SwabExpress: An End-to-End Protocol for Extraction-Free COVID-19 Testing.

BACKGROUND: The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse-transcription PCR (RT-qPCR). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce. To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction. METHODS: We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral inactivation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance. RESULTS: After optimization, SwabExpress has a low limit of detection at 2-4 molecules/µL, 100% sensitivity, and 99.4% specificity when compared side by side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time. CONCLUSION: SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.

Purification/Amplification-Free RNA Detection Platform for Rapid and Multiplex Diagnosis of Plant Viral Infections.

Genetic tests are highly sensitive, and quantitative methods for diagnosing human viral infections, including COVID-19, are also being used to diagnose plant diseases in various agricultural settings. Conventional genetic tests for plant viruses are mostly based on methods that require purification and amplification of viral genomes from plant samples, which generally take several hours in total, making it difficult to use them in rapid detection at point-of-care testing (POCT). In this study, we developed Direct-SATORI, a rapid and robust genetic test that eliminates the purification and amplification processes of viral genomes by extending the recently developed amplification-free digital RNA detection platform called SATORI, allowing the detection of various plant viral genes in a total of less than 15 min with a limit of detection (LoD) of 98 ∼ copies/µL using tomato viruses as an example. In addition, the platform can simultaneously detect eight plant viruses directly from ∼1 mg of tomato leaves with a sensitivity of 96% and a specificity of 99%. Direct-SATORI can be applied to various infections related to RNA viruses, and its practical use is highly anticipated as a versatile platform for plant disease diagnostics in the future.

SARS-Cov-2 Spike-S1 Antigen Test Strip with High Sensitivity Endowed by High-Affinity Antibodies and Brightly Fluorescent QDs/Silica Nanospheres.

The extensive research into developing novel strategies for detecting respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in clinical specimens, especially the sensitive point-of-care testing method, is still urgently needed to reach rapid screening of viral infections. Herein, a new lateral flow immunoassay (LFIA) platform was reported for the detection of SARS-CoV-2 spike-S1 protein antigens, in which four sensitive and specific SARS-CoV-2 mouse monoclonal antibodies (MmAbs) were tailored by using quantum dot (QD)-loaded dendritic mesoporous silica nanoparticles modified further for achieving the -COOH group surface coating (named Q/S-COOH nanospheres). Importantly, compact QD adsorption was achieved in mesoporous channels of silica nanoparticles on account of highly accessible central-radial pores and electrostatic interactions, leading to significant signal amplification. As such, a limit of detection for SARS-CoV-2 spike-S1 testing was found to be 0.03 ng/mL, which is lower compared with those of AuNPs-LFIA (traditional colloidal gold nanoparticles, Au NPs) and enzyme-linked immunosorbent assay methods. These results show that optimizing the affinity of antibody and the intensity of fluorescent nanospheres simultaneously is of great significance to improve the sensitivity of LFIA.

Optimal allocation of scarce PCR tests during the COVID-19 pandemic.

BACKGROUND/AIM: During the coronavirus disease (COVID-19) pandemic, Germany and various other countries experienced a shortage of polymerase chain reaction (PCR) laboratory tests due to the highly transmissible SARS-CoV-2 Omicron variant that drove an unprecedented surge of infections. This study developed a mathematical model that optimizes diagnostic capacity with lab-based PCR testing. METHODS: A mathematical model was constructed to determine the value of PCR testing in relation to the pre-test probability of COVID-19. Furthermore, the model derives the lower and upper bounds for the threshold pre-test probability of the designated priority group. The model was applied in a German setting using the PCR test-positivity rate at the beginning of February 2022. RESULTS: The value function of PCR testing is bell-shaped with respect to the pre-test probability, reaching a maximum at a pre-test probability of 0.5. Based on a PCR test-positivity rate of 0.3 and assuming that at least two thirds of the tested population have a pre-test probability below, lower and higher pre-test probability thresholds are ≥ 0.1 and 0.7, respectively. Therefore, individuals who have a 25% likelihood of testing positive because they exhibit symptoms should be a higher priority for PCR testing. Furthermore, a positive rapid antigen test in asymptomatic individuals with no known exposure to COVID-19 should be confirmed using PCR. Yet, symptomatic individuals with a positive RAT should be excluded from PCR testing. CONCLUSION: A mathematical model that allows for the optimal allocation of scarce PCR tests during the COVID-19 pandemic was developed.

