Molecular Microbiological and Immune Characterization of a Cohort of Patients Diagnosed with Early Lyme Disease

Abstract

Lyme disease is a tick-borne infection caused by the bacteria Borrelia burgdorferi. Current diagnosis of early Lyme disease relies heavily on clinical criteria, including the presence of an erythema migrans rash. The sensitivity of current gold-standard diagnostic tests relies upon antibody formation, which is typically delayed and thus of limited utility in early infection. We conducted a study of blood and skin biopsy specimens from 57 patients with a clinical diagnosis of erythema migrans. Samples collected at the time of diagnosis were analyzed using an ultra-sensitive, PCR-based assay employing an isothermal amplification step and multiple primers. In 75.4% of patients, we directly detected one or more B. burgdorferi genotypes in the skin. Two-tier testing showed that 20 (46.5%) of those found to be PCR positive remained serologically negative at both acute and convalescent time points. Multiple genotypes were found in 3 (8%) of those where a specific genotype could be identified. The 13 participants who lacked PCR and serologic evidence for exposure to B. burgdorferi could be differentiated as a group from PCR positive participants by their levels of several immune markers as well as by clinical descriptors such as their number of acute symptoms and the pattern of their erythema migrans rash. These results suggest that within a Mid-Atlantic cohort, patient subgroups can be identified using PCR-based direct detection approaches. This may be particularly useful in future research such as vaccine trials and public health surveillance of tick-borne disease patterns.

Predicting Lyme Disease From Patients' Peripheral Blood Mononuclear Cells Profiled With RNA-Sequencing

“…Direct evidence of infection was obtained using a sensitive PCR/ESI-MS approach that detected Borrelia burgdorferi in the skin or blood of most of our patients. Interestingly, there was a subgroup of cases that were PCR/ESI-MS negative and who failed to seroconvert on standard twotier antibody tests [ Figure 2, as previously identified in (13)]. Remarkably, these patients generally cluster within the other cases, suggesting that these patients may have LD, but current assays are not capable of detecting the presence of Borrelia in their skin or blood.…”

Immunosequencing of the T-cell receptor repertoire reveals signatures specific for diagnosis and characterization of early Lyme disease

“…This observation, as well as the increased sensitivity of the assay over STTT, suggests that TCR testing may be able to identify Lyme disease even in the absence of acute (IgM) to convalescent (IgG) seroconversion, a common occurrence among individuals treated early in the course of disease (33), and supports the role of T-cell-based testing as both an alternative and complementary method for diagnosis. Importantly, 11 of 12 individuals who presented with EM rash, but were both STTT-negative and B. burgdorferi PCR-negative, were also TCRnegative, calling into question the diagnosis of Lyme disease in these individuals and suggesting that T-cell-based testing may aid in the differential diagnosis of Lyme disease and similar tickborne illnesses with overlapping manifestations, such as STARI (48). persistent T-cell responses in ongoing disease (55,56).…”

“…2A). Of note, PCR testing results for blood and/or skin biopsy were available for a subgroup of 57 individuals in the JHU cohort; of those, 12 individuals tested negative by both STTT and PCR, raising the possibility that these individuals had a non-Lyme tick-borne illness, such as Southern tick-associated rash illness (STARI) (48). The majority (11/12) of these individuals were TCRnegative; excluding them from the analysis did not appreciably alter performance characteristics (sensitivities of 59% versus 32% for TCR assay and STTT, respectively).…”

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.…”