Immunohistochemistry and real-time PCR as diagnostic tools for detection of Borrelia burgdorferi sensu lato in ticks collected from humans.

The objective of this study was to evaluate different methods used for detection of Borrelia burgdorferi sensu lato (s.l.) in ticks: immunohistochemistry followed by focus floating microscopy (FFM) and real-time polymerase chain reaction (real-time PCR) targeting the ospA and hbb genes. Additionally, an optimized ospA real-time PCR assay was developed with an integrated internal amplification control (IAC) for the detection of inhibition in the PCR assay and was validated as an improved screening tool for B. burgdorferi. One hundred and thirty-six ticks collected from humans in a hospital from Cluj-Napoca, Romania, were investigated regarding genus, stage of development and sex, and then tested by all three assays. A poor quality of agreement was found between FFM and each of the two real-time PCR assays, as assessed by concordance analysis (Cohen's kappa), whereas the agreement between the two real-time PCR assays was moderate. The present study argues for a low sensitivity of FFM and underlines that discordant results of different assays used for detection of B. burgdorferi in ticks are frequent.

Assessing the risk of human granulocytic anaplasmosis and lyme borreliosis after a tick bite in Bavaria, Germany.

To date, only isolated incidences of human granulocytic anaplasmosis (HGA) have been reported in Europe. However, entomological studies in Bavaria, Germany showed a prevalence of Anaplasma phagocytophilum of between 2 and 9.5% in the tick vector Ixodes ricinus. In this study we assessed the risk of pathogenic A. phagocytophilum infection after a tick bite in Bavaria. The seroprevalence of anti-Borrelia burgdorferi sensu lato (s.l.) antibodies was investigated as an indicator of past exposure, seroconversion as actual exposure of participants to ticks. Patients with a tick bite in the preceding four weeks were recruited by participating doctors. Questionnaires on demographics, tick exposure and clinical signs were completed by patients and doctors, respectively. Two blood samples, taken at an interval of two weeks, were tested for antibodies against A. phagocytophilum and B. burgdorferi s.l. One of 107 recruited patients showed serological evidence of an acute infection of A. phagocytophilum but had no clinical signs. Four out of six patients with serological evidence of an acute B. burgdorferi s.l. infection, presented with erythema migrans. A seroprevalence of 7.5% for A. phagocytophilum and 13.1% for B. burgdorferi s.l. was detected. The comparatively high seroprevalence of B. burdorferi s.l. and A. phagocytophilum antibodies indicate frequent past exposure of participants to ticks. The finding of one acute infection of A. phagocytophilum in the absence of clinical signs, supports entomological evidence that the strains of A. phagocytophilum predominantly present in the Bavarian tick population may cause transient infections but are of low pathogenicity in humans.

Development and validation of a multi-target TaqMan qPCR method for detection of Borrelia burgdorferi sensu lato.

Reliable detection of bacteria belonging to the Borrelia burgdorferi sensu lato species complex in vertebrate reservoirs, tick vectors, and patients is key to answer questions regarding Lyme borreliosis epidemiology. Nevertheless, the description of characteristics of qPCRs for the detection of B. burgdorferi s. l. are often limited. This study covers the development and validation of two duplex taqman qPCR assays used to target four markers on the chromosome of genospecies of B. burgdorferi s. l. Analytical specificity was determined with a panel of spirochete strains. qPCR characteristics were specified using water or tick DNA spiked with controlled quantities of the targeted DNA sequences of B. afzelii, B. burgdorferi sensu stricto or B. bavariensis. The effectiveness of detection results was finally evaluated using DNA extracted from ticks and biopsies from mammals whose infectious status had been determined by other detection assays. The developed qPCR assays allow exclusive detection of B. burgdorferi s. l. with the exception of the M16 marker which also detect relapsing fever Borreliae. The limit of detection is between 10 and 40 copies per qPCR reaction depending on the sample type, the B. burgdorferi genospecies and the targeted marker. Detection tests performed on various kind of samples illustrated the accuracy and robustness of our qPCR assays. Within the defined limits, this multi-target qPCR method allows a versatile detection of B. burgdorferi s. l., regardless of the genospecies and the sample material analyzed, with a sensitivity that would be compatible with most applications and a reproducibility of 100% under measurement conditions of limits of detection, thereby limiting result ambiguities.

