Assessing the change in the epidemiology of seasonal respiratory viruses with the onset of the COVID-19 pandemic.

Background. Respiratory tract infections are among the most important causes of mortality and morbidity in children worldwide. The COVID-19 pandemic has affected the distribution of seasonal respiratory viruses as in all areas of life. In this study, we have aimed to evaluate the changes in the rates of seasonal respiratory viruses with the onset of the pandemic.Methods. This study included patients who were admitted to the Pediatrics Clinic of Eskisehir Osmangazi University Faculty of Medicine Hospital between December 2018 and February 2022 with respiratory tract infections and in whom pathogens were detected from nasopharyngeal swab samples analysed by multiplex PCR method.Results. A total of 833 respiratory tract pathogens were detected in 684 cases consisting of male (55.3 %), and female (44.7 %), patients with a total mean age of 42 months. Single pathogen was revealed in 550, and multiple pathogens in 134 cases. Intensive care was needed in 14 % of the cases. Most frequently influenza A/B, rhinovirus and respiratory syncytial virus (RSV) were detected during the pre-pandemic period, while rhinovirus, RSV, and adenovirus were observed during the lockdown period. In the post-lockdown period, the incidence rates of rhinovirus, RSV, human bocavirus (HboV) (12 %), influenza virus infections increased, and patients with RSV and bocavirus infections required intensive care hospitalization.Conclusion. It is thought that the COVID-9 pandemic lockdown measures may have an impact on the distribution of seasonal respiratory viruses, especially RSV and influenza. Current, prospective and large case series regarding the mechanism of action and dynamics are needed.

Complications of Nasopharyngeal Swabs and Safe Procedures for COVID-19 Testing Based on Anatomical Knowledge.

Nasopharyngeal swabs have been widely to prevent the spread of coronavirus disease 2019 (COVID-19). Nasopharyngeal COVID-19 testing is a generally safe and well-tolerated procedure, but numerous complications have been reported in the media. Therefore, the present study aimed to review and document adverse events and suggest procedural references to minimize preventable but often underestimated risks. A total of 27 articles were selected for the review of 842 related documents in PubMed, Embase, and KoreaMed. The complications related to nasopharyngeal COVID-19 testing were reported to be rarely happened, ranging from 0.0012 to 0.026%. Frequently documented adverse events were retained swabs, epistaxis, and cerebrospinal fluid leakage, often associated with high-risk factors, including severe septal deviations, pre-existing skull base defects, and previous sinus or transsphenoidal pituitary surgery. Appropriate techniques based on sufficient anatomical knowledge are mandatory for clinicians to perform nasopharyngeal COVID-19 testing. The nasal floor can be predicted by the line between the nostril and external ear canal. For safe testing, the angle of swab insertion in the nasal passage should remain within 30° of the nasal floor. The swab was gently inserted along the nasal septum just above the nasal floor to the nasopharynx and remained on the nasopharynx for several seconds before removal. Forceful insertion should be attempted, and alternative examinations should be considered, especially in vulnerable patients. In conclusion, patients and clinicians should be aware of rare but possible complications and associated high-risk factors. The suggested procedural pearls enable more comfortable and safe nasopharyngeal COVID-19 testing for both clinicians and patients.

Portable real-time colorimetric LAMP-device for rapid quantitative detection of nucleic acids in crude samples.

Loop-mediated isothermal amplification is known for its high sensitivity, specificity and tolerance to inhibiting-substances. In this work, we developed a device for performing real-time colorimetric LAMP combining the accuracy of lab-based quantitative analysis with the simplicity of point-of-care testing. The device innovation lies on the use of a plastic tube anchored vertically on a hot surface while the side walls are exposed to a mini camera able to take snapshots of the colour change in real time during LAMP amplification. Competitive features are the rapid analysis (< 30 min), quantification over 9 log-units, crude sample-compatibility (saliva, tissue, swabs), low detection limit (< 5 copies/reaction), smartphone-operation, fast prototyping (3D-printing) and ability to select the dye of interest (Phenol red, HNB). The device's clinical utility is demonstrated in cancer mutations-analysis during the detection of 0.01% of BRAF-V600E-to-wild-type molecules from tissue samples and COVID-19 testing with 97% (Ct < 36.8) and 98% (Ct < 30) sensitivity when using extracted RNA and nasopharyngeal-swabs, respectively. The device high technology-readiness-level makes it a suitable platform for performing any colorimetric LAMP assay; moreover, its simple and inexpensive fabrication holds promise for fast deployment and application in global diagnostics.

