Molecular Detection of SARS-CoV-2 From Throat Swabs Performed With or Without Specimen Collection From the Tonsils: Protocol for a Multicenter Randomized Controlled Trial.

BACKGROUND: Testing for SARS-CoV-2 is essential to provide early COVID-19 treatment for people at high risk of severe illness and to limit the spread of infection in society. Proper upper respiratory specimen collection is the most critical step in the diagnosis of the SARS-CoV-2 virus in public settings, and throat swabs were the preferred specimens used for mass testing in many countries during the COVID-19 pandemic. However, there is still a discussion about whether throat swabs have a high enough sensitivity for SARS-CoV-2 diagnostic testing, as previous studies have reported a large variability in the sensitivity from 52% to 100%. Many previous studies exploring the diagnostic accuracy of throat swabs lack a detailed description of the sampling technique, which makes it difficult to compare the different diagnostic accuracy results. Some studies perform a throat swab by only collecting specimens from the posterior oropharyngeal wall, while others also include a swab of the palatine tonsils for SARS-CoV-2 testing. However, studies suggest that the palatine tonsils could have a tissue tropism for SARS-CoV-2 that may improve the SARS-CoV-2 detection during sampling. This may explain the variation of sensitivity reported, but no clinical studies have yet explored the differences in sensitivity and patient discomfort whether the palatine tonsils are included during the throat swab or not. OBJECTIVE: The objective of this study is to examine the sensitivity and patient discomfort of a throat swab including the palatine tonsils compared to only swabbing the posterior oropharyngeal wall in molecular testing for SARS-CoV-2. METHODS: We will conduct a randomized controlled study to compare the molecular detection rate of SARS-CoV-2 by a throat swab performed from the posterior oropharyngeal wall and the palatine tonsils (intervention group) or the posterior oropharyngeal wall only (control group). Participants will be randomized in a 1:1 ratio. All participants fill out a baseline questionnaire upon enrollment in the trial, examining their reason for being tested, symptoms, and previous tonsillectomy. A follow-up questionnaire will be sent to participants to explore the development of symptoms after testing. RESULTS: A total of 2315 participants were enrolled in this study between November 10, 2022, and December 22, 2022. The results from the follow-up questionnaire are expected to be completed at the beginning of 2024. CONCLUSIONS: This randomized clinical trial will provide us with information about whether throat swabs including specimens from the palatine tonsils will improve the diagnostic sensitivity for SARS-CoV-2 molecular detection. These results can, therefore, be used to improve future testing recommendations and provide additional information about tissue tropism for SARS-CoV-2. TRIAL REGISTRATION: ClinicalTrials.gov NCT05611203; https://clinicaltrials.gov/study/NCT05611203. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/47446.

A Literature Review on the Relative Diagnostic Accuracy of Chest CT Scans versus RT-PCR Testing for COVID-19 Diagnosis.

BACKGROUND: Reverse transcription polymerase chain reaction (RT-PCR) is the main technique used to identify COVID-19 from respiratory samples. It has been suggested in several articles that chest CTs could offer a possible alternate diagnostic tool for COVID-19; however, no professional medical body recommends using chest CTs as an early COVID-19 detection modality. This literature review examines the use of CT scans as a diagnostic tool for COVID-19. METHOD: A comprehensive search of research works published in peer-reviewed journals was carried out utilizing precisely stated criteria. The search was limited to English-language publications, and studies of COVID-19-positive patients diagnosed using both chest CT scans and RT-PCR tests were sought. For this review, four databases were consulted: these were the Cochrane and ScienceDirect catalogs, and the CINAHL and Medline databases made available by EBSCOhost. FINDINGS: In total, 285 possibly pertinent studies were found during an initial search. After applying inclusion and exclusion criteria, six studies remained for analysis. According to the included studies, chest CT scans were shown to have a 44 to 98% sensitivity and 25 to 96% specificity in terms of COVID-19 diagnosis. However, methodological limitations were identified in all studies included in this review. CONCLUSION: RT-PCR is still the suggested first-line diagnostic technique for COVID-19; while chest CT is adequate for use in symptomatic patients, it is not a sufficiently robust diagnostic tool for the primary screening of COVID-19.

