explains that labs that are negative have no relation to Lyme diagnosis in a person and refers to the CDC that lab monitoring and testing with Elisa and Western Blot was "developed for national reporting of Lyme disease: it is not appropriate for clinical diagnosis."

Some bands may be fairly specific to Lyme: 12, 22, 23/25, 31, 34, 35, 37, 39, 83

Finally, some feel the PCR test is the best test. Most PCR tests are performed by laboratory which almost never find it in positive people. However, the PCR test should be done by IGeneX, Medical Diagnostic Labs or another tick disease specialty lab, it is fairly useless. PCR testing can have a false negative of 30% in those with positive Lyme. It is also good to test the PCR from blood serum, whole blood and urine, so they have more ways to look for the illness.

Excerpts from a book in manuscript by: Dan Kinderlehrer, MD
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 16:50

J. G. Brown -syöpäinstituutin tutkijat ovat löytäneet uuden menetelmän jolla voidaan testata vaikeasti diagnosoitavia sairauksia kuten borrelioosia, reumaa, lupusta jne. analysoimalla elimistön nesteitä, erityisesti veren plasmaa.

UofL Researchers Achieve Diagnostic Breakthrough Using New Plasma Test

University of Louisville James Graham Brown Cancer Center researchers have found a simple new way to detect hard-to-diagnose conditions by analyzing body fluids, especially blood plasma. Louisville Bioscience Inc. (LBI) has licensed the disease-screening breakthrough, which it believes could prove lucrative.
When heated, blood proteins create a unique visual map that provides clues to specific diseases, researchers learned. UofL scientists Jonathan Chaires, Nichola Garbett, A. Bennett Jenson and LBI believe the technology will lead to a future where doctors diagnose complex diseases with a simple blood test based on differential scanning calorimetry. Every disease examined so far leaves a distinctive signature on the test result, explains Chaires, who led the research team.

?This is in contrast to healthy people, for whom the test results look pretty much the same,? Chaires said.

In preliminary studies, blood plasma from individuals with rheumatoid arthritis, Lyme disease and lupus show test results that differ significantly from those of healthy individuals.
Alustavissa tutkimuksissa on huomattu että esim. Borrelioosia sairastavien veren plasma poikkeaa huomattavasti terveiden ihmisten plasmasta.
Eventually, the technology may be adopted for use in doctor?s offices and hospitals, providing physicians with an easy, fast way to test for hard-to-diagnose diseases and disorders.

LBI is uniquely positioned to develop this new technology for market applications, starting with early stage diagnostic medical tests. LBI?s team, which includes CEO Steve Benight, has over 20 years experience studying the thermodynamic binding properties of biological materials.

The exclusive licensing agreement, negotiated by UofL?s Office of Technology Transfer, became effective Oct. 1.

?We are extremely excited about the huge opportunity this technology offers to revolutionize medical diagnostics and make personalized medicine a reality,? Benight said.
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 17:36

Ihoon muodostuva rengasmainen jyvässolujen aiheuttama ihomuutos l. granuloma annulare näyttäisi olevan borreliabakteerin aiheuttama. Saksalaisessa tutkimuksessa ihosta otettu PCR-testi ei osoittautunut kovin luotettavaksi (46,7 %), Se sijaan mikroskooppinen tutkimus (flow-focus microscopy, FFM) osoittautui huomattavasti luotettavammaksi (93,3 %).

Suom.huom. Kyseinen ihomuutos ja siitä tehty tutkimus saattaa olla merkityksellinen sillä näyttää siltä, että ihomuutoksesta kärsivät eivät välttämättä saa oikeaa diagnoosia ja hoitoa.

"Törmäsin" sattumalta esim. seuraavaan nettikeskusteluun:

http://keskustelu.suomi24.fi/terveys_sh ... 0010536217


Granuloma annulare - a manifestation of infection with Borrelia?

Ziemer M, Grabner T, Eisendle K, Baltaci M, Zelger B.

Department of Dermatology and Venereology, Friedrich-Schiller- University, Jena, Germany.

Background:
Among the theories of origin of granuloma annulare (GA) are those of infection. Reports gave raise to the assumption that there is evidence for Borrelia as the causing agent. Methods: To assess the evidence for infection with Borrelia in GA, tissue sections were stained with a polyclonal Borrelia antibody. With focus-floating microscopy (FFM), slides were scanned at a 200- to 400-fold magnification. Part of the material was also investigated with a Borrelia-specific polymerase chain reaction (PCR). Results: A total of 157 biopsies of GA have been investigated. Using FFM, Borrelia were detected in 127 cases of GA (80.9%). Borrelia were more prominent in localized (85.2%) than in diffuse GA (62.1%). In 27 cases of GA analysed by PCR, Borrelia-specific DNA could be detected in only one case (3.7%), but was positive in 21 cases by FFM (77.8%). About 93.3% of 15 control cases of borreliosis were positive with FFM and 46.7% with PCR, while all controls other than borreliosis remained negative for spirochetes.

Conclusion:
FFM is a reliable method to show Borrelia in tissue sections of GA, which is more sensitive than PCR. This underlines the possibility that Borrelia are involved specifically in the aetiology and pathogenesis of GA.

PMID: 18616764 [PubMed - as supplied by publisher]
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 17:41

Tutkimustulokset: C3a ja C4a -arvot olivat huomattavan korkealla kaikilla varhaisvaiheen borrelioosia sairastavilla. Kyseinen testi on tällä hetkellä ainoa laboratoriodiagnostinen menetelmä varhaisvaiheen borrelioosin toteamiseksi. Testi kykeni toteamaan arvojen nousun jopa 14 tuntia sen jälkeen kun henkilö oli saanut tartunnan.

Shoemakerin ym tutkimus: http://biotoxin.info/docs/C3a_C4a_Acute ... y_2006.pdf

-----------------------------------------------------
There Is Hope for Newer Lyme Testing
http://www.endfatigue.com/tools-support ... sting.html

Using a combination of C3a and C4a blood testing may diagnose Lyme Disease more accurately.

By Ritch Shoemaker, M.D.
Pocomoke, MD

The International Archives of Allergy and Immunology has accepted our paper on use of elevated C3a and C4a as diagnostic tools for acute Lyme. Patients were seen within 48 hours of tick bite who did or didn't have ECM and who did or didn't have symptoms. The C3a and C4a levels in affected groups were markedly higher than in non-tick bites controls. ECM patients had higher levels than non-ECM. Alan Barbour gave us pure cultures of B. burgorferi and B. hermseii; when added to cell-free plasma, the same elevation of C3a and C4a was seen. Innate immune responses involving complement are seemingly non-specific, but this work provides a basis for comparison. The earliest we saw a rise in C4a was 14 hours after a tick bite. Sure beats taking two doses of doxycycline and guessing.

