Point-of-care testing for COVID-19: a simple two-step molecular diagnostic development and validation during the SARS-CoV-2 pandemic.

BACKGROUND: During the coronavirus disease 19 (COVID-19) pandemic, diagnostic testing of the general population proved challenging due to limitations of the gold-standard diagnostic procedure using reverse transcription real-time polymerase chain reaction (RT-qPCR) for large-scale testing on the centralised model, especially in low-resource areas. OBJECTIVES: To address this, a point-of-care (PoC) diagnostic protocol for COVID-19 was developed, providing fast, reliable, and affordable testing, particularly for low-mid develop areas. METHODS: The PoC diagnostic process combines a simple paper-based RNA extraction method housed within a 3D-printed plastic device with a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. Nasopharyngeal/oropharyngeal swabs (NOS) and saliva samples were tested between 2020 and 2021, with the assistance of Santa Catarina's State Health Secretary, Brazil. FINDINGS: The developed diagnostic protocol showed a limit of detection of 9,900 copies and an overall diagnostic specificity of 98% for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from 1,348 clinical analysed samples. The diagnostic sensitivity was 95% for NOS samples, 85% for early morning saliva, and 69% for indiscriminate saliva. MAIN CONCLUSIONS: In conclusion, the developed device successfully extracted SARS-CoV-2 viral RNA from swabs and saliva clinical samples. When combined with colorimetric RT-LAMP, it provides results within 45 min using minimal resources, thus delivering a diagnostic kit protocol that is applicable in large-scale sampling.

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

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

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.

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.

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.

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

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

SARS-CoV-2 and the ocular surface: test accuracy and viral load.

PURPOSE: This study aimed to evaluate the pre-sence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the ocular surface of individuals clinically suspected of coronavirus disease 2019 (COVID-19) and determine the accuracy of different approaches of molecular testing on the ocular surface based on the nasopharyngeal positivity status for COVID-19. METHODS: A total of 152 individuals with suspected COVID-19 symptoms who simultaneously underwent nasopharyngeal and two different tear film collection techniques for quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) were included. Tears were collected and randomized: one eye had the filter strip for the Schirmer test and the contralateral eye had conjunctival swab/cytology in the inferior fornix. All patients underwent slit lamp biomicroscopy. The accuracy of various ocular surface collection techniques used for the detection of SARS-CoV-2 RNA was determined. RESULTS: Of the 152 patients enrolled in the study, 86 (56.6%) had COVID-19 confirmed by nasopharyngeal PCR. Both tear film collection techniques detected viral particles: the Schirmer test was positive in 16.3% (14/86) and the conjunctival swab/cytology in 17.4% (15/86), with no statistically significant differences. No positive ocular tests were found among those with negative nasopharyngeal PCR tests. The overall agreement of the ocular tests was 92.7%, and in combination, the sensitivity would increase to 23.2%. The mean cycle threshold values in the nasopharyngeal, Schirmer, and conjunctival swab/cytology tests were 18.2 ± 5.3, 35.6 ± 1.4, and 36.4 ± 3.9, respectively. Compared with the nasopharyngeal test, the Schirmer (p=0.001) and conjunctival swab/cytology (p<0.001) tests had significantly different Ct values. CONCLUSION: The Schirmer (16.3%) and conjunctival swab (17.4%) tests were comparably capable of detecting SARS-CoV-2 RNA in the ocular surface by RT-PCR accurately based on nasopharyngeal status and demonstrated indistinct sensitivity and specificity. Simultaneous specimen sampling and processing from the nasopharyngeal, Schirmer, and conjunctival swab/cytology tests demonstrated significantly lower viral load in both ocular surface approaches than in the nasopharyngeal test. Ocular manifestations detected by slit lamp biomicroscopy were not associated with ocular RT-PCR positivity.