Detection of SARS-CoV-2 infection prevalence in 860 cancer patients with a combined screening procedure including triage, molecular nasopharyngeal swabs and rapid serological test. A report from the first epidemic wave.

INTRODUCTION: Even if now we have available the weapon of vaccination against SARS-CoV-2, the patients with cancer remains a very frail population in which frequently the immunologic response to vaccination may be impaired. In this setting, the SARS-CoV-2 infection screening retains a great value. However, there are still limited data on the feasibility and efficacy of combined screening procedures to assess the prevalence of SARS-CoV-2 infection (including asymptomatic cases) in cancer outpatients undergoing antineoplastic therapy. PATIENTS AND RESULTS: From May 1, 2020, to June 15, 2020, during the first wave of SARS-CoV-2 pandemic, 860 consecutive patients, undergoing active anticancer therapy, were evaluated and tested for SARS-CoV-2 with a combined screening procedure, including a self-report questionnaire, a molecular nasopharyngeal swab (NPS) and a rapid serological immunoassay (for anti-SARS-CoV-2 IgG/IgM antibodies). The primary endpoint of the study was to estimate the prevalence of SARS-CoV-2 infection (including asymptomatic cases) in consecutive and unselected cancer outpatients by a combined screening modality. A total of 2955 SARS-CoV-2 NPS and 860 serological tests, in 475 patients with hematologic cancers and in 386 with solid tumors, were performed. A total of 112 (13%) patients self-reported symptoms potentially COVID-19 related. In 1/860 cases (< 1%) SARS-CoV-2 NPS was positive and in 14 cases (1.62%) the specific serological test was positive (overall prevalence of SARS-CoV-2 infection 1.62%). Of the 112 cases who declared symptoms potentially COVID-19-related, only 2.7% (3/112) were found SARS-CoV-2 positive. CONCLUSIONS: This is the largest study reporting the feasibility of a combined screening procedure (including triage, NPS and serologic test) to evaluate the prevalence of SARS-CoV-2 infection in cancer patients receiving active therapy, during the first epidemic wave and under the restrictive lockdown measures, in one of the active areas of the SARS-CoV-2 circulation. Lacking specific recommendations for the detection of asymptomatic SARS-CoV-2 cases, a combined diagnostic screening might be more effective to detect the exact prevalence of SARS-CoV-2 in neoplastic patient population. The prevalence can obviously change according to the territorial context, the entity of the restrictive measures adopted and the phase of the epidemic curve. However, its exact and real-time knowledge could be important to balance risks/benefits of oncologic treatments, avoiding (if the prevalence is low) the reduction of dose intensity or the selection of less intensive (but also less effective) anti-cancer therapies.

A Community Study of SARS-CoV-2 Detection by RT-PCR in Saliva: A Reliable and Effective Method.

Efficient, wide-scale testing for SARS-CoV-2 is crucial for monitoring the incidence of the infection in the community. The gold standard for COVID-19 diagnosis is the molecular analysis of epithelial secretions from the upper respiratory system captured by nasopharyngeal (NP) or oropharyngeal swabs. Given the ease of collection, saliva has been proposed as a possible substitute to support testing at the population level. Here, we used a novel saliva collection device designed to favour the safe and correct acquisition of the sample, as well as the processivity of the downstream molecular analysis. We tested 1003 nasopharyngeal swabs and paired saliva samples self-collected by individuals recruited at a public drive-through testing facility. An overall moderate concordance (68%) between the two tests was found, with evidence that neither system can diagnose the infection in 100% of the cases. While the two methods performed equally well in symptomatic individuals, their discordance was mainly restricted to samples from convalescent subjects. The saliva test was at least as effective as NP swabs in asymptomatic individuals recruited for contact tracing. Our study describes a testing strategy of self-collected saliva samples, which is reliable for wide-scale COVID-19 screening in the community and is particularly effective for contact tracing.