To do C3a and C4a testing, Dr. Shoemaker recommends you go to LabCorp. LabCorp will forward frozen plasma to National Jewish (NJC) in Denver. You must ask for the RIA (NJC) and not let LabCorp send the specimen to Cambridge where they do an ELISA.
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 17:46

Immunosciences-laboratorio on kehitellyt uuden IVIAT-menetelmän jonka avulla borrelioosin diagnostiikkaan luvataan parannusta.

Ps. Tutkin asiaa nopeasti ja heidän kehittämänsä testin kerrotaan tunnistavan bakteerien proteiinit infektion aikana (in vivo) eikä vasta viljelyvaiheessa (in vitro).



-http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0001824
-http://www.pnas.org/content/100/14/8508.abstract


Evid Based Complement Alternat Med. 2007 Oct 15; [Epub ahead of print]

Novel Diagnosis of Lyme Disease: Potential for CAM Intervention.

Vojdani A, Hebroni F, Raphael Y, Erde J, Raxlen B.

Immunosciences Lab., Inc., 8693 Wilshire Blvd., Suite 200, Beverly Hills, CA
90211, USA. drari@msn.com.

Lyme disease (LD) is the most common tick-borne disease in the northern hemisphere, producing a wide range of disabling effects on multiple human targets, including the skin, the nervous system, the joints and the heart.

Insufficient clinical diagnostic methods, the necessity for prompt antibiotic treatment along with the pervasive nature of infection impel the development and establishment of new clinical diagnostic tools with increased accuracy, sensitivity and specificity. The goal of this article is 4-fold: (i) to detail LD infection and pathology, (ii) to review prevalent diagnostic methods, emphasizing inherent problems, (iii) to introduce the usage of in vivo induced antigen technology (IVIAT) in clinical diagnostics and (iv) to underscore the relevance of a novel comprehensive LD diagnostic approach to practitioners of Complementary and Alternative Medicine (CAM).

Utilization of this analytical method will increase the accuracy of the diagnostic process and abridge the time to treatment, with antibiotics, herbal medicines and nutritional supplements, resulting in improved quality of care and disease prognosis.

PMID: 18955246 [PubMed - as supplied by publisher]
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Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 17:47

Borrelioositestien herkkyys on useissa tutkimuksissa osoittautunut huonoksi. Seuraavassa tutkimuksessa arvioitiin LIAISON vasta-ainetestin (VIsE) herkkyyttä. Se oli n. 70 %.

Clin Vaccine Immunol. 2008 Oct 22; [Epub ahead of print]

Evaluation of the LIAISON(R) Borrelia burgdorferi Test, a Recombinant VlsE-based Chemiluminescence Immunoassay for the Diagnosis of Lyme Disease.

Ledue TB, Collins MF, Young J, Schriefer ME.

Foundation for Blood Research, Scarborough, ME, USA; Centers for Disease Control and Prevention, Diagnostics and Reference Section, Division of Vector-Borne Infectious Diseases, Ft. Collins, CO, USA.

Recent efforts to improve the serologic diagnosis of Lyme disease have included the use of a synthetic peptide (C6) that reproduces the sequence of invariable region 6 of VlsE, the variable surface antigen of Borrelia burgdorferi. In the present study, the diagnostic performance of DiaSorin's recombinant VlsE-based chemiluminescence immunoassay was evaluated in 1,947 human serum samples.

Sensitivity was determined using two serum panels from the CDC. For

Panel I, we observed sensitivities of 68.4% and 75.6% for subjects with early, localized (n = 19) or disseminated disease (n = 41), respectively.

For panel II, we observed sensitivities of 61.5% and 100% for subjects with early (n = 26) or late stage disease (n = 11), respectively.

We observed a specificity of 99.5% for healthy donors (n = 600) living either in endemic or non-endemic regions of the U.S. Overall, specificity among 207 potentially cross-reactive sera from subjects with other spirochetal infections, non-spirochetal infections including bacterial and viral infections, autoimmune or neurologic disease, or who were positive for rheumatoid factor, anti-mouse antibodies, or were previously vaccinated for Lyme disease was 93.7%.

In a direct comparison of 1,038 prospectively collected samples for Lyme disease testing we observed a relative sensitivity of 70%, a relative specificity of 99.1%, and an overall agreement of 97.1% between the DiaSorin recombinant VlsE chemiluminescence immunoassay and the Immunetics(R) peptide-based C6 enzyme- inked immunosorbent assay.

PMID: 18945880 [PubMed - as supplied by publisher]
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ViestiKirjoittaja soijuv » Su Tammi 25, 2009 17:48

Itävaltalainen tutkimus: Virtsan PCR-testi osoittautui herkkyydeltään luotettavammaksi borrelioosin diagnostiikassa (ihomuutosvaihe) kuin verestä otettavat näytteet. Testistä saattaa olla hyötyä myös sellaisissa seropositiivisissa tapauksissa joissa oireet eivät ole ns. borrelioosille tyypillisiä.

Acta Derm Venereol. 2007 ;87 (1):39-42 17225014 (P,S,G,E,B)
Course of Borrelia burgdorferi DNA Shedding in Urine after Treatment.
[My paper] Elisabeth Aberer, Andreas R Bergmann, Anna-Maria Derler, Bruno Schmidt
Department of Dermatology, Medical University of Graz, Auenbruggerplatz 8.

Diagnosis of Lyme borreliosis by urine polymerase chain reaction (PCR) has been recognized as having better diagnostic sensitivity in patients with erythema migrans than serological methods. We made serial tests with 192 urine specimens from 70 patients with erythema migrans and 60 urine specimens from 21 patients with acrodermatitis chronica atrophicans to evaluate the course of positive urine PCR after antibiotic treatment. Before treatment, urine samples from patients with erythema migrans showed a positive PCR in 27/34 samples (79%), and those from patients with acrodermatitis chronica atrophicans in 7/11 (63%). The specificity of bands was proven by hybridization with GEN-ETI-KTM-DEIA kit in 40/41 samples. Borrelia DNA in urine decreased gradually within the observation period of one year in both patients with erythema migrans and acrodermatitis chronica atrophicans, and persisted without clinical symptoms in 4/45 patients with erythema migrans (8%) after 12 months. Urine PCR can serve as a diagnostic method in early Lyme borreliosis and also in seropositive patients with unclear clinical symptoms.
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Su Tammi 25, 2009 17:50

Borrelioosia esiintyy eri puolilla Venäjää. Venäläiset tutkivat 90 henkilön borrelioosivasta-aineita. Henkilöillä oli ollut ihomuutos + erilaisia yleisoireita kuten väsymys, kuume, päänsärky, luusto-/lihaksistokivut jne. 2 viikon - 4 kk:n kuluessa tartunnasta 58 henkilölle kehittyi erilaisia neurologisia- ja/tai niveloireita.