Uma análise dos principais diagnósticos de Sars-cov-2 disponíveis no Brasil: uma revisão de literatura / Diagnosis and main types of coronavirus in humans: a narrative review / Diagnóstico y principales tipos de coronavirus en humanos: una revisión narrativa

Introdução: Com a emergência do SARS-CoV-2 foi disponibilizado uma grande quantidade de ferramentas de diagnóstico. Neste contexto, a falta de vacina, de tratamento e o grande número de casos graves e morte, possibilitou a aprovação emergencial de diversos testes, que ainda necessitam de estudos populacionais para seu registro definitivo. Objetivo: Realizar uma revisão de literatura para avaliar as metodologias de diagnóstico disponíveis no Brasil, de acordo com a realidade local de saúde, explorando o momento epidemiológico a complexidade do teste e a finalidade da sua aplicação. Metodologia: Trata-se de um estudo bibliográfico, descritivo do tipo revisão de literatura. Foram utilizadas as seguintes bases de dados científicos para buscas: PUBMED, MEDLINE, LILACS E COCHRANE LIBRARY, através de descritores selecionados na plataforma DECS. Resultados: O cenário de diversos ensaios, baseados em diferentes metodologias, como os testes baseados em RNA viral, em detecção de antígenos virais ou de anticorpos, associados ao conhecimento da história natural do vírus, possibilita uma análise crítica do melhor diagnóstico de acordo com a clínica do paciente, os epidemiológicos, o objetivo do diagnóstico e a acurácia do ensaio. Atualmente, há mudança no padrão imunológico da população e a descrição de tipos e subtipos de SARS-CoV-2 com mudanças gênicas, que podem levar a mudanças na acurácia diagnóstica ou a re-emergência em surtos de doença grave. Conclusão: Ainda é incerto o caminho evolutivo da história natural da Covid-19 e os ensaios diagnósticos estão em diferentes estágios de desenvolvimento, validação e produção e cada tipo de teste tem suas próprias vantagens e desvantagens distintas inerentes a plataforma tecnológica de origem e uma combinação de tipos de testes usados em momentos diferentes pode ser útil para a condução clínica dos pacientes e no controle da pandemia por SARS-CoV-2.

Introduction: With the emergence of SARS-CoV-2, a large number of diagnostic tools were made available. In this context, the lack of vaccine, treatment and the large number of severe cases and death, allowed the emergency approval of several tests, which still require population studies for their definitive registration. Objective: To carry out a literature review to evaluate the diagnostic methodologies available in Brazil, according to the local health reality, exploring the epidemiological moment, the complexity of the test and the purpose of its application. Methodology: This is a bibliographic, descriptive study of the literature review type. The following scientific databases were used for searches: PUBMED, MEDLINE, LILACS AND COCHRANE LIBRARY, through selected descriptors on the DECS platform. Results: The scenario of several tests, based on different methodologies, such as tests based on viral RNA, on detection of viral antigens or antibodies, associated with knowledge of the natural history of the virus, allows a critical analysis of the best diagnosis according to the patient's clinical, epidemiological, diagnostic objective and assay accuracy. Currently, there is a change in the immune pattern of the population and the description of types and subtypes of SARS-CoV-2 with genetic changes, which can lead to changes in diagnostic accuracy or the re-emergence in outbreaks of severe disease. Conclusion: The evolutionary path of the natural history of Covid-19 is still uncertain and diagnostic assays are at different stages of development, validation and production and each type of test has its own distinct advantages and disadvantages inherent in the technology platform of origin and a combination of types of tests used at different times can be useful for the clinical management of patients and in the control of the SARS-CoV-2 pandemic.

