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.

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.

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.

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.

The potential of tailed amplicons for SARS-CoV-2 detection in Nucleic Acid Lateral Flow Assays.

Nucleic Acid Lateral Flow Assays (NALFAs) are a promising solution for the point-of-care detection of viruses like SARS-CoV-2. However, they show some drawbacks, such as the great dependency on the use of antibodies and the need for post-amplification protocols that enable the preparation of amplicons for effective readings, as well as low sensitivity. Here, we developed amplicons of a specific SARS-CoV-2 gene tailed with single-strand DNA (ssDNA) sequences to hybridize with DNA probes immobilized on the NALFA strips, thus overcoming the aforementioned problems. Results have shown that tailed primers have not compromised the amplification efficiency and allowed the correct detection of the amplicons in the lateral flow strip. This approach has presented a limit of detection (LOD) of 25 RNA copies /reaction mix (1 copy/µL) and the test of cross-reactivity with other related viruses has not shown any cross-reactivity. Twenty clinical samples were evaluated by NALFA and simultaneously compared with the gold standard RT-qPCR protocol, originating equal results. Although the number of clinical specimens tested being relatively small, this indicates a sensitivity and specificity both of 100%. In short, an alternative NALFA was successfully implemented, rendering an accurate route for SARS-CoV-2 diagnosis, compatible with low-resource settings.

Logistic regression analysis of the value of biomarkers, clinical symptoms, and imaging examinations in COVID-19 for SARS-CoV-2 nucleic acid detection.

The detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) nucleic acid detection provides a direct basis for diagnosing Coronavirus Disease 2019. However, nucleic acid test false-negative results are common in practice and may lead to missed diagnosis. Certain biomarkers, clinical symptoms, and imaging examinations are related to SARS-CoV-2 nucleic acid detection and potential predictors. We examined nucleic acid test results, biomarkers, clinical symptoms, and imaging examination data for 116 confirmed cases and asymptomatic infections in Zhuhai, China. Patients were divided into nucleic acid-positive and -false-negative groups. Predictive values of biomarkers, symptoms, and imaging for the nucleic acid-positive rate were calculated by Least Absolute Shrinkage and Selection Operators regression analysis and binary logistic regression analysis, and areas under the curve of these indicators were calculated. Hemoglobin (OR = 1.018, 95% CI: 1.006-1.030; P = .004) was higher in the respiratory tract-positive group than the nucleic acid-negative group, but platelets (OR = 0.996, 95% CI: 0.993-0.999; P = .021) and eosinophils (OR = 0.013, 95% CI: 0.001-0.253; P = .004) were lower; areas under the curve were 0.563, 0.614, and 0.642, respectively. Some biomarkers can predict SARS-CoV-2 viral nucleic acid detection rates in Coronavirus Disease 2019 and are potential auxiliary diagnostic tests.

Development and evaluation of a lyophilization protocol for colorimetric RT-LAMP diagnostic assay for COVID-19.

Molecular diagnostics involving nucleic acids (DNA and RNA) are regarded as extremely functional tools. During the 2020 global health crisis, efforts intensified to optimize the production and delivery of molecular diagnostic kits for detecting SARS-CoV-2. During this period, RT-LAMP emerged as a significant focus. However, the thermolability of the reagents used in this technique necessitates special low-temperature infrastructure for transport, storage, and conservation. These requirements limit distribution capacity and necessitate cost-increasing adaptations. Consequently, this report details the development of a lyophilization protocol for reagents in a colorimetric RT-LAMP diagnostic kit to detect SARS-CoV-2, facilitating room-temperature transport and storage. We conducted tests to identify the ideal excipients that maintain the molecular integrity of the reagents and ensure their stability during room-temperature storage and transport. The optimal condition identified involved adding 5% PEG 8000 and 75 mM trehalose to the RT-LAMP reaction, which enabled stability at room temperature for up to 28 days and yielded an analytical and diagnostic sensitivity and specificity of 83.33% and 90%, respectively, for detecting SARS-CoV-2. This study presents the results of a lyophilized colorimetric RT-LAMP COVID-19 detection assay with diagnostic sensitivity and specificity comparable to RT-qPCR, particularly in samples with high viral load.