A colorimetric reverse transcription-loop mediated isothermal amplification (RT-LAMP) method for the rapid detection of SARS-CoV-2 in Thailand.

COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global health threat. Timely identification of infected cases is important for appropriate patient management and the control of viral spread. Simple and cost-effective tests are required to increase access to testing and early case detection. Here, we describe a colorimetric reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method to detect SARS-CoV-2. The RT-LAMP could amplify the orf1ab sequence detectable by visual color change within 45 min at 63 °C. The limit of detection (LoD) for SARS-CoV-2 RNA was less than 100 copies (13.36) per reaction with no cross-amplification with other related viruses. Clinical evaluation using leftover RNA samples extracted from 163 nasopharyngeal swab specimens showed perfect agreement in negative (n = 124) and positive samples with cycle thresholds (Ct) < 34 cycles (n = 33) detected by real-time reverse transcription-polymerase chain reaction (RT-PCR), targeting RdRp and N genes as a reference. Overall, the diagnostic accuracy, sensitivity, specificity, positive and negative predictive values of RT-LAMP in testing were 96.32% (95% CI: 92.16-98.64%), 84.62% (95% CI: 68.47-94.14%), 100% (95% CI: 97.07-100.0%), 100% (95% CI: 89.42-100.0%), and 95.38% (95% CI: 90.22-98.29), respectively. This RT-LAMP assay is simple and reliable, with the potential to be an alternative for the rapid detection of SAR-CoV-2 with minimal time and fewer resources compared to real-time RT-PCR.

Evaluation of self-collected nasal, urine, and saliva samples for molecular detection of SARS-CoV-2 using an EUA approved RT-PCR assay and a laboratory developed LAMP SARS-CoV-2 test.

As the SARS-CoV-2 virus spread throughout the world, millions of positive cases of COVID-19 were registered and, even though there are millions of people already vaccinated against SARS-CoV-2, a large part of the global population remains vulnerable to contracting the virus. Massive nasopharyngeal sample collection in Puerto Rico at the beginning of the pandemic was limited by the scarcity of trained personnel and testing sites. To increase SARS-CoV-2 molecular testing availability, we evaluated the diagnostic accuracy of self-collected nasal, saliva, and urine samples using the TaqPath reverse transcription polymerase chain reaction (RT-PCR) COVID-19 kit to detect SARS-CoV-2. We also created a colorimetric loop-mediated isothermal amplification (LAMP) laboratory developed test (LDT) to detect SARS-CoV-2, as another strategy to increase the availability of molecular testing in community-based laboratories. Automated RNA extraction was performed in the KingFisher Flex instrument, followed by PCR quantification of SARS-CoV-2 on the 7500 Fast Dx RT-PCR using the TaqPath RT-PCR COVID-19 molecular test. Data was interpreted by the COVID-19 Interpretive Software from Applied Biosystems and statistically analyzed with Cohen's kappa coefficient (k). Cohen's kappa coefficient (k) for paired nasal and saliva samples showed moderate agreement (0.52). Saliva samples exhibited a higher viral load. We also observed 90% concordance between LifeGene-Biomarks' SARS-CoV-2 Rapid Colorimetric LAMP LDT and the TaqPath RT-PCR COVID-19 test. Our results suggest that self-collected saliva is superior to nasal and urine samples for COVID-19 testing. The results also suggest that the colorimetric LAMP LDT is a rapid alternative to RT-PCR tests for the detection of SARS-CoV-2. This test can be easily implemented in clinics, hospitals, the workplace, and at home; optimizing the surveillance and collection process, which helps mitigate global public health and socioeconomic upheaval caused by airborne pandemics.

RT-qPCR investigation of post-mortem tissues during COVID-19.

