Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.

A pooled testing strategy for identifying SARS-CoV-2 at low prevalence.

Suppressing infections of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will probably require the rapid identification and isolation of individuals infected with the virus on an ongoing basis. Reverse-transcription polymerase chain reaction (RT-PCR) tests are accurate but costly, which makes the regular testing of every individual expensive. These costs are a challenge for all countries around the world, but particularly for low-to-middle-income countries. Cost reductions can be achieved by pooling (or combining) subsamples and testing them in groups1-7. A balance must be struck between increasing the group size and retaining test sensitivity, as sample dilution increases the likelihood of false-negative test results for individuals with a low viral load in the sampled region at the time of the test8. Similarly, minimizing the number of tests to reduce costs must be balanced against minimizing the time that testing takes, to reduce the spread of the infection. Here we propose an algorithm for pooling subsamples based on the geometry of a hypercube that, at low prevalence, accurately identifies individuals infected with SARS-CoV-2 in a small number of tests and few rounds of testing. We discuss the optimal group size and explain why, given the highly infectious nature of the disease, largely parallel searches are preferred. We report proof-of-concept experiments in which a positive subsample was detected even when diluted 100-fold with negative subsamples (compared with 30-48-fold dilutions described in previous studies9-11). We quantify the loss of sensitivity due to dilution and discuss how it may be mitigated by the frequent re-testing of groups, for example. With the use of these methods, the cost of mass testing could be reduced by a large factor. At low prevalence, the costs decrease in rough proportion to the prevalence. Field trials of our approach are under way in Rwanda and South Africa. The use of group testing on a massive scale to monitor infection rates closely and continually in a population, along with the rapid and effective isolation of people with SARS-CoV-2 infections, provides a promising pathway towards the long-term control of coronavirus disease 2019 (COVID-19).

PACRAT: pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription.

The SARS-CoV-2 pandemic underscored the need for early, rapid, and widespread pathogen detection tests that are readily accessible. Many existing rapid isothermal detection methods use the recombinase polymerase amplification (RPA), which exhibits polymerase chain reaction (PCR)-like sensitivity, specificity, and even higher speed. However, coupling RPA to other enzymatic reactions has proven difficult. For the first time, we demonstrate that with tuning of buffer conditions and optimization of reagent concentrations, RPA can be cascaded into an in vitro transcription reaction, enabling detection using fluorescent aptamers in a one-pot reaction. We show that this reaction, which we term PACRAT (pathogen detection with aptamer-observed cascaded recombinase polymerase amplification-in vitro transcription) can be used to detect SARS-CoV-2 RNA with single-copy detection limits, Escherichia coli with single-cell detection limits, and 10-min detection times. Further demonstrating the utility of our one-pot, cascaded amplification system, we show PACRAT can be used for multiplexed detection of the pathogens SARS-CoV-2 and E. coli, along with multiplexed detection of two variants of SARS-CoV-2.

Predicting the presence of infectious virus from PCR data: A meta-analysis of SARS-CoV-2 in non-human primates.

Researchers and clinicians often rely on molecular assays like PCR to identify and monitor viral infections, instead of the resource-prohibitive gold standard of viral culture. However, it remains unclear when (if ever) PCR measurements of viral load are reliable indicators of replicating or infectious virus. The recent popularity of PCR protocols targeting subgenomic RNA for SARS-CoV-2 has caused further confusion, as the relationships between subgenomic RNA and standard total RNA assays are incompletely characterized and opinions differ on which RNA type better predicts culture outcomes. Here, we explore these issues by comparing total RNA, subgenomic RNA, and viral culture results from 24 studies of SARS-CoV-2 in non-human primates (including 2167 samples from 174 individuals) using custom-developed Bayesian statistical models. On out-of-sample data, our best models predict subgenomic RNA positivity from total RNA data with 91% accuracy, and they predict culture positivity with 85% accuracy. Further analyses of individual time series indicate that many apparent prediction errors may arise from issues with assay sensitivity or sample processing, suggesting true accuracy may be higher than these estimates. Total RNA and subgenomic RNA showed equivalent performance as predictors of culture positivity. Multiple cofactors (including exposure conditions, host traits, and assay protocols) influence culture predictions, yielding insights into biological and methodological sources of variation in assay outcomes-and indicating that no single threshold value applies across study designs. We also show that our model can accurately predict when an individual is no longer infectious, illustrating the potential for future models trained on human data to guide clinical decisions on case isolation. Our work shows that meta-analysis of in vivo data can overcome longstanding challenges arising from limited sample sizes and can yield robust insights beyond those attainable from individual studies. Our analytical pipeline offers a framework to develop similar predictive tools in other virus-host systems, including models trained on human data, which could support laboratory analyses, medical decisions, and public health guidelines.

