Ruotsalainen tutkimusprojekti

Valvojat: Borrelioosiyhdistys, Bb, Jatta1001

Ruotsalainen tutkimusprojekti

ViestiKirjoittaja soijuv » Ma Helmi 28, 2011 17:48

Ruotsi. Käynnissä oleva tutkimus, Pia Forsberg: Punkkien välityksellä saatujen tautien määrät lisääntyvät jatkuvasti. Ruotsissa 20% kerätyistä punkeista oli Bb positiivisia. Aikuisista punkeista bakteeria löytyi muita useammin. Bakteerin alalajeja on tähän mennessä löytynyt 6; B. afzelii (61%), B. garinii (21%), B. valaisiana (12 %), B. burgdorferi senu stricto (2 %), B. lusitaniae (2 %), and B. miyamotoi-like (2 %).

Tutkimukseen etsitään parhaillaan osallistujia. Tutkimuksessa selvitetään Bb:n esiintymistä punkeissa, lisäinfektioita, tartuntariskiä jne.
Pia Forsberg
Tick-borne infections - interaction between vector, pathogen and human

Tick-borne infections are increasing (1) probably at least partly, depending on the climate changes. Ticks are often carriers of both bacteria and viruses, which can be transmitted to humans. Several of these microorganisms, Borrelia (B) burgdorferi (2,3,4,5,6), Anaplasma (A) phagocytophilum (4), Rickettsia (R) Helvetica (7) and tick-borne encephalitis ? virus (TBEV) (4,5,8), are all known to cause disease in human. This is expensive for the healthcare, particularly the worry about the borrelia infection and TBE, that many people feel.

It is not unusual that individuals want antibiotic therapy after a known tick-bite already before a disease is prominent, which might increase the development of resistance among bacteria and other adverse effects in the host. The risk to be infected by these microbes after a single tick-bite is not known. However, estimations have been done from the occurrence of infected ticks that have been collected from the nature.

Indeed it has been investigated the frequency of infections in ticks which have been BITTEN the humans (9) but as far as we know, the symptoms and the immune-reactivity in the bitten human has never been connected to the confirmed microbe in the tick. Thus, it is unknown how often ticks which have bitten individuals are infected and how the interaction is taking place between the microbial agent and the host (the human). It is also unknown why the course of the disease is different in different patients, particularly in TBE (8) and borreliosis (6, 10, 11 and 12). It is thus of importance to clarify the risk for disease after the bite of an infected tick.

Specific objectives and questions

We will in this project create better instruments for studies of tick-borne diseases by using newly developed molecular biological techniques for the detection and quantification of the microorganisms. The bitten individuals will be followed up for three months in order to survey the symptoms and their immune-reactivity of their blood. The infected tick will be connected to the individual?s symptoms and disease development and also the development of the host immune-reactivity.

One considerable aim is to reduce the fear of tick-borne diseases in our country.

The objective with the study is to answer the following questions:

1. The interaction between the microbe and tick which has bitten the human:

* How often are the ticks which bite the individuals in the different study areas infected with B. burgdorferi, TBEV, A. phagocytophilum and/or R. Helvetica?
* What are the geographical differences?
* Are there completely new variants of bacteria/viruses which are spread by ticks? Have the new dangerous TBEV already reached Sweden?
* How often are the ticks co-infected with two or more strains of the pathogen?
* How often are the ticks co-infected with one or more microbes?

2. The interaction between the microbe and the bitten individual=human:

* What is the risk to get a disease after a bite of an infected tick with these microbes? Are there asymptomatic infections after a bite by a confirmed infected tick?

* Is there any connection between the risk of being infected and the time period the tick has been sucking blood?
* Is there any connection between the number of bacteria/viruses in the tick and the development of disease?
* Is there any connection between the genetically variant/subtype of Borrelia or TBEV and the clinical prognosis of the infection?
* How is the immune-reactivity in the bitten individual connected to the clinical prognosis and to the tick?
* Is there any connection between co-infected ticks and the prognosis of the diseases?