Assessing the comparability of cycle threshold values derived from five external quality assessment rounds for omicron nucleic acid testing.

BACKGROUND: A variety of open-system real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays for several acute respiratory syndrome coronavirus 2 are currently in use. This study aimed to ensure the quality of omicron nucleic acid testing and to assess the comparability of cycle threshold (Ct) values derived from RT-PCR. METHODS: Five external quality assessment (EQA) rounds using the omicron virus-like particles were organized between February 2022 and June 2022. RESULTS: A total of 1401 qualitative EQA reports have been collected. The overall positive percentage agreement was 99.72%, the negative percentage agreement was 99.75%, and the percent agreement was 99.73%. This study observed a significant variance in Ct values derived from different test systems. There was a wide heterogeneity in PCR efficiency among different RT-PCR kits and inter-laboratories. CONCLUSION: There was strong concordance among laboratories performing qualitative omicron nucleic acid testing. Ct values from qualitative RT-PCR tests should not be used for clinical or epidemiological decision-making to avoid the potential for misinterpretation of the results.

A rapid and highly sensitive multiple detection of human adenovirus type 3, type 7 and respiratory syncytial virus by recombinase-aided reverse transcription PCR.

BACKGROUND: Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase-aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology. METHODS: In this study, we developed and validated the multiplex reverse transcription recombinase-aided PCR (multiplex RT-RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process. RESULTS: Using recombinant plasmids, the sensitivity of multiplex RT-RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT-RAP showed no cross-reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT-RAP and the results were found to be consistent with those of corresponding RT-qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT-RAP was two to eightfold higher than that of corresponding RT-qPCR. CONCLUSION: We conclude the multiplex RT-RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load.

Standardization of in-house anti-IgG and IgA ELISAs for the detection of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). RT-PCR detection of viral RNA represents the gold standard method for diagnosis of COVID-19. However, multiple diagnostic tests are needed for acute disease diagnosis and assessing immunity during the COVID-19 outbreak. Here, we developed in-house anti-RBD IgG and IgA enzyme-linked immunosorbent assays (ELISAs) using a well-defined serum sample panel for screening and identification of human SARS-CoV-2 infection. We found that our in-house anti-SARS-CoV-2 IgG ELISA displayed a 93.5% sensitivity and 98.8% specificity whereas our in-house anti-SARS-CoV-2 IgA ELISA provided assay sensitivity and specificity at 89.5% and 99.4%, respectively. The agreement kappa values of our in-house anti-SARS-CoV-2 IgG and IgA ELISA assays were deemed to be excellent and fair, respectively, when compared to RT-PCR and excellent for both assays when compared to Euroimmun anti-SARS-CoV-2 IgG and IgA ELISAs. These data indicate that our in-house anti-SARS-CoV-2 IgG and IgA ELISAs are compatible performing assays for the detection of SARS-CoV-2 infection.

Predicting missed health care visits during the COVID-19 pandemic using machine learning methods: evidence from 55,500 individuals from 28 European countries.

BACKGROUND: Pandemics such as the COVID-19 pandemic and other severe health care disruptions endanger individuals to miss essential care. Machine learning models that predict which patients are at greatest risk of missing care visits can help health administrators prioritize retentions efforts towards patients with the most need. Such approaches may be especially useful for efficiently targeting interventions for health systems overburdened during states of emergency. METHODS: We use data on missed health care visits from over 55,500 respondents of the Survey of Health, Ageing and Retirement in Europe (SHARE) COVID-19 surveys (June - August 2020 and June - August 2021) with longitudinal data from waves 1-8 (April 2004 - March 2020). We compare the performance of four machine learning algorithms (stepwise selection, lasso, random forest, and neural networks) to predict missed health care visits during the first COVID-19 survey based on common patient characteristics available to most health care providers. We test the prediction accuracy, sensitivity, and specificity of the selected models for the first COVID-19 survey by employing 5-fold cross-validation, and test the out-of-sample performance of the models by applying them to the data from the second COVID-19 survey. RESULTS: Within our sample, 15.5% of the respondents reported any missed essential health care visit due to the COVID-19 pandemic. All four machine learning methods perform similarly in their predictive power. All models have an area under the curve (AUC) of around 0.61, outperforming random prediction. This performance is sustained for data from the second COVID-19 wave one year later, with an AUC of 0.59 for men and 0.61 for women. When classifying all men (women) with a predicted risk of 0.135 (0.170) or higher as being at risk of missing care, the neural network model correctly identifies 59% (58%) of the individuals with missed care visits, and 57% (58%) of the individuals without missed care visits. As the sensitivity and specificity of the models are strongly related to the risk threshold used to classify individuals, the models can be calibrated depending on users' resource constraints and targeting approach. CONCLUSIONS: Pandemics such as COVID-19 require rapid and efficient responses to reduce disruptions in health care. Based on characteristics available to health administrators or insurance providers, simple machine learning algorithms can be used to efficiently target efforts to reduce missed essential care.