Influenza A(H1N1)pdm09 Virus but Not Respiratory Syncytial Virus Interferes with SARS-CoV-2 Replication during Sequential Infections in Human Nasal Epithelial Cells.

The types of interactions between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses are not well-characterized due to the low number of co-infection cases described since the onset of the pandemic. We have evaluated the interactions between SARS-CoV-2 (D614G mutant) and influenza A(H1N1)pdm09 or respiratory syncytial virus (RSV) in the nasal human airway epithelium (HAE) infected simultaneously or sequentially (24 h apart) with virus combinations. The replication kinetics of each virus were determined by RT-qPCR at different post-infection times. Our results showed that during simultaneous infection, SARS-CoV-2 interferes with RSV-A2 but not with A(H1N1)pdm09 replication. The prior infection of nasal HAE with SARS-CoV-2 reduces the replication kinetics of both respiratory viruses. SARS-CoV-2 replication is decreased by a prior infection with A(H1N1)pdm09 but not with RSV-A2. The pretreatment of nasal HAE with BX795, a TANK-binding kinase 1 inhibitor, partially alleviates the reduced replication of SARS-CoV-2 or influenza A(H1N1)pdm09 during sequential infection with both virus combinations. Thus, a prior infection of nasal HAE with SARS-CoV-2 interferes with the replication kinetics of A(H1N1)pdm09 and RSV-A2, whereas only A(H1N1)pdm09 reduces the subsequent infection with SARS-CoV-2. The mechanism involved in the viral interference between SARS-CoV-2 and A(H1N1)pdm09 is mediated by the production of interferon.

Non-pharmaceutical interventions during the COVID-19 epidemic changed detection rates of other circulating respiratory pathogens in Japan.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has circulated worldwide and causes coronavirus disease 2019 (COVID-19). At the onset of the COVID-19 pandemic, infection control measures were taken, such as hand washing, mask wearing, and behavioral restrictions. However, it is not fully clear how the effects of these non-pharmaceutical interventions changed the prevalence of other pathogens associated with respiratory infections. In this study, we collected 3,508 nasopharyngeal swab samples from 3,249 patients who visited the Yamanashi Central Hospital in Japan from March 1, 2020 to February 28, 2021. We performed multiplex polymerase chain reaction (PCR) using the FilmArray Respiratory Panel and singleplex quantitative reverse transcription PCR targeting SARS-CoV-2 to detect respiratory disease-associated pathogens. At least one pathogen was detected in 246 (7.0%) of the 3,508 samples. Eleven types of pathogens were detected in the samples collected from March-May 2020, during which non-pharmaceutical interventions were not well implemented. In contrast, after non-pharmaceutical interventions were thoroughly implemented, only five types of pathogens were detected, and the majority were SARS-CoV-2, adenoviruses, or human rhinoviruses / enteroviruses. The 0-9 year age group had a higher prevalence of infection with adenoviruses and human rhinoviruses / enteroviruses compared with those 10 years and older, while those 10 years and older had a higher prevalence of infection with SARS-CoV-2 and other pathogens. These results indicated that non-pharmaceutical interventions likely reduced the diversity of circulating pathogens. Moreover, differences in the prevalence of pathogens were observed among the different age groups.

Coupling immuno-magnetic capture with LC-MS/MS(MRM) as a sensitive, reliable, and specific assay for SARS-CoV-2 identification from clinical samples.

Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 104 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC-MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC-MS analysis. A sensitive and specific LC-MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well.

Secondary organizing pneumonia after recovery of mild COVID-19 infection.

A 36-year-old male with diffuse large B-cell lymphoma on maintenance rituximab therapy presented to the emergency department with high fever and fatigue. A chest X-ray showed a lobar infiltrate, 40 days before admission the patient suffered from a mild coronavirus disease 2019 (COVID-19) infection and fully recovered. PCR nasopharyngeal swab was negative for COVID-19. Comprehensive biochemical, radiological, and pathological evaluation including 18-fluorodeoxyglucose positron emission tomography with computed tomography and transbronchial lung biopsy found no pathogen or lymphoma recurrence. Treatment for pneumonia with antibiotic and antifungal agents was nonbeneficial. A diagnosis of secondary organizing pneumonia (OP) was made after pneumonia migration and a rapid response to corticosteroids. OP secondary to a viral respiratory infection has been well described. Raising awareness for post-COVID-19 OP has therapeutic and prognostic importance because those patients benefit from steroid therapy. We believe the condition described here is underdiagnosed and undertreated by doctors worldwide. Because of the ongoing global pandemic we are now encountering a new kind of patient, patients that have recovered from COVID-19. We hope that this case may contribute to gaining more knowledge about this growing patient population.