In 2020, there were numerous cases in Kazakhstan with clinical symptoms of COVID-19 but negative PCR results in nasopharyngeal and oropharyngeal swabs. The diagnosis was confirmed clinically and by CT scans (computed tomography). The problem with such negative PCR results for SARS-CoV-2 infection confirmation still exists and indicates the need to confirm the diagnosis in the bronchoalveolar lavage in such cases. There is also a lack of information about confirmation of SARS-CoV-2 infection in deceased patients. In this study, various tissue materials, including lungs, bronchi, and trachea, were examined from eight patients who died, presumably from SARS-CoV-2 infection, between 2020 and 2022. Naso/oropharyngeal swabs taken from these patients in hospitals tested PCR negative for SARS-CoV-2. This study presents a modified RNA isolation method based on a comparison of the most used methods for RNA isolation in laboratories: QIAamp Viral RNA Mini Kit and TRIzol-based method. This modified nucleic acid extraction protocol can be used to confirm SARS-CoV-2 infection by RT-qPCR in the tissues of deceased patients in disputed cases. RT-qPCR with RNA of SARS-CoV-2 re-extracted with such method from post-mortem tissues that were stored at -80 °C for more than 32 months still demonstrated high-yielding positive results.

Direct Detection of SARS-CoV-2 RNA in Saliva with Colorimetric RT-LAMP.

RT-LAMP is an effective alternative to RT-PCR-based diagnostics, offering high specificity, sensitivity, and rapid results. One notable advantage is the robustness of its enzymes, allowing for direct amplification from crude samples without the need for prior isolation of RNA. Colorimetric LAMP is particularly attractive as it eliminates the need for complex instrumentation, making it suitable for point-of-care applications. Here, we present a comprehensive step-by-step protocol for establishing an RT-LAMP-based test for direct detection of SARS-CoV-2 genomic RNA in saliva samples using different colorimetric detection methods. Importantly, this versatile test can be easily adapted to detect emerging pathogens.

Hand-held all-in-one (HAO) self-test kit for rapid and on-site detection of SARS-CoV-2 with colorimetric LAMP.

Throughout the COVID-19 pandemic, individuals potentially infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were forcibly recalled to local or central hospitals, where the diagnostic results were obtained a couple of days after the liquid biopsies were subjected to conventional polymerase chain reaction (PCR). This slow output of such a complex and time-consuming laboratory procedure hindered its widespread application. To overcome the limitations associated with such a centralized diagnostic system, we developed a hand-held and all-in-one type test kit in which the analytical results can be obtained in only 30 min. The test kit consists of three major steps for on-site SARS-CoV-2 RNA detection: 1) virus lysis by heat, 2) RNA enrichment by membrane, and 3) real-time detection by colorimetric loop-mediated isothermal amplification (c-LAMP). The proposed device operates in a sample-to-answer format, is fully automated, and reduces dependence on traditional laboratory settings, facilitating large-scale population screening.

Consecutive positive nucleic acid tests in COVID-19 patients: a retrospective cohort study in Fangcang hospitals, Shanghai.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) spread rapidly in Shanghai in February 2022. Patients with asymptomatic and mild symptoms were admitted to Fangcang shelter hospitals for centralized quarantine. METHODOLOGY: A total of 5,217 non-severe patients hospitalized in the Longyao Fangcang and Shilong Fangcang hospitals were included in the study. Demographic and clinical characteristics, comorbidity, exposure history, treatment and disease duration were analyzed. Univariate analysis and binomial logistic regression analysis were performed to identify the factors influencing nucleic acid change from positive to negative over 14 days. RESULTS: Consecutive positive nucleic acid test results (days) were significantly associated with advanced age (OR = 1.343, 95% CI 1.143 to 1.578, p < 0.001), smoking (OR = 0.510, 95% CI 0.327 to 0.796, p = 0.003) and vaccination (OR = 0.728, 95% CI 0.641 to 0.827, p < 0.001). However, there was no significant difference between asymptomatic and mild symptomatic patients (p = 0.187). In univariate analysis, comorbidities including diabetes, hypertension, cardiovascular system, malignant tumors, autoimmune diseases and cerebral apoplexy were associated with consecutive positive nucleic acid test results, but there was no significant difference in binomial logistics regression analysis. CONCLUSIONS: Aging and comorbid conditions lead to the prolongation of positive nucleic acid test results for several days. Improving vaccination coverage is beneficial for prevention and control of the epidemic. The management and treatment methods of Shanghai Fangcang shelter hospitals had important referential significance, which can provide valuable guidance for the prevention and control of the COVID-19 epidemic in the future.

