Development and evaluation of a two-step multiplex TaqMan real-time PCR assay for detection/quantification of different genospecies of Borrelia burgdorferi sensu lato

Nowadays, at least four clinically important B. burgdorferi sensu lato (s.l.) genospecies (B. afzelii, B. garinii, B. burgdorferi sensu stricto (s.s.) and B. lusitaniae) circulate in Portugal. Each genospecies has a different tropism that resuls in a diverse array of clinical manifestations. The standard diagnostic procedure used is normally simple, nevertheless, during the "window-period" phase, in which specific antibodies cannot yet be detected, diagnosis becomes difficult, and calls for reliable, sensitive and specific laboratory methods, such as molecular tests. The aim of this study was to develop and evaluate a multiplex TaqMan real-time PCR assay to infer the presence of B. burgdorferi s.l. genospecies in clinical and vector-derived samples. The assay consists of two steps: (i) a first duplex real-time PCR targeting both flaB of B. burgdorferi s.l., and an internal control (18S rDNA for tick samples or the mammal β-actin gene for clinical samples); and (ii) a second tetraplex real-time PCR targeting the flaB gene of B. afzelii, B. garinii, B. burgdorferi s.s. and B. lusitaniae. The first step revealed a high specificity and sensitivity, allowing the detection of as low as 20 genome equivalents (GE) of B. burgdorferi s.l. from isolated cultures, clinical samples and ticks. The second step revealed high specificity, but a slightly lower sensitivity (2×102 GE) for detection of B. afzelii, B. garinii, B. burgdorferi s.s. and B. lusitaniae in purified DNA extracts, and particularly when testing cerebrospinal fluid (CSF) samples. Nonetheless, both real-time PCR protocols were developed to be applied at the beginning of the infection, to improve early diagnosis of Lyme borreliosis (LB), where detection of Borrelia should not rely on the use of CSF samples. The assay here described is of special interest for the analysis of both environmental and clinical samples, being advantageous in the former phase screening of Lyme borreliosis, when the efficiency of serologically based diagnoses may be seriously compromised.