Vasta-ainetestit olivat epäluotettavia: Ne olivat positiiviset vain kahdella kymmenestä ihomuutosvaiheessa, 15:llä 21:sta neurologisia oireita kärsivistä ja kahdella neljästä niveloireisesta. Ps. Tutkimus on vuodelta 1988 ja yksi tutkijoista oli Steere.

J Infect Dis. 1988 Oct;158(4):748-53.
Lyme borreliosis in the Soviet Union: a cooperative US-USSR report.

Dekonenko EJ, Steere AC, Berardi VP, Kravchuk LN.
Poliomyelitis Institute, Moscow, Union of Soviet Socialist Republics.


We identified 90 patients with tick-borne erythema migrans in the Union of Soviet Socialist Republics (USSR) in areas from the western Baltic Republics to the Maritime Territory on the Pacific Ocean. Symptoms associated with the erythema included fever, malaise and fatigue, headache, myalgias, arthralgias, or regional lymphadenopathy.


Within two weeks to four months, 58 (64%) of the patients developed neurological abnormalities, particularly radicular pain, cranial neuritis, or lymphocytic meningitis, and four (4%) patients developed monoarticular or oligoarticular arthritis. We tested the sera from 35 Soviet patients by using an isolate from the United States. The serological data showed elevated IgM and/or IgG antibody titers to Borrelia burgdorferi in 2 of 10 patients with erythema migrans, 15 of 21 with neurological abnormalities, and 2 of 4 with arthritis. Our observations suggest that Lyme borreliosis occurs in diverse areas of the USSR.

PMID: 3171226 [PubMed - indexed for MEDLINE
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja borrelioosi » Ti Tammi 27, 2009 11:05

Punkkien levittämien tautien diagnostiikkaan on kehitetty Reverse line blotting - testimenetelmä.

http://www.ncbi.nlm.nih.gov/entrez/quer ... &DB=pubmed

Arch Inst Pasteur Tunis. 2004;81(1-4):47-50.

Reverse line blotting: a new technique for the sensitive detection of tick borne pathogens.

* Tait A,
* Oura C.

Department of Veterinary Parasitology, Institute of Comparative Medicine, University of Glasgow, Bearsden Rd, Glasgow G61 1QH, UK.

Recently, a new molecular diagnostic technique for tick borne pathogens has been developed and is called Reverse Line Blotting. This paper outlines the basis of this method and then illustrates its use for the analysis of blood samples obtained from Ugandan cattle and buffalo. The sensitivity and the specificity are discussed in relation to the use of this technique in the Maghreb

PMID: 16929766 [PubMed - indexed for MEDLINE
borrelioosi
 

ViestiKirjoittaja soijuv » Ke Tammi 28, 2009 18:23

Unkari: Neuroborrelioosin diagnostiikassa käytetään yleensä selkäydinnesteen vasta-aineiden määritystä. Unkarilaisessa tutkimuksessa selvitettiin yhtä aikaa sekä seerumin että selkäydinnesteen vasta-ainepitoisuuksia (ELISA + immunoblotti). Testin herkkyys oli 80 %. Vasta-aineiden tuotannossa esiintyi laadullisia eroja joten vaikuttaa siltä että keskushermostossa tapahtuu omaa vasta-aineiden muodostusta eivätkä veren vasta-aineeet vuoda veri-aivoesteen läpi keskushermostoon. Näyttää myös siltä että toisistaan poikkeavat B-solut huolehtivat vasta-aineiden tuotannosta veressä ja keskushermostossa

Different B-cell populations are responsible for the peripheral and intrathecal antibody production in neuroborreliosis. Lakos A, Ferenczi E, Komoly S, Granström M.Center for Tick-borne Diseases, Visegrádi 14, H-1132 Budapest, Hungary. alakos@axelero.hu

The diagnosis of neuroborreliosis (NB)--a serious complication of Lyme disease--relies on demonstration of intrathecal borrelia antibody production. We hypothesized that if a qualitative difference between the cerebrospinal fluid and the serum immunoblot-banding patterns was observed, then the borrelia antibodies found in the CSF could not be the result of leakage of serum antibodies to the CSF due to blood-brain barrier damage, but rather had to be produced intrathecally.

CSF/serum pairs from 69 NB patients and from 85 control patients with other neurological disorders were investigated. All samples were tested blindly by immunoblot and a commercial capture ELISA kit for NB. The concordance between the two methods was 85.7%.

When using the other method as reference, the accuracy of the two assays in the two patient materials was similar: 80% for sensitivity and 95% for specificity. Four types of comparative immunoblot-banding patterns that reflected intrathecal borrelia ant!ibody synthesis were distinguished. The study showed that a simple comparison between the immunoblot pattern of serum and CSF samples allows for a reliable diagnosis of NB by demonstration of intrathecal antibody production. This is the first study to show that a qualitative difference of the antibody response between the immune response of serum and CSF is a rule.

The findings also imply that partly different B-cell populations are responsible for the antibody production in the blood and in the central nervous system. In addition, our observation provides possible implications for other infectious diseases with CNS involvement.

PMID: 16303786 [PubMed - indexed for MEDLINE]
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Pe Tammi 30, 2009 18:40

Borrelioositestit saattavat joidenkin lähteiden mukaan jättää virheellisesti huomioimatta jopa 75 - 90 % borrelioositapauksista (Johns Hopkins/ILADS). Alla olevan tutkimuksen mukaan myös tutkittujen kolmen bartonellatestin (kissanraapimatauti) herkkyys/spesifisyys olivat alhaiset. Ne antavat virheellisiä tuloksia jopa 50 -75 %:ssa tapauksia.

Lyme tests miss 75-90 percent of the people infected (Hopkins/ILADS). Bartonella tests miss 50 - 75 percent of those infected (Tufts).

Am J Clin Pathol. 2009 Feb;131(2):250-6. Links
Evaluation of immunohistochemistry in identifying Bartonella henselae in cat-scratch disease.
Caponetti GC, Pantanowitz L, Marconi S, Havens JM, Lamps LW, Otis CN.
Department of Pathology, Baystate Medical Center, Tufts University School of Medicine, Springfield, MA, USA.