Introducción: Con la aparición del SARS-CoV-2, se dispuso de un gran número de herramientas diagnósticas. En este contexto, la falta de vacuna, tratamiento y el gran número de casos graves y muerte, permitieron la aprobación de urgencia de varias pruebas, que aún requieren estudios poblacionales para su registro definitivo. Objetivo: Realizar una revisión bibliográfica para evaluar las metodologías diagnósticas disponibles en Brasil, de acuerdo con la realidad sanitaria local, explorando el momento epidemiológico, la complejidad de la prueba y la finalidad de su aplicación. Metodología: Se trata de un estudio bibliográfico, descriptivo, del tipo revisión de literatura. Para las búsquedas se utilizaron las siguientes bases de datos científicas PUBMED, MEDLINE, LILACS Y COCHRANE LIBRARY, a través de descriptores seleccionados en la plataforma DECS. Resultados: El escenario de varias pruebas, basadas en diferentes metodologías, como pruebas basadas en el ARN viral, en la detección de antígenos virales o anticuerpos, asociado al conocimiento de la historia natural del virus, permite un análisis crítico del mejor diagnóstico de acuerdo con la clínica del paciente, epidemiológica, objetivo diagnóstico y precisión de la prueba. Actualmente, hay un cambio en el patrón inmunológico de la población y la descripción de tipos y subtipos de SARS-CoV-2 con cambios genéticos, que pueden conducir a cambios en la precisión diagnóstica o la reaparición en brotes de enfermedad grave. Conclusiones: El camino evolutivo de la historia natural del Covid-19 es aún incierto y los ensayos de diagnóstico se encuentran en diferentes etapas de desarrollo, validación y producción y cada tipo de prueba tiene sus propias ventajas y desventajas distintas inherentes a la plataforma tecnológica de origen y una combinación de tipos de pruebas utilizadas en diferentes momentos puede ser útil para el manejo clínico de los pacientes y en el control de la pandemia de SARS- CoV-2.

Distinct Outcomes in COVID-19 Patients with Positive or Negative RT-PCR Test.

Identification of the SARS-CoV-2 virus by RT-PCR from a nasopharyngeal swab sample is a common test for diagnosing COVID-19. However, some patients present clinical, laboratorial, and radiological evidence of COVID-19 infection with negative RT-PCR result(s). Thus, we assessed whether positive results were associated with intubation and mortality. This study was conducted in a Brazilian tertiary hospital from March to August of 2020. All patients had clinical, laboratory, and radiological diagnosis of COVID-19. They were divided into two groups: positive (+) RT-PCR group, with 2292 participants, and negative (-) RT-PCR group, with 706 participants. Patients with negative RT-PCR testing and an alternative most probable diagnosis were excluded from the study. The RT-PCR(+) group presented increased risk of intensive care unit (ICU) admission, mechanical ventilation, length of hospital stay, and 28-day mortality, when compared to the RT-PCR(-) group. A positive SARS-CoV-2 RT-PCR result was independently associated with intubation and 28 day in-hospital mortality. Accordingly, we concluded that patients with a COVID-19 diagnosis based on clinical data, despite a negative RT-PCR test from nasopharyngeal samples, presented more favorable outcomes than patients with positive RT-PCR test(s).

Genomic surveillance of SARS-CoV-2 Spike gene by sanger sequencing.

The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike gene. Monitoring the S gene mutations is crucial for successful controlling measures and detecting variants that can evade vaccine immunity. Even after the costs reduction resulting from the pandemic, the new generation sequencing methodologies remain unavailable to a large number of scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S gene, this work describes a new feasible protocol for complete nucleotide sequencing of the S gene using the Sanger technique. Such a methodology could be easily adopted by any laboratory with experience in sequencing, adding to effective surveillance of SARS-CoV-2 spreading and evolution.

Prolonged SARS-CoV-2 Positivity in Immunocompetent Patients: Virus Isolation, Genomic Integrity, and Transmission Risk.

