Why do some PCR-negative patients test positive on COVID-19 blood tests?

Serological assays for the coronavirus disease 2019 (COVID-19) sometimes show contradictory findings when compared with nasopharyngeal swab polymerase chain reaction (PCR) tests, according to a recent Singapore study. In particular, even PCR-negative patients with no known exposure could test positive for antibodies.

“We need to understand the possibility of cross reactivity to other as yet unknown viruses, possibly immune activation from other infections which could explain consistently positive results on epitope assays, and be mindful of the use of serological testing in settings where the prevalence of COVID-19 is low,” the researchers said.

Fifty-one patients (median age 54 years, 62.7 percent men) hospitalized for acute respiratory illnesses, all of whom were PCR-negative for SARS-CoV-2, participated in the study. Four assays were used for serological assessment: cPass, Elecsys, S-protein flow-based (SFB), and epitope-based enzyme-linked immunosorbent assay (ELISA).

Patients presented to the hospital after a median of 2–3 days after symptom onset and majority had either fever (86.3 percent) or cough (70.6 percent) at presentation. Twenty-four percent hand pneumonia on chest X-ray, while 28 percent had lymphopaenia. More than half had normal X-ray findings. [J Med Virol 2022;doi:10.1002/jmv.27656]

None of the patients had any proven or suspected contact with confirmed COVID-19 cases. Moreover, none were part of any confirmed clusters in Singapore or came in close contact with persons under quarantine. While majority (80 percent) were in the country at the time of the first SARS epidemic, none reported having had the disease or even a history of the middle-east respiratory syndrome.

Despite such history and negative PCR tests, the B-cell epitope assay found five patients (9.8 percent) positive for SARS-CoV-2-specific antibodies, particularly recognizing epitopes on the spike and nucleocapsid proteins. One participant was positive for two epitopes. Similarly, the SFB assay found three participants with elevated immunoglobulin G1 levels against the full-length spike proteins. No patient tested positive on Elecsys and cPass.

Though none were healthcare workers, 62.5 percent of patients who were serology-positive for SARS-CoV-2 worked in occupations deemed high-risk for infection, a proportion significantly higher than that in serology-negative comparators (p=0.039). Bacterial infection (p=0.028) and neutrophilia (p=0.013) during acute hospitalization were likewise significantly more common among serology-positive patients.

“It is plausible that the patients in our cohort had polyclonal B-cell activation [in response to bacterial infection] resulting in the production of antibodies that had some have cross-reactivity to SARS-CoV-2 virus, which was seen in some of the assays used,” the researchers explained.

Another reason for the findings could be the higher risk of assay false positivity due to low COVID-19 prevalence in the community.

“The positive predictive value (PPV) for the B-cell Epitope and SFB assays is low for the low-risk community cases enrolled into our study, but the PPV would have been higher if used amongst high-risk groups such as in a migrant worker dormitory,” the researchers said.