Autocoleta de swab nasofaríngeo e teste molecular em pool testing como estratégias para detecção de coronavírus da síndrome respiratória aguda grave 2 (SARS-CoV-2): viabilidade em estudantes de medicina da Universidade Federal de Minas Gerais, 2021 / Autocolección de hiopados nasofaríngeos y pruebas moleculares en pool testing como estrategias para la detección del Coronavirus Síndrome Respiratorio Agudo Severo 2 (SARS-CoV-2): viabilidad en estudiantes de medicina de la Universidad Federal de Minas Gerais, Brasil, 2021 / Self-collected nasopharyngeal swab and molecular test using pool testing as strategies to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): feasibility in medical students at the Universidade Federal de Minas Gerais, Brazil, 2021

Objetivo: Demonstrar a viabilidade da utilização combinada da autocoleta de swab nasofaríngeo e pool testing para detecção do SARS-CoV-2 em inquéritos epidemiológicos. Métodos: A experiência envolveu amostra de 154 estudantes da Universidade Federal de Minas Gerais, que realizaram a autocoleta do swab nasofaríngeo em cabines individuais e sem supervisão. O teste molecular foi realizado utilizando-se a técnica de pool testing. Resultados: A obtenção de amostras durou cerca de 5 minutos por pessoa. Realizou-se análise para detecção de RNA endógeno em 40 amostras e os resultados indicaram que não houve falhas decorrentes da autocoleta. Nenhum dos pools detectou presença de RNA viral. O custo da realização do teste molecular (RT-PCR) por pool testing com amostras obtidas por autocoleta foi cerca de dez vezes menor do que nos métodos habituais. Conclusão: As estratégias investigadas mostraram-se economicamente viáveis e válidas para a pesquisa de SARS-CoV-2 em inquéritos epidemiológicos.

Objetivo: Demostrar la viabilidad del uso combinado de la auto recolección de swabs nasofaríngeos y tests por agrupamiento (pool testing) para la detección del SARS-CoV-2 en encuestas epidemiológicas. Métodos: La prueba involucró a una muestra de 154 estudiantes de la Universidade Federal de Minas Gerais, quienes realizaron e autorecolectado del hisopo nasofaríngeo en cabinas individuales sin supervisión. La prueba molecular se realizó utilizando la técnica de prueba de grupo. Resultados: La obtención de muestras duró unos 5 minutos por persona. Se realizó un análisis para detectar ARN endógeno en 40 muestras y los resultados indicaron que no hubo fallas derivadas de la autorecolección. Ninguno de los grupos detectó la presencia de ARN viral. El costo de realizar una prueba molecular (RT-PCR) por pool con muestras obtenidas por auto-recolección fue aproximadamente 10 veces menor que con los métodos habituales. Conclusión: Las estrategias investigadas demostraron ser económicamente viables y válidas para la investigación del SARS-CoV-2 en encuestas epidemiológicas.

Objective: To show the feasibility of the combined use of self-collected nasopharyngeal swab and pool testing to detect SARS-CoV-2 in epidemiological surveys. Methods: This experience included a sample of 154 students at the Universidade Federal de Minas Gerais, who performed self-collected nasopharyngeal swab in individual cabins and without supervision. The molecular test was performed using the pool testing technique. Results: It took each person 5 minutes to collect the sample. An analysis was performed to detect endogenous RNA in 40 samples. The results showed that there were no failures resulting from self-collection. None of the pools detected the presence of viral RNA. The cost of molecular testing (RT-PCR), by pool testing, with samples obtained by self-collection was about ten times lower than the usual methods. Conclusion: The strategies that were investigated proved to be economically feasible and valid for the research on SARS-CoV-2 in epidemiological surveys.

