Different interpretations of inconclusive results of SARS-CoV-2 real-time RT PCR.

We investigated whether inconclusive results could be interpreted differently depending on the situation. First, data from retesting of the initial samples from subjects without a confirmed COVID-19 history were analyzed. And by analyzing the results of consecutive tests with new specimens after receiving inconclusive results between arrivals and locals for 2 periods. As a result, 179 of 219 cases (81.7%) showed still inconclusive or weakly positive results. If contamination is well controlled in a general laboratory, the effectiveness of retesting with the same sample is limited. The rate of the subsequently positive patient was significantly higher in locals than in arrivals and periods with a higher positive rate. The inconclusive results could be interpreted differently depending on the epidemiologic background and the positive rate at that time.

Borderline SARS-CoV-2 patients: the trace behind.

BACKGROUND: Effective and precise SARS-CoV-2 detection assays are crucial for maintaining regular hospital routines and identifying infected hospital employees and infected patients before hospital admission. Inconclusive PCR test results of potentially infectious borderline SARS-CoV-2 patients can confuse clinicians and delay appropriate infection control. OBJECTIVES AND STUDY DESIGN: In this retrospective study, we followed up borderline SARS-CoV-2 patients who were tested (from the second sample with the same method) at the Clinical Department of Clinical Microbiology. We aimed to determine the positivity conversion ratio within 7 days after inconclusive PCR test results. RESULTS: Out of 247 borderline patients, who were resampled and retested in the same laboratory, 60 patients (29.4%) showed conversion of the borderline viral load (inconclusive RT-PCR test) to a positive RT-PCR test result. CONCLUSIONS: Our results highlight the need for retesting of borderline patients with inconclusive SARS-CoV-2 results. Follow-up testing of inconclusive PCR results within 7 days can identify additional positive results and reduce the potential risk of intrahospital transmission.

Follow-up COVID-19 PCR result up to day 5 with clinical features predicts positivity for inconclusive results.

Introduction: False-positive inconclusive polymerase chain reaction (PCR) results against severe acute respiratory syndrome coronavirus 2 were not low and have potentially harmful effects. We aimed to find parameters to differentiate positive cases from false-positive ones, and suggest an optimal scheme and follow-up period for inconclusive results. Methods: Cases with inconclusive PCR tests among healthcare personnel from February 2020 to June 2021 were classified as confirmed positive, clinically positive, and clinically negative groups, which were compared. The diagnostic accuracy of follow-up tests and composites of clinical and laboratory data were analyzed. Results: Symptoms, contact history, and lower cycle threshold of the N gene were more common in the COVID-19 positive group. The scoring schemes combining symptom and contact history with follow-up PCR results had higher sensitivities than the PCR tests only modality. Follow-up tests up to 5 days combined with symptoms and contact history could discriminate between positive and negative cases. Conclusion: A follow-up PCR test up to day 5 with clinical features might predict positivity and shorten the quarantine period in most healthcare personnel.

Optimal decision theory for diagnostic testing: Minimizing indeterminate classes with applications to saliva-based SARS-CoV-2 antibody assays.

In diagnostic testing, establishing an indeterminate class is an effective way to identify samples that cannot be accurately classified. However, such approaches also make testing less efficient and must be balanced against overall assay performance. We address this problem by reformulating data classification in terms of a constrained optimization problem that (i) minimizes the probability of labeling samples as indeterminate while (ii) ensuring that the remaining ones are classified with an average target accuracy X. We show that the solution to this problem is expressed in terms of a bathtub-type principle that holds out those samples with the lowest local accuracy up to an X-dependent threshold. To illustrate the usefulness of this analysis, we apply it to a multiplex, saliva-based SARS-CoV-2 antibody assay and demonstrate up to a 30 % reduction in the number of indeterminate samples relative to more traditional approaches.

Incidence of COVID-19 infection and its variation with demographic and clinical profile: lessons learned at a COVID-19 RT-PCR laboratory in Nagpur, India.

Introduction. The coronavirus disease 2019 (COVID-19) pandemic emerged as a global health crisis in 2020. The first case in India was reported on 30 January 2020 and the disease spread throughout the country within months. Old persons, immunocompromised patients and persons with co-morbidities, especially of the respiratory system, have a more severe and often fatal outcome to the disease. In this study we have analysed the socio-demographic trend of the COVID-19 outbreak in Nagpur and adjoining districts. Methods. The study was conducted from April to December 2020. Nasopharyngeal and oropharyngeal swabs collected from suspected cases of COVID-19 were tested using reverse-transcription polymerase chain reaction (RT-PCR) at a diagnostic molecular laboratory at a tertiary care hospital in central India. Patient-related data on demographic profile and indication for testing were obtained from laboratory requisition forms. The results of the inconclusive repeat samples were also noted. The data were analysed using SPSS v24.0. Results. A total of 46 898 samples were received from April to December 2020, of which 41 410 were included in the study; 90.6 % of samples belonged to adults and 9.4 % belonged to children. The overall positivity rate in the samples was 19.3 %, although it varied over the period. The yield was significantly high in the elderly age group (25.5 %) and symptomatic patients (22.6 %). On repeat testing of patients whose first test was inconclusive, 17.1% were positive. There was a steady increase of both the number of tests and the rate of positivity in the initial period of the study, followed by a sharp decline. Conclusion. We can conclude that rigorous contact tracing and COVID-appropriate behaviour (wearing a mask, social distancing and hand hygiene) are required to break the chain of transmission. Elderly people are more susceptible to infection and should follow stringent precautions. It is also important to perform repeat testing of those individuals whose tests are inconclusive with fresh samples so that no positive cases are missed. Understanding of demographics is crucial for better management of this crisis and proper allocation of resources.