Cat-scratch disease (CSD) is largely due to infection with Bartonella henselae. Microbiologic detection is difficult, and molecular testing is not readily available. A monoclonal antibody (mAB) to B henselae has become commercially available.

We evaluated the usefulness of immunohistochemical analysis (IHC) for diagnosing CSD on surgical specimens and compared these results with polymerase chain reaction (PCR) detection and serologic testing for B henselae. We studied 24 formalin-fixed, paraffin-embedded (FFPE) cases of lymphadenitis with histologic and/or clinical suspicion of CSD.

Control cases included 14 cases of lymphadenopathy other than CSD. FFPE tissue sections were evaluated with an mAB to B henselae, Steiner silver stain (SSS), and PCR that targeted B henselae and Bartonella quintana. Positive cases were as follows: SSS, 11 (46%); PCR, 9 (38%); and IHC, 6 (25%). Only 2 cases (8%) were positive for all 3 studies. All control cases were negative for IHC and PCR.

The diagnostic sensitivity of these 3 tests is low for CSD. SSS seems to be the most sensitive test but is the least specific. PCR is more sensitive than IHC and may, therefore, serve as a helpful second-line test on all IHC- cases.
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Pe Tammi 30, 2009 21:20

"Erilaiset borrelioositestit antavat erilaisia testituloksia. Testien standardoinnilla on kiire."

Our study revealed that different approaches for direct demonstration of borrelial infection give distinct results, that there is an urgent need for standardization of the methods for direct detection of borrelial infection,

J Clin Microbiol. 2008 Oct;46(10):3375-9. Epub 2008 Aug 20. Links

Validation of cultivation and PCR methods for diagnosis of Lyme neuroborreliosis.

Cerar T, Ogrinc K, Cimperman J, Lotric-Furlan S, Strle F, Ruzić-Sabljić E.
University of Ljubljana, Faculty of Medicine, Institute of Microbiology and Immunology, Zaloska 4, 1105 Ljubljana, Slovenia. tjasa.cerar@mf.uni-lj.si

Borrelial infection may manifest with a wide range of clinical signs, and in many cases, microbiological findings are essential for a proper diagnosis. This study included 48 patients with a working clinical diagnosis of Lyme neuroborreliosis, 45 patients with a working clinical diagnosis of suspected Lyme neuroborreliosis, and a control group comprising 42 patients with tick-borne encephalitis and 21 neurosurgical patients. The aim of the study was to analyze and compare findings of two PCR methods and Borrelia burgdorferi sensu lato culture results by examination of prospectively collected cerebrospinal fluid (CSF) and blood specimens from patients with clinical features of Lyme neuroborreliosis. Borrelial DNA was detected with at least one of the PCR approaches in 16/135 (11.9%) blood samples and 24/156 (15.4%) CSF samples. Using MseI restriction of PCR products of the amplified rrf-rrl region, we identified the majority of strains as Borrelia afzelii. Borreliae were isolated from 1/135 (0.7%) blood samples and from 5/156 (3.2%) CSF specimens. Using MluI restriction for characterization of isolated strains, Borrelia garinii was identified in all CSF isolates.

Our study revealed that different approaches for direct demonstration of borrelial infection give distinct results, that there is an urgent need for standardization of the methods for direct detection of borrelial infection, and that the design of studies evaluating the validation of such methods should include appropriate control group(s) to enable assessment of both sensitivity and specificity.

PMID: 18716226 [PubMed - in process]


-------------------------------------------
MluI-LRFP represents a highly specific and reproducible method for Borrelia identification.

Res Microbiol. 2008 Jul-Aug;159(6):441-8. Epub 2008 Jun 6. Links

Characterization of Borrelia burgdorferi sensu lato isolates by pulsed-field gel electrophoresis after MluI restriction of genomic DNA.

Ruzić-Sabljić E, Zore A, Strle F.
Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000 Ljubljana, Slovenia. eva.ruzic-sabljic@mf.uni-lj.si

At least three Borrelia species (Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto) cause disease in humans, but Borrelia spielmanii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii have also been reported to be rare or potential causes of human disease in Europe. Pulsed-field gel electrophoresis after MluI restriction of the genomic DNA (MluI large restriction fragment patterns, LRFPs) represents one of several approaches that have been used to assess Borrelia genotypic characteristics. The aim of the present report was to analyze the value of MluI-LRFP for identification of B. burgdorferi sensu lato at a species level and for further species subtype delineation. Results of the present study are based on 1487 B. afzelii strains, 285 B. garinii strains, 29 B. burgdorferi sensu stricto strains, 23 B. valaisiana strains, 8 B. spielmanii strains and 3 B. lusitaniae strains. Using MluI-LRFP, we were able to delineate all Borrelia species included in the study. Each of the six examined Borrelia species displayed unique MluI-LRFPs that enabled straightforward separation of strains into particular species, and also of strains within species. The subtypes of B. afzelii (Mla2 and Mla3), B. spielmanii (Mls1 and Mls2) and B. lusitaniae (Mll1 and Mll2) uncovered in the present analysis have not been reported previously. MluI-LRFP represents a highly specific and reproducible method for Borrelia identification.
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Pe Tammi 30, 2009 21:25

Oikea-aikaisessa borrelioosi PCR -testissä positiiviset tulokset vaihtelivat. Ne olivat korkeimmat nivelneste- ja kudosnäytteissä ja alhaisimmat veri-ja selkäydinnestenäytteissä.

Diagnostic Microbiology and Infectious Disease

Ngolela Esther Babady, Lynne M. Sloan, Emily A. Vetter, Robin Patel, and Matthew J. Binnicker

Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA Division of Infectious Diseases, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA


Abstract
We performed a retrospective review of 23,777 specimens tested by our Lyme real-time polymerase chain reaction assay to determine the percent positive rates by specimen source. The percent positive rates were highest in synovial fluid (6.4%) and tissue (6.5%), and lowest in blood (0.1%) and cerebrospinal fluid (0.09%).

Keywords: Lyme; PCR; Blood; Cerebrospinal fluid; Synovial fluid
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Pe Tammi 30, 2009 21:34

Neuroborrelioosia sairastavista 36,7 %:lla, iho-oireisista 17,6 %:lla ja niveloireisista 54,5 %:lla löydettiin hoitojen jälkeenkin borreliabakteerin DNA:ta PCR-menetelmällä. (Tsekki)

Int J Dermatol. 2008 Oct;47(10):1004-10.