Lateral flow strip assay of a gene segment in the COVID-19 virus with combined dual readout mode and preliminary multisite hybrid chain reaction amplification.

The past and present scenario of COVID-19 has revealed the necessity of simple point-of-care tests. When combined with the great advantages of amplification, lateral flow assay nucleic acid analysis represents a more sensitive molecular diagnostic technique compared to universal protein analysis. Room temperature operation, an enzyme-free nature, and in situ elongation make hybrid chain reaction amplification (HCR) a good candidate for amplified combined lateral flow assays (LFAs). Since dual modes of detection can not only satisfy different application scenarios, but also reduce the false-negative rate, in this paper, visual and fluorescent detection based on labelling with colloidal gold nanoparticles and fluorescence labelling were incorporated into a HCR integrated with a LFA. The detection assay was finished in 30 minutes. The linear relationship between the signal and the concentration of the characteristic segment in the COVID-19 ORF gene was demonstrated. The obtained detection limits of as low as 10 fM (6.02 × 103 copies per mL) and 1 fM (6.02 × 102 copies per mL), respectively, were comparable with those in the literature. The multi-site HCR amplification integrated with LFA of a 1053 bp nucleic acid chain was also preliminarily studied, and tri-site amplification was found to exhibit higher signal intensity than single-site amplification. This study provides a promising strategy for simple, sensitive, and wide-ranging detection of pathogenic bacteria.

Original article: novelty of Canadian manufacture nasopharyngeal swabs for collection of samples being tested for SARS-CoV-2 in a pandemic setting.

Objectives: The COVID-19 pandemic caused a global shortage of nasopharyngeal (NP) swabs, required for RT-PCR testing. Canadian manufacturers were contacted to share NP swab innovations. The primary objective was to determine whether novel NP test swabs were comparable to commercially available swabs regarding user characteristics, ability to collect a specimen, and diagnostic performance using RT-PCR testing. Methods: Participants were randomized by swab (test/control) and nostril (left/right). A calculated positive percent agreement ≥90% was considered successful. Mean Ct values of viral genes and housekeeping gene (RNase P) were considered similar if a Ct difference ≤ 2 between control and test group was obtained. There also was a qualitative assessment of swabs usability. Results: 647 participants were enrolled from Huaycan Hospital in Lima, Peru, distributed over 8 NP swabs brands. Seven brands agreed to share their results. There were no statistically significant differences between the test swabs of these 7 brands and control swabs. Conclusion: All the seven brands are comparable to the commercially available flocked swabs used for SARS-CoV-2 regarding test results agreement, ability to collect a specimen, and user characteristics.

A Fast and Sensitive One-Tube SARS-CoV-2 Detection Platform Based on RTX-PCR and Pyrococcus furiosus Argonaute.

Since SARS-CoV-2 is a highly transmissible virus, alternative reliable, fast, and cost-effective methods are still needed to prevent virus spread that can be applied in the laboratory and for point-of-care testing. Reverse transcription real-time fluorescence quantitative PCR (RT-qPCR) is currently the gold criteria for detecting RNA viruses, which requires reverse transcriptase to reverse transcribe viral RNA into cDNA, and fluorescence quantitative PCR detection was subsequently performed. The frequently used reverse transcriptase is thermolabile; the detection process is composed of two steps: the reverse transcription reaction at a relatively low temperature, and the qPCR performed at a relatively high temperature, moreover, the RNA to be detected needs to pretreated if they had advanced structure. Here, we develop a fast and sensitive one-tube SARS-CoV-2 detection platform based on Ultra-fast RTX-PCR and Pyrococcus furiosus Argonaute-mediated Nucleic acid Detection (PAND) technology (URPAND). URPAND was achieved ultra-fast RTX-PCR process based on a thermostable RTX (exo-) with both reverse transcriptase and DNA polymerase activity. The URPAND can be completed RT-PCR and PAND to detect nucleic acid in one tube within 30 min. This method can specifically detect SARS-CoV-2 with a low detection limit of 100 copies/mL. The diagnostic results of clinical samples with one-tube URPAND displayed 100% consistence with RT-qPCR test. Moreover, URPAND was also applied to identify SARS-CoV-2 D614G mutant due to its single-nucleotide specificity. The URPAND platform is rapid, accurate, tube closed, one-tube, easy-to-operate and free of large instruments, which provides a new strategy to the detection of SARS-CoV-2 and other RNA viruses.