Comparison of the diagnostic accuracy of the Pluslife Mini Dock RHAM technology with Abbott ID Now and Cepheid GenXpert: A retrospective evaluation study.

Rapid and sensitive detection of pathogens is critical in interrupting the transmission chain of infectious diseases. Currently, real-time (RT-)PCR represents the gold standard for the detection of SARS-CoV-2. RNase HII-assisted amplification (RHAM) is a promising technology, enabling reliable point-of-care (PoC) testing; however, its diagnostic accuracy has not yet been investigated. The present study compared the Pluslife Mini Dock (RHAM technology), with Abbott ID Now and Cepheid GeneXpert IV. The positive percent agreement (PPA) and negative percent agreement (NPA) were determined in 100 SARS-CoV-2 positive and 210 SARS-CoV-2 negative samples. Further, the reliability of the Pluslife Mini Dock was investigated in different SARS-CoV-2 variants (Delta and Omicron subvariants). The PPA was 99.00% for Pluslife, 100.00% for Abbott ID Now, and 99.00% for Cepheid GeneXpert, with an NPA of 100.00%, 98.90%, and 93.72%, respectively. Abbott ID Now demonstrated the highest rate of invalid results. All SARS-CoV-2 analysed variants were detected by the Pluslife device. Altogether, the Pluslife Mini Dock demonstrated a PPA of 99.16% (235/237) for CT < 36 and an NPA of 100.00% (313/313), respectively. In conclusion, the Pluslife Mini Dock demonstrated better analytical performance than Abbott ID Now and Cepheid GeneXpert IV, representing a highly accurate and rapid PoC alternative to RT-PCR.

Rapid Determination of SARS-CoV-2 Integrity and Infectivity by Using Propidium Monoazide Coupled with Digital Droplet PCR.

SARS-CoV-2 is a highly infectious virus responsible for the COVID-19 pandemic. Therefore, it is important to assess the risk of SARS-CoV-2 infection, especially in persistently positive patients. Rapid discrimination between infectious and non-infectious viruses aids in determining whether prevention, control, and treatment measures are necessary. For this purpose, a method was developed and utilized involving a pre-treatment with 50 µM of propidium monoazide (PMAxx, a DNA intercalant) combined with a digital droplet PCR (ddPCR). The ddPCR method was performed on 40 nasopharyngeal swabs (NPSs) both before and after treatment with PMAxx, revealing a reduction in the viral load at a mean of 0.9 Log copies/mL (SD ± 0.6 Log copies/mL). Furthermore, six samples were stratified based on the Ct values of SARS-CoV-2 RNA (Ct < 20, 20 < Ct < 30, Ct > 30) and analyzed to compare the results obtained via a ddPCR with viral isolation and a negative-chain PCR. Of the five samples found positive via a ddPCR after the PMAxx treatment, two of the samples showed the highest post-treatment SARS-CoV-2 loads. The virus was isolated in vitro from both samples and the negative strand chains were detected. In three NPS samples, SARS CoV-2 was present post-treatment at a low level; it was not isolated in vitro, and, when detected, the strand was negative. Our results indicate that the established method is useful for determining whether the SARS-CoV-2 within positive NPS samples is intact and capable of causing infection.

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.

Development of a novel Colorimetric Assay for the rapid diagnosis of Coronavirus disease 2019 from nasopharyngeal samples.