Testing for SARS-CoV-2: lessons learned and current use cases.

SUMMARYThe emergence and worldwide dissemination of SARS-CoV-2 required both urgent development of new diagnostic tests and expansion of diagnostic testing capacity on an unprecedented scale. The rapid evolution of technologies that allowed testing to move out of traditional laboratories and into point-of-care testing centers and the home transformed the diagnostic landscape. Four years later, with the end of the formal public health emergency but continued global circulation of the virus, it is important to take a fresh look at available SARS-CoV-2 testing technologies and consider how they should be used going forward. This review considers current use case scenarios for SARS-CoV-2 antigen, nucleic acid amplification, and immunologic tests, incorporating the latest evidence for analytical/clinical performance characteristics and advantages/limitations for each test type to inform current debates about how tests should or should not be used.

Test accuracy of rapid diagnostic tests and reverse-transcription polymerase chain reaction against virus isolation in cell culture for assessing SARS-CoV-2 infectivity: Systematic review and meta-analysis.

We aimed to assess the performance of Ag-RDT and RT-qPCR with regard to detecting infectious SARS-CoV-2 in cell cultures, as their diagnostic test accuracy (DTA) compared to virus isolation remains largely unknown. We searched three databases up to 15 December 2021 for DTA studies. The bivariate model was used to synthesise the estimates. Risk of bias was assessed using QUADAS-2/C. Twenty studies (2605 respiratory samples) using cell culture and at least one molecular test were identified. All studies were at high or unclear risk of bias in at least one domain. Three comparative DTA studies reported results on Ag-RDT and RT-qPCR against cell culture. Two studies evaluated RT-qPCR against cell culture only. Fifteen studies evaluated Ag-RDT against cell culture as reference standard in RT-qPCR-positive samples. For Ag-RDT, summary sensitivity was 93% (95% CI 78; 98%) and specificity 87% (95% CI 70; 95%). For RT-qPCR, summary sensitivity (continuity-corrected) was 98% (95% CI 95; 99%) and specificity 45% (95% CI 28; 63%). In studies relying on RT-qPCR-positive subsamples (n = 15), the summary sensitivity of Ag-RDT was 93% (95% CI 92; 93%) and specificity 63% (95% CI 63; 63%). Ag-RDT show moderately high sensitivity, detecting most but not all samples demonstrated to be infectious based on virus isolation. Although RT-qPCR exhibits high sensitivity across studies, its low specificity to indicate infectivity raises the question of its general superiority in all clinical settings. Study findings should be interpreted with caution due to the risk of bias, heterogeneity and the imperfect reference standard for infectivity.

Comparative Diagnostic Utility of SARS-CoV-2 Rapid Antigen and Molecular Testing in a Community Setting.

BACKGROUND: SARS-CoV-2 antigen-detection rapid diagnostic tests (Ag-RDTs) have become widely utilized but longitudinal characterization of their community-based performance remains incompletely understood. METHODS: This prospective longitudinal study at a large public university in Seattle, WA utilized remote enrollment, online surveys, and self-collected nasal swab specimens to evaluate Ag-RDT performance against real-time reverse transcription polymerase chain reaction (rRT-PCR) in the context of SARS-CoV-2 Omicron. Ag-RDT sensitivity and specificity within 1 day of rRT-PCR were evaluated by symptom status throughout the illness episode and Orf1b cycle threshold (Ct). RESULTS: From February to December 2022, 5757 participants reported 17 572 Ag-RDT results and completed 12 674 rRT-PCR tests, of which 995 (7.9%) were rRT-PCR positive. Overall sensitivity and specificity were 53.0% (95% confidence interval [CI], 49.6%-56.4%) and 98.8% (95% CI, 98.5%-99.0%), respectively. Sensitivity was comparatively higher for Ag-RDTs used 1 day after rRT-PCR (69.0%), 4-7 days after symptom onset (70.1%), and Orf1b Ct ≤20 (82.7%). Serial Ag-RDT sensitivity increased with repeat testing ≥2 (68.5%) and ≥4 (75.8%) days after an initial Ag-RDT-negative result. CONCLUSIONS: Ag-RDT performance varied by clinical characteristics and temporal testing patterns. Our findings support recommendations for serial testing following an initial Ag-RDT-negative result, especially among recently symptomatic persons or those at high risk for SARS-CoV-2 infection.

The Infectious Diseases Society of America Guidelines on the Diagnosis of COVID-19: Molecular Diagnostic Testing (December 2023).

