Prevalence druhů Borrelia burgdorferi sensu latou pacientů v České republice; přímá sekvenačníanalýza a polymerázová řetězová reakce v reálném čase

Prevalence of Borreliaburgdorferi sensu lato Species among Patients in the Czech Republic; Direct SequencingAnalysis and Real-time Polymerase Chain Reaction

The spread of borreliosis depends on geographical, environmental and climatic factors as well ason the pathogenesis of the causative agent of the group of Borrelia burgdorferi sensu lato. The risein the incidence of the disease and emergence of new symptoms are of concern. Relationshipsbetween genospecies and symptoms, their geographical spread and possible interference of otherpathogens are the subject of the present study. Eighty-seven patients with borreliosis from Centraland Eastern Bohemia and Moravia were enrolled in the study. Forty-nine patients of group 1 showedclinical positivity, 21 patients of group 2 tested positive at PCR screening and 17 patients of group3 were culture positive. Forty-eight patients and 17 isolated strains showed positivity for plasmidsand the Borrelia burgdorferi sensu lato genome in conventional nested PCR. Borrelial genotypesand subtypes were detected by direct sequencing of OspA and OspC products. Quantitative datawere determined from specific product melting temperature curves for real time PCR.Based on sequencing of the OspA gene, B. garinii (subtypes 6, 5, 4 and 3), B. burgdorferi s.s. and B.afzelii were detected in 14 (51.8 %), 8 (29.6 %) and 5 (18.5 %) out of 27 Central Bohemian patients,respectively. Eastern Bohemian patients showed predominance of B. garinii subtype 5 and co-infectionwith Anaplasma phagocytophilum in 7.6 %. The predominant causative agent in 25 Moravianpatients was B. afzelii (11 patients, i.e. 44 %), followed by B. burgdorferi s.s. (9 patients, 36 %) and B.garinii 5 patients, i.e. 20 %). Sequences of two hypervariable regions of the OspA and OspC genesand distances in phylogenetic trees showed differences not only between genospecies and subtypesbut also between wild strains detected by direct sequencing from patient specimens and in vitrocultured strains. The greatest differences were found for patients with long-termborrelial infection.

Key words:
borreliosis – real time PCR – sequencing analysis.