Development of a validated molecular analytical method to determine the viral safety of F(AB´)2 products: A novel application for a well-known technique.

The immunotherapy agents derived from horses are biological products that allow the neutralization of clinically relevant immunogens, such as the SARS-CoV-2 virus that causes COVID-19, or the neutralization of toxins present in the venoms of snakes, spiders, and other poisonous animals. Due to their importance, detecting adventitious viruses in equine hyperimmune serum (raw material in industrial processes) is a critical step to support the safety of products for human use, and, in consequence, it is a requirement for commercialization and distribution. The safety of the finished product is based on three complementary approaches: (i) testing of the source material (horse serum) donations, (ii) release of the starting material (i.e., pool of horse serum) based on non-reactivity for a range of human infectious or pathogenic viruses, and (iii) validate (selected) steps of the manufacturing process for their capacity to inactivate and/or remove a wide range of viruses potentially present in the starting material. Orthogonal approaches to reduce viral contamination risk include implementing a reliable and validated system for detecting adventitious viruses. Thus, it is necessary to establish trustworthy and sufficiently sensitive analytical methods to evidence the lack of viruses to assure the safety of the therapeutic product. Therefore, in this research, an analytical method based on end-point Reverse Transcription Polymerase Chain Reaction (RT-PCR) was developed, implemented, and validated in hyperimmune equine serum samples to detect Venezuelan equine encephalitis virus, West Nile virus, and Rabies virus.

Authorized SARS-CoV-2 molecular methods show wide variability in the limit of detection.

On February 29th, 2020, the U.S. Food and Drug Administration issued the first Emergency Use Authorization (EUA) for a SARS-CoV-2 assay outside of the U.S. Centers for Disease Control and Prevention. As of May 3rd, 2021, 289 total EUAs have been granted. Like influenza, there is no standard for defining limit of detection (LoD), but rather guidance that analytical sensitivity/LoD be established as the level that gives a 95% detection rate in at least 20 replicates. Here we compare the performance characteristics of SARS-CoV-2 tests receiving EUA by standardizing sensitivity to a common unit of measure and assess the variability in LoD between tests. Additionally, we looked at factors that may impact sensitivities due to lack of standardization of the test development process and compare results for a standardized reference panel for comparative analysis within a subset of EUA tests offered by the U.S. Food and Drug Administration.

Clinical Diagnostic Point-of-Care Molecular Assays for SARS-CoV-2.

Laboratories faced many challenges throughout the COVID-19 pandemic. Point-of-care (POC) SARS-CoV-2 nucleic acid amplification tests (NAATs) provided a key solution to the need for rapid turnaround time in select patient populations and were implemented at the POC but also within laboratories to supplement traditional molecular assays. Clinical Laboratory Improvement Amendments-waived rapid POC SARS-CoV-2 NAATs offer the benefit of reduced educational requirements for operators and can be performed by non-laboratory-trained individuals. However, these methods must be validated to ensure the manufacturer's performance specifications are met and they are found to be fit-for-purpose in the clinical workflows they are implemented.

Exploring beyond the limit: How comparative stochastic performance affects retesting outcomes in six commercial SARS CoV-2 nucleic acid amplification tests.

Objectives: To examine the comparative stochasticity profile of six commercial SARS-CoV-2 nucleic acid amplification tests (NAATs) and how this may affect retesting paradigms. Methods: Commercial quality control (QC) material was serially diluted in viral transport media to create a panel covering 10-10,000 copies/ml. The panel was tested across six commercial NAATs. A subset of high cycle threshold results was retested on a rapid PCR assay to simulate retesting protocols commonly used to discriminate false positives. Results: Performance beyond the LOD differed among assays, with three types of stochasticity profiles observed. The ability of the rapid PCR assay to reproduce a true weak positive specimen was restricted to its own stochastic performance at the corresponding viral concentration. Conclusion: Stochastic performance of various NAATs overlap across low viral concentrations and affect retesting outcomes. Relying on retesting alone to discriminate false positives risk missing true positives even when a more sensitive assay is deployed for confirmatory testing.

The PRONTO study: Clinical performance of ID NOW in individuals with compatible SARS-CoV-2 symptoms in walk-in centres-accelerated turnaround time for contact tracing.

BACKGROUND: This PRONTO study investigated the clinical performance of the Abbott ID NOWTM (IDN) COVID-19 diagnostic assay used at point of care and its impact on turnaround time for divulgation of test results. METHODS: Prospective study conducted from December 2020 to February 2021 in acute symptomatic participants presenting in three walk-in centres in the province of Québec. RESULTS: Valid paired samples were obtained from 2,372 participants. A positive result on either the IDN or the standard-of-care nucleic acid amplification test (SOC-NAAT) was obtained in 423 participants (prevalence of 17.8%). Overall sensitivity of IDN and SOC-NAAT were 96.4% (95% CI: 94.2-98.0%) and 99.1% (95% CI: 97.6-99.8), respectively; negative predictive values were 99.2% (95% CI: 98.7-99.6%) and 99.8% (95% CI: 99.5-100%), respectively. Turnaround time for positive results was significantly faster on IDN. CONCLUSION: In our experience, IDN use in symptomatic individuals in walk-in centres is a reliable sensitive alternative to SOC-NAAT without the need for subsequent confirmation of negative results. Such deployment can accelerate contact tracing, reduce the burden on laboratories and increase access to testing.