Accurate molecular diagnostic tests are necessary for confirming a diagnosis of coronavirus disease 2019 (COVID-19) and for identifying asymptomatic carriage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The number of available SARS-CoV-2 nucleic acid detection tests continues to increase as does the COVID-19 diagnostic literature. Thus, the Infectious Diseases Society of America (IDSA) developed an evidence-based diagnostic guideline to assist clinicians, clinical laboratorians, patients, and policymakers in decisions related to the optimal use of SARS-CoV-2 nucleic acid amplification tests. In addition, we provide a conceptual framework for understanding molecular diagnostic test performance, discuss nuances of test result interpretation in a variety of practice settings, and highlight important unmet research needs related to COVID-19 diagnostic testing. IDSA convened a multidisciplinary panel of infectious diseases clinicians, clinical microbiologists, and experts in systematic literature review to identify and prioritize clinical questions and outcomes related to the use of SARS-CoV-2 molecular diagnostics. Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology was used to assess the certainty of evidence and make testing recommendations. The panel agreed on 12 diagnostic recommendations. Access to accurate SARS-CoV-2 nucleic acid testing is critical for patient care, hospital infection prevention, and the public health response to COVID-19 infection. Information on the clinical performance of available tests continues to grow, but the quality of evidence of the current literature to support this updated molecular diagnostic guideline remains moderate to very low. Recognizing these limitations, the IDSA panel weighed available diagnostic evidence and recommends nucleic acid testing for all symptomatic individuals suspected of having COVID-19. In addition, testing is suggested for asymptomatic individuals with known or suspected contact with a COVID-19 case when the results will impact isolation/quarantine/personal protective equipment (PPE) usage decisions. Evidence in support of rapid testing and testing of upper respiratory specimens other than nasopharyngeal swabs, which offer logistical advantages, is sufficient to warrant conditional recommendations in favor of these approaches.

Rapid, Portable, and Electricity-free Sample Extraction Method for Enhanced Molecular Diagnostics in Resource-Limited Settings.

The COVID-19 pandemic has highlighted the need for rapid and reliable diagnostics that are accessible in resource-limited settings. To address this pressing issue, we have developed a rapid, portable, and electricity-free method for extracting nucleic acids from respiratory swabs (i.e. nasal, nasopharyngeal and buccal swabs), successfully demonstrating its effectiveness for the detection of SARS-CoV-2 in residual clinical specimens. Unlike traditional approaches, our solution eliminates the need for micropipettes or electrical equipment, making it user-friendly and requiring little to no training. Our method builds upon the principles of magnetic bead extraction and revolves around a low-cost plastic magnetic lid, called SmartLid, in combination with a simple disposable kit containing all required reagents conveniently prealiquoted. Here, we clinically validated the SmartLid sample preparation method in comparison to the gold standard QIAamp Viral RNA Mini Kit from QIAGEN, using 406 clinical isolates, including 161 SARS-CoV-2 positives, using the SARS-CoV-2 RT-qPCR assays developed by the US Centers for Disease Control and Prevention (CDC). The SmartLid method showed an overall sensitivity of 95.03% (95% CI: 90.44-97.83%) and a specificity of 99.59% (95% CI: 97.76-99.99%), with a positive agreement of 97.79% (95% CI: 95.84-98.98%) when compared to QIAGEN's column-based extraction method. There are clear benefits to using the SmartLid sample preparation kit: it enables swift extraction of viral nucleic acids, taking less than 5 min, without sacrificing significant accuracy when compared to more expensive and time-consuming alternatives currently available on the market. Moreover, its simplicity makes it particularly well-suited for the point-of-care where rapid results and portability are crucial. By providing an efficient and accessible means of nucleic acid extraction, our approach aims to introduce a step-change in diagnostic capabilities for resource-limited settings.

From Lab to Home: Ultrasensitive Rapid Detection of SARS-CoV-2 with a Cascade CRISPR/Cas13a-Cas12a System Based Lateral Flow Assay.

Currently, CRISPR/Cas-based molecular diagnostic techniques usually rely on the introduction of nucleic acid amplification to improve their sensitivity, which is usually more time-consuming, susceptible to aerosol contamination, and therefore not suitable for at-home molecular testing. In this research, we developed an advanced CRISPR/Cas13a-Cas12a-based lateral flow assay that facilitated the ultrasensitive and rapid detection of SARS-CoV-2 RNA directly from samples, without the need for nucleic acid amplification. This method was called CRISPR LFA enabling at-home RNA testing (CLEAR). CLEAR used a novel cascade mechanism with specially designed probes that fold into hairpin structures, enabling visual detection of SARS-CoV-2 sequences down to 1 aM sensitivity levels. More importantly, CLEAR had a positive coincidence rate of 100% and a negative coincidence rate of 100% for clinical nasopharyngeal swabs from 16 patients. CLEAR was particularly suitable for at-home molecular testing, providing a low-cost, user-friendly solution that can efficiently distinguish between different SARS-CoV-2 variants. CLEAR overcame the common limitations of high sensitivity and potential contamination associated with traditional PCR-based systems, making it a promising tool for widespread public health application, especially in environments with limited access to laboratory resources.