Performance evaluation of a newly developed 2019-nCoV nucleic acid detection kit based on Ion Proton sequencing platform and its comparison with the MGI Tech (DNBSEQ-G99) platform.

PURPOSE: To evaluate the performance of a newly developed 2019-nCoV nucleic acid detection kit based on Ion Proton sequencing platform and make comparation with MGI Tech (DNBSEQ-G99) platform. METHODS: References and clinical samples were used to evaluate the precision, agreement rate, limit of detection (LOD), anti-interference ability and analytical specificity. Twenty-seven clinical specimens were used to make comparison between two platforms. RESULTS: The kit showed good intra-assay, inter-assay, inter-day precision between different operators and laboratories, fine agreement rate with references, a relatively low LOD of 1 × 103 copies/ml, anti-interference capability of 5 % whole blood and 1mg/ml mucin and no cross reaction with twenty-nine common clinical pathogens. Consistency of variant classification was observed between two platforms. The WGS from Ion Proton tended to have higher coverage and less missing data. CONCLUSIONS: The newly developed kit has shown satisfactory performances and excellent consistency with DNBSEQ-G99, making it a good alternative choice clinically.

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.

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.

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.

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.

Taqman PACMAN: a simple molecular approach for positive rapid antigen test confirmation during periods of low prevalence.

Antigen-based rapid diagnostic tests (Ag-RDTs) were widely deployed to enhance SARS-CoV-2 testing capacity during the COVID-19 pandemic. Consistent with national guidance for low prevalence settings, positive Ag-RDTs were confirmed using nucleic acid amplification tests (NAATs) to avoid false positive results. However, increasing demands for positive Ag-RDT confirmation competed with other testing priorities in clinical laboratories. This work hypothesized that real-time RT-PCR without nucleic acid extraction (NAE) would be sufficiently sensitive to support positive Ag-RDT confirmation. Ag-RDT and NAAT results from community-based asymptomatic testing sites prior to the omicron variant wave were compared to calculate the weekly false positive rate (FPR) and false detection rate (FDR). Real-time RT-PCR was compared with and without NAE using 752 specimens previously tested positive for SARS-CoV-2 using commercial NAATs and 344 specimens from Ag-RDT-positive individuals. The impact of SARS-CoV-2 prevalence on laboratory resources required to sustain Ag-RDT confirmation was modeled for the RT-PCR with and without NAE. Overall, FPR was low [0.07% (222/330,763)] in asymptomatic testing sites, but FDR was high [30.7% (222/724)]. When RT-PCR was compared with and without NAE, 100% concordance was obtained with NAAT-positive specimens, including those from Ag-RDT-positive individuals. NAE-free RT-PCR significantly reduced time to results, human resources, and overall costs. A 30.7% FDR reaffirms the need for NAAT-based confirmation of positive Ag-RDT results during low SARS-CoV-2 prevalence. NAE-free RT-PCR was shown to be a simple and cost-sparing NAAT-based solution for positive Ag-RDT confirmation, and its implementation supported data-driven broader Ag-RDT deployment into communities, workplaces, and households. IMPORTANCE: Rapid antigen testing for SARS-CoV-2 was widely deployed during the COVID-19 pandemic. In settings of low prevalence, national guidance recommends that positive antigen test results be confirmed with molecular testing. Given the high testing burden on clinical laboratories during the COVID-19 pandemic, the high volume of positive antigen tests submitted for confirmatory testing posed challenges for laboratory workflow. This study demonstrated that a simple PCR method without prior nucleic acid purification is an accurate and cost-effective solution for positive rapid antigen test confirmation. Implementing this method allowed molecular confirmatory testing for positive antigen tests to be sustained as antigen testing was expanded into large populations such as workplaces, schools, and households.

