Laboratory Methods for Detection of Infectious Agents and Serological Response in Humans With Tick-Borne Infections: A Systematic Review of Evaluations Based on Clinical Patient Samples.

Background: For the most important and well-known infections spread by Ixodes ticks, Lyme borreliosis (LB) and tick-borne encephalitis (TBE), there are recommendations for diagnosis and management available from several health authorities and professional medical networks. However, other tick-borne microorganisms with potential to cause human disease are less known and clear recommendations on diagnosis and management are scarce. Therefore, we performed a systematic review of published studies and reviews focusing on evaluation of laboratory methods for clinical diagnosis of human tick-borne diseases (TBDs), other than acute LB and TBE. The specific aim was to evaluate the scientific support for laboratory diagnosis of human granulocytic anaplasmosis, rickettsiosis, neoehrlichiosis, babesiosis, hard tick relapsing fever, tularemia and bartonellosis, as well as tick-borne co-infections and persistent LB in spite of recommended standard antibiotic treatment. Methods: We performed a systematic literature search in 11 databases for research published from 2007 through 2017, and categorized potentially relevant references according to the predefined infections and study design. An expert group assessed the relevance and eligibility and reviewed the articles according to the QUADAS (diagnostic studies) or AMSTAR (systematic reviews) protocols, respectively. Clinical evaluations of one or several diagnostic tests and systematic reviews were included. Case reports, non-human studies and articles published in other languages than English were excluded. Results: A total of 48 studies fulfilled the inclusion criteria for evaluation. The majority of these studies were based on small sample sizes. There were no eligible studies for evaluation of tick-borne co-infections or for persistent LB after antibiotic treatment. Conclusions: Our findings highlight the need for larger evaluations of laboratory tests using clinical samples from well-defined cases taken at different time-points during the course of the diseases. Since the diseases occur at a relatively low frequency, single-center cross-sectional studies are practically not feasible, but multi-center case control studies could be a way forward.

Molecular detection of Borrelia burgdorferi sensu lato - An analytical comparison of real-time PCR protocols from five different Scandinavian laboratories.

INTRODUCTION: Lyme borreliosis (LB) is the most common tick transmitted disease in Europe. The diagnosis of LB today is based on the patient´s medical history, clinical presentation and laboratory findings. The laboratory diagnostics are mainly based on antibody detection, but in certain conditions molecular detection by polymerase chain reaction (PCR) may serve as a complement. AIM: The purpose of this study was to evaluate the analytical sensitivity, analytical specificity and concordance of eight different real-time PCR methods at five laboratories in Sweden, Norway and Denmark. METHOD: Each participating laboratory was asked to analyse three different sets of samples (reference panels; all blinded) i) cDNA extracted and transcribed from water spiked with cultured Borrelia strains, ii) cerebrospinal fluid spiked with cultured Borrelia strains, and iii) DNA dilution series extracted from cultured Borrelia and relapsing fever strains. The results and the method descriptions of each laboratory were systematically evaluated. RESULTS AND CONCLUSIONS: The analytical sensitivities and the concordance between the eight protocols were in general high. The concordance was especially high between the protocols using 16S rRNA as the target gene, however, this concordance was mainly related to cDNA as the type of template. When comparing cDNA and DNA as the type of template the analytical sensitivity was in general higher for the protocols using DNA as template regardless of the use of target gene. The analytical specificity for all eight protocols was high. However, some protocols were not able to detect Borrelia spielmanii, Borrelia lusitaniae or Borrelia japonica.

A prospective study on the incidence of Borrelia burgdorferi sensu lato infection after a tick bite in Sweden and on the Åland Islands, Finland (2008-2009).

Lyme borreliosis (LB) is a common and increasing tick-borne disease in Europe. The risk of acquiring a Borrelia infection after a tick bite is not fully known. Therefore, we investigated the incidence of Borrelia infection after a bite by a Borrelia-infected tick and if the Borrelia load and/or the duration of tick-feeding influenced the risk of infection. During 2008-2009, ticks and blood samples were collected from 1546 tick-bitten persons from Sweden and the Åland Islands, Finland. Follow-up blood samples were taken 3 months after the tick bite. The duration of tick feeding was microscopically estimated and Borrelia was detected and quantified in ticks by real-time PCR. Anti-Borrelia antibodies were detected in sera using ELISA tests and immunoblot. Five percent (78/1546) of the study participants developed Borrelia infection (LB diagnosis and/or seroconversion) after a tick bite (45% bitten by Borrelia-infected ticks and 55% bitten by uninfected ticks). Of these, 33 developed LB (whereof 9 also seroconverted) while 45 participants seroconverted only. Experience of non-specific symptoms was more frequently reported by Borrelia-infected participants compared to uninfected participants. All who seroconverted removed "their" ticks significantly later than those who did not. The Borrelia load in the ticks did not explain the risk of seroconversion. Regional and sex differences in the Borrelia seroprevalence were found. The risk of developing a Borrelia infection after a bite by a Borrelia-infected tick is small but increases with the duration of tick feeding.

Invasive Mycobacterium marinum infection of the hand.

Mycobacterium marinum infection of the hand is rare. We report the case of a 39-year-old man with M marinum infection that resulted in a chronic soft tissue infection, extensor tendon synovitis, and arthritis of the metacarpophalangeal (MCP) joints. The cause was probably tropical freshwater fishes.

Clinical appearance of erythema migrans caused by Borrelia afzelii and Borrelia garinii--effect of the patient's sex.

