Molecular Detection of Borrelia Bacteria in Cerebrospinal Fluid-Optimisation of Pre-Analytical Sample Handling for Increased Analytical Sensitivity.

Malin Lager,Per-Eric Lindgren,Anna J Henningsson,Peter Wilhelmsson,Andreas Matussek

doi:10.3390/diagnostics11112088

Abstract

The main tools for clinical diagnostics of Lyme neuroborreliosis (LNB) are based on serology, i.e., detection of antibodies in cerebrospinal fluid (CSF). In some cases, PCR may be used as a supplement, e.g., on CSF from patients with early LNB. Standardisation of the molecular methods and systematic evaluation of the pre-analytical handling is lacking. To increase the analytical sensitivity for detection of Borrelia bacteria in CSF by PCR targeting the 16S rRNA gene, parameters were systematically evaluated on CSF samples spiked with a known amount of cultured Borrelia bacteria. The results showed that the parameters such as centrifugation time and speed, the use of complementary DNA as a template (in combination with primers and a probe aiming at target gene 16S rRNA), and the absence of inhibitors (e.g., erythrocytes) had the highest impact on the analytical sensitivity. Based on these results, a protocol for optimised handling of CSF samples before molecular analysis was proposed. However, no clinical evaluation of the proposed protocol has been done so far, and further investigations of the diagnostic sensitivity need to be performed on well-characterised clinical samples from patients with LNB.

Highlights

Lyme neuroborreliosis (LNB) is a disease caused by tick-borne Borrelia bacteria and constitutes about 3–12% of all borreliosis cases in Europe and in the USA [1]
(2017) [16], we have shown that the analytical sensitivities, specificities, and concordance among eight different PCR protocols used in laboratories in Scandinavia are high
The results showed that the parameters with the highest impact on the analytical sensitivity were the centrifugation time and speed, the use of complementary DNA (cDNA) as template, and the absence of erythro‐

Summary

Introduction

Lyme neuroborreliosis (LNB) is a disease caused by tick-borne Borrelia bacteria and constitutes about 3–12% of all borreliosis cases in Europe and in the USA [1]. The most common clinical manifestations of LNB in Europe are lymphocytic meningitis, facial palsy, and radicular pain (Bannwarth’s syndrome). Diagnosis of LNB is based on the patients medical history and clinical signs and symptoms together with leucocytosis in the cerebrospinal fluid (CSF). An elevated anti-Borrelia antibody index as an indication of intrathecal production of specific antibodies [2]. The B. miyamotoi bacteria can be detected in both serum and CSF by serological or molecular analysis [3,4]. B. miyamotoi infection is rarely detected in serological assays used for detection of Borrelia burgdorferi sensu lato (s.l.), and specific serological tests are not commercially available

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