Optimisation and evaluation of viral genomic sequencing of SARS-CoV-2 rapid diagnostic tests: a laboratory and cohort-based study.

BACKGROUND: Sequencing of SARS-CoV-2 from rapid diagnostic tests (RDTs) can bolster viral genomic surveillance efforts; however, approaches to maximise and standardise pathogen genome recovery from RDTs remain underdeveloped. We aimed to systematically optimise the elution of genetic material from RDT components and to evaluate the efficacy of RDT sequencing for outbreak investigation. METHODS: In this laboratory and cohort-based study we seeded RDTs with inactivated SARS-CoV-2 to optimise the elution of genomic material from RDT lateral flow strips. We measured the effect of changes in buffer type, time in buffer, and rotation on PCR cycle threshold (Ct) value. We recruited individuals older than 18 years residing in the greater Boston area, MA, USA, from July 18 to Nov 5, 2022, via email advertising to students and staff at Harvard University, MA, USA, and via broad social media advertising. All individuals recruited were within 5 days of a positive diagnostic test for SARS-CoV-2; no other relevant exclusion criteria were applied. Each individual completed two RDTs and one PCR swab. On Dec 29, 2022, we also collected RDTs from a convenience sample of individuals who were positive for SARS-CoV-2 and associated with an outbreak at a senior housing facility in MA, USA. We extracted all returned PCR swabs and RDT components (ie, swab, strip, or buffer); samples with a Ct of less than 40 were subject to amplicon sequencing. We compared the efficacy of elution and sequencing across RDT brands and components and used RDT-derived sequences to infer transmission links within the outbreak at the senior housing facility. We conducted metagenomic sequencing of negative RDTs from symptomatic individuals living in the senior housing facility. FINDINGS: Neither elution duration of greater than 10 min nor rotation during elution impacted viral titres. Elution in Buffer AVL (Ct=31·4) and Tris-EDTA Buffer (Ct=30·8) were equivalent (p=0·34); AVL outperformed elution in lysis buffer and 50% lysis buffer (Ct=40·0, p=0·0029 for both) as well as Universal Viral Transport Medium (Ct=36·7, p=0·079). Performance of RDT strips was poorer than that of matched PCR swabs (mean Ct difference 10·2 [SD 4·3], p<0·0001); however, RDT swabs performed similarly to PCR swabs (mean Ct difference 4·1 [5·2], p=0·055). No RDT brand significantly outperformed another. Across sample types, viral load predicted the viral genome assembly length. We assembled greater than 80% complete genomes from 12 of 17 RDT-derived swabs, three of 18 strips, and four of 11 residual buffers. We generated outbreak-associated SARS-CoV-2 genomes using both amplicon and metagenomic sequencing and identified multiple introductions of the virus that resulted in downstream transmission. INTERPRETATION: RDT-derived swabs are a reasonable alternative to PCR swabs for viral genomic surveillance and outbreak investigation. RDT-derived lateral flow strips yield accurate, but significantly fewer, viral reads than matched PCR swabs. Metagenomic sequencing of negative RDTs can identify viruses that might underlie patient symptoms. FUNDING: The National Science Foundation, the Hertz Foundation, the National Institute of General Medical Sciences, Harvard Medical School, the Howard Hughes Medical Institute, the US Centers for Disease Control and Prevention, the Broad Institute and the National Institute of Allergy and Infectious Diseases.

Paper-based loop-mediated isothermal amplification and CRISPR integrated platform for on-site nucleic acid testing of pathogens.