[b]Examination of specific DNA by PCR in patients with different forms of Lymeborreliosis. [/b]

Picha D, Moravcova L, Holeckova D, Zd'arsky E, Valesova M, Maresova V, HercogovaJ, Vanousova D. Charles University, 2nd Medical School, 1st Clinic for Infectious Diseases,Teaching Hospital Bulovka, Prague, Czech Republic. dusan.picha@fnb.cz

BACKGROUND: Borrelial specific DNA was examined in a group of 62 patients withdifferent forms of Lyme borreliosis (LB) (32 patients suffered fromneuroborreliosis, 19 manifested erythema migrans, and 11 joint involvement).

METHODS: Nested-PCR system with five newly derived primers was used in parallel.The study was organized prospectively, the presence of DNA was tested forplasma, CSF, joint fluid and urine before treatment, and plasma, joint fluid andurine were examined after treatment.

RESULTS: Before therapy, 36 patients(58.1%) were DNA positive on the whole; 21 positive patients (65.6%) were foundin the group of neuroborreliosis, 8 (42.1%) showed signs of skin involvement,and 7 (63.6%) were positive in arthritis.

After treatment, 11 patients (36.7%)were positive in neuroborreliosis, 3 (17.6%) in skin form, and 6 (54.5%) injoint form of LB.
Among 97 positive amplifications the most frequent target wasfound in primer corresponding with 16S rDNA (50 samples, 51.5%). Lower but verysimilar results were obtained with primers for OspA (18 positive amplifications;18.6%), OspC (13 positive amplifications; 13.4%), and flagellin (13 positiveamplifications; 13.4%). There were 11 patients in whom only DNA and no specificantibodies were found.
CONCLUSIONS: Specific DNA was found in all clinicalgroups of LB with similar sensitivity. Examination of the borrelial DNA in urinedisplayed the same sensitivity as in CSF and had a two times higher sensitivitythan in plasma. Publication Types:Research Support, Non-U.S. Gov't PMID: 18986344 [PubMed - in process]
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Ke Helmi 04, 2009 09:18

Seuraavissa tutkimuksissa kolmen borrelioositestin herkkyys oli melko alhainen (47 - 68 %). Testit ristireagoivat esim. Epstein Barr ja sytomegaloviruksen sekä syfiliksen aiheuttajan (T.pallidum) kanssa. (Venäjä)

Antibiot Khimioter. 2004;49(8-9):25-8.Links
[Preparations for serodiagnosis of diseases due to causative agents of ixode tick-borne Borreliosis (Lyme disease). Communication 2. Comparative study of recombinant enzyme immunoassay test-systems (rELISA) for serological diagnosis of ixode tick-borne Borreliosis]
[Article in Russian]


Manzeniuk IN, Vorob'eva MS, Nikitiuk NM, Arumova EA, Anan'eva LP, Baranova SG, Viatkina TG, Masiago AV, Kozarenko AA, Andreĭchuk IuA.
One foreign and two Russian recombinant enzyme immunoassay test-systems were comparatively investigated under conditions of an encoded experiment. Sensitivity in the experiment with the Russian test-systems Borreliosis-ELISA-IgG and Lyme Best was 63.8 and 68.8% respectively. As for the test-system Borrelia IgG Recombinant, it was 47.5%.
All the test-systems were highly specific (94.4 to 99.5%). The test-systems Lyme Best and Borrelia IgG Recombinant revealed partial cross reactions with sera from patients with systemic lupus erythematosus and leptospirosis.

PMID: 15727142 [PubMed - indexed for MEDLINE

----------------------------------------------------------

Antibiot Khimioter. 2004;49(6):10-4.Links
[Preparations for serodiagnosis of diseases due to causative agents of ixode tick-borne borreliosis (Lyme disease). Communication 1. Comparative study of the 1st generation enzyme immunoassay test-systems for detection of antibodies to Borrelia burkdorferi sensu lato]
[Article in Russian]


Manzeniuk IN, Vorob'eva MS, Arumova EA, Barabova EV, Evsegneev SI, Anan'eva LP.
Four foreign and one Russian 1st generation test-systems for detecting class G antibodies or summary antibodies to Borrelia burkdorferi sensu lato were comparitively investigated with the use of the clinical material under conditions of an encoded experiment. Cross reactions with sera from patients with syphilis, Epstein-Barr infection, cytomegalovirus infection and systemic lupus erythematosus were observed. The best specificity and sensitivity parameters were provided by the Enzygnost Borreliosis test-system.

PMID: 15628796 [PubMed - indexed for MEDLINE

[Russians do it better?]
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Ke Helmi 04, 2009 15:14

Int J Dermatol. 2008 Oct;47(10):1004-10.

Examination of specific DNA by PCR in patients with different forms of Lymeborreliosis.

Picha D, Moravcova L, Holeckova D, Zd'arsky E, Valesova M, Maresova V, HercogovaJ, Vanousova D. BACKGROUND: Borrelial specific DNA was examined in a group of 62 patients withdifferent forms of Lyme borreliosis (LB) (32 patients suffered fromneuroborreliosis, 19 manifested erythema migrans, and 11 joint involvement). METHODS: Nested-PCR system with five newly derived primers was used in parallel.The study was organized prospectively, the presence of DNA was tested forplasma, CSF, joint fluid and urine before treatment, and plasma, joint fluid andurine were examined after treatment.

RESULTS: Before therapy, 36 patients(58.1%) were DNA positive on the whole; 21 positive patients (65.6%) were foundin the group of neuroborreliosis, 8 (42.1%) showed signs of skin involvement,and 7 (63.6%) were positive in arthritis. After treatment, 11 patients (36.7%)were positive in neuroborreliosis, 3 (17.6%) in skin form, and 6 (54.5%) injoint form of LB. Among 97 positive amplifications the most frequent target wasfound in primer corresponding with 16S rDNA (50 samples, 51.5%). Lower but verysimilar results were obtained with primers for OspA (18 positive amplifications;18.6%), OspC (13 positive amplifications; 13.4%), and flagellin (13 positiveamplifications; 13.4%). There were 11 patients in whom only DNA and no specificantibodies were found.

