Utility of Whole Blood Real-Time PCR Testing for the Diagnosis of Early Lyme Disease

George W. Pratt, PhD; Mihae Platt, MD, PhD; Ana Velez, MSc; Lokinendi V. Rao, PhD

Disclosures

Am J Clin Pathol. 2022;158(3):327-330. 

In This Article

Abstract and Introduction

Abstract

Objectives: Whole blood real-time polymerase chain reaction (WB-RTPCR) detection of Borrelia burgdorferi is not currently recommended for diagnosing Lyme disease. This study aims to elucidate the utility of WB-RTPCR as a diagnostic aid for early Lyme disease (ELD), defined as either positive PCR or positive immunoglobulin M with negative immunoglobulin G immunoblot.

Methods: A retrospective analysis was performed on 33,199 blood specimens evaluated concurrently by WB-RTPCR and antibody-capture serology (ACEIA) methods (group A). Fifty-six pairs of specimens from a separate data set were retrospectively identified and analyzed at initial and follow-up time points to monitor for seroconversion (group B). Also, a separate data set of 2,526 specimens concurrently assessed by molecular and modified two-tiered enzyme-linked immunosorbent assay serology methods was analyzed (group C).

Results: Group A yielded 1,379 specimens consistent with ELD when tested by ACEIA and WB-RTPCR. In total, 131 (9.5% of positive results) were identified by WB-RTPCR, with negative serology. Group C identified 358 samples compatible with ELD, with 31 (8.7% of positive results) identified by RTPCR alone.

Conclusions: When used concurrently with serologic testing, WB-RTPCR testing increases diagnostic sensitivity in cases of ELD.

Introduction

Borrelia burgdorferi is the leading causative agent of Lyme disease in the United States. Approximately 35,000 cases are reported to the Centers for Disease Control and Prevention (CDC) each year. Estimates suggest about 476,000 people in the United States are treated for Lyme disease every year.[1,2] The CDC recommends two-tiered serologic testing to diagnose Lyme disease.[3] The first tier is a sensitive enzyme immunoassay (EIA) targeting B burgdorferi–reactive antibodies in patient sera. The second tier is an immunoblot specific for B burgdorferi immunoglobulin M (IgM) or immunoglobulin G (IgG) antibodies. Tier 2 immunoblot can also be replaced with an orthogonal EIA in a modified two-tiered testing (MTTT) algorithm. Two-tiered serologic testing provides 66% to 78% sensitivity and 95% to 99% specificity for Lyme disease diagnosis.[4] Other serologic methods, such as antibody-capture EIAs (ACEIAs), have also been used to test for Lyme disease.[5] The serologic testing methodology does not directly detect the presence of the pathogen, and sensitivity of the assays is lacking during the early stages of Lyme disease when measurable antibody levels are not yet present in sera.

Direct molecular testing for B burgdorferi DNA in whole blood has high specificity and can help diagnose early Lyme disease (ELD) during spirochetemia.[6,7] However, spirochetemia in ELD is a relatively brief event, usually several days, after the initial infection. As a result, whole blood real-time polymerase chain reaction (WB-RTPCR) was found to have low sensitivity in previous studies when considering the overall course of Lyme disease, whereas WB-RTPCR may provide better sensitivity if used during early stages before seroconversion.[8,9]

Concurrent molecular and serologic testing could broaden the diagnostic window for ELD. It is difficult to define very early stages to capture the right window for PCR positivity. Therefore, we explored a simultaneous molecular and serologic testing model for Lyme disease. We examined the results of concurrent WB-RTPCR and serology testing with a laboratory-developed ACEIA or MTTT to detect ELD from a national reference laboratory.

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