Highly sensitive extraction-free saliva-based molecular assay for rapid diagnosis of SARS-CoV-2.

The COVID-19 pandemic highlighted the necessity of fast, sensitive, and efficient methods to test large populations for respiratory viruses. The "gold standard" molecular assays for detecting respiratory viruses, such as quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR), rely on invasive swab samples and require time-consuming and labor-intensive extraction processes. Moreover, the turnaround time for RT-qPCR-based assays is too lengthy for rapid screening. Extraction-free saliva-based methods provide a non-invasive sampling process with a fast turnaround time and are suitable for high-throughput applications. However, when used with a standard RT-qPCR system, the absence of extraction significantly reduces the assays' sensitivity. Here, using a novel optical modulation biosensing (OMB) platform, we developed a rapid and highly sensitive extraction-free saliva-based molecular assay. We blindly tested 364 paired nasopharyngeal swabs and saliva samples from suspected SARS-CoV-2 cases in Israel. Compared with the gold standard swab-based RT-qPCR assay, the sensitivity of the extraction-free saliva-based OMB assay is 90.7%, much higher than the sensitivity of extraction-free saliva-based RT-qPCR assay (77.8%) with similar specificity (95.3% and 97.6%, respectively). Moreover, out of 12 samples identified by the OMB-based assay as positive, 8 samples were collected from hospitalized patients in a COVID-19 ward and were verified to be SARS-CoV-2-positive upon admission, indicating that the actual clinical sensitivity and specificity of the OMB assay are higher. Considering its user-friendly saliva-based protocol, short and cost-effective extraction-free process, and high clinical accuracy, the OMB-based molecular assay is very suitable for high-throughput testing of large populations for respiratory viruses. IMPORTANCE: Three years after the SARS-CoV-2 outbreak, there are no molecular tests that combine low-cost and straightforward sample preparation, effective sample handling, minimal reagent and disposable requirements, high sensitivity, and high throughput required for mass screening. Existing rapid molecular techniques typically sacrifice certain requirements to meet others. Yet, localized outbreaks of novel viral diseases happen daily in different parts of the world. In this context, respiratory diseases are of specific importance, as they are frequently airborne and highly contagious, with the potential for a rapid global spread. The widely accepted opinion is that another pandemic is just a question of time. To ensure that the containment efforts for the upcoming "disease X" are successful, introducing rapid, high-throughput, and highly sensitive diagnostic methods for detecting and identifying pathogens is critical. A few months into the pandemic, saliva was suggested as a diagnostic matrix for SARS-CoV-2 detection. The collection of saliva does not require swabs and is minimally invasive. In particular, extraction-free saliva-based assays require fewer reagents and disposables, and therefore are faster and cheaper, offering an appealing alternative for low-income countries. Unfortunately, current extraction-free saliva-based detection methods, such as direct RT-qPCR or isothermal amplification, have either low sensitivity or low throughput. Therefore, we believe that the presented highly sensitive ht-OMBi platform and the extraction-free saliva-based molecular assay can become an essential tool in the infectious disease monitoring toolbox.

Use of strips of rapid diagnostic tests as a source of ribonucleic acid for genomic surveillance of viruses: an example of SARS-CoV-2.

BACKGROUND: This study aimed to demonstrate that the genomic material of SARS-CoV-2 can be isolated from strips of COVID-19 rapid diagnostic test cassettes. METHOD: It was a prospective cross-sectional study involving patients admitted to treatment centers and sampling sites in the city of Conakry, Guinea. A total of 121 patients were double sampled, and 9 more patients were tested only for RDT. PCR was conducted according to the protocol of the RunMei kit. Sequencing was performed by using the illumina COVIDSeq protocol. Nine COVID-19 RDTs without nasopharyngeal swabs were in addition tested. RESULT: Among the 130 COVID-19 RDTs, forty-seven were macroscopically positive, whereas seventy-two were positive according to PCR using RDT strip, while among the 121 VTM swabs, sixty-four were positive. Among eighty-three negative COVID-19 RDTs, twenty-seven were positive by PCR using RDT strip with a geometric mean Ct value of 32.49 cycles. Compared to those of PCR using VTM, the sensitivity and specificity of PCR using RDT strip were estimated to be 100% and 85.96%, respectively, with 93.39% test accuracy. Among the fifteen COVID-19 RDT extracts eligible for sequencing, eleven had sequences identical to those obtained via the standard method, with coverage between 75 and 99.6%. CONCLUSION: These results show that COVID-19 RDTs can be used as biological material for the genomic surveillance of SARS-CoV-2.