Borrelia bavariensis sp. nov. is widely distributed in Europe and Asia.

Since the original description of Borrelia bavariensis sp. nov. in 2009, additional samples available from humans and ticks from Europe and Mongolia, respectively, have been used to further characterize Borrelia strains belonging to this group of spirochaetes that utilize rodents as reservoir hosts. These investigations suggested the presence of related strains in Europe and Asia and confirmed their status as representing a distinct species. Furthermore, samples that were investigated by researchers from China and Japan confirm the ecological relationship of members of this proposed species with rodents and suggest that it has a wide distribution in Eurasia. Here, we use phylogenetic and genetic distance analyses to validate B. bavariensis sp. nov. as a species within the Borrelia burgdorferi sensu lato species complex. The type strain is PBi(T) ( = DSM 23469(T) = BAA-2496(T)).

Presence of Borrelia spp. DNA in ticks, but absence of Borrelia spp. and of Leptospira spp. DNA in blood of fever patients in Madagascar.

The occurrence of tick-borne relapsing fever and leptospirosis in humans in Madagascar remains unclear despite the presence of their potential vectors and reservoir hosts. We screened 255 Amblyomma variegatum ticks and 148 Rhipicephalus microplus ticks from Zebu cattle in Madagascar for Borrelia-specific DNA. Borrelia spp. DNA was detected in 21 Amblyomma variegatum ticks and 2 Rhipicephalus microplus ticks. One Borrelia found in one Rhipicephalus microplus showed close relationship to Borrelia theileri based on genetic distance and phylogenetic analyses on 16S rRNA and flaB sequences. The borreliae from Amblyomma variegatum could not be identified due to very low quantities of present DNA reflected by high cycle threshold values in real-time-PCR. It is uncertain whether these low numbers of Borrelia spp. are sufficient for transmission of infection from ticks to humans. In order to determine whether spirochaete infections are relevant in humans, blood samples of 1009 patients from the highlands of Madagascar with fever of unknown origin were screened for Borrelia spp. - and in addition for Leptospira spp. - by real-time PCR. No target DNA was detected, indicating a limited relevance of these pathogens for humans in the highlands of Madagascar.

Clinical and serological one-year follow-up of patients after the bite of Ixodes ricinus ticks infected with Borrelia burgdorferi sensu lato.

BACKGROUND: The risk of developing Lyme borreliosis (LB) after the bite of a Borrelia (B.) burgdorferi sensu lato (s.l.) infected tick in Romania is unknown. METHODS: The present prospective study, performed in 2010-2011 in a hospital in Romania, has followed-up clinical and serological outcome of patients that presented with B. burgdorferi positive Ixodes (I.) ricinus bite. A second group of patients, including age, sex and residence-matched individuals bitten by B. burgdorferi negative ticks, was followed-up as a control group. The subjects' outcome was evaluated one year after the tick bite. RESULTS: Forty-three out of 389 ticks detached from patients were positive by hbb Real-Time PCR (RT-PCR) for B. burgdorferi s.l. (mainly B. afzelii, but also B. garinii, B. burgdorferi sensu stricto, B. spielmanii/B. valaisiana and B. lusitaniae). Twenty patients bitten by B. burgdorferi positive ticks and twenty matched control patients returned for the one year follow-up. Two patients from the B. burgdorferi positive group developed clinical manifestations of acute LB (erythema migrans) and 5 patients seroconverted (two from the B. burgdorferi positive group and three from the B. burgdorferi negative group). Borrelia afzelii was identified in ticks collected from persons that developed erythema migrans (EM). Comparing the two groups of patients, no statistical significant differences were found regarding presence of clinical symptoms or seroconversion. CONCLUSIONS: No outcome differences were found between the group of patients bitten by B. burgdorferi positive ticks and the group of patients bitten by B. burgdorferi negative ticks.

Long-term in vitro cultivation of Borrelia miyamotoi.

Borrelia are fastidious bacteria some of which are difficult to grow in vitro. Here, we report a method for successful continuous in vitro cultivation of the emerging pathogen Borrelia miyamotoi. The type and quantity of serum as well as the atmosphere were critical for successful in vitro cultivation. Optimal growth was achieved using 50% pooled human serum and an atmosphere of 6% CO2.

Low prevalence of Borrelia bavariensis in Ixodes ricinus ticks in southeastern Austria.