CONCLUSIONS: Specific DNA was found in all clinicalgroups of LB with similar sensitivity. Examination of the borrelial DNA in urinedisplayed the same sensitivity as in CSF and had a two times higher sensitivity than in plasma.
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Ma Maalis 09, 2009 13:07

Uusi menetelmä vähäistenkin borreliabakteerimäärien löytämiseksi elimistöstä (molecular beacons). 2009

http://www.biomedcentral.com/1471-2180/9/43/abstract

Methodology article

Detection and quantification of Lyme spirochetes using sensitive andspecific molecular beacon probes

Diana S Saidac , Salvatore A E Marras and Nikhat Parveen BMC Microbiology 2009, 9:43doi:10.1186/1471-2180-9-43 Published: 24 February 2009Abstract (provisional)

BackgroundLyme disease, caused by Borrelia burgdorferi, affects a large number ofpeople in both the USA and Europe. The mouse is a natural host for this spirochete and is widely used as a model system to study Lyme pathogenesis mechanisms. Since disease manifestations often depend upon the spirochete burden in a particular tissue, it is critical to accurately measure thebacterial number in infected tissues. The current methods either lacksensitivity and specificity (SYBR Green), or require independent analysis ofsamples in parallel to quantitate host and bacterial DNA (TaqMan).

We havedeveloped a novel molecular beacon-based convenient multiplex real-timequantitative PCR assay to identify and detect small numbers of B.burgdorferi in infected mouse tissues. Results We show here that molecular beacons are effective, sensitive and specificprobes for detecting and estimating wide-ranging numbers of B. burgdorferiin the presence of mouse DNA. In our assays, the spirochete recA and themouse nidogen gene amplicons were detected simultaneously using molecularbeacons labeled with different fluorophores. We further validated theapplication of these probes by quantifying the wild-type strain andbgp-defective mutant of B. burgdorferi. The bgp-defective mutant shows aten-fold reduction in the level of spirochetes present in various tissues.


ConclusionsThe high sensitivity and specificity of molecular beacons makes them superior probes for the detection of small numbers of B. burgdorferi.Furthermore, the use of molecular beacons can be expanded for the simultaneous detection and quantification of multiple pathogens in the infected hosts, including humans, and in the arthropod vectors.
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Ke Maalis 11, 2009 18:45

Selkäydinnesteen serotonin määritys kroonista borrelioosia sairastavilta. 16 borrelioosia sairastavalla esiintyi mm. masennusta ja kognitiivisia häiriöitä (Puola):

Przegl Epidemiol. 2008;62(4):793-800.

[Evaluation of cerebrospinal fluid serotonin (5-HT) concentration in patientswith post-Lyme disease syndrome--preliminary study] [Article in Polish] Kepa L, Oczko-Grzesik B, Badura-Glombik T. Oddzial Chorob Zakaznych Slaskiego Uniwersytetu Medycznego w Bytomiu. The aim of the study was evaluation of usefulness of cerebrospinal fluid (CSF)serotonin level examination in diagnostics of post-Lyme disease syndrome. The study was performed in 16 subjects. In all individuals CSF serotoninconcentration was estimated on the 1st day of hospitalization. In patients with depressive and cognitive impairments, proved in neuropsychological tests, -group I--mean CSF serotonin concentration was 1,26 ng/ml, whereas in subjectswithout abnormalities in tests--group II--respectively--3,87 ng/ml. Thedifference of mean CSF serotonin levels was statistically significant (p<0,01).

The obtained results indicate usefulness of this CSF parameter, besidesneuropsychological tests, in objective evaluation of clinical state in patientswith post-Lyme disease syndrome. Publication Types:English Abstract PMID: 19209742 [PubMed - in process]
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » To Huhti 16, 2009 20:43

Lähes kaikilla borrelioosin myöhäisvaihetta sairastavista (niveltulehdusta sairastavia) esiintyi voimakasta IgG-reaktiivisuutta borreliabakteerin (B.burgdorferi) glykolipidejä kohtaan. (USA 2009)

Clin Immunol. 2009 Mar 31; [Epub ahead of print] Strong IgG antibody responses to Borrelia burgdorferi glycolipids in patientswith Lyme arthritis, a late manifestation of the infection.

Jones KL, Seward RJ, Ben-Menachem G, Glickstein LJ, Costello CE, Steere AC. Division of Rheumatology, Allergy, and Immunology, Center for Immunology andInflammatory Diseases, Massachusetts General Hospital, Harvard Medical School,Boston, MA 02114, USA.

In this study, the membrane lipids of B. burgdorferi were separated into 16fractions; the components in each fraction were identified, and theimmunogenicity of each fraction was determined by ELISA using sera from Lymedisease patients. Only the 2 glycolipids, acylated cholesteryl galactoside (ACG,BbGL-I) and monogalactosyl diacylglycerol (MgalD, BbGL-II), were immunogenic.Early in the infection, 24 of 84 patients (29%) who were convalescent fromerythema migrans and 19 of the 35 patients (54%) with neuroborreliosis had weakIgG responses to purified MgalD, and a smaller percentage of patients had earlyresponses to synthetic ACG.

However, almost all of 75 patients with Lymearthritis, a late disease manifestation, had strong IgG reactivity with bothglycolipids. Thus, almost all patients with Lyme arthritis have strong IgGantibody responses to B. burgdorferi glycolipid antigens.

PMID: 19342303 [PubMed - as supplied by publisher]
soijuv
 
Viestit: 3097
Liittynyt: Ke Tammi 21, 2009 14:16

ViestiKirjoittaja soijuv » Ma Huhti 20, 2009 11:18

"Borreliabakteeri on vakava uhka terveydelle. Oireiden ja kliinisen kuvan moninaisuuden vuoksi testejä tulee suorittaa eri testejä käyttäen toistuvasti." (Romania)

Rev Med Chir Soc Med Nat Iasi. 2008 Apr-Jun;113(2):496-501.

[Results of etiologic diagnosis in clinical syndrome consistent with acute andchronic borreliosis] [Article in Romanian]

Perseca T, Feder A, Molnar GB. Institutul de Sanatate Publica, Prof. Dr. Iuliu Moldovan" Cluj Napoca.

Borreliosis is a multisystem infection, which in the absence of adequatediagnosis and clinical management, may develop towards various clinical forms ofchronic pathology. Due to the heterogeneity of clinical manifestations it isknown under more names: erythema migrans, Lyme disease, neuroborreliosis etc.

MATERIAL AND METHOD: Taking into account the present interest and the weight inpathology of syndromes consistent with the suspicion of a Borrelia spp.infection, since 2002 we applied in current practice the investigation of thisetiology. There have been investigated 481 subjects, clinically suspected ofBorrelia spp. infection that had historical risk of tick bite and cases ofserous meningitis, after exclusion of usual etiology. Tests were performed onELISA kits with standardised immunoreagents and recently, for result validation,on Western immunoblot kits (WB).