Emergence of Coronavirus disease 2019 (COVID-19) pandemic has posed a huge threat to public health. Rapid and reliable test to diagnose infected subjects is crucial for disease spread control. We developed a colorimetric test for COVID-19 detection using a Colorimetric Assay based on thiol-linked RNA modified gold nanoparticles (AuNPs) and oligonucleotide probes. This method was conducted on RNA from 200 pharyngeal swab samples initially tested by Real-Time polymerase chain reaction (RT-PCR) as gold standard. A specific oligonucleotide probe designed based on ORF1ab of COVID-19 was functionalized with AuNPs-probe conjugate. The exposure of AuNP-probe to isolated RNA samples was tested using hybridization. In this comparative study, the colorimetric functionalized AuNPs assay exhibited a detection limit of 25 copies/µL. It was higher in comparison to the RT-PCR method, which could only detect 15 copies/µL. The results demonstrated 100% specificity and 96% sensitivity for the developed method. Herein, we developed an incredibly rapid, simple and cost-effective Colorimetric Assay lasting approximately 30 min which could process considerably higher number of COVID-19 samples compared to the RT-PCR. This AuNP-probe conjugate colorimetric method could be considered the optimum alternatives for conventional diagnostic tools especially in over-populated and/or low-income countries.

Ensuring accuracy in the development and application of nucleic acid amplification tests (NAATs) for infectious disease.

Diagnostic tests were heralded as crucial during the Coronavirus disease (COVID-19) pandemic with most of the key methods using bioanalytical approaches that detected larger molecules (RNA, protein antigens or antibodies) rather than conventional clinical biochemical techniques. Nucleic Acid Amplification Tests (NAATs), like the Polymerase Chain Reaction (PCR), and other molecular methods, like sequencing (that often work in combination with NAATs), were essential to the diagnosis and management during COVID-19. This was exemplified both early in the pandemic but also later on, following the emergence of new genetic SARS-CoV-2 variants. The 100 day mission to respond to future pandemic threats highlights the need for effective diagnostics, therapeutics and vaccines. Of the three, diagnostics represents the first opportunity to manage infectious diseases while also being the most poorly supported in terms of the infrastructure needed to demonstrate effectiveness. Where performance targets exist, they are not well served by consensus on how to demonstrate they are being met; this includes analytical factors such as limit of detection (LOD) false positive results as well as how to approach clinical evaluation. The selection of gold standards or use of epidemiological factors such as predictive value, reference ranges or clinical thresholds are seldom correctly considered. The attention placed on molecular diagnostic tests during COVID-19 illustrates important considerations and assumptions on the use of these methods for infectious disease diagnosis and beyond. In this manuscript, we discuss state-of-the-art approaches to diagnostic evaluation and explore how they may be better tailored to diagnostic techniques like NAATs to maximise the impact of these highly versatile bioanalytical tools, both generally and during future outbreaks.

Accelerated diagnosis: a crosslinking catalytic hairpin assembly system for rapid and sensitive SARS-CoV-2 RNA detection.

The COVID-19 pandemic has underscored the urgent need for rapid and reliable strategies for early detection of SARS-CoV-2. In this study, we propose a DNA nanosphere-based crosslinking catalytic hairpin assembly (CCHA) system for the rapid and sensitive SARS-CoV-2 RNA detection. The CCHA system employs two DNA nanospheres functionalized with catalytic hairpin assembly (CHA) hairpins. The presence of target SARS-CoV-2 RNA initiated the crosslinking of DNA nanospheres via CHA process, leading to the amplification of fluorescence signals. As a result, the speed of SARS-CoV-2 diagnosis was enhanced by significantly increasing the local concentration of the reagents in a crosslinked DNA product, leading to a detection limit of 363 fM within 5 min. The robustness of this system has been validated in complex environments, such as fetal bovine serum and saliva. Hence, the proposed CCHA system offers an efficient and simple approach for rapid detection of SARS-CoV-2 RNA, holding substantial promise for enhancing COVID-19 diagnosis.

