By Richard R. Watkins, MD, MS, FACP, FIDSA, FISAC
Professor of Internal Medicine, Northeast Ohio Medical University, Rootstown, OH
SYNOPSIS: A prospective case-control study from the Netherlands evaluated three cellular tests for diagnosing Lyme disease. All three had low specificity compared to serological testing, leading to an unacceptably high number of false-positive results.
SOURCE: Baarsma ME, van de Schoor FR, Gauw SA, et al. Diagnostic parameters of cellular tests for Lyme borreliosis in Europe (VICTORY study): A case-control study. Lancet Infect Dis 2022;22:1388-1396.
The standard method for diagnosing Lyme disease in clinical practice is by two-tiered serological testing. However, because of ill-defined and long-lasting symptoms, some patients seek out alternative Lyme testing. Tests that focus on the cellular response of the immune system (cellular tests) have been growing in popularity as an alternative to serological testing. These measure the memory T-cell-mediated immune response after ex-vivo stimulation of whole blood or peripheral blood mononuclear cells (PBMCs) with specific pathogenic antigens. An example is interferon-gamma release assays for tuberculosis. Notably, some cellular tests can be performed without a physician’s order. Baarsma and colleagues sought to assess the diagnostic performance of three cellular tests for Lyme disease compared to standard two-tier testing (STTT).
The study was a prospective, case-control analysis that included subjects with confirmed Lyme disease (defined in Table 1) based on healthy controls, and those with potentially cross-reactive conditions (CRCs), including autoimmune disease, syphilis, leptospirosis, or cytomegalovirus. Subjects were adults living in the Netherlands and recruited between May 2018 and March 2020. Antibiotic therapy was allowed for up to seven days before subjects were ruled ineligible to participate. Three cellular assays were assessed: Spirofind Revised (an INFγ release assay), iSpot Lyme (another INFγ release assay), and LTT-MELISA (a lymphocyte transformation test). STTT was used as a comparator. Clinical data were collected at baseline and 12 weeks after study enrollment in patients with Lyme disease, and blood samples were collected at baseline, six weeks, and 12 weeks. Controls had baseline clinical and laboratory assessments only.
Table 1. Definition of Confirmed Lyme Disease1 |
Inclusion criteria:
Exclusion criteria:
|
There were 271 subjects with Lyme disease, 228 healthy controls, and 41 controls with CRCs. Among the cellular tests, the iSpot Lyme had the highest sensitivity at baseline (54.3%; 95% confidence interval [CI], 44.5% to 63.7%), but this dropped to 11.7% (95% CI, 5.5% to 18.6%) when equivocal results were counted as negative. The sensitivity of LTT-MELISA was 30.3% (95% CI, 23.8% to 36.7%), which dropped to 19.3% (95% CI, 14.9% to 25.0%) when equivocal results were counted as negative, and the sensitivity of Spirofind was 43.1% (95% CI, 36.4% to 50.4%). Spirofind does not produce equivocal results by design. STTT had the lowest sensitivity (28.1%, 95% CI, 23.0% to 33.6%), which primarily was due to equivocal results not confirmed by immunoblot. No differences in the sensitivities of the cellular tests were found between patients with erythema migrans, disseminated Lyme, or healthy controls.
Regarding specificity, the STTT was the highest at 94.7% (95% CI, 91.5% to 97.7%). All of the cellular tests had low specificity. Spirofind had a specificity of 80.6% (95% CI, 75.3% to 85.5%), iSpot was 31.1% (95% CI, 21.5% to 40.3%), and LTT-MELISA was 52.6% (95% CI, 44.9% to 60.3%). For controls with CRCs, specificity was similar for STTT (100%, 95% CI, 91.4% to 100%) and Spirofind (94.4%, 95% CI, 86.2% to 100%), but low for LTT-MELISA (57.9%, 95% CI, 42.1% to 72.9%) and iSpot (27.3%, 95% CI, 10.5% to 44.6%).
COMMENTARY
STTT starts with an enzyme-linked assay (EIA) and, if positive, is followed by immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against B. burgdorferi. This approach is fairly sensitive (87%) and very specific (99%) for disseminated Lyme disease.2 The present study by Baarsma et al found a lower sensitivity (28.1%) but a similar specificity (94.7%). The reason for the discrepancy in sensitivities is unclear. It could be related to antibiotic therapy or that 75% of the sera was obtained within three weeks after symptom onset, when serology is known to yield a low sensitivity. However, the chance of a false-positive test is of heightened concern with Lyme disease because cellular tests often are requested for patients with a low pre-test probability. This makes specificity more important in the positive predictive value of the test than sensitivity. Thus, the findings from the study render cellular tests for Lyme disease unfit for clinical use.
Cellular tests have been hypothesized to be tests of cure. Their poor diagnostic parameters found in the present study do not support this approach. The low sensitivities and specificities mean there is a substantial chance that a result at follow-up is either a false-positive or a false-negative, thus precluding any reliable conclusion about whether the infection is cured or still present.
There were a few limitations to the study. Some of the confidence intervals were wide, reflecting a relatively small sample size. Cellular tests are dependent on functional immune cells, which make them more susceptible to non-valid results compared to STTT. Finally, patients who get cellular tests typically have longer lasting symptoms, which was not taken into account in the present study.
The study by Baarsma and colleagues is an important addition to the literature on the diagnosis of Lyme disease. It likely will play a role in future versions of Lyme disease guidelines and inform further research on diagnosing this common yet vexing infection. Cellular tests are interesting and may play a role in the future for diagnosing Lyme disease but are not recommended for clinical use at the present time.
REFERENCES
- van de Schoor FR, Baarsma ME, Gauw SA, et al. Validation of cellular tests for Lyme borreliosis (VICTORY) study. BMC Infect Dis 2019;19:732.
- Hinckley AF, Connally NP, Meek JI, et al. Lyme disease testing by large commercial laboratories in the United States. Clin Infect Dis 2014;59:676-681.
A prospective case-control study from the Netherlands evaluated three cellular tests for diagnosing Lyme disease. All three had low specificity compared to serological testing, leading to an unacceptably high number of false-positive results.
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