RESULTS: Our results revealed the Borreliaetiology in 32% of cases (27.96-36.29% CI = 95%) at the screening, valueexpressed by the persistent positivity of the specific immunoglobulins (Ig) IgM(80.5%) and IgM+IgG (19.5%). Historic infection, represented exclusively by IgGpositivity, was present in 8.6% (5.87-11.98% CI = 95%) from the cases that werenegative for IgM (68%, 63.71-72.04%, CI = 95%). This weight is superposable withthe results obtained in investigating a comparable sample of healthy individuals(193 subjects with 6.74% historical IgG, 3.79-10.96%, CI = 95%). Based on theseresults, it can be considered that ELISA procedure is useful and of reliableprognosis value for screening the Borrelia spp. etiology, the next step, takinginto account the higher sensitivity of WB, being WB procedure which is usefulfor confirmation of ELISA positive cases and for treatment efficiencysurveillance.

The results prove that Borrelia spp. infections are a publichealth issue, which due to the diversity of clinical manifestations and diagnosis difficulties need repeated and complex laboratory investigations.


Publication Types:English Abstract PMID: 19295026 [PubMed - in process]
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ViestiKirjoittaja soijuv » Ke Huhti 29, 2009 16:21

Milloin tulisi tehdä immunoblottaus ja miten tuloksia tulkitaan? Borrelioosin laboratoriodiagnostiikassa käytetään edelleen ensisijassa kustannustehokkaita vasta-ainetestejä kuten Elisa ja immunoblottaus. (Saksa 2009)

Curr Probl Dermatol. 2009;37:167-177. Epub 2009 Apr 8.

When Is the Best Time to Order a Western Blot and How Should It Be Interpreted?

Hunfeld KP, Kraiczy P. Institute of Medical Microbiology and Infection Control, Hospital of the JohannWolfgang Goethe-University Frankfurt, Frankfurt am Main, Germany.

Despite significant progress in the diagnostics of Lyme borreliosis, includingmolecular methods, the detection of a specific antibody response remains themainstay in the laboratory diagnosis of the disease. Current guidelines proposethe combination of highly sensitive screening assays, such as ELISAs, with veryspecific confirmatory tests, such as immunoblots, to guarantee a cost-effective,sensitive and specific diagnostic approach. For a correct interpretation of theserological findings, the investigator must always consider a whole series ofclinical and laboratory facts. Here, we summarize current laboratory algorithmsin the diagnosis of Lyme borreliosis, with a special emphasis on when to order aWestern blot and how to interpret it correctly in the context of additionalclinical and laboratory information. Copyright (c) 2009 S. Karger AG, Basel. PMID: 19367101 [PubMed - as supplied by publisher]
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ViestiKirjoittaja soijuv » Ke Huhti 29, 2009 16:25

Onko serologisilla testeillä merkitystä ihossa esiintyvän borrelioosin seurannassa? Toistuvilla seurantatesteillä on hyvin rajoitetusti arvoa arvioitaessa hoidon tehokkuutta. Siksi niitä ei suositella tehtäväksi. Seurannan tulee keskittyä kliiniseen kuvaan. (Itävalta 2009)

Curr Probl Dermatol. 2009;37:178-182. Epub 2009 Apr 8.

Is Serological Follow-Up Useful for Patients with Cutaneous Lyme Borreliosis?

Mullegger RR, Glatz M. Department of Dermatology, State Hospital Wiener Neustadt, Wiener Neustadt,Austria.

Serologic follow-up examinations are frequently performed in patients witherythema migrans, borrelial lymphocytoma, and acrodermatitis chronicaatrophicans (the 3 dermatoborrelioses) to evaluate treatment efficacy. There is,however, substantial proof in the literature that antibody titer developmentafter therapy is unpredictable and variable, and moreover it is largelyuncorrelated with the clinical course and mode of antibiotic treatment. Forexample, persistent positive IgG and/ or IgM antibody titers do not indicatetreatment failure.

Thus, repeated serologic testing is of very limited value forassessing therapy efficacy, and therefore not recommended in the follow-up of dermatoborrelioses patients. Since cultivation of the etiologic agent, Borreliaburgdorferi sensu lato, and polymerase chain reaction are also inadequate forthis purpose, the assessment of patients with cutaneous manifestations of Lymeborreliosis in the follow-up rests primarily on the clinical picture. Copyright(c) 2009 S. Karger AG, Basel. PMID: 19367102 [PubMed - as supplied by publisher]
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ViestiKirjoittaja soijuv » To Huhti 30, 2009 11:00

Artikkeli lumbaalipunktion käytöstä ja eri borrelioositestien rajoituksista. Artikkelissa mainitaan lumbaalipunktiota tarvittavan nimenomaan neuroborrelioosin diagnostiikassa.

Allaolevassa tiivistelmässä testien rajoituksia ei tuoda esille - asiasta kiinnostuneiden tulee etsiä käsiinsä koko artikkeli.


Curr Probl Dermatol. 2009;37:200-206. Epub 2009 Apr 8. What Are the Indications for Lumbar Puncture in Patients with Lyme Disease? Rupprecht TA, Pfister HW. Department of Neurology, Ludwig-Maximilians University, Munich, Germany.

Lyme neuroborreliosis (LNB) is a tick-borne disease of the nervous system,caused by the spirochete Borrelia burgdorferi. Having entered the host at thesite of the tick bite, the spirochetes can initially cause a local inflammatoryreaction, called erythema migrans. If left untreated, the Borrelia candisseminate in the second stage of the disease and invade the central nervoussystem, causing LNB. The diagnosis of LNB is based on a compatible clinicalpicture (meningitis, cranial neuritis or radiculoneuritis), lymphocyticpleocytosis in the cerebrospinal fluid (CSF) and intrathecal Borreliaburgdorferi-specific antibody production. As the clinical picture of LNB may beunspecific, a lumbar puncture to analyze the CSF is usually mandatory forconfirmation of the suspected diagnosis. The indications for a lumbar punctureand the limitations of the different diagnostic procedures are the main topicsof this review. In addition, a short overview of the epidemiology and thetherapeutic principles of LNB is given.

Copyright (c) 2009 S. Karger AG, Basel. PMID: 19367105 [PubMed - as supplied by publisher]
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ViestiKirjoittaja soijuv » To Huhti 30, 2009 11:03

C6 Lyme ELISA -testin herkkyys osoittautui huonoksi selkäydinnesteestä verrattuna immunoblottaukseen.

Diagn Microbiol Infect Dis. 2009 Apr 17; [Epub ahead of print] The C6 Lyme antibody test has low sensitivity for antibody detection incerebrospinal fluid. Vermeersch P, Resseler S, Nackers E, Lagrou K. Laboratory Medicine, University Hospitals Leuven, B-3000 Leuven, Belgium.