SARS-CoV-2 N-Antigen Quantification in Respiratory Tract, Plasma and Urine: Kinetics and Association with RT-qPCR Results.

Qualitative SARS-CoV-2 antigen assays based on immunochromatography are useful for mass diagnosis of COVID-19, even though their sensitivity is poor in comparison with RT-PCR assays. In addition, quantitative assays could improve antigenic test performance and allow testing with different specimens. Using quantitative assays, we tested 26 patients for viral RNA and N-antigen in respiratory samples, plasma and urine. This allowed us to compare the kinetics between the three compartments and to compare RNA and antigen concentrations in each. Our results showed the presence of N-antigen in respiratory (15/15, 100%), plasma (26/59, 44%) and urine (14/54, 28.9%) samples, whereas RNA was only detected in respiratory (15/15, 100%) and plasma (12/60, 20%) samples. We detected N-antigen in urine and plasma samples until the day 9 and day 13 post-inclusion, respectively. The antigen concentration was found to correlate with RNA levels in respiratory (p < 0.001) and plasma samples (p < 0.001). Finally, urinary antigen levels correlated with plasma levels (p < 0.001). Urine N-antigen detection could be part of the strategy for the late diagnosis and prognostic evaluation of COVID-19, given the ease and painlessness of sampling and the duration of antigen excretion in this biological compartment.

Dynamics of SARS-CoV-2 seroassay sensitivity: a systematic review and modelling study.

BackgroundSerological surveys have been the gold standard to estimate numbers of SARS-CoV-2 infections, the dynamics of the epidemic, and disease severity. Serological assays have decaying sensitivity with time that can bias their results, but there is a lack of guidelines to account for this phenomenon for SARS-CoV-2.AimOur goal was to assess the sensitivity decay of seroassays for detecting SARS-CoV-2 infections, the dependence of this decay on assay characteristics, and to provide a simple method to correct for this phenomenon.MethodsWe performed a systematic review and meta-analysis of SARS-CoV-2 serology studies. We included studies testing previously diagnosed, unvaccinated individuals, and excluded studies of cohorts highly unrepresentative of the general population (e.g. hospitalised patients).ResultsOf the 488 screened studies, 76 studies reporting on 50 different seroassays were included in the analysis. Sensitivity decay depended strongly on the antigen and the analytic technique used by the assay, with average sensitivities ranging between 26% and 98% at 6 months after infection, depending on assay characteristics. We found that a third of the included assays departed considerably from manufacturer specifications after 6 months.ConclusionsSeroassay sensitivity decay depends on assay characteristics, and for some types of assays, it can make manufacturer specifications highly unreliable. We provide a tool to correct for this phenomenon and to assess the risk of decay for a given assay. Our analysis can guide the design and interpretation of serosurveys for SARS-CoV-2 and other pathogens and quantify systematic biases in the existing serology literature.

Impedimetric Sensing: An Emerging Tool for Combating the COVID-19 Pandemic.

The COVID-19 pandemic revealed a pressing need for the development of sensitive and low-cost point-of-care sensors for disease diagnosis. The current standard of care for COVID-19 is quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). This method is sensitive, but takes time, effort, and requires specialized equipment and reagents to be performed correctly. This make it unsuitable for widespread, rapid testing and causes poor individual and policy decision-making. Rapid antigen tests (RATs) are a widely used alternative that provide results quickly but have low sensitivity and are prone to false negatives, particularly in cases with lower viral burden. Electrochemical sensors have shown much promise in filling this technology gap, and impedance spectroscopy specifically has exciting potential in rapid screening of COVID-19. Due to the data-rich nature of impedance measurements performed at different frequencies, this method lends itself to machine-leaning (ML) algorithms for further data processing. This review summarizes the current state of impedance spectroscopy-based point-of-care sensors for the detection of the SARS-CoV-2 virus. This article also suggests future directions to address the technology's current limitations to move forward in this current pandemic and prepare for future outbreaks.