Persons who are interested in participating in the study will be asked, when they were bitten by a tick and to bring the tick to the primary health care centre (PHC). The health care personal will take blood samples from the individual and the individual will be asked to fill in a questionnaire which is related to tick-borne infections. The person will also be asked to collect any further ticks which bites the person during the study-period (3 months), and come back for a second visit which will take place at the PHC 12+/-2 weeks after the tick-bite. Blood will then be drawn again and the individual will be asked to fill in a new questionnaire regarding the symptoms. If symptoms of a tick-borne infection would appear during the study-period, the patients must contact their PHC for actions. Then a new blood sample is drawn and a copy of the relevant record is sent to the main investigator (PF). Those who will get tick-borne infections will also be followed up through blood sampling and clinical observations longer than the default study-period until they are healthy.

From the collected ticks RNA will be extracted. For the detection and quantification of infectious agents (B.burgdorferi, TBEV, A.phagocytophilum and R.Helvetica), RNAt will be analysed with real-time PCR (qPCR). Group or species specific primers for these microorganisms will be used. When primer designing and optimizing has been completed, one must balance specificity contra sensitivity. For typing and epidemiological tracing of different clones of microorganisms, the extracted RNAt will be amplified by conventional PCR followed by electrophoresis analysis of the PCR-products (13, 14). Then information will be received if certain clones of microorganisms more often will infect persons than others and if several multiclonal infections are present. These methods are now already established on the Department of Medial Microbiology, University Hospital in Linköping regarding B. burgdorferi and TBEV, and A. phagocytophilum. The next step is to establish methods for R. Helvetica.

The ticks are photographed and scutal-index and coxal index are measured to calculate an approximate time for the duration of the tick bite. Thus, the scutal and coxal index are used to confirm how long time the tick has sucked blood.


RNA extraction:

Ticks are homogenized with TissueLyser (Qiagen, Hilden, Germany) and RNA was extracted, using MagAttract® RNA Tissue Mini M48 Kit with the BioRobot M48 Workstation (Qiagen). The RNA was converted into cDNA using Illustra? Ready-to-Go RT-PCR Beads kit (GE Healthcare, Amersham Place, UK). The use of cDNA derived from tick extracted RNA (from 2008 and onwards) offers greater sensitivity due to the presence of multiple copies of 16S rRNA in each bacterial cell.

PCR B.burgdorferi: For detection and quantification of Borrelia spp., real-time PCR analyses for the 16S rRNA gene were performed (Wilhelmsson, P., P. Forsberg, L. Fryland, J. Nordgren, S. Börjesson, C. Ekerfelt, J. Ernerudh, S. Bergström, and P-E. Lindgren. Prevalence and diversity of Borrelia species in ticks that have bitten humans in Sweden. To be. published). This is a LUX assay and the detection limit is 10 copies and therefore it is possible to detect a single bacterial cell. All Borrelia positive samples were species determined by sequencing the PCR-products from the intergenic spacer 5S-23S (15).

PCR TBE-virus: In order to be able to use as optimal tools for the molecular genetic work as possible, primers for detection, quantification and subtyping of TBEV were designed for use with real-time PCR. The primers were designed from scratch based on multiple alignments of 74 sequences (15 complete genomes, 59 covering the E-gene) of TBEV currently present in the GenBank database. Two approaches were used: a) primers for simultaneous detection of all three subtypes using SybrGreenTM, b) primers for subtype identification designed for Light upon eXtension (LUXTM) to be used in a multiplex real-time PCR with one common primer (for the three subtypes) without fluorophore, and one LUXTM modified primer with different fluorophores, specific for each subtype. This PCR-based approach for detection, quantification and subtyping of TBEV strains have recently been successfully evaluated using TBEV-strains of all three subtypes.

