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Two-tier testing no better than a flip of a coin: Molecular Microbiological and Immune Characterization of a Cohort of Patients Diagnosed with Early Lyme Disease


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 ultrasensitive,
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 re-
mained serologically negative at both acute and convalescent time points. Multiple

genotypes were found in three (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 the
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 identi-
fied 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 dis-
ease patterns.

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