The preoperative evaluation of post-COVID-19 patients scheduled for elective surgery - What is important not to miss!

Continuous rise in the number of COVID-19 cases, since it was first diagnosed in 2019, forced the entire medical fraternity to delay elective surgeries. The preoperative evaluation guidelines that were used in the pre-COVID-19 era underwent significant changes, adding modifications to meet the post-COVID patients' specific criteria and requirements. Currently, all patients before or at the time of hospital admission were tested using a nasopharyngeal swab, by RT-PCR for SARS-CoV-2. Apart from this, for a patient undergoing elective surgery in their post-COVID-19 period, it is mandatory to obtain a detailed history of COVID-19 disease/SARS-CoV-2 infection, to identify residual symptoms or any organ dysfunction the infection might have caused. As well as the functional optimization of the patient to achieve the best clinical and biological status before the surgery. After all the systems have been thoroughly investigated, the risk-benefit ratio needs to be calculated, keeping in mind the cytokine storm and inflammatory responses encountered postoperatively. A mere negative RT-PCR test cannot be considered as the only decisive factor to operate, as the post-COVID-19 phase can influence postoperative outcome of the patient. Hence, the pre-operative evaluation protocols of post-COVID patients should be set and followed thoroughly, in order to avoid post-surgical complications. For better surgical and post-surgical management of post-COVID-19 patients, conducting clinical tests, assessing previously administered medications, evaluating the need for deep venous thrombosis prophylaxes, and identifying subclinical inflammatory state are the measures that should be taken.

Evaluation of Three Multiplex Real-time Reverse Transcription PCR Assays for Simultaneous Detection of SARS-CoV-2, Influenza A/B, and Respiratory Syncytial Virus in Nasopharyngeal Swabs.

BACKGROUND: In the coronavirus disease 2019 (COVID-19) pandemic era, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus (Flu), and respiratory syncytial virus (RSV) is important in the rapid differential diagnosis in patients with respiratory symptoms. Three multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been recently developed commercially in Korea: PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech); STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor); and Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene). We evaluated the analytical and clinical performances of these kits. METHODS: A limit of detection tests were performed and cross-reactivity analysis was executed using clinical respiratory samples. Ninety-seven SARS-CoV-2-positive, 201 SARS-CoV-2-negative, 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive nasopharyngeal swabs were tested using the three assays. The AdvanSure™ respiratory viruses rRT-PCR assay (AdvanSure; LG Life Sciences) was used as a comparator assay for RSV. RESULTS: Except in influenza B, in SARS-CoV-2 and influenza A, there were no significant differences in detecting specific genes of the viruses among the three assays. All three kits did not cross-react with common respiratory viruses. All three kits had greater than 92% positive percent agreement and negative percent agreement and ≥ 0.95 kappa value in the detection of SARS-CoV-2 and flu A/B. Allplex detected RSV more sensitively than AdvanSure. CONCLUSION: The overall performance of three multiplex rRT-PCR assays for the concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. These kits will promote prompt differential diagnosis of COVID-19, influenza, and RSV infection in the COVID-19 pandemic era.

Clinical evaluation of the SD Biosensor SARS-CoV-2 saliva antigen rapid test with symptomatic and asymptomatic, non-hospitalized patients.

BACKGROUND: Performance of the SD Biosensor saliva antigen rapid test was evaluated at a large designated testing site in non-hospitalized patients, with or without symptoms. METHOD: All eligible people over 18 years of age presenting for a booked appointment at the designated SARS-CoV-2 testing site were approached for inclusion and enrolled following verbal informed consent. One nasopharyngeal swab was taken to carry out the default antigen rapid test from which the results were reported back to the patient and one saliva sample was self-taken according to verbal instruction on site. This was used for the saliva antigen rapid test, the RT-PCR and for virus culture. Sensitivity of the saliva antigen rapid test was analyzed in two ways: i, compared to saliva RT-PCR; and ii, compared to virus culture of the saliva samples. Study participants were also asked to fill in a short questionnaire stating age, sex, date of symptom onset. Recommended time of ≥30mins since last meal, drink or cigarette if applicable was also recorded. The study was carried out in February-March 2021 for 4 weeks. RESULTS: We could include 789 people with complete records and results. Compared to saliva RT-PCR, overall sensitivity and specificity of the saliva antigen rapid test was 66.1% and 99.6% which increased to 88.6% with Ct ≤30 cutoff. Analysis by days post onset did not result in higher sensitivities because the large majority of people were in the very early phase of disease ie <3 days post onset. When breaking down the data for symptomatic and asymptomatic individuals, sensitivity ranged from 69.2% to 50% respectively, however the total number of RT-PCR positive asymptomatic participants was very low (n = 5). Importantly, almost all culture positive samples were detected by the rapid test. CONCLUSION: Overall, the potential benefits of saliva antigen rapid test, could outweigh the lower sensitivity compared to nasopharyngeal antigen rapid test in a comprehensive testing strategy, especially for home/self-testing and in vulnerable populations like elderly, disabled or children where in intrusive testing is either not possible or causes unnecessary stress.