Self-swabbing versus assisted swabbing for viral detection by qRT-PCR: the experience from SARS-CoV-2 based on a meta-analysis of six prospectively designed evaluations conducted in a UK setting.

PURPOSE: In April 2020, the UK Government implemented NHS Test and Trace to provide SARS-CoV-2 quantitative reverse transcription polymerase chain reaction (qRT-PCR) testing for the public, with nose-and-throat swabbing for samples performed by trained staff. Self-swabbing (SS) would allow rapid scale-up of testing capacity and access. Six studies were undertaken to determine whether SS was as effective for detecting SARS-CoV-2 as swabbing performed by trained staff. METHODS: Six prospective studies were conducted between April-October 2020, using six swab/media combinations. Differences between assisted swabbing (AS) and SS were evaluated for concordance, positivity, sensitivity, cycle threshold (Ct) values and void rates. Statistical analysis was performed using 95% confidence intervals (CIs), paired t-tests and model-based methods. RESULTS: Overall, 3,253 individuals were recruited (median age 37 years, 49% female), with 2,933 having valid paired qRT-PCR results. Pooled concordance rate was 98% (95% CI: 96%, 99%). Positivity rate differences for SS (8.1%) and AS (8.4%) and differences in pooled sensitivities between SS (86%; 95% CI: 78%, 92%) and AS (91%; 95% CI: 78%, 96%) were nonsignificant. Both types of swabbing led to pooled void rates below 2% and strongly correlated Ct values. Age, sex and previous swabbing experience did not have a significant impact on concordance or sensitivity. CONCLUSION: The UK adopted a policy to promote self-testing for SARS-CoV-2 based on data demonstrating equivalence of SS versus AS. Positive outcomes with SS are likely generalisable to testing for other respiratory pathogens, and we consider self-sampling and self-testing essential for future pandemic preparedness.

Diagnostic performance, stability, and acceptability of self-collected saliva without additives for SARS-CoV-2 molecular diagnosis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the coronavirus disease 2019 (COVID-19), leading to a global pandemic. The molecular diagnosis of this virus is mostly performed by collecting upper respiratory samples, which has many disadvantages, including patient discomfort and the need for trained healthcare professionals. Although saliva has emerged as a more comfortable sample, the use of additives to preserve viral RNA is expensive and, in some cases, difficult for self-collection. METHOD: This study evaluated the diagnostic performance by RT-PCR and stability of self-collected saliva using wide-mouth specimen collection cups without stabilization and/or inactivation buffers for SARS-CoV-2 detection, compared to nasopharyngeal samples and saliva collected with additives. Additionally, the study assessed the acceptability of this sample collection method among participants and healthcare personnel. RESULTS: The study included 1281 volunteers with a 24.6% positive infection rate. Saliva demonstrated comparable diagnostic performance to nasopharyngeal samples, with a sensitivity of 87.6% and specificity of 99.6%, for a total percent agreement of 96.4%. The study also showed that viral RNA in saliva remained stable for at least 72 h at different temperatures. Notably, saliva samples without additives exhibited a lower RdRp Ct compared to samples with additives, suggesting that the absence of stabilization and/or inactivation buffers does not significantly affect its performance. The study highlighted the acceptability of saliva among patients and healthcare personnel due to its noninvasive nature and ease of collection. CONCLUSIONS: This research supports the implementation of self-collected saliva as a comfortable and user-friendly alternative sample for SARS-CoV-2 diagnosis.

CRISPR-Cas12a-based ultrasensitive assay for visual detection of SARS-CoV-2 RNA.

The development of ultrasensitive and visual methods is of great significance for molecular diagnosis at the point-of-care. In this study, we have integrated recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system to design an ultrasensitive strategy for visual nucleic acid testing. RPA is utilized to amplify the target nucleic acid, producing amplicons that activate the single-stranded DNase property of CRISPR-Cas12a. The activated CRISPR-Cas12a then degrades the single-stranded DNA on magnetic nanoparticles (MNPs), releasing immobilized GOx from the MNPs which catalyses the chromogenic substrate. The developed method exhibits remarkable sensitivity, successfully detecting as low as 10 aM (∼6 copies per µL) of the target nucleic acid by visual colour changes in solution. The instrumental limit of detection is calculated to be 2.86 aM (∼2 copies per µL), comparable to the sensitivity of polymerase chain reaction (PCR). Importantly, this approach only requires isothermal incubation operation and does not involve costly instruments. The method has been validated by visually detecting the SARS-CoV-2 RNA gene fragment within 50 minutes. With its ultrasensitivity, simplicity of operation, and potential for integration into a point-of-care detection kit, this strategy holds great promise for nucleic acid testing in various settings.