An outlook on coronavirus disease 2019 detection methods.

Diagnostic testing plays a fundamental role in the mitigation and containment of coronavirus disease 2019 (COVID-19), as it enables immediate quarantine of those who are infected and contagious and is essential for the epidemiological characterization of the virus and estimating the number of infected cases worldwide. Confirmation of viral infections, such as COVID-19, can be achieved through two general approaches: nucleic acid amplification tests (NAATs) or molecular tests, and serological or antibody-based tests. The genetic material of the pathogen is detected in NAAT, and in serological tests, host antibodies produced in response to the pathogen are identified. Other methods of diagnosing COVID-19 include radiological imaging of the lungs and in vitro detection of viral antigens. This review covers different approaches available to diagnosing COVID-19 by outlining their advantages and shortcomings, as well as appropriate indications for more accurate testing.

Risk factors for recurrent positive results of the nucleic acid amplification test for COVID-19 patients: a retrospective study.

Positive retests of COVID-19 represent a public health concern because of the increased risk of transmission. This study explored whether factors other than the nucleic acid amplification test (NAAT) contribute to positive retest results. Patients with COVID-19 admitted to the Guanggu district of the Hubei Maternal and Child Health Hospital between February 17 and March 28, 2020, were retrospectively included. The patients were grouped into the negative (n = 133) and positive (n = 51) retest groups. The results showed that the proportion of patients presenting with cough was higher (P < 0.001) and the proportion of patients with dyspnea was lower (P = 0.018) in the positive than in the negative retest group. The positive retest group showed shorter durations between symptom onset and hospitalization (P < 0.001) and symptom onset and the first positive NAAT (P = 0.033). The positive retest group had higher basophil counts (P = 0.023) and direct bilirubin (P = 0.032) and chlorine concentrations (P = 0.023) but lower potassium concentrations (P = 0.001) than the negative retest group. Multivariable regression analysis showed that coughing (OR = 7.59, 95% CI 2.28-25.32, P = 0.001) and serum chloride concentrations (OR = 1.38, 95% CI 1.08-1.77, P = 0.010) were independently associated with a positive retest result. Coughing and serum chloride concentrations were independent risk factors for positive NAAT retest results. Patients with a hospital stay of < 2 weeks or a short incubation period should stay in isolation and be monitored to reduce transmission. These results could help identify patients who require closer surveillance.

Longitudinal evaluation of the Abbott ARCHITECT SARS-CoV-2 IgM and IgG assays in a pediatric population.

BACKGROUND: Clinical laboratory testing has been an essential part of COVID-19 management. Serology can provide valuable information regarding a patient's exposure to virus, and may have a larger role to play as vaccines becomes available. Limited data is available on the serological response in pediatric patients. Here we investigate the use of one manufacturer's commercial assays for detecting IgM and IgG in an exclusively pediatric population. METHODS: Abbott SARS-CoV-2 IgM and IgG assays were performed on an Abbott ARCHITECT i1000. For specificity studies, we tested 78 patient specimens collected before the COVID-19 pandemic, and 66 specimens from patients who tested negative for SARS-CoV-2 nucleic acid amplification test (NAAT) during the COVID-19 pandemic. For sensitivity we tested 181 specimens from 41 patients with a positive NAAT result. Precision data was acquired for 20 days. RESULTS: For IgM, the highest qualitative positive agreement with molecular results was observed to be 15-30 days after a positive NAAT result or after symptom onset. For IgG, the highest positive agreement was 31-60 days after a positive NAAT result or 61-90 days after the start of symptoms. IgM started to decline 30 days after NAAT results and faded by 90 days. IgG started to decrease 60 days after a positive NAAT result. CONCLUSION: The Abbott IgM and IgG assays have negative agreements of 98.7-100% relative to NAAT results. The IgM and IgG levels assayed by these methods start to decline months after positive molecular results and onset of symptoms in a pediatric population.

Nucleic acid amplification tests on respiratory samples for the diagnosis of coronavirus infections: a systematic review and meta-analysis.

