Molecular detection of Borrelia burgdorferi sensu lato – An analytical comparison of real-time PCR protocols from five different Scandinavian laboratories

Abstract

IntroductionLyme borreliosis (LB) is the most common tick transmitted disease in Europe. The diagnosis of LB today is based on the patient´s medical history, clinical presentation and laboratory findings. The laboratory diagnostics are mainly based on antibody detection, but in certain conditions molecular detection by polymerase chain reaction (PCR) may serve as a complement.AimThe purpose of this study was to evaluate the analytical sensitivity, analytical specificity and concordance of eight different real-time PCR methods at five laboratories in Sweden, Norway and Denmark.MethodEach participating laboratory was asked to analyse three different sets of samples (reference panels; all blinded) i) cDNA extracted and transcribed from water spiked with cultured Borrelia strains, ii) cerebrospinal fluid spiked with cultured Borrelia strains, and iii) DNA dilution series extracted from cultured Borrelia and relapsing fever strains. The results and the method descriptions of each laboratory were systematically evaluated.Results and conclusionsThe analytical sensitivities and the concordance between the eight protocols were in general high. The concordance was especially high between the protocols using 16S rRNA as the target gene, however, this concordance was mainly related to cDNA as the type of template. When comparing cDNA and DNA as the type of template the analytical sensitivity was in general higher for the protocols using DNA as template regardless of the use of target gene. The analytical specificity for all eight protocols was high. However, some protocols were not able to detect Borrelia spielmanii, Borrelia lusitaniae or Borrelia japonica.

Direct Molecular Detection and Genotyping of Borrelia burgdorferi Sensu Lato in Cerebrospinal Fluid of Children with Lyme Neuroborreliosis

“…All samples were analyzed by two independent PCR assays, and no sample was classified as positive for B. burgdorferi sensu lato DNA based on a single PCR. We consider the risk of false-positive results with these methods to be low, in accordance with the previous validation of these assays (26,27).…”

“…The following thermocycling parameters were used on a LightCycler 480 system: 2 min at 40°C followed by 10 min at 95°C and 47 cycles of 15 s at 95°C, 30 s at 60°C, and 20 s at 72°C. The ospA and 16S rRNA gene real-time assays used in this study have been validated against several panels of Borrelia strains in order to document the ability to detect strains relevant to human disease (26). The specificity of the assays has been challenged by testing DNA extracted from Ͼ130 CSF samples from adult non-LB controls (27) without generating any false-positive results.…”

Molecular and serological tools for clinical diagnostics of Lyme borreliosis - can the laboratory analysis be improved?

“…The ospA gene is also a commonly used target for PCRanalysis. However, previous studies have shown that protocols for detecting ospA are unable to detect the species B. spielmanii and B. miyamotoi, both of which are known to be pathogenic to humans 189 .…”

“…Assays based in flagellin should therefore preferably be used as a recombinant truncated internal fragment 22,50 . The flaB gene is also used as a target in PCRanalysis and has been proven to detect all pathogenic strains 189 .…”

“…(2017) 189 showed that an increase in analytical sensitivity is correlated to the use of different target genes. For example, for increased analytical sensitivity in samples extracted for total NA The conclusion of paper IV is presented in the fact box in Figure 21.…”