Rapid one-pot isothermal amplification reassembled of fluorescent RNA aptamer for SARS-CoV-2 detection.

The outbreak of SARS-CoV-2 poses a serious threat to human life and health. A rapid nucleic acid tests can effectively curb the spread of the disease. With the advantages of fluorescent RNA aptamers, low background and high sensitivity. A variety of fluorescent RNA aptamer sensors have been developed for the detection of nucleic acid. Here, we report a hypersensitive detection platform in which SARS-CoV-2 initiates RTF-EXPAR to amplify trigger fragments. This activation leads to the reassembled of the SRB2 fluorescent RNA aptamer, restoring its secondary structure for SR-DN binding and turn-on fluorescence. The platform completes the assay in 30 min and all reactions occur in one tube. The detection limit is as low as 116 aM. Significantly, the platform's quantitative analyses were almost identical to qPCR results in simulated tests of positive samples. In conclusion, the platform is sensitive, accurate and provides a new protocol for point-of-care testing of viruses.

Diagnostic performance of a point-of-care molecular system for the detection of SARS-CoV-2 in Peru. / Rendimiento diagnóstico de un sistema molecular de uso en el punto de atención para la detección de SARS-CoV-2 en Perú.

The present study assessed the diagnostic performance of the Xpert®Xpress SARS-CoV-2 test in comparison with the Charité protocol real-time RT PCR for the detection of SARS-CoV-2 in Peruvian patients. This was a diagnostic test study that included 100 nasal and pharyngeal swab samples. We obtained an overall concordance of 98.70% (95%CI: 92.98-99.97), with a kappa coefficient of 0.97 (95%CI: 0.86-1.00) and sensitivity and relative specificity rates of 100% and 96.15%, respectively. Additionally, the percentage of the area under the ROC curve was 98.08% in both cases, and an analytical specificity rate of 100% was obtained for the different respiratory viruses evaluated. In conclusion, the Xpert®Xpress SARS-CoV-2 test, by using nasal and pharyngeal swab samples, was highly sensitive and specific, and the kappa coefficient showed an excellent correlation when compared to the reference test. Motivation for the study. To describe and evaluate a closed molecular platform, easy to use and of importance in Peru for the management of diseases of public health priority, now implemented for the detection of SARS-CoV-2. Main findings. Highly sensitive and specific molecular test, with excellent correlation compared to the reference test for detecting SARS-CoV-2. Implications. Can be used in point-of-care laboratories for rapid molecular detection of different infectious agents, including SARS-CoV-2. Little expertise and minimal infrastructure are required to implement it.

En el presente estudio se estimó el rendimiento diagnóstico de la prueba Xpert®Xpress SARS-CoV-2 en comparación con la RT PCR en tiempo real-protocolo Charité, para la detección de SARS-CoV-2 en pacientes peruanos. Se trató de un diseño de prueba diagnóstica que incluyó 100 muestras de hisopado nasal y faríngeo. Se obtuvo una concordancia global de 98,70% (IC95%: 92,98-99,97), con un coeficiente kappa de 0,97 (IC95%: 0,86-1.00); se estimó una sensibilidad y especificad relativa de 100% y 96,15%, respectivamente. Adicionalmente, el porcentaje del área bajo la curva ROC fue 98,08% en ambos casos y se obtuvo una especificidad analítica del 100% para los diferentes virus respiratorios evaluados. En conclusión, la prueba Xpert®Xpress SARS-CoV-2 a partir de muestras de hisopado nasal y faríngeo fue altamente sensible y específica, así mismo el coeficiente kappa mostró una excelente correlación, al compararla con la prueba de referencia. Motivación para realizar el estudio. Descripción y evaluación de una plataforma molecular cerrada, de fácil uso y de importancia en el Perú para el manejo de enfermedades de prioridad en salud pública, ahora implementada para la detección de SARS-CoV-2. Principales hallazgos. Prueba molecular altamente sensible y específica, con una correlación excelente con respecto al referente para detectar SARS-CoV-2. Implicancias. Puede ser utilizada en los laboratorios que se encuentran en los puntos de atención del paciente para la detección molecular rápida de diferentes agentes infecciosos, incluido el SARS-CoV-2. Se necesita poca experticia y mínima infraestructura para poder implementarla.