BACKGROUND: Management and control of coronavirus disease 2019 (COVID-19) relies on reliable diagnostic testing. OBJECTIVES: To evaluate the diagnostic test accuracy (DTA) of nucleic acid amplification tests (NAATs) for the diagnosis of coronavirus infections. DATA SOURCES: PubMed, Web of Science, the Cochrane Library, Embase, Open Grey and conference proceeding until May 2019. PubMed and medRxiv were updated for COVID-19 on 31st August 2020. STUDY ELIGIBILITY: Studies were eligible if they reported on agreement rates between different NAATs using clinical samples. PARTICIPANTS: Symptomatic patients with suspected upper or lower respiratory tract coronavirus infection. METHODS: The new NAAT was defined as the index test and the existing NAAT as reference standard. Data were extracted independently in duplicate. Risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 tool. Confidence regions (CRs) surrounding summary sensitivity/specificity pooled by bivariate meta-analysis are reported. Heterogeneity was assessed using meta-regression. RESULTS: Fifty-one studies were included, 22 of which included 10 181 persons before COVID-19 and 29 including 8742 persons diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The overall summary sensitivity was 89.1% (95%CR 84.0-92.7%) and specificity 98.9% (95%CR 98.0-99.4%). Nearly all the studies evaluated different PCRs as both index and reference standards. Real-time RT PCR assays resulted in significantly higher sensitivity than other tests. Reference standards at high risk of bias possibly exaggerated specificity. The pooled sensitivity and specificity of studies evaluating SARS-COV-2 were 90.4% (95%CR 83.7-94.5%) and 98.1% (95%CR 95.9-99.2), respectively. SARS-COV-2 studies using samples from the lower respiratory tract, real-time RT-PCR, and tests targeting the N or S gene or more than one gene showed higher sensitivity, and assays based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), especially when targeting only the RNA-dependent RNA polymerase (RdRp) gene, showed significantly lower sensitivity compared to other studies. CONCLUSIONS: Pooling all studies to date shows that on average 10% of patients with coronavirus infections might be missed with PCR tests. Variables affecting sensitivity and specificity can be used for test selection and development.

Performance of Nucleic Acid Amplification Tests for Detection of Severe Acute Respiratory Syndrome Coronavirus 2 in Prospectively Pooled Specimens.

Pooled nucleic acid amplification tests for severe acute respiratory syndrome coronavirus 2 could increase availability of testing at decreased cost. However, the effect of dilution on analytical sensitivity through sample pooling has not been well characterized. We tested 1,648 prospectively pooled specimens by using 3 nucleic acid amplification tests for severe acute respiratory syndrome coronavirus 2: a laboratory-developed real-time reverse transcription PCR targeting the envelope gene, and 2 commercially available Panther System assays targeting open reading frame 1ab. Positive percent agreement (PPA) of pooled versus individual testing ranged from 71.7% to 82.6% for pools of 8 and from 82.9% to 100.0% for pools of 4. We developed and validated an independent stochastic simulation model to estimate effects of dilution on PPA and efficiency of a 2-stage pooled real-time reverse transcription PCR testing algorithm. PPA was dependent on the proportion of tests with positive results, cycle threshold distribution, and assay limit of detection.

Molecular Diagnosis of Coronavirus Disease 2019.

OBJECTIVES: To review molecular diagnostics for coronavirus disease 2019. The world is in the midst of a coronavirus disease 2019 pandemic. Containing the spread of the severe acute respiratory distress coronavirus is critical. Instrumental to the future success is the ability to reliably and reproducibly detect this inciting pathogen to inform public health containment policies and treatment decisions. DATA SOURCES: Molecular diagnostics focusing on molecular detection methodologies for detection of the virus and the presence of the disease. STUDY SELECTION: Narrative review. DATA EXTRACTION: Literature, PubMed, Scopus, and official government documents. DATA SYNTHESIS: Diagnosing severe acute respiratory syndrome coronavirus is done through real-time reverse transcriptase-polymerase chain reaction tests, cell culture, and serology. For patients, diagnostics are an integral part of a full medical history, physical examinations, blood tests, and diagnostic imaging. CONCLUSIONS: Here, we review current approaches to the molecular diagnosis of coronavirus disease 2019.

A Comparison of Five SARS-CoV-2 Molecular Assays With Clinical Correlations.

OBJECTIVES: Comparative assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) molecular assays that have been operationalized through the US Food and Drug Administration's Emergency Use Authorization process are warranted to assess real-world performance. Characteristics such as sensitivity, specificity, and false-negative rate are important to inform clinical use. METHODS: We compared five SARS-CoV-2 assays using nasopharyngeal and nasal swab specimens submitted in transport media; we enriched this cohort for positive specimens, since we were particularly interested in the sensitivity and false-negative rate. Performance of each test was compared with a composite standard. RESULTS: The sensitivities and false-negative rates of the 239 specimens that met inclusion criteria were, respectively, as follows: Centers for Disease Control and Prevention 2019 nCoV Real-Time RT-PCR Diagnostic Panel, 100% and 0%; TIB MOLBIOL/Roche z 480 Assay, 96.5% and 3.5%; Xpert Xpress SARS-CoV-2 (Cepheid), 97.6% and 2.4%; Simplexa COVID-19 Direct Kit (DiaSorin), 88.1% and 11.9%; and ID Now COVID-19 (Abbott), 83.3% and 16.7%. CONCLUSIONS: The assays that included a nucleic acid extraction followed by reverse transcription polymerase chain reaction were more sensitive than assays that lacked a full extraction. Most false negatives were seen in patients with low viral loads, as extrapolated from crossing threshold values.