Current guidelines for patient isolation in COVID-19 cases recommend a symptom-based approach, averting the use of control real-time reverse transcription PCR (rRT-PCR) testing. However, we hypothesized that patients with persistently positive results by RT-PCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be potentially infectious for a prolonged time, even if immunocompetent and asymptomatic, which would demand a longer social isolation period than presently recommended. To test this hypothesis, 72 samples from 51 mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2 were tested for their infectiousness in cell culture. The serological response of samples from those patients and virus genomic integrity were also analyzed. Infectious viruses were successfully isolated from 34.38% (22/64) of nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation up to 128 days. Complete SARS-COV-2 genome integrity was demonstrated, suggesting the presence of replication-competent viruses. No correlation was found between the isolation of infectious viruses and rRT-PCR cycle threshold values or the humoral immune response. These findings call attention to the need to review current isolation guidelines, particularly in scenarios involving high-risk individuals. IMPORTANCE In this study, we evaluated mildly symptomatic immunocompetent patients with long-lasting positive rRT-PCR results for SARS-CoV-2. Infectious viruses were successfully isolated in cell cultures from nasopharynx samples obtained 14 days or longer after symptom onset. Indeed, we observed successful virus isolation for up to 128 days. Moreover, SARS-CoV-2 genome integrity was demonstrated by sequencing, suggesting the presence of replication-competent viruses. These data point out the risk of continuous SARS-CoV-2 transmission from patients with prolonged detection of SARS-CoV-2 in the upper respiratory tract, which has important implications for current precaution guidelines, particularly in settings where vulnerable individuals may be exposed (e.g., nursing homes and hospitals).

Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting.

The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.

High sensitivity-low cost detection of SARS-CoV-2 by two steps end point RT-PCR with agarose gel electrophoresis visualization.

More than one year since Coronavirus disease 2019 (COVID-19) pandemic outbreak, the gold standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection is still the RT-qPCR. This is a limitation to increase testing capacities, particularly at developing countries, as expensive reagents and equipment are required. We developed a two steps end point RT-PCR reaction with SARS-CoV-2 Nucleocapsid (N) gene and Ribonuclease P (RNase P) specific primers where viral amplicons were verified by agarose gel electrophoresis. We carried out a clinical performance and analytical sensitivity evaluation for this two-steps end point RT-PCR method with 242 nasopharyngeal samples using the CDC RT-qPCR protocol as a gold standard technique. With a specificity of 95.8%, a sensitivity of 95.1%, and a limit of detection of 20 viral RNA copies/uL, this two steps end point RT-PCR assay is an affordable and reliable method for SARS-CoV-2 detection. This protocol would allow to extend COVID-19 diagnosis to basic molecular biology laboratories with a potential positive impact in surveillance programs at developing countries.

Evaluation of a COVID-19 Vaccine Campaign and SARS-CoV-2 Infection and Mortality Among Adults Aged 60 Years And Older in a Middle-Income Country.