Evaluations of modes of pooling specimens for COVID-19 screened by quantitative PCR and droplet digital PCR.

Though pooling samples for SARS-CoV-2 detection has effectively met the need for rapid diagnostic and screening tests, many factors can influence the sensitivity of a pooled test. In this study, we conducted a simulation experiment to evaluate modes of pooling specimens and aimed at formulating an optimal pooling strategy. We focussed on the type of swab, their solvent adsorption ability, pool size, pooling volume, and different factors affecting the quality of preserving RNA by different virus solutions. Both quantitative PCR and digital PCR were used to evaluate the sampling performance. In addition, we determined the detection limit by sampling which is simulated from the virus of different titers and evaluated the effect of sample-storage conditions by determining the viral load after storage. We found that flocked swabs were better than fibre swabs. The RNA-preserving ability of the non-inactivating virus solution was slightly better than that of the inactivating virus solution. The optimal pooling strategy was a pool size of 10 samples in a total volume of 9 mL. Storing the collected samples at 4 °C or 25 °C for up to 48 h had little effect on the detection sensitivity. Further, we observed that our optimal pooling strategy performed equally well as the single-tube test did. In clinical applications, we recommend adopting this pooling strategy for low-risk populations to improve screening efficiency and shape future strategies for detecting and managing other respiratory pathogens, thus contributing to preparedness for future public health challenges.

An Immiscible-Phase Filtration Device for Isolation, Amplification, and Detection of Nucleic Acids for Clinical Diagnostics.

Clinical diagnostics of infectious diseases via nucleic acid amplification tests (NAATs) depend on a separate step of isolation of nucleic acids from cells/viruses embedded in complex biological matrices. The most recent example has been reverse transcription polymerase chain reaction (RT-PCR) for amplification and detection of SARS-CoV-2 RNA for COVID-19 diagnostics. Kits for RNA extraction and purification are commercially available; however, their integration with amplification systems is generally lacking, resulting in two separate steps, i.e., sample preparation and amplification. This makes NAATs more time-consuming, requiring skilled personnel, and can increase the likelihood of contamination. Here, we describe a setup and methodology to perform the quick extraction and detection of nucleic acids in an integrated manner. In particular, we focus on the use of an immiscible filtration device for capture, isolation, concentration, amplification, and colorimetric detection of SARS-CoV-2 RNA.

Reliability of the rapid antigen test for diagnosis of SARS-CoV-2 in Tunisia.

Background: Early and accurate diagnosis is crucial for preventing the spread of SARS-CoV-2 infection. The rapid antigen test was developed for testing infection, and it was necessary to assess its performance before widespread use in Tunisia. Aim: To evaluate the effectiveness of a rapid antigen test for the detection of SARS-CoV-2 in nasopharyngeal swabs in Tunisia. Methods: Nasopharyngeal samples were taken from COVID-19 suspected cases between October and December 2020 and tested using the Standard Q COVID-19 Ag test (SD-Biosensor, Republic of Korea) and real-time reverse transcription polymerase chain reaction (RT­PCR). Results: Overall, 4539 patients were tested. Of the total study population (N = 4539), 82.5% of positive samples remained positive with the rapid antigen test, while 20.2% (470/2321) of samples that were negative with rapid antigen test were confirmed positive with RT-PCR, giving a negative predictive value of 79.8% for the rapid antigen test. The sensitivity and negative predictive value of the rapid antigen test were 70.2% and 65.8%, respectively. These results improved to 96.4% and 92.8%, respectively, when considering the cycle threshold value by RT-PCR below 25. Conclusion: Although the rapid antigen test was less sensitive than RT-PCR, its ability to rapidly detect individuals with high viral loads makes it suitable for use during an epidemic.

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.