Diagnostic performance of rapid antigen tests (RAT) for COVID-19 and factors associated with RAT-negative results among RT-PCR-positive individuals during Omicron BA.2, BA.5 and XBB.1 predominance.

BACKGROUND: While numerous studies have evaluated the real-world performance of rapid antigen tests (RATs), data on the effect of Omicron sublineages such as XBB and reinfections on RAT performance is limited. We assessed the performance of RATs and factors associated with RAT-negative results among individuals who tested SARS-CoV-2-positive by reverse transcription-polymerase chain reaction (RT-PCR). METHODS: We conducted a retrospective study among Singapore residents who underwent testing for SARS-CoV-2 with RAT (Acon Flowflex or SD Biosensor) and RT-PCR in the same clinical encounter between 9 May 2022 and 21 November 2022. RT-PCR served as a reference standard for RAT performance. Logistic regression was used to estimate the odds ratios (OR) of factors associated with negative RAT results among RT-PCR-positive cases. RESULTS: Of 8,620 clinical encounters analysed, 3,519 (40.8%) were SARS-CoV-2-positive on RT-PCR. Overall sensitivity and specificity of RAT was 84.6% (95% CI 83.3-85.7%) and 99.4% (95% CI 99.1-99.6%) respectively. Acon Flowflex consistently achieved higher sensitivity and specificity than SD Biosensor test kit. Among RT-PCR-positive cases, individuals who had a previous documented SARS-CoV-2 infection, coinfection with another respiratory pathogen or tested ≥ 6 days from symptom onset had higher odds of testing RAT-negative, but the associations were attenuated after adjustment for cycle threshold values (proxy for viral load). There was no significant difference in RAT performance between Omicron sublineages BA.2, BA.5 and XBB.1. CONCLUSION: Diagnostic performance of RAT was not affected by changes in predominant circulating Omicron sublineages. However, reinfection cases may be under ascertained by RAT. In individuals with a previous SARS-CoV-2 infection episode or symptom onset ≥ 6 days prior to testing, a confirmatory RT-PCR may be considered if there is high clinical suspicion.

Evaluation and comparison of one-step real-time PCR and one-step RT-LAMP methods for detection of SARS-CoV-2.

BACKGROUND: There is an increasing disease trend for SARS-COV-2, so need a quick and affordable diagnostic method. It should be highly accurate and save costs compared to other methods. The purpose of this research is to achieve these goals. METHODS: This study analyzed 342 samples using TaqMan One-Step RT-qPCR and fast One-Step RT-LAMP (Reverse Transcriptase Loop-Mediated Isothermal Amplification). The One-Step LAMP assay was conducted to assess the sensitivity and specificity. RESULTS: The research reported positive samples using two different methods. In the RT-LAMP method, saliva had 92 positive samples (26.9%) and 250 negative samples (73.09%) and nasopharynx had 94 positive samples (27.4%) and 248 negative samples (72.51%). In the RT-qPCR method, saliva had 86 positive samples (25.1%) and 256 negative samples (74.8%) and nasopharynx had 93 positive samples (27.1%) and 249 negative samples (72.8%). The agreement between the two tests in saliva and nasopharynx samples was 93% and 94% respectively, based on Cohen's kappa coefficient (κ) (P < 0.001). The rate of sensitivity in this technique was reported at a dilution of 1 × 101 and 100% specificity. CONCLUSIONS: Based on the results of the study the One-Step LAMP assay has multiple advantages. These include simplicity, cost-effectiveness, high sensitivity, and specificity. The One-Step LAMP assay shows promise as a diagnostic tool. It can help manage disease outbreaks, ensure prompt treatment, and safeguard public health by providing rapid, easy-to-use testing.

Correlation of COVID-19 vaccination and RT-PCR ct value among cases in Addis Ababa, Ethiopia: implication for future preparedness.