Performances of Dried Blood Spots and Point-of-Care Devices to Identify Virological Failure in HIV-Infected Patients: A Systematic Review and Meta-Analysis.

To broaden access to HIV viral load monitoring (VLM), the use of blood samples from dried blood spots (DBS) or point-of-care (POC) devices, could be of great help in settings where plasma is not easily accessible. The variety of assays available makes the choice complex. This systematic review and meta-analysis aims to estimate the sensitivity and specificity of DBS and POC devices to identify patients in virological failure using World Health Organization (WHO) recommendations (viral load ≥1000 copies/mL), compared with plasma, for the assays currently available. Four databases were searched for articles, and two reviewers independently identified articles reporting sensitivity and specificity of DBS and/or POC to identify patients in virological failure. We excluded articles that used other thresholds as well as articles with a total number of participants below 50 to avoid reporting bias. Heterogeneity and factors associated with assays' performances were assessed by I2 statistics and metaregression. The protocol of this review follows the PRISMA guidelines. Out of 941 articles, 47 were included: 32 DBS evaluations and 16 POC evaluations. Overall, when using DBS, the Abbott RT HIV-1, Roche CAP-CTM, NucliSENS BioMerieux and Aptima assays presented sensitivity and specificity exceeding 85%, but reported results were highly heterogeneous. Factors associated with better performances were high volume of blood and the use of the same assay for DBS and plasma VLM. Regarding the POC devices, SAMBA I, SAMBA II, and GeneXpert devices presented high sensitivity and specificity exceeding 90%, with less heterogeneity. DBS is suitable VLM, but performances can vary greatly depending on the protocols, and should be performed in trained centers. POC is suitable for VLM with less risk of heterogeneity but is more intensive in costs and logistics.

Rapid and Sensitive Diagnosis of COVID-19 Using an Electricity-Free Self-Testing System.

Rapid and sensitive detection of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for early diagnosis and effective treatment. Nucleic acid testing has been considered the gold standard method for the diagnosis of COVID-19 for its high sensitivity and specificity. However, the polymerase chain reaction (PCR)-based method in the central lab requires expensive equipment and well-trained personnel, which makes it difficult to be used in resource-limited settings. It highlights the need for a sensitive and simple assay that allows potential patients to detect SARS-CoV-2 by themselves. Here, we developed an electricity-free self-testing system based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that allows for rapid and accurate detection of SARS-CoV-2. Our system employs a heating bag as the heat source, and a 3D-printed box filled with phase change material (PCM) that successfully regulates the temperature for the RT-LAMP. The colorimetric method could be completed in 40 min and the results could be read out by the naked eye. A ratiometric measurement for exact readout was also incorporated to improve the detection accuracy of the system. This self-testing system is a promising tool for point-of-care testing (POCT) that enables rapid and sensitive diagnosis of SARS-CoV-2 in the real world and will improve the current COVID-19 screening efforts for control and mitigation of the pandemic.

A CRISPR-Cas12a-based platform for ultrasensitive rapid highly specific detection of Mycobacterium tuberculosis in clinical application.

Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific sdaA gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the sdaA gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis mycobacterium (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions.

Updates on the Biofunctionalization of Gold Nanoparticles for the Rapid and Sensitive Multiplatform Diagnosis of SARS-CoV-2 Virus and Its Proteins: From Computational Models to Validation in Human Samples.

Since the outbreak of the pandemic respiratory virus SARS-CoV-2 (COVID-19), academic communities and governments/private companies have used several detection techniques based on gold nanoparticles (AuNPs). In this emergency context, colloidal AuNPs are highly valuable easy-to-synthesize biocompatible materials that can be used for different functionalization strategies and rapid viral immunodiagnosis. In this review, the latest multidisciplinary developments in the bioconjugation of AuNPs for the detection of SARS-CoV-2 virus and its proteins in (spiked) real samples are discussed for the first time, with reference to the optimal parameters provided by three approaches: one theoretical, via computational prediction, and two experimental, using dry and wet chemistry based on single/multistep protocols. Overall, to achieve high specificity and low detection limits for the target viral biomolecules, optimal running buffers for bioreagent dilutions and nanostructure washes should be validated before conducting optical, electrochemical, and acoustic biosensing investigations. Indeed, there is plenty of room for improvement in using gold nanomaterials as stable platforms for ultrasensitive and simultaneous "in vitro" detection by the untrained public of the whole SARS-CoV-2 virus, its proteins, and specific developed IgA/IgM/IgG antibodies (Ab) in bodily fluids. Hence, the lateral flow assay (LFA) approach is a quick and judicious solution to combating the pandemic. In this context, the author classifies LFAs according to four generations to guide readers in the future development of multifunctional biosensing platforms. Undoubtedly, the LFA kit market will continue to improve, adapting researchers' multidetection platforms for smartphones with easy-to-analyze results, and establishing user-friendly tools for more effective preventive and medical treatments.