Diagnostic Performance of Self-Collected Saliva Versus Nasopharyngeal Swab for the Molecular Detection of SARS-CoV-2 in the Clinical Setting.

Coronavirus disease 19 (COVID-19)-caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-has spread rapidly around the world. The global shortage of equipment and health care professionals, diagnostic cost, and difficulty in collecting nasopharyngeal swabs (NPSs) necessitate the use of an alternative specimen type for SARS-CoV-2 diagnosis. In this study, we investigated the use of saliva as an alternative specimen type for SARS-CoV-2 detection. Participants presenting COVID-19 symptoms and their contacts were enrolled at the COVID-19 Screening Unit of Dhaka Hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), from July to November 2020. Paired NPS and saliva specimens were collected from each participant. Reverse transcription-quantitative PCR (RT-qPCR) was performed to detect SARS-CoV-2. Of the 596 suspected COVID-19-positive participants, 231 (38.7%) were detected as COVID-19 positive by RT-qPCR from at least 1 specimen type. Among the positive cases, 184 (79.6%) patients were identified to be positive for SARS-CoV-2 based on NPS and saliva samples, whereas 45 (19.65%) patients were positive for SARS-CoV-2 based on NPS samples but negative for SARS-CoV-2 based on the saliva samples. Two (0.5%) patients were positive for SARS-CoV-2 based on saliva samples but negative for SARS-CoV-2 based on NPS samples. The sensitivity and specificity of the saliva samples were 80.3% and 99.4%, respectively. SARS-CoV-2 detection was higher in saliva (85.1%) among the patients who visited the clinic after 1 to 5 days of symptom onset. A lower median cycle threshold (CT) value indicated a higher SARS-CoV-2 viral load in NPS than that in saliva for target genes among the positive specimens. The study findings suggest that saliva can be used accurately for diagnosis of SARS-CoV-2 early after symptom onset in clinical and community settings. IMPORTANCE As the COVID-19 pandemic erupted, the WHO recommended the use of nasopharyngeal or throat swabs for the detection of SARS-CoV-2 etiology of COVID-19. The collection of NPS causes discomfort because of its invasive collection procedure. There are considerable risks to health care workers during the collection of these specimens. Therefore, an alternative, noninvasive, reliable, and self-collected specimen was explored in this study. This study investigated the feasibility and suitability of saliva versus NPS for the detection of SARS-CoV-2. Here, we showed that the sensitivity of saliva specimens was 80.35%, which meets the WHO criteria. Saliva is an easy-to-get, convenient, and low-cost specimen that yields better results if it is collected within the first 5 days of symptom onset. Our study findings suggest that saliva can be used in low-resource countries, community settings, and vulnerable groups, such as children and elderly people.