Our aim was to evaluate the performance of the commercial Immunetics C6 Lyme ELISA assay as a screening assay for anti-Borrelia burgdorferi antibodies incerebrospinal fluid (CSF). Sensitivity of C6 ELISA was determined in 28consecutive patients who were diagnosed with neuroborreliosis and had evidencefor intrathecal antibody synthesis on immunoblot analysis. The presence ofadditional bands in CSF or of bands with a stronger intensity in CSF comparedwith serum was considered evidence of intrathecal synthesis. All 28 patientstested borderline or positive with C6 ELISA on serum. Of the 28 CSF samples, 17(61%) and 19 (68%) tested positive with C6 ELISA using a threshold of 0.9 and0.5 (optical density/cutoff).

The C6 Lyme ELISA tested has a low sensitivity forantibody detection in cerebrospinal fluid compared with immunoblot analysis.
PMID: 19376674 [PubMed - as supplied by publisher]
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ViestiKirjoittaja soijuv » To Touko 14, 2009 21:51

Borrelioosia sairastavien seerumin ja selkäydinnesteen sICAM-1 and sVCAM-1 pitoisuudet olivat koholla. (Puola)

Pol Arch Med Wewn. 2009 Apr;119(4):200-4. Levels of sVCAM-1 and sICAM-1 in patients with lyme disease.

Biesiada G, Czepiel J, Sobczyk-Krupiarz I, Salamon D, Garlicki A, Mach T. Department of Gastroenterology, Hepatology and Infectious Diseases, JagiellonianUniversity Medical College, Krakow, Poland. gbiesiada@op.pl


INTRODUCTION: Lyme disease is a multi-organ animal-borne disease caused by thespirochete Borrelia burgdorferi (Bb).

OBJECTIVES: As the pathogenesis of Lymeborreliosis is not fully understood, the study has been designed to examinelevels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and solubleintercellular adhesion molecule-1 (sICAM-1) in serum and the cerebrospinal fluid(CSF) of patients with Lyme borreliosis and their associations with clinicalsigns and symptoms and anti-Borrelia burgdorferi (anti-Bb) antibody titers.

PATIENTS AND METHODS: Sixty-four patients were enrolled in the study, including39 patients treated for Lyme borreliosis and 25 without the disease (controlgroup). In both groups sVCAM-1 and sICAM-1 levels were determined in serum andthe CSF. RESULTS: Mean serum sICAM-1 and sVCAM-1 levels were higher in patientswith Lyme borreliosis than in the control group. Serum sICAM-1 levels weresignificantly lower among patients with results positive for immunoglobulin Mseroreactivity with Bb than among those with negative antibody responses. Inpatients with Bb-specific serum immunoglobulin G (IgG) antibodies, significantlyhigher serum sICAM-1 levels were found. Higher sVCAM-1 and sICAM-1 levels in theCSF were observed in patients positive for anti-Bb IgG antibody titers in theCSF.

CONCLUSIONS: In patients with Lyme borreliosis, endothelial cell activation results in elevated levels of sICAM-1 and sVCAM-1 in serum and the CSF.



PMID: 19413177 [PubMed - in process]
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ViestiKirjoittaja soijuv » Ke Kesä 10, 2009 21:53

Slovakia 2009. 15 potilaalla oli diagnosoitu borrelioosi kliinisesti. 9 potilaalla serologiset testit olivat positiiviset, yhdellä oli positiivinen selkäydinnestelöydös.

Klin Mikrobiol Infekc Lek. 2009 Apr;15(2):40-43. [Evidence of Borrelia burgdorferi spirochetes in patients with early disseminated lyme borreliosis.] [Article in Slovak] Schwarzova K, Holeckova K, Kostanova Z. Institute of Virology, Slovak Academy of Science, Bratislava, Slovak Republic,virukats@savba.sk.

Fifteen patients with clinically documented early disseminated lyme borreliosiswere screened for the presence of spirochetes. In vitro trials of isolation ofthe pathogen were positive in 8 blood and 1 cerebrospinal fluid (CSF) samples.The positivity was further confirmed by electron microscopy, Western blotanalysis and PCR. However, succesfull DNA extraction after using Borreliaspecific primers proved the positivity in 9 blood and 1 CSF samples (67 %).Restriction fragment length polymorphism (RFLP) method using MseI restriction ofPCR products of the amplified rrf-rrl region, allowed the identification ofthree species, namely the Borrelia garinii, B. afzelii and B. burgdorferi s.s. PMID: 19488960 [PubMed - as supplied by publisher]
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ViestiKirjoittaja soijuv » Su Kesä 14, 2009 19:04

Borrelioosin diagnostiikka: Immunoblottauksessa (Western Blot) merkittäviä proteiineja ovat esim. 94, 83, 75, 66, 60, 55, 46, 41, 39, 34, 31, 29, 22 ja 17. Proteiineista 39kDa:n nousua tavataan muita useammin borrelioosia sairastavilla.

Vol. 30, No. 2
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1992, p. 370-376
0095-1137/92/020370-07$02.00/0
Copyright © 1992, American Society for Microbiology

Serodiagnosis of Lyme Borreliosis by Western Immunoblot:
Reactivity of Various Significant Antibodies against Borrelia burgdorferi

BINGNAN MA,* BEYAT CHRISTEN,t DANTON LEUNG,t AND CARMEN VIGO-PELFREYt
Whittaker Bioproducts, Inc., Walkersville, Maryland 21793-0127
Received 26 July 1991/Accepted 23 October 1991

The significance of various antibodies against Borrelia burgdorferi was studied by Western blot (immunoblot) by using 578 human serum samples. The proteins regularly detected by using samples from patients with Lyme borreliosis were those with bands with molecular masses of 94, 83, 75, 66, 60, 55, 46, 41, 39, 34, 31, 29, 22, and 17 kDa. The detectable frequencies of most of these proteins appeared to be significantly different between the sera from patients with Lyme borreliosis and those from normal control individuals as well as from the group with syphilis.

The 39-kDa protein band recognized by polyvalent antibody was found to be the most significant marker for Lyme borreliosis.
Furthermore, an anti-39-kDa immunoglobulin M response was detected in the samples from patients with early-stage Lyme borreliosis.

Results from the use of monoclonal antibodies and patient sera revealed that the 39- and 41-kDa proteins may be structurally related but are immunologically distinct antigens. The significance of antibody reactivities to the 41-, 94-, 22-, 31-, and 34-kDa protein bands is also discussed.
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