We report the development and initial validation of a paper-based nucleic acid testing platform that integrates Loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR) technology, referred to as PLACID (Paper-based LAMP-CRISPR Integrated Diagnostics). LAMP eliminates the need for thermal cycling, resulting in simplified instrumentation, and the CRISPR-associated protein (Cas 12a) system eliminates false positive signals from LAMP products, resulting in highly selective and sensitive assays. We optimized the assay to perform both amplification and detection entirely on paper, eliminating the need for complex fluid handling steps and lateral flow assay transfers. Additionally, we engineered a smartphone-operated system that includes a low-powered, non-contact IR heating chamber to actuate paper-based LAMP and CRISPR reactions and enable the detection of fluorescent signals from the paper. The platform demonstrates high specificity and sensitivity in detecting nucleic acid targets with a limit of detection of 50 copies/µL. We integrate an equipment-free sample preparation separation technology designed to streamline the preparation of crude samples prior to nucleic acid testing. The practical utility of our platform is demonstrated by the successful detection of spiked SARS-CoV-2 RNA fragments in saliva, E. Coli in soil, and pathogenic E. Coli in clinically fecal samples of infected patients. Furthermore, we demonstrate that the paper-based LAMP CRISPR chips employed in our assays possess a shelf life of several weeks, establishing them as viable candidates for on-site diagnostics.

Soft interface confined DNA walker for sensitive and specific detection of SARS-CoV-2 variants.

Nucleic acid detection is conducive to preventing the spread of COVID-19 pandemic. In this work, we successfully designed a soft interface confined DNA walker by anchoring hairpin reporter probes on cell membranes for the detection of SARS-CoV-2 variants. In the presence of target RNA, the cyclic self-assembly reaction occurred between hairpin probes H1 and H2, and the continuous walking of target RNA on cell membranes led to the gradual amplification of fluorescence signal. The enrichment of H1 on membranes and the unique fluidity of membranes promoted the collision efficiency between DNA strands in the reaction process, endowing this method with high sensitivity. In addition, the double-blind test of synthetic RNA in 5% normal human serum demonstrated the good stability and anti-interference in complex environment of this method, which exhibited great potential in clinical diagnostics.

Evaluation of the analytical and clinical performance of two RT-PCR based point-of-care tests; Cepheid Xpert® Xpress CoV-2/Flu/RSV plus and SD BioSensor STANDARD™ M10 Flu/RSV/SARS-CoV-2.

BACKGROUND: Rapid and accurate detection of viral respiratory infections is important for infection control measures. This study compares the analytical and clinical performance of the Xpert® Xpress CoV-2/Flu/RSV plus test ("Xpert", Cepheid) and the STANDARD™ M10 Flu/RSV/SARS-CoV-2 test ("M10", SD Biosensor). Both tests are quadruplex RT-PCR assays for rapid diagnosis of SARS-CoV-2, influenza A/B and RSV. STUDY DESIGN: Analytical sensitivities were determined by limit of detection for SARS-CoV-2, influenza A, influenza B and RSV, respectively. Additionally, the clinical performance of the Xpert and the M10 tests was evaluated against standard-of-care RT-PCR by testing of 492 clinical specimens. RESULTS: The analytical sensitivities for Xpert versus M10 test was 10, 50, 50 and 300 versus 300, 200, 800 and 1500 copies/mL for SARS-CoV-2, influenza A, influenza B and RSV, respectively. Clinical sensitivity for the Xpert test was superior across all four pathogens compared to the M10 test. Xpert showed clinical sensitivity of 100 % in all Ct-ranges for all four pathogens whereas M10 showed clinical sensitivity of 100 % in the 25-30 Ct-range, 84-100 % in the 30-35 Ct-range and 47-67 % in the >35 Ct-range across the four pathogens. Translating into real-life clinical sensitivity, the Xpert would detect 100 % of all four pathogens, whereas M10 would detect 92.1, 92.4, 84.8 and 94.7 % for SARS-CoV-2, influenza A, influenza B and RSV. CONCLUSION: This study demonstrates improved analytical and clinical performance of Xpert Xpress CoV-2/Flu/RSV plus compared to STANDARD M10 Flu/RSV/SARS-CoV-2, which is important for ensuring accuracy of diagnosis at all stages of a respiratory infection.