Changes in peripheral blood in SARS CoV-2 patients and its clinico-pathological correlation: A prospective cross-sectional study.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) caused by SARS-CoV2 can present from mild flu-like symptoms to acute respiratory distress syndrome. There is multi-organ involvement; particularly, hematopoietic system can be associated with morphological changes in blood cells of COVID-19 patients. METHOD: We conducted a cross-sectional study on a cohort of 50 COVID-19 patients, confirmed on RT-PCR with documented cycle threshold (Ct) value. Peripheral blood sample of these patients was collected and examined for complete blood counts (CBC) on automated haematological analyser as well as Leishman-stained blood smears to look for morphological changes in blood cells. Morphological changes were evaluated with reference to clinical severity and Ct value. Additionally, association between Ct value and clinical severity was also performed. Statistical tests were performed, and P value <.05 was considered significant. RESULTS: Mean age of our study group was 42.16 ± 15.55 years, with male preponderance. Most commonly observed peripheral blood changes were hypolobation (P value = .002) and toxic granules (P value = .005) in neutrophils, atypical granules with nucleolar prominence in lymphocytes, cytoplasmic granulation with clumped nuclear chromatin in monocytes, giant platelets and thrombocytopenia and normocytic normochromic anaemia. CONCLUSION: No association was found between clinical severity and Ct value as well as peripheral blood morphological changes with Ct value. We conclude that examination of peripheral smear coupled with complete blood count (CBC) is only partially supportive of disease pathogenesis and to assess the viral load other parameters should be utilised instead of relying solely on Ct value.

Multisite Clinical Validation of Isothermal Amplification-Based SARS-CoV-2 Detection Assays Using Different Sampling Strategies.

Isothermal amplification-based tests have been introduced as rapid, low-cost, and simple alternatives to real-time reverse transcriptase PCR (RT-PCR) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. The clinical performance of two isothermal amplification-based tests (Atila Biosystems iAMP coronavirus disease of 2019 [COVID-19] detection test and OptiGene COVID-19 direct plus RT-loop-mediated isothermal amplification [LAMP] test) was compared with that of clinical RT-PCR assays using different sampling strategies. A total of 1,378 participants were tested across 4 study sites. Compared with standard of care RT-PCR testing, the overall sensitivity and specificity of the Atila iAMP test for detection of SARS-CoV-2 were 76.2% and 94.9%, respectively, and increased to 88.8% and 89.5%, respectively, after exclusion of an outlier study site. Sensitivity varied based on the anatomic site from which the sample was collected. Sensitivity for nasopharyngeal sampling was 65.4% (range across study sites, 52.8% to 79.8%), for midturbinate was 88.2%, for saliva was 55.1% (range across study sites, 42.9% to 77.8%), and for anterior nares was 66.7% (range across study sites, 63.6% to 76.5%). The specificity for these anatomic collection sites ranged from 96.7% to 100%. Sensitivity improved in symptomatic patients (overall, 82.7%) and those with a higher viral load (overall, 92.4% for cycle threshold [CT] of ≤25). Sensitivity and specificity of the OptiGene direct plus RT-LAMP test, which was conducted at a single study site, were 25.5% and 100%, respectively. The Atila iAMP COVID test with midturbinate sampling is a rapid, low-cost assay for detecting SARS-CoV-2, especially in symptomatic patients and those with a high viral load, and could be used to reduce the risk of SARS-CoV-2 transmission in clinical settings. Variation of performance between study sites highlights the need for site-specific clinical validation of these assays before clinical adoption. IMPORTANCE Numerous SARS-CoV-2 detection assays have been developed and introduced into the market under emergency use authorizations (EUAs). EUAs are granted primarily based on small studies of analytic sensitivity and specificity with limited clinical validations. A thorough clinical performance evaluation of SARS-CoV-2 assays is important to understand the strengths, limitations, and specific applications of these assays. In this first large-scale multicentric study, we evaluated the clinical performance and operational characteristics of two isothermal amplification-based SARS-CoV-2 tests, namely, (i) iAMP COVID-19 detection test (Atila BioSystems, USA) and (ii) COVID-19 direct plus RT-LAMP test (OptiGene Ltd., UK), compared with those of clinical RT-PCR tests using different sampling strategies (i.e., nasopharyngeal, self-sampled anterior nares, self-sampled midturbinate, and saliva). An important specific use for these isothermal amplification-based, rapid, low-cost, and easy-to-perform SARS-CoV-2 assays is to allow for a safer return to preventive clinical encounters, such as cancer screening, particularly in low- and middle-income countries that have low SARS-CoV-2 vaccination rates.

Quantifying SARS-CoV-2 nucleocapsid antigen in oropharyngeal swabs using single molecule array technology.