Importance: Although there are reports of COVID-19 vaccine implementation in real-world populations, these come from high-income countries or from experience with messenger RNA technology vaccines. Data on outcomes of vaccine deployment in low- or middle-income countries are lacking. Objective: To assess whether the pragmatic application of the 3 COVID-19 vaccines available in Argentina, 2 of which have no reports of evaluation in real-world settings to date, were associated with a reduction in morbidity, all-cause mortality, and mortality due to COVID-19. Design, Setting, and Participants: This cohort study used individual and ecological data to explore outcomes following vaccination with rAd26-rAd5, ChAdOx1, and BBIBP-CorV. To correct for differences in exposure times, results are shown using incidence density per 100 000 person-days from the start of the vaccination campaign (December 29, 2020) to the occurrence of an event or the end of follow-up (May 15, 2021). Participants included 663 602 people aged at least 60 years residing in the city of Buenos Aires, Argentina. Statistical analysis was performed from June 1 to June 15, 2021. Main Outcomes and Measures: Diagnosis of COVID-19 confirmed by reverse transcription-polymerase chain reaction, death from all causes, and death within 30 days of a diagnosis of COVID-19. Poisson regression models were fitted to estimate associations with all 3 outcomes. Results: Among 663 602 residents of the city of Buenos Aires included in the study, 540 792 (81.4%) were vaccinated with at least 1 dose, with 457 066 receiving 1 dose (mean [SD] age, 74.5 (8.9) years; 61.5% were female [n = 281 284]; 68.0% [n = 310 987] received the rAd26-rAd5 vaccine; 29.5% [n = 135 036] received ChAdOx1; 2.4% [n = 11 043] received BBIBP-CorV) and 83 726 receiving 2 doses (mean [SD] age, 73.4 [6.8] years; 63.5% were female [n = 53 204]). The incidence density of confirmed COVID-19 was 36.25 cases/100 000 person-days (95% CI, 35.80-36.70 cases/100 000 person-days) among those who did not receive a vaccine, 19.13 cases/100 000 person-days (95% CI, 18.63-19.62 cases/100 000 person-days) among those who received 1 dose, and 4.33 cases/100 000 person-days (95% CI, 3.85-4.81 cases/100 000 person-days) among those who received 2 doses. All-cause mortality was 11.74 cases/100 000 person-days (95% CI, 11.51-11.96 cases/100 000 person-days), 4.01 cases/100 000 person-days (95% CI, 3.78-4.24 cases/100 000 person-days) and 0.40 cases/100 000 person-days (95% CI, 0.26-0.55 cases/100 000 person-days). COVID-19-related-death rate was 2.31 cases/100 000 person-days (95% CI, 2.19-2.42 cases/100 000 person-days), 0.59 cases/100 000 person-days (95% CI, 0.50-0.67 cases/100 000 person-days), and 0.04 cases/100 000 person-days (95% CI, 0.0-0.09 cases/100 000 person-days) among the same groups. A 2-dose vaccination schedule was associated with an 88.1% (95% CI, 86.8%-89.2%) reduction in documented infection, 96.6% (95% CI, 95.3%-97.5%) reduction in all-cause death, and 98.3% (95% CI, 95.3%-99.4%) reduction in COVID-19-related death. A single dose was associated with a 47.2% (95% CI, 44.2%-50.1%) reduction in documented infection, 65.8% (95% CI, 61.7%-69.5%) reduction in all-cause death, and 74.5% (95% CI, 66%-80.8%) reduction in COVID-19-related death. Conclusions and Relevance: This study found that within the first 5 months after the start of the vaccination campaign, vaccination was associated with a significant reduction in COVID-19 infection as well as a reduction in mortality.

Nested pool testing strategy for the diagnosis of infectious diseases.

The progress of the SARS-CoV-2 pandemic requires the design of large-scale, cost-effective testing programs. Pooling samples provides a solution if the tests are sensitive enough. In this regard, the use of the gold standard, RT-qPCR, raises some concerns. Recently, droplet digital PCR (ddPCR) was shown to be 10-100 times more sensitive than RT-qPCR, making it more suitable for pooling. Furthermore, ddPCR quantifies the RNA content directly, a feature that, as we show, can be used to identify nonviable samples in pools. Cost-effective strategies require the definition of efficient deconvolution and re-testing procedures. In this paper we analyze the practical implementation of an efficient hierarchical pooling strategy for which we have recently derived the optimal, determining the best ways to proceed when there are impediments for the use of the absolute optimum or when multiple pools are tested simultaneously and there are restrictions on the throughput time. We also show how the ddPCR RNA quantification and the nested nature of the strategy can be combined to perform self-consistency tests for a better identification of infected individuals and nonviable samples. The studies are useful to those considering pool testing for the identification of infected individuals.

Diagnostic accuracy and acceptability of molecular diagnosis of COVID-19 on saliva samples relative to nasopharyngeal swabs in tropical hospital and extra-hospital contexts: The COVISAL study.

A prospective study was conducted among different intra and extra-hospital populations of French Guiana to evaluate the performance of saliva testing compared to nasopharyngeal swabs. Persons aged 3 years and older with mild symptoms suggestive of COVID-19 and asymptomatic persons with a testing indication were prospectively enrolled. Nasopharyngeal and salivary samples were stored at 4°C before analysis. Both samples were analyzed with the same Real-time PCR amplification of E gene, N gene, and RdRp gene. Between July 22th and October 28th, 1159 persons were included, of which 1028 were analyzed. When only considering as positives those with 2 target genes with Ct values <35, the sensitivity of RT-PCR on saliva samples was 100% relative to nasopharyngeal samples. Specificity positive and negative predictive values were above 90%. Across a variety of cultures and socioeconomic conditions, saliva tests were generally much preferred to nasopharyngeal tests and persons seemed largely confident that they could self-sample. For positive patients defined as those with the amplification of 2 specific target genes with Ct values below 35, the sensitivity and specificity of RT-PCR on saliva samples was similar to nasopharyngeal samples despite the broad range of challenging circumstances in a tropical environment.