Performance of rapid antigen tests in identifying Omicron BA.4 and BA.5 infections in South Africa.

BACKGROUND: Concerns around accuracy and performance of rapid antigen tests continue to be raised with the emergence of new SARS-CoV-2 variants. OBJECTIVE: To evaluate the performance of two widely used SARS-CoV-2 rapid antigen tests during BA.4/BA.5 SARS-CoV-2 wave in South Africa (May - June 2022). STUDY DESIGN: A prospective field evaluation compared the SARS-CoV-2 Antigen Rapid test from Hangzhou AllTest Biotech (nasal swab) and the Standard Q COVID-19 Rapid Antigen test from SD Biosensor (nasopharyngeal swab) to the Abbott RealTime SARS-CoV-2 assay (nasopharyngeal swab) on samples collected from 540 study participants. RESULTS: Overall 28.52% (154/540) were SARS-CoV-2 RT-PCR positive with median cycle number value of 12.30 (IQR 9.30-19.40). Out of the 99 successfully sequenced SARS-CoV-2 positive samples, 18 were classified as BA.4 and 56 were classified as BA.5. The overall sensitivities of the AllTest SARS-CoV-2 Ag test and Standard Q COVID-19 Ag test were 73.38% (95% CI 65.89-79.73) and 74.03% (95% CI 66.58-80.31) and their specificities were 97.41% (95% CI 95.30-98.59) and 99.22% (95% CI 97.74-99.74) respectively. Sensitivity was >90% when the cycle number value was <20. The sensitivity of both rapid tests was >90% in samples infected with Omicron sub-lineage BA.4 and BA.5. CONCLUSION: Accuracy of tested rapid antigen tests that target the nucleocapsid SARS-CoV-2 protein, were not adversely affected by BA.4 and BA.5 Omicron sub-variants.

Community-based COVID-19 active case finding and rapid response in the Democratic Republic of the Congo: Improving case detection and response.

A community-based coronavirus disease (COVID-19) active case-finding strategy using an antigen-detecting rapid diagnostic test (Ag-RDT) was implemented in the Democratic Republic of Congo (DRC) to enhance COVID-19 case detection. With this pilot community-based active case finding and response program that was designed as a clinical, prospective testing performance, and implementation study, we aimed to identify insights to improve community diagnosis and rapid response to COVID-19. This pilot study was modeled on the DRC's National COVID-19 Response Plan and the COVID-19 Ag-RDT screening algorithm defined by the World Health Organization (WHO), with case findings implemented in 259 health areas, 39 health zones, and 9 provinces. In each health area, a 7-member interdisciplinary field team tested the close contacts (ring strategy) and applied preventive and control measures to each confirmed case. The COVID-19 testing capacity increased from 0.3 tests per 10,000 inhabitants per week in the first wave to 0.4, 1.6, and 2.2 in the second, third, and fourth waves, respectively. From January to November 2021, this capacity increase contributed to an average of 10.5% of COVID-19 tests in the DRC, with 7,110 positive Ag-RDT results for 40,226 suspected cases and close contacts who were tested (53.6% female, median age: 37 years [interquartile range: 26.0-50.0)]. Overall, 79.7% (n = 32,071) of the participants were symptomatic and 7.6% (n = 3,073) had comorbidities. The Ag-RDT sensitivity and specificity were 55.5% and 99.0%, respectively, based on reverse transcription polymerase chain reaction analysis, and there was substantial agreement between the tests (k = 0.63). Despite its limited sensitivity, the Ag-RDT has improved COVID-19 testing capacity, enabling earlier detection, isolation, and treatment of COVID-19 cases. Our findings support the community testing of suspected cases and asymptomatic close contacts of confirmed cases to reduce disease spread and virus transmission.

Metagenomics characterization of respiratory viral RNA pathogens in children under five years with severe acute respiratory infection in the Free State, South Africa.