This study aimed to develop a highly sensitive SARS-CoV-2 nucleocapsid antigen assay using the single molecule array (Simoa) technology and compare it with real time RT-PCR as used in routine clinical practice with the ambition to achieve a comparative technical and clinical sensitivity. Samples were available from 148 SARS-CoV-2 real time RT-PCR positive and 73 SARS-CoV-2 real time RT-PCR negative oropharyngeal swabs. For determination of technical sensitivity SARS-CoV-2 virus culture material was used. The samples were treated with lysis buffer and analyzed using both an in-house and a pre-commercial SARS-CoV-2 nucleocapsid antigen assay on Simoa. Both nucleocapsid antigen assays have a technical sensitivity corresponding to around 100 SARS-CoV-2 RNA molecules/mL. Using a cut-off at 0.1 pg/mL the pre-commercial SARS-CoV-2 nucleocapsid antigen assay had a sensitivity of 96% (95% CI 91.4-98.5%) and specificity of 100% (95% CI 95.1-100%). In comparison the in-house nucleocapsid antigen assay had sensitivity of 95% (95% CI 89.3-98.1%) and a specificity of 100% (95% CI 95.1-100%) using a cut-off at 0.01 pg/mL. The two SARS-CoV-2 nucleocapsid antigen assays correlated with r = 0.91 (P < 0.0001). The in-house and the pre-commercial SARS-CoV-2 nucleocapsid antigen assay demonstrated technical and clinical sensitivity comparable to real-time RT-PCR methods for identifying SARS-CoV-2 infected patients and thus can be used clinically as well as serve as a reference method for antigen Point of Care Testing.

Routine, Cost-Effective SARS-CoV-2 Surveillance Testing Using Pooled Saliva Limits Viral Spread on a Residential College Campus.

Routine testing for SARS-CoV-2 is rare for institutes of higher education due to prohibitive costs and supply chain delays. During spring 2021, we routinely tested all residential students 1 to 2 times per week using pooled, RNA-extraction-free, reverse transcription quantitative PCR (RT-qPCR) testing of saliva at a cost of $0.43/sample with same-day results. The limit of detection was 500 copies/ml on individual samples, and analysis indicates 1,000 and 2,500 copies/ml in pools of 5 and 10, respectively, which is orders of magnitude more sensitive than rapid antigen tests. Importantly, saliva testing flagged 83% of semester positives (43,884 tests administered) and was 95.6% concordant with nasopharyngeal diagnostic results (69.0% concordant on the first test when the nucleocapsid gene (N1) cycle threshold (CT) value was >30). Moreover, testing reduced weekly cases by 59.9% in the spring despite far looser restrictions, allowing for more normalcy while eliminating outbreaks. We also coupled our testing with a survey to clarify symptoms and transmissibility among college-age students. While only 8.5% remained asymptomatic throughout, symptoms were disparate and often cold-like (e.g., only 37.3% developed a fever), highlighting the difficulty with relying on symptom monitoring among this demographic. Based on reported symptom progression, we estimate that we removed 348 days of infectious individuals by routine testing. Interestingly, viral load (CT value) at the time of testing did not affect transmissibility (R2 = 0.0085), though those experiencing noticeable symptoms at the time of testing were more likely to spread the virus to close contacts (31.6% versus 14.3%). Together, our findings support routine testing for reducing the spread of SARS-CoV-2. Implementation of cost- and resource-efficient approaches should receive strong consideration in communities that lack herd immunity. IMPORTANCE This study highlights the utility of routine testing for SARS-CoV-2 using pooled saliva while maintaining high sensitivity of detection (under 2,500 copies/ml) and rapid turnaround of high volume (up to 930 samples in 8 h by two technicians and one quantitative PCR [qPCR] machine). This pooled approach allowed us to test all residential students 1 to 2 times per week on our college campus during the spring of 2021 and flagged 83% of our semester positives. Most students were asymptomatic or presented with symptoms mirroring common colds at the time of testing, allowing for removal of infectious individuals before they otherwise would have sought testing. To our knowledge, the total per-sample consumable cost of $0.43 is the lowest to date. With many communities still lagging in vaccination rates, routine testing that is cost-efficient highlights the capacity of the laboratory's role in controlling the spread of SARS-CoV-2.