Validation of conventional PCR-like alternative to SARS-CoV-2 detection with target nucleocapsid protein gene in naso-oropharyngeal samples.

SARS-CoV-2 has spread worldwide and has become a global health problem. As a result, the demand for inputs for diagnostic tests rose dramatically, as did the cost. Countries with inadequate infrastructure experience difficulties in expanding their qPCR testing capacity. Therefore, the development of sensitive and specific alternative methods is essential. This study aimed to develop, standardize, optimize, and validate conventional RT-PCR targeting the N gene of SARS-CoV-2 in naso-oropharyngeal swab samples compared to qPCR. Using bioinformatics tools, specific primers were determined, with a product expected to be 519 bp. The reaction conditions were optimized using a commercial positive control, and the detection limit was determined to be 100 fragments. To validate conventional RT-PCR, we determined a representative sampling of 346 samples from patients with suspected infection whose diagnosis was made in parallel with qPCR. A sensitivity of 92.1% and specificity of 100% were verified, with an accuracy of 95.66% and correlation coefficient of 0.913. Under current Brazilian conditions, this method generates approximately 60% savings compared to qPCR costs. Conventional RT-PCR, validated herein, showed sufficient results for the detection of SARS-CoV-2 and can be used as an alternative for epidemiological studies and interspecies correlations.

Covid-19 diagnosis by combining RT-PCR and pseudo-convolutional machines to characterize virus sequences.

The Covid-19 pandemic, a disease transmitted by the SARS-CoV-2 virus, has already caused the infection of more than 120 million people, of which 70 million have been recovered, while 3 million people have died. The high speed of infection has led to the rapid depletion of public health resources in most countries. RT-PCR is Covid-19's reference diagnostic method. In this work we propose a new technique for representing DNA sequences: they are divided into smaller sequences with overlap in a pseudo-convolutional approach and represented by co-occurrence matrices. This technique eliminates multiple sequence alignment. Through the proposed method, it is possible to identify virus sequences from a large database: 347,363 virus DNA sequences from 24 virus families and SARS-CoV-2. When comparing SARS-CoV-2 with virus families with similar symptoms, we obtained [Formula: see text] for sensitivity and [Formula: see text] for specificity with MLP classifier and 30% overlap. When SARS-CoV-2 is compared to other coronaviruses and healthy human DNA sequences, we obtained [Formula: see text] for sensitivity and [Formula: see text] for specificity with MLP and 50% overlap. Therefore, the molecular diagnosis of Covid-19 can be optimized by combining RT-PCR and our pseudo-convolutional method to identify DNA sequences for SARS-CoV-2 with greater specificity and sensitivity.

Design and in silico validation of polymerase chain reaction primers to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Accurate designing of polymerase chain reaction (PCR) primers targeting conserved segments in viral genomes is desirable for preventing false-negative results and decreasing the need for standardization across different PCR protocols. In this work, we designed and described a set of primers and probes targeting conserved regions identified from a multiple sequence alignment of 2341 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genomes from the Global Initiative on Sharing All Influenza Data (GISAID). We subsequently validated those primers and probes in 211,833 SARS-CoV-2 whole-genome sequences. We obtained nine systems (forward primer + reverse primer + probe) that potentially anneal to highly conserved regions of the virus genome from these analyses. In silico predictions also demonstrated that those primers do not bind to nonspecific targets for human, bacterial, fungal, apicomplexan, and other Betacoronaviruses and less pathogenic sub-strains of coronavirus. The availability of these primer and probe sequences will make it possible to validate more efficient protocols for identifying SARS-CoV-2.