Sequential analysis and real-time LightCycler polymerase chain reaction in the identification of Borrelia and Ehrlichia spp
Abstract
Purpose of the study: To differentiate the genotypes and subtypes of Borrelia burgdorferi sensu lato and Ehrlichia spp. in cultures, in samples of patients' cerebrospinal fluid and blood and in animal tissues. The study focuses on southern and eastern Bohemia and on Moravia.
Material: The investigation involved 16 borrelia strains isolated from the blood and CSF of patients and further 49 samples of CSF and blood of borreliosis patients, tissues from 80 pieces of game animals and blood from 55 cows. Methods: The differentiation and quantification of the two pathogenes was done using real-time LightCycler PCR with the primers 16S rRNA, OpA, recA genes and direct PCR sequential analysis of the products.
Results: We demonstrated 11 strains of Borrelia garinii, OspA types 5, 6, 4 and 5 strains of B. afzelii. B. burgdorferi sensu stricto was not demonstrated in the cultures.
The sequential analysis PCR of the products from 49 CSF samples demonstrated B. burgdorferi s.s. in 20.4 %, B. afzelii in 12.2 %, B. garnii subtypes 6, 5, 4 and 3 in 61.3 % - out of these 8.2 % of the samples contained combinations similar to OspA types. Co-infection with Ehrlichia phagocytophila was found in 6.1 % of samples.
E. phagocytophila subtype Frankonia I and II was demonstrated in 12.5 % of game animals and in 9 % of cattle. B. burgdorferi sensu stricto and B. garinii were identified in 17.5 % of game.
Real-time LightCycler PCR was less sensitive in patients' fluids than in cultures and animal tissues. Conclusions: We demonstrated that all three genotypes of B. burgdorferi sensu lato are capable of causing neuroborreliosis.
The course of the infection and its symptoms may be affected by various OspA subtypes.