Development of a sensitive molecular diagnostic assay for detecting Borrelia burgdorferi DNA from the blood of Lyme disease patients by digital PCR

Abstract

Lyme disease patients would greatly benefit from a timely, sensitive, and specific molecular diagnostic test that can detect the causal agent Borrelia burgdorferi at the onset of symptoms. Currently available diagnostic methods recommended by the Centers for Disease Control and Prevention for Lyme disease involve indirect serological tests that rely on the detection of a host-antibody response, which often takes more than three weeks to develop. With this process, many positive cases are not detected within a timely manner, preventing a complete cure. In this study, we have developed a digital polymerase chain reaction (PCR) assay that detects Lyme disease on clinical presentation with a sensitivity two-fold higher than that of the currently available diagnostic methods, using a cohort of patient samples collected from the Lyme disease endemic state of Connecticut, USA, in 2016–2018. Digital PCR technology was chosen as it is more advanced and sensitive than other PCR techniques in detecting rare targets. The analytical detection sensitivity of this diagnostic assay is approximately three genome copies of B. burgdorferi. The paucity of spirochetes in the bloodstream of Lyme disease patients has hindered the clinical adoption of PCR-based diagnostic tests. However, this drawback was overcome by using a comparatively larger sample volume, applying pre-analytical processing to the blood samples, and implementing a pre-amplification step to enrich for B. burgdorferi-specific gene targets before the patient samples are analyzed via digital PCR technology. Pre-analytical processing of blood samples from acute patients revealed that the best sample type for Lyme disease detection is platelet-rich plasma rather than whole blood. If detected in a timely manner, Lyme disease can be completely cured, thus limiting antibiotic overuse and associated morbidities.

Capture Sequencing Enables Sensitive Detection of Tick-Borne Agents in Human Blood

“…Direct molecular tests can detect infection earlier than serologic tests, but their clinical utility for B. burgdorferi is limited by low sensitivity in blood, the most frequently tested specimen for TBD samples ( Moore et al, 2016 ). The development of alternative molecular approaches, such as multi-locus PCR and electrospray ionization mass spectrometry (PCR/ESI-MS), and digital PCR, have led to improvements in sensitivity ( Eshoo et al, 2012 ; Das et al, 2020 ; Mosel et al, 2020 ). In previous work, we demonstrated the improvement in sensitivity and genomic analyses of tick-borne agents that can be achieved using TBDCapSeq.…”

“…Our tests were performed with extracts from only 400 μl of blood. Achieving higher sensitivity often requires testing of larger blood volumes (1–20 ml) because of the low spirochete burden present in the blood of infected patients ( Das et al, 2020 ; Mosel et al, 2020 ). In addition, sensitivity may also be influenced on the blood fraction tested ( Das et al, 2020 ).…”

“…Achieving higher sensitivity often requires testing of larger blood volumes (1–20 ml) because of the low spirochete burden present in the blood of infected patients ( Das et al, 2020 ; Mosel et al, 2020 ). In addition, sensitivity may also be influenced on the blood fraction tested ( Das et al, 2020 ). Finally, our pooling strategy for sequencing resulted in reduced read allocation for each sample.…”

Development of a capture sequencing assay for enhanced detection and genotyping of tick-borne pathogens

“…For example, increasing the sample volume may partially offset the paucity of spirochetes in liquid specimens and enhance detection. In our experiments, we used nucleic acids extracted from only 200 μl of whole blood, in contrast to other molecular studies of B. burgdorferi s.s. that typically employ much greater sample volumes for spirochete detection (20 ml of whole blood or 1 ml of platelet-rich plasma) 44 , 46 . Increasing sequencing depth may also result in greater yield of pathogen-specific reads.…”

Diagnosis of Lyme Borreliosis With a Novel, Seminested Real-Time Polymerase Chain Reaction Targeting the 5S-23S Intergenic Spacer Region: Clinical Features, Histopathology, and Immunophenotype in 44 Patients

“…In the past few years, several studies provided new or modified PCR assays for detection of Borrelia. [21][22][23][24][25][26][27] A fragment of the groEl gene, which encodes a highly conserved 60 kDa heat shock protein of B. burgdorferi s.l., was used for detection in ticks. 21 Others focused on identification of Borrelia in blood samples, for example, by way of a quantitative real-time PCR targeting ribosomal genes (16SrRNA), which identified Borrelia infections while IgM serology was still negative 22 or by using specific DNA hybridization to purify B. burgdorferi DNA from blood followed by PCR amplification of flagellin and OspA gene fragments.…”