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.

Lyme disease patients would benefit greatly 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. This results in nondetection of many genuine cases on a timely basis, preventing complete cure. In this study we have developed a digital PCR (polymerase chain reaction) assay that detects Lyme disease on clinical presentation at twice the sensitivity 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 and the lower limit of detection of this diagnostic assay was found to be three genome copies of B. burgdorferi. The paucity of spirochetes in the bloodstream of Lyme disease patients that hinders the clinical adoption of PCR-based diagnostic tests, was overcome by using a comparatively larger sample volume, pre-analytical processing of blood samples and a pre-amplification step to enrich for B. burgdorferi-specific gene targets before using the digital PCR technology to analyze patient samples. Pre-analytical processing of blood samples from acute patients revealed that the best sample type for Lyme disease detection is platelet-rich plasma and not whole blood. If detected on time, Lyme disease can be cured completely limiting the overuse of antibiotics and associated morbidities.

The world is currently facing an unprecedented pandemic caused by the novel coronavirus SARS-CoV-2 (COVID-19) which was first reported in late 2019 by China to the World Health Organization (WHO). The containment strategy for COVID-19, which has non-specific flu-like symptoms and where upwards of 80% of the affected has either mild or no symptoms, is critically centered upon diagnostic testing, tracking and isolation. Thus, the development of specific and sensitive diagnostic tests for COVID-19 is key towards the first successful step of disease management. Public health organizations like the WHO and the US-based Centers for Disease Control and Prevention (CDC) have developed real-time PCR (RT-PCR) based diagnostic tests to aid in the detection of acute infection. In this study we sought to modify the CDC RT-PCR diagnostic assay protocol to increase its sensitivity and to make the assay directly portable to health care providers in a community-based hospital setting. A number of modifications to the original protocol were tested. Increasing the RT-PCR annealing temperature by 7°C to 62°C was associated with the most significant improvement in sensitivity, wherein the cycle-threshold (Ct) value for the N2 assay was reduced by ~3 units, in effect both reducing the overall number of inconclusive results and yielding N1/N2 assays to have similar Ct values. The limit of detection of the modified assay was also improved (0.86 RNA copies/μl for both nCoV 2019_N1/N2 assays) compared to the CDC RT-PCR diagnostic assay (1 and 3.16 RNA copies/μl for nCoV 2019_N1 and N2 assay, respectively). Using this modification, there was no significant effect on SARS-CoV-2 detection rate when viral RNA extraction was performed either manually or through an automated extraction method. We believe this modified protocol allows for more sensitive detection of the virus which in turn will be useful for pandemic management.