PCR A. phagocytophilum: The detection and quantification of A. phagocytophilum is based on a real-time PCR assay targeting a fragment of the 16S rRNA gene. The assay utilizes a species specific primer pair generating a 150 bp product (16) and a hydrolysis probe. The amplification is carried out in a Rotorgene thermal cycler. The method has been optimized to ensure very good efficiency, linearity and curve fitting for reliable and reproducible results. The assay?s quantification limit is 10 copies of template per reaction, however the detection limit is as low as 1 copy. A 860 bp fragment of the 16S rRNA is amplified from all positive samples using a nested PCR assay for the purpose of species confirmation and strain determination.

Antibody analysis: Parallel with the microbiological analysis, antibodies and biomarkers in blood will be determined by Luminex xMap technique. Antibody analysis (17) and immunological analysis in blood, i.e. cytokines and chemokines, will be performed at the Department of Clinical Immunology, University Hospital in Linköping.

Serum from the study subjects from both the time of tick bite and the second visit to the PHC will be analysed for both anti-flagellum and anti-C6 antibodies with the enzyme-linked immunosorbent assays (ELISA) IDEIA? Borrelia IgG (Oxoid, Cambs, UK) and C6 Lyme ELISA? kit (Immunetics Inc., Cambridge,MA, USA), respectively. Seroconversion, i.e. the development of new antibodies, indicating a current infection, was defined as conversion from sero-negative to ?positive, and in the case of an initially positive test as at least a twofold increase of the arbitrary units calculated with Oxoid?s formula for increased antibody titre (anti-flagellum antibodies) or with Immunetics? Lyme Index (anti-C6 antibodies). All serum samples from study subjects who showed seroconversion in any or both ELISAs will be further analysed with the line immunoblot recomLine Borrelia IgG (Mikrogen, Neuried, Germany), detecting antibodies against several different Borrelia antigens, in order to confirm conversion. ELISA positives verified by seroconversion also in the line immunoblot assay, will be considered as having a probable current borreliosis. Serological findings will be compared with self-reporting of symptoms possibly associated with Lyme Borreliosis and scrutinized medical records. (Linda Fryland, Peter Wilhelmsson, Per-Eric Lindgren, Dag Nyman, Christina Ekerfelt, Pia Forsberg. Low risk of developing Borrelia burgdorferi infection in the South East of Sweden after a bite by a Borrelia burgdorferi-infected tick. Submitted, under review).

In order to obtain valid knowledge within this research-area, we estimate that 10 000 ticks are needed to be analysed together with the bitten individuals blood and their disease history. Therefore the project must be running during several years, uncertain of how many, depending on the success of the tick-collections and must also incorporate many different areas in Sweden and also Åland, which is an high-endemic area.

Significance of the project

The study aim is to elucidate the connection between infected ticks and the risk for a human to be infected and diseased after a tick-bite. Furthermore we will scrutinize quantitative aspects/dose of infective agents in relation to symptomatic respectively asymptomatic infections. Another part of the project will deal with clonality ? are certain clones of the microbe more aggressive than others? It is very important to further scrutinize these questions in order to, in a more adequate way evaluate the risk-judgement for infection and in order to take proper measures. Therefore the results will directly be applied in the healthcare settings regarding risk-judgement, diagnostics and the strategy of therapies when infection is confirmed. This new knowledge will also be useful for the development of new therapies, in particularly in borreliosis. We think that it is an urgent investigation which has never earlier been done. The results can also create opportunities to increase the possibilities of quick, valid and reproducible diagnostics based on PCR of tick-borne pathogenic microorganisms in patients as well as in the tick itself. Furthermore, possibilities will be created for epidemiological studies in different geographical areas, i.e. to follow different populations of tick-borne microorganisms in humans and also in other hosts.

It is important for the healthcare to receive knowledge about the spread of infected ticks, the risk for the transmission of infection and the risk for developing symptoms after that the microbe has reached the patient. We also hope to receive more knowledge about why certain persons become more diseased than others.