Prompt detection of viral respiratory pathogens is crucial in managing respiratory infection including severe acute respiratory infection (SARI). Metagenomics next-generation sequencing (mNGS) and bioinformatics analyses remain reliable strategies for diagnostic and surveillance purposes. This study evaluated the diagnostic utility of mNGS using multiple analysis tools compared with multiplex real-time PCR for the detection of viral respiratory pathogens in children under 5 years with SARI. Nasopharyngeal swabs collected in viral transport media from 84 children admitted with SARI as per the World Health Organization definition between December 2020 and August 2021 in the Free State Province, South Africa, were used in this study. The obtained specimens were subjected to mNGS using the Illumina MiSeq system, and bioinformatics analysis was performed using three web-based analysis tools; Genome Detective, One Codex and Twist Respiratory Viral Research Panel. With average reads of 211323, mNGS detected viral pathogens in 82 (97.6%) of the 84 patients. Viral aetiologies were established in nine previously undetected/missed cases with an additional bacterial aetiology (Neisseria meningitidis) detected in one patient. Furthermore, mNGS enabled the much needed viral genotypic and subtype differentiation and provided significant information on bacterial co-infection despite enrichment for RNA viruses. Sequences of nonhuman viruses, bacteriophages, and endogenous retrovirus K113 (constituting the respiratory virome) were also uncovered. Notably, mNGS had lower detectability rate for severe acute respiratory syndrome coronavirus 2 (missing 18/32 cases). This study suggests that mNGS, combined with multiple/improved bioinformatics tools, is practically feasible for increased viral and bacterial pathogen detection in SARI, especially in cases where no aetiological agent could be identified by available traditional methods.

A review of current effective COVID-19 testing methods and quality control.

COVID-19 is a highly infectious disease caused by the SARS-CoV-2 virus, which primarily affects the respiratory system and can lead to severe illness. The virus is extremely contagious, early and accurate diagnosis of SARS-CoV-2 is crucial to contain its spread, to provide prompt treatment, and to prevent complications. Currently, the reverse transcriptase polymerase chain reaction (RT-PCR) is considered to be the gold standard for detecting COVID-19 in its early stages. In addition, loop-mediated isothermal amplification (LMAP), clustering rule interval short palindromic repeats (CRISPR), colloidal gold immunochromatographic assay (GICA), computed tomography (CT), and electrochemical sensors are also common tests. However, these different methods vary greatly in terms of their detection efficiency, specificity, accuracy, sensitivity, cost, and throughput. Besides, most of the current detection methods are conducted in central hospitals and laboratories, which is a great challenge for remote and underdeveloped areas. Therefore, it is essential to review the advantages and disadvantages of different COVID-19 detection methods, as well as the technology that can enhance detection efficiency and improve detection quality in greater details.

A novel anterior nasal swab to detect respiratory viruses: a prospective study of diagnostic accuracy.

Detection of respiratory viruses requires testing of the upper respiratory tract to obtain specimens for analysis. However, nasal and throat swabs can cause discomfort and procedural anxiety in children. Respiratory sampling methods which are accurate and less invasive are needed. We aim to determine the positive and negative percentage agreement of a novel anterior nasal swab (ANS) compared with the combined throat and anterior nasal swab (CTN), the reference standard, for detection of respiratory viruses. Children 5 - 18 years of age presenting to a tertiary paediatric hospital with respiratory symptoms were tested with both swabs in randomised order. Respiratory samples were tested on a multiplex RT-PCR panel. Viral detections, RT-PCR cycle-threshold values and child/parent/clinician experience of the swab were recorded. There were 157 viral detections from 249 participant CTN swabs. In comparison with the CTN, the overall positive and negative percentage agreement of ANS for detection of respiratory viruses was 96.2% (95% CI, 91.8-98.3%) and 99.8% (95% CI, 99.6-99.9%), respectively. The ANS was "extremely comfortable", or only a "little uncomfortable" for 90% of children compared with 48% for CTN. 202 children (84%) rated the ANS as the preferred swab, and 208 (87%) indicated they would prefer ANS for future testing. The ANS required additional laboratory handling processes compared to the CTN. The ANS has high positive percentage agreement and is comparable to the current standard of care. The high acceptability from the less invasive ANS provides a more comfortable method for respiratory virus testing in children.Trial registrationClinicalTrials.gov ID NCT05043623.