| Abstract: | Members of the genus Borrelia are among the most common infectious agents causing tick-borne disease in humans worldwide. Here, we developed a Light Upon eXtension (LUX) real-time PCR assay that can detect and quantify Borrelia species in ticks that have fed on humans, and we applied the assay to 399 such ticks. Borrelia PCR-positive ticks were identified to species level by sequencing the products of conventional PCR performed using Borrelia group-specific primers. There was a 19% prevalence of Borrelia spp. in the detached ticks, and the number of spirochetes per Borrelia PCR-positive tick ranged from 2.0 × 102 to 4.9 × 105, with a median of 7.8 × 103 spirochetes. Adult ticks had a significantly larger number of spirochetes, with a median of 8.4 × 104 compared to the median of nymphs of 4.4 × 104. Adult ticks also exhibited a higher prevalence of Borrelia (33%) than nymphs (14%). Among the identified species, Borrelia afzelii was found to predominate (61%) and was followed by B. garinii (23%), B. valaisiana (13%), B. burgdorferi sensu stricto (1%), B. lusitaniae (1%), and B. miyamotoi-like (1%). Also, 3% of the ticks were coinfected with multiple strains of B. afzelii. Notably, this is the first report of B. lusitaniae being detected in ticks in Sweden. Our LUX real-time PCR assay proved to be more sensitive than a corresponding TaqMan assay. In conclusion, the novel LUX real-time PCR method is a rapid and sensitive tool for detection and quantification of Borrelia spp. in ticks.Lyme borreliosis (LB) is the most common tick-borne disease in humans in Europe (26), and it is caused by spirochetes belonging to the Borrelia burgdorferi sensu lato complex. That group comprises the species B. burgdorferi sensu stricto, B. afzelii, and B. garinii, which are usually transmitted by the vector Ixodes ricinus. Furthermore, there have been reports of B. valaisiana, B. lusitaniae, and B. spielmanii being detected in samples of human skin and cerebrospinal fluid (5, 7, 30), which suggests that those three species can also give rise to LB. It is often hard to distinguish the clinical symptoms of LB from those of other diseases (10), and hence, it can be difficult to establish a correct diagnosis, especially if the patient is unable to recall having a tick bite.Today, diagnosis is based mainly on serological tests, although some PCR-based approaches, such as the TaqMan real-time PCR assay (3, 12), have been developed to detect Borrelia species in clinical samples. Even if real-time PCR is not yet considered to be a routine method in clinical practice, it can nonetheless provide valuable information about Borrelia infections, with regard to species type and the number of spirochetes present. Additional major advantages of PCR in this context are its simplicity, sensitivity, robustness, and speed. Other assays besides the TaqMan assay include a method based on SYBR green dye chemistry (37) and another using Light Upon eXtension (LUX) (Invitrogen Corporation). Compared to the SYBR green real-time PCR assay, the LUX assay offers the benefit of using a self-quenched primer with a hairpin loop structure, which makes it more specific; that is, it entails less unspecific binding and primer-dimer formation. Furthermore, the fluorophore is attached to the hairpin loop in the LUX setup, and thus, in contrast to the TaqMan assay, this PCR technique does not need an internal probe and is therefore a better choice if broader specificity is required. The LUX assay also has the capacity for melting curve analysis, which offers the possibility of discriminating between PCR products with different base pair compositions (23) and thereby revealing false-positive samples.Ixodes ricinus has been found in 23 of the 25 provinces in Sweden (9), but it is most common in the southern and central parts of the country and along the northeastern coast (14). Various investigators have described the prevalence and diversity of Borrelia in ticks collected in the field in Sweden (4, 8, 9, 14), and to date, five species of these bacteria have been recorded: B. afzelii, B. garinii, B. valaisiana, B. burgdorferi sensu stricto, and also one that is closely related to B. miyamotoi, which is known to be associated with relapsing fever. According to the cited studies, the prevalence of Borrelia spp. in Sweden varies between 3% and 23%. However, detection was not achieved by real-time PCR in those investigations, and thus, no attempts were made to quantify the Borrelia spirochetes in the ticks. To our knowledge, no quantification of Borrelia spirochetes in ticks detached from humans has ever been performed.Our aim was to study the prevalence of Borrelia and to quantify Borrelia cells in ticks that had fed on humans, and we developed a LUX real-time PCR assay for that purpose. In addition, we examined possible geographical differences in prevalence, and we also studied the temporal and spatial distribution of Borrelia species. |