AIM: The aim in this survey was to study the clinical characteristics of infections caused by Borrelia genospecies in patients with erythema migrans where borrelial origin was confirmed by polymerase chain reaction. The aim was also to study factors influencing the clinical appearance of erythema migrans. METHODS: The study was conducted in southern Sweden from May 2001 to December 2003 on patients 18 years and older attending with erythema migrans at outpatient clinics. All erythema migrans were verified by polymerase chain reaction, photographed and categorized as "annular" or "non-annular" lesions. A logistic regression model was used to analyze relations between the appearance of the erythema migrans (i.e. annular or non-annular) and factors that influenced its clinical appearance. RESULTS: A total of 118 patients, 54 women (45.8%) and 64 men (54.2%), fulfilled the inclusion criteria. Of these patients, 74% were infected by B. afzelii and 26% by B. garinii (p < 0.001). A total of 45% (38/85) of the erythema migrans were annular, 46% (39/85) were nonannular and 9.4% (8/85) were atypical. For men infected by B. afzelii, the odds ratio of developing non-annular erythema migrans was 0.09 (95% CI: 0.03-0.33) in comparison with women with the same infection. CONCLUSIONS: In this prospective study of a large series of erythema migrans, where infecting genospecies were confirmed by polymerase chain reaction, the sex of patients infected with B. afzelii had a strong influence on the appearance of the rash. Patients infected by B. garinii more often had non-annular erythema migrans and a more virulent infection with more individuals presenting with fever, raised levels of C-reactive protein and seroreactivity in the convalescence sera.

A reverse transcriptase-polymerase chain reaction assay of Borrelia burgdorferi 16S rRNA for highly sensitive quantification of pathogen load in a vector.

We developed a real-time quantitative detection assay for the pathogen Borrelia burgdorferi, a Lyme borreliosis (LB) agent, using reverse transcription-polymerase chain reaction (RT-PCR) with primers and probe for a Borrelia genus-specific region of 16S ribosomal RNA. The standard curve of the assay was linear by semi-log plot over more than five orders of magnitude, and the detection limit of the assay was one thousandth of a single cell of B. burgdorferi. The minimum target level for detection using the RT-PCR assay for 16S RNA was 40-fold lower than the RT-PCR assay for messenger RNA of ospA, a highly expressed, plasmid-borne gene, and 1600-fold lower than the RT-PCR assay for messenger RNA of p66, a chromosome-borne gene of B. burgdorferi. The 16S rRNA assay was then applied in an experimental setting for monitoring the spirochetal load in B. burgdorferi-infected Ixodes scapularis ticks before and after they fed on Peromyscus leucopus mice immunized with recombinant OspA. Unfed infected ticks had a mean of 2,240 spirochetes per tick, and after feeding on non-immunized mice and engorgement, the mean number of spirochetes increased to 223,900 per tick. In contrast, there were either no or

Differential immune response to the variable surface loop antigen of P66 of Borrelia burgdorferi sensu lato species in geographically diverse populations of lyme borreliosis patients.

We have studied the immune response to a variable surface-exposed loop region of the P66 outer membrane protein from Borrelia burgdorferi sensu lato by using an enzyme immunoassay. Lyme borreliosis populations found in North America and Sweden were preferentially more seroreactive to P66 from their respective regional species, namely, B. burgdorferi sensu stricto and B. garinii and B. afzelii, respectively.

5-y Follow-up study of patients with neuroborreliosis.

The objective of this follow-up study was to determine the long-term outcome of strictly classified cases of neuroborreliosis treated with antibiotics. A 1-y prospective population-based survey of Lyme borreliosis was conducted in southern Sweden between 1992 and 1993. A total of 349 identified cases with suspected neuroborreliosis were followed up 5 y later. Medical records were reviewed and all participants filled in a questionnaire. Of those patients classified with definite neuroborreliosis, 114/130 completed the follow-up, of whom 111 had completed the initial antibiotic treatment. Of the 114 patients followed up, 86 (75%) had recovered completely and 70 (61%) had recovered within 6 months. Residual neurological symptoms, such as facial palsy, concentration disorder, paresthesia and/or neuropathy, were reported by 28/114 patients. No significant differences between different antibiotic treatments were observed in terms of the occurrence of sequelae. To conclude, we found that 25% (95% confidence interval 17-33%) of the patients suffered from residual neurological symptoms 5 y post-treatment. However, the clinical outcome of treated neuroborreliosis is favorable as only 14/114 (12%) patients had sequelae that influenced their daily activities post-treatment. Early diagnosis and treatment would seem to be of great importance in order to avoid such sequelae.

Three major Lyme Borrelia genospecies (Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii) identified by PCR in cerebrospinal fluid from patients with neuroborreliosis in Sweden.

The Lyme Borrelia genospecies Borrelia afzelii and B. garinii have previously been isolated using a culture method in Swedish patients with Lyme borreliosis (LB). There are reports suggesting that the genospecies distribution in human tissue specimens as determined by molecular methods is different from that obtained by culture. In the present study, we developed a nested PCR for detection of Lyme Borrelia-specific DNA in cerebrospinal fluid from Swedish patients with LB. The genospecies were subsequently identified by sequence analysis in a total of 7 PCR-positive patients. Two sequences were identified as B. burgdorferi sensu stricto (s. s.), 1 as B. afzelii and 4 as B. garinii. These are the first reported cases in which B. burgdorferi s. s. has been shown to be the causative agent of human LB in Sweden. The results of our study confirm that the use of direct molecular analytical methods for Borrelia genospecies identification in clinical specimens can provide epidemiological information additional to that obtained by culture.