BACKGROUND: The COVID-19 disease requires accurate diagnosis to effectively manage infection rates and disease progression. The study aims to assess the relationship between vaccination status and RT-PCR cycle threshold (Ct) values by comparing clinical, RDT and RT-PCR results. METHODS: A total of 453 suspected COVID-19 cases were included in this study. Nasopharyngeal swabs were collected for both RDT and RT-PCR testing, with RDTs conducted on-site and RT-PCR at the Ethiopian Public Health Institute (EPHI) genomics laboratory. Detailed clinical, RDT, and RT-PCR results were analyzed. Data analysis included descriptive statistics, cross-tabulation, and Chi-Square tests to investigate the connections between diagnostic outcomes and vaccination status, with a focusing on Ct values. RESULTS: RDT results showed 34.0% negative and 65.8% positive, while RT-PCR results indicated 35.8% negative and 64.2% positive cases. The discrepancies between RDT and RT-PCR results emphasize the importance of thorough testing. No significant association was found between vaccination status and viral load, as indicated by Ct values. Among RT-PCR positive cases, 49.8% had been vaccinated, suggesting challenges in interpreting results among vaccinated individuals. Further analysis revealed that vaccination (first or second dose) had minimal impact on Ct values, indicating limited influence of vaccination status on viral load dynamics in infected individuals. CONCLUSIONS: The study highlights the significant differences between RDT and RT-PCR outcomes, underscoring the need for a comprehensive testing approach. Additionally, the findings suggest that vaccination status does not significantly impact RT-PCR Ct values, complicating the interpretation of diagnostic results in vaccinated individuals, especially in breakthrough infections and potential false positives.

Laboratory-based molecular test alternatives to RT-PCR for the diagnosis of SARS-CoV-2 infection.

BACKGROUND: Diagnosing people with a SARS-CoV-2 infection played a critical role in managing the COVID-19 pandemic and remains a priority for the transition to long-term management of COVID-19. Initial shortages of extraction and reverse transcription polymerase chain reaction (RT-PCR) reagents impaired the desired upscaling of testing in many countries, which led to the search for alternatives to RNA extraction/purification and RT-PCR testing. Reference standard methods for diagnosing the presence of SARS-CoV-2 infection rely primarily on real-time reverse transcription-polymerase chain reaction (RT-PCR). Alternatives to RT-PCR could, if sufficiently accurate, have a positive impact by expanding the range of diagnostic tools available for the timely identification of people infected by SARS-CoV-2, access to testing and the use of resources. OBJECTIVES: To assess the diagnostic accuracy of alternative (to RT-PCR assays) laboratory-based molecular tests for diagnosing SARS-CoV-2 infection. SEARCH METHODS: We searched the COVID-19 Open Access Project living evidence database from the University of Bern until 30 September 2020 and the WHO COVID-19 Research Database until 31 October 2022. We did not apply language restrictions. SELECTION CRITERIA: We included studies of people with suspected or known SARS-CoV-2 infection, or where tests were used to screen for infection, and studies evaluating commercially developed laboratory-based molecular tests for the diagnosis of SARS-CoV-2 infection considered as alternatives to RT-PCR testing. We also included all reference standards to define the presence or absence of SARS-CoV-2, including RT-PCR tests and established clinical diagnostic criteria. DATA COLLECTION AND ANALYSIS: Two authors independently screened studies and resolved disagreements by discussing them with a third author. Two authors independently extracted data and assessed the risk of bias and applicability of the studies using the QUADAS-2 tool. We presented sensitivity and specificity, with 95% confidence intervals (CIs), for each test using paired forest plots and summarised results using average sensitivity and specificity using a bivariate random-effects meta-analysis. We illustrated the findings per index test category and assay brand compared to the WHO's acceptable sensitivity and specificity threshold for diagnosing SARS-CoV-2 infection using nucleic acid tests. MAIN RESULTS: We included data from 64 studies reporting 94 cohorts of participants and 105 index test evaluations, with 74,753 samples and 7517 confirmed SARS-CoV-2 cases. We did not identify any published or preprint reports of accuracy for a considerable number of commercially produced NAAT assays. Most cohorts were judged at unclear or high risk of bias in more than three QUADAS-2 domains. Around half of the cohorts were considered at high risk of selection bias because of recruitment based on COVID status. Three quarters of 94 cohorts were at high risk of bias in the reference standard domain because of reliance on a single RT-PCR result to determine the absence of SARS-CoV-2 infection or were at unclear risk of bias due to a lack of clarity about the time interval between the index test assessment and the reference standard, the number of missing results, or the absence of a participant flow diagram. For index tests categories with four or more evaluations and when summary estimations were possible, we found that: a) For RT-PCR assays designed to omit/adapt RNA extraction/purification, the average sensitivity was 95.1% (95% CI 91.1% to 97.3%), and the average specificity was 99.7% (95% CI 98.5% to 99.9%; based on 27 evaluations, 2834 samples and 1178 SARS-CoV-2 cases); b) For RT-LAMP assays, the average sensitivity was 88.4% (95% CI 83.1% to 92.2%), and the average specificity was 99.7% (95% CI 98.7% to 99.9%; 24 evaluations, 29,496 samples and 2255 SARS-CoV-2 cases); c) for TMA assays, the average sensitivity was 97.6% (95% CI 95.2% to 98.8%), and the average specificity was 99.4% (95% CI 94.9% to 99.9%; 14 evaluations, 2196 samples and 942 SARS-CoV-2 cases); d) for digital PCR assays, the average sensitivity was 98.5% (95% CI 95.2% to 99.5%), and the average specificity was 91.4% (95% CI 60.4% to 98.7%; five evaluations, 703 samples and 354 SARS-CoV-2 cases); e) for RT-LAMP assays omitting/adapting RNA extraction, the average sensitivity was 73.1% (95% CI 58.4% to 84%), and the average specificity was 100% (95% CI 98% to 100%; 24 evaluations, 14,342 samples and 1502 SARS-CoV-2 cases). Only two index test categories fulfil the WHO-acceptable sensitivity and specificity requirements for SARS-CoV-2 nucleic acid tests: RT-PCR assays designed to omit/adapt RNA extraction/purification and TMA assays. In addition, WHO-acceptable performance criteria were met for two assays out of 35 when tests were used according to manufacturer instructions. At 5% prevalence using a cohort of 1000 people suspected of SARS-CoV-2 infection, the positive predictive value of RT-PCR assays omitting/adapting RNA extraction/purification will be 94%, with three in 51 positive results being false positives, and around two missed cases. For TMA assays, the positive predictive value of RT-PCR assays will be 89%, with 6 in 55 positive results being false positives, and around one missed case. AUTHORS' CONCLUSIONS: Alternative laboratory-based molecular tests aim to enhance testing capacity in different ways, such as reducing the time, steps and resources needed to obtain valid results. Several index test technologies with these potential advantages have not been evaluated or have been assessed by only a few studies of limited methodological quality, so the performance of these kits was undetermined. Only two index test categories with enough evaluations for meta-analysis fulfil the WHO set of acceptable accuracy standards for SARS-CoV-2 nucleic acid tests: RT-PCR assays designed to omit/adapt RNA extraction/purification and TMA assays. These assays might prove to be suitable alternatives to RT-PCR for identifying people infected by SARS-CoV-2, especially when the alternative would be not having access to testing. However, these findings need to be interpreted and used with caution because of several limitations in the evidence, including reliance on retrospective samples without information about the symptom status of participants and the timing of assessment. No extrapolation of found accuracy data for these two alternatives to any test brands using the same techniques can be made as, for both groups, one test brand with high accuracy was overrepresented with 21/26 and 12/14 included studies, respectively. Although we used a comprehensive search and had broad eligibility criteria to include a wide range of tests that could be alternatives to RT-PCR methods, further research is needed to assess the performance of alternative COVID-19 tests and their role in pandemic management.