Another advantage with this study is that a frozen bank of genetic materials from ticks, which have been bitten humans, will be collected and saved for identification of new or more severe tick-borne infections that might be established in Sweden according to the climate changes in the future.

Preliminary Results

The study started in the spring of 2007 and until now we have collected 2 500 ticks and 2 serum samples (day1 and day 90) from the 2 500 tick-bitten individuals and their history of tick associated disease during the study period. Thirty-seven PHCs from different areas in Sweden are now incorporated in the study and this year (2010) a further six PHCs will participate. From Åland there are six islands involved.

Twenty percent of the ticks (164 of 770) were Borrelia positive and the Borrelia cell quantity

ranged from 200 to 490 000 cells per Borrelia positive tick. The Borrelia prevalence between

the PHCs varied from 11 to 33 %. Adult ticks were more often Borrelia

positive than the nymphs and the adults had a significantly higher number of Borrelia cells.

So far, we have determined a total of six different Borrelia species; B. afzelii (61%), B. garinii (21%), B. valaisiana (12 %), B. burgdorferi senu stricto (2 %), B. lusitaniae (2 %), and B. miyamotoi-like (2 %). Three percent of the ticks were shown to be co-infected with multiple strains of Borrelia species. (Wilhelmsson, P., P. Forsberg, L. Fryland, J. Nordgren, S. Börjesson, C. Ekerfelt, J. Ernerudh, S. Bergström, and P-E. Lindgren. Prevalence and diversity of Borrelia species in ticks that have bitten humans in Sweden. To be published).

Among the 380 DNA samples analysed from the ticks collected in 2007, 29 gave a positive amplification curve, indicating a possible prevalence of 7,6 %. However, some of the values obtained were below the quantification limit and therefore must be further confirmed. The 16S rRNA gene fragments from all positive samples have been amplified and prepared for sequencing (18). The results of sequencing will allow for the determination of the true prevalence of A. phagocytophilum infection among these ticks and provide insight on strain diversity.

Sera from 217 study subjects have been analysed for presence of anti-Borrelia antibodies. Only ten (~5%) of these study subjects had developed new antibodies against Borrelia during the three month study period. Four of the ten subjects who had seroconverted experienced symptoms possibly associated with Lyme Borreliosis during the study period according to self-reports from the study questionnaires. However, only one of the four attended PHC during the study time (Linda Fryland, Peter Wilhelmsson, Per-Eric Lindgren, Dag Nyman, Christina Ekerfelt, Pia Forsberg. Low risk of developing Borrelia burgdorferi infection in the South East of Sweden after a bite by a Borrelia burgdorferi-infected tick. Submitted, under review).

The preliminary results indicate that in spite of that the tick is infected with Borrelia, it is very unusual that the tick-bitten individual consults a doctor ? indicating that the symptoms are relatively mild.

Ethical considerations

Drn: M132-06. 2006-09-06.

The study might develop some anxiety for tick-borne infections by the participants in the study. We will recruit participants by announcing in newspapers, television, radio and on local spots. Thus, it is a risk to further worry people who are afraid of tick-bites. On the other hand, there are already people who are worried in the community and the study will give further knowledge about the risks for tick-bites and in the future we might be able to calm down this worry. The study will cause minimal damage and the risk with the blood-sample collections will be very low.

The relative advantage for the participants in the study is that tick-borne infection will be confirmed earlier and the person will get treatment at once. The contact with the healthcare will also ensure safety among the participants. Furthermore, the knowledge from the study might lead to better therapy, vaccination strategies and diagnostics regarding tick-borne infections and this is also an advantage in a perspective for the community. Some other aspects are that it is important to avoid unnecessary, potentially risky antibiotic treatments and expensive investigations (laboratory and X-ray) of unexplained symptoms.

Because the risks of the study are minimal and the participants might be advantaged when being included into the study and that important knowledge about tick-borne infections will be reached, we think that the usefulness of the study is predominan
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