Borrelia bavariensis was recently described as a distinct genospecies among the B. burgdorferi sensu lato complex. The prevalence of B. bavariensis in Austria, a highly endemic area for tick-transmitted pathogens, is scarcely characterized. To investigate the prevalence of B. bavariensis in Ixodes ricinus ticks we reevaluated the results of a study conducted in 518 ticks from southeastern Austria collected in 2002 and 2003. The presence of B. burgdorferi s.l.-specific DNA in ticks was analyzed by a PCR for the outer surface protein A (ospA) gene. Borrelia species were differentiated by restriction fragment length polymorphism (RFLP) analysis, and samples positive for B. bavariensis were further analyzed by multilocus sequence analysis. Two of 133 (1.5%) B. burgdorferi s.l.-positive I. ricinus ticks were infected with B. bavariensis. Both specimens were coinfected with the OspA serotype 5 of B. garinii. Borrelia bavariensis is present; however, seem to be rare in I. ricinus ticks in southeastern Austria.

Real-time PCR-based identification of Borrelia burgdorferi sensu lato species in ticks collected from humans in Romania.

The aims of our study were to determine (i) which tick species bite humans in Romania and (ii) the prevalence of Borrelia (B.) burgdorferi genospecies in these ticks. All ticks collected from patients who presented to the Clinic of Infectious Diseases Cluj Napoca in spring/summer 2010 were morphologically identified by an entomologist and tested for B. burgdorferi genospecies prevalence by a real-time PCR assay targeting the hbb gene and melting curve analysis. Out of 532 ticks, 518 were Ixodes ricinus, 10 Dermacentor marginatus, and 3 Haemaphysalis spp. ticks, and one unidentified tick due to destruction. Since evaluation of the hbb PCR revealed that it was not possible to differentiate between B. spielmanii/B. valaisiana and B. garinii/B. bavariensis, sequencing of an 800-bp fragment of the ospA gene was performed in these cases. Out of 389 investigated ticks, 43 were positive by hbb PCR for B. burgdorferi sensu lato. The positive samples were 42 Ixodes ricinus (11.1% B. burgdorferi sensu lato prevalence) and the one unidentified tick. Species identification revealed the presence of mainly B. afzelii, but also of B. garinii, B. burgdorferi sensu stricto, B. valaisiana, and B. lusitaniae. In 4 samples, differentiation between B. spielmanii/B. valaisiana was impossible. Our study shows that the most relevant human pathogenic B. burgdorferi genospecies - predominantly B. afzelii - are present in ticks collected from Romanian patients.

Prevalence of Borrelia burgdorferi s.l. OspA types in Ixodes ricinus ticks from selected localities in Slovakia and Poland.

In this study, 746 questing Ixodes (I.) ricinus ticks from eastern Slovakia and 187 ticks from southern Poland were investigated for infection with Borrelia (B.) burgdorferi sensu lato and different outer surface protein A (OspA) types by an improved restriction fragment length polymorphism (RFLP) analysis of the ospA gene. The method enables differentiation of both single and multiple infections with B. burgdorferi s.s. (OspA type 1), B. afzelii (OspA type 2), B. garinii (OspA types 3-8), B. valaisiana (subgroups I and II), B. lusitaniae, B. bissettii, and the recently described genospecies A14S. Broad heterogeneity in B. burgdorferi s.l. was found including all species and subtypes except for B. lusitaniae, B. bissettii, and genospecies A14S. Regional prevalence of B. burgdorferi s.l. varied between 8% and 22.5%. The most frequent species were B. garinii (45.4%) and, notably, B. burgdorferi s.s. (31.3%). I. ricinus nymphs harbored almost exclusively B. burgdorferi s.s. and B. garinii OspA type 4, while in adults a broad variety of B. burgdorferi s.l. types was found. Mixed infections were significantly more often in nymphs than in adult ticks. In all mixed infected nymphs, B. burgdorferi s.s. with OspA type 4 was present. These data strongly suggest that B. burgdorferi s.s. and B. garinii OspA type 4 are maintained in these areas by specific transmission cycles involving a so far undetermined vertebrate host which is frequently fed on by I. ricinus larvae. This improved method provides a reliable tool for epidemiological studies on the heterogeneity of B. burgdorferi species and OspA types, an important prerequisite for improved local risk assessment and for test- and vaccine development for Europe.