Strategic Retesting to Reduce False Positive SARS-CoV-2 PCR Test Results.

BACKGROUND: Nucleic acid amplification testing is the gold standard for SARS-CoV-2 diagnostics, although it may produce a certain number of false positive results. There has not been much published about the characteristics of false positive results. In this study, based on retesting, specimens that initially tested positive for SARS-CoV-2 were classified as true or false positive groups to characterize the distribution of cycle threshold (CT) values for N1 and N2 targets and number of targets detected for each group. METHODS: Specimens that were positive for N-gene on retesting and accompanied with S-gene were identified as true positives (true positive based on retesting, rTP), while specimens that retested negative were classified as false positives (false positive based on retesting, rFP). RESULTS: Of the specimens retested, 85/127 (66.9%) were rFP, 16/47 (34.0%) specimens with both N1 and N2 targets initially detected were rFP, and the CT values for each target was higher in rFP than in rTP. ROC curve analysis showed that optimal cutoff values of CT to differentiate between rTP and rFP were 34.8 for N1 and 33.0 for N2. With the optimal cutoff values of CT for each target, out of the 24 specimens that were positive for both N1 and N2 targets and classified as rTP, 23 (95.8%) were correctly identified as true positives. rFP specimens had a single N1 target in 52/61 (85.2%) and a single N2 target in 17/19 (89.5%). Notably, no true positive results were obtained from any specimens with only N2 target detected. CONCLUSIONS: These results suggest that retesting should be performed for positive results with a CT value greater than optimal cutoff value for each target or with a single N1 target amplified, considering the possibility of a false positive. This may provide guidance on indications to perform retesting to minimize the number of false positives.