Clinical and epidemiologic characteristics of inconclusive results in SARS-CoV-2 RT-PCR assays.

BACKGROUND: Inconclusive results in SARS-CoV-2 molecular assays cause confusion among clinicians and delay appropriate infection prevention and control. In this study, we aimed to characterize the respiratory specimens associated with inconclusive SARS-CoV-2 molecular assay results. METHODS: We re-evaluated inconclusive specimens by 3 additional RT-PCR assays and attempted to detect subgenomic RNA (sgRNA) in these specimens. RESULTS: Among follow-up tests from confirmed SARS-CoV-2 cases, 36.3% of the inconclusive results were classified as presumptive positive results (45/124). However, none of the specimens from 36 screening cases was classified as a presumptive positive result. Among 160 inconclusive specimens, sgRNAs were detected in 78 samples (48.8%): 58 were confirmed cases (58/124, 46.8%) and 20 were screening cases (20/36, 55.6%). CONCLUSIONS: The results of our study suggest the recommendation of considering inconclusive results as positive results for confirmed SARS-CoV-2 cases. In screening cases, viral remnants could be partially amplified in PCR assays, and these inconclusive results could be related to previous infections. In addition, sgRNAs were detected in about half of the inconclusive specimens; however, the clinical significance of sgRNA is not yet clear.

A novel ORF1a-based SARS-CoV-2 RT-PCR assay to resolve inconclusive samples.

BACKGROUND: India bears the second largest burden of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. A multitude of reverse transcription polymerase chain reaction (RT-PCR) detection assays with disparate gene targets, including automated high-throughput platforms, are available. Varying concordance and interpretation of diagnostic results in this setting can result in significant reporting delays, leading to suboptimal disease management. This article reports the development of a novel ORF1a-based SARS-CoV-2 RT-PCR assay - Viroselect - that shows high concordance with conventional assays and the ability to resolve inconclusive results generated during the peak of the epidemic in Mumbai, India. METHODS: A unique target region within SARS-CoV-2 ORF1a - the non-structural protein 3 (nsp3) region - was used to design and develop the assay. This hypervariable region (1923-3956) between SARS-CoV-2, SARS-CoV-1 and Middle East respiratory syndrome coronavirus was utilized to design the primers and probes for the RT-PCR assay. The concordance of this assay with commonly used emergency use authorization (US Food and Drug Administration) manual kits and an automated high-throughput testing platform was evaluated. Further, a retrospective analysis was carried out using Viroselect on samples reported as 'inconclusive' between April and October 2020. RESULTS: In total, 701 samples were tested. Concordance analysis of 477 samples demonstrated high overall agreement of Viroselect with both manual (87.6%) and automated (84.7%) assays. Also, in the retrospective analysis of 224 additional samples reported as 'inconclusive', Viroselect was able to resolve 100% (19/19) and 93.7% (192/205) of samples which had inconclusive results on manual and automated high-throughput platforms, respectively. CONCLUSION: Viroselect had high concordance with conventional assays, both manual and automated, and has potential to resolve inconclusive samples.

Clinical and Epidemiologic Evaluation of Inconclusive COVID-19 PCR Results Using a Quantitative Algorithm.

OBJECTIVES: The inconclusive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR) result causes confusion and delay for infection prevention precautions and patient management. We aimed to develop a quantitative algorithm to assess and interpret these inconclusive results. METHODS: We created a score-based algorithm by combining laboratory, clinical, and epidemiologic data to evaluate 69 cases with inconclusive coronavirus disease 2019 (COVID-19) PCR results from the Centers for Disease Control and Prevention (CDC) assay (18 cases) and the TaqPath assay (51 cases). RESULTS: We determined 5 (28%) of 18 (CDC assay) and 20 (39%) of 51 (TaqPath assay) cases to be false positive. Lowering the cycle threshold cutoff from 40 to 37 in the TaqPath assay resulted in a dramatic reduction of the false-positive rate to 14%. We also showed testing of asymptomatic individuals is associated with a significantly higher probability of having a false-positive result. CONCLUSIONS: A substantial percentage of inconclusive SARS-CoV-2 PCR results can be false positive, especially among asymptomatic patients. The quantitative algorithm we created was shown to be effective and could provide a useful tool for clinicians and hospital epidemiologists to interpret inconclusive COVID-19 PCR results and provide clinical guidance when additional PCR or antibody test results are available.