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).

Viral Micro-RNAs Are Detected in the Early Systemic Response to Injury and Are Associated With Outcomes in Polytrauma Patients.

OBJECTIVES: To evaluate early activation of latent viruses in polytrauma patients and consider prognostic value of viral micro-RNAs in these patients. DESIGN: This was a subset analysis from a prospectively collected multicenter trauma database. Blood samples were obtained upon admission to the trauma bay (T0), and trauma metrics and recovery data were collected. SETTING: Two civilian Level 1 Trauma Centers and one Military Treatment Facility. PATIENTS: Adult polytrauma patients with Injury Severity Scores greater than or equal to 16 and available T0 plasma samples were included in this study. Patients with ICU admission greater than 14 days, mechanical ventilation greater than 7 days, or mortality within 28 days were considered to have a complicated recovery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Polytrauma patients (n = 180) were identified, and complicated recovery was noted in 33%. Plasma samples from T0 underwent reverse transcriptase-quantitative polymerase chain reaction analysis for Kaposi's sarcoma-associated herpesvirus micro-RNAs (miR-K12_10b and miRK-12-12) and Epstein-Barr virus-associated micro-RNA (miR-BHRF-1), as well as Luminex multiplex array analysis for established mediators of inflammation. Ninety-eight percent of polytrauma patients were found to have detectable Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus micro-RNAs at T0, whereas healthy controls demonstrated 0% and 100% detection rate for Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus, respectively. Univariate analysis revealed associations between viral micro-RNAs and polytrauma patients' age, race, and postinjury complications. Multivariate least absolute shrinkage and selection operator analysis of clinical variables and systemic biomarkers at T0 revealed that interleukin-10 was the strongest predictor of all viral micro-RNAs. Multivariate least absolute shrinkage and selection operator analysis of systemic biomarkers as predictors of complicated recovery at T0 demonstrated that miR-BHRF-1, miR-K12-12, monocyte chemoattractant protein-1, and hepatocyte growth factor were independent predictors of complicated recovery with a model complicated recovery prediction area under the curve of 0.81. CONCLUSIONS: Viral micro-RNAs were detected within hours of injury and correlated with poor outcomes in polytrauma patients. Our findings suggest that transcription of viral micro-RNAs occurs early in the response to trauma and may be associated with the biological processes involved in polytrauma-induced complicated recovery.

Repositive RT-PCR test in discharged COVID-19 patients during medical isolation observation.

Objectives: The epidemiological and clinical characteristics of patients with coronavirus disease 2019 (COVID-19) have been researched. However, the prevalence of repositivity by real-time PCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains unclear. Methods: A retrospective study was conducted involving 599 discharged patients with COVID-19 in a single medical centre. The clinical features of patients during their hospitalization and 14-day post-discharge quarantine were collected. Results: A total of 122 patients (20.4%) out of 599 patients retested positive after discharge. Specifically, 94 (15.7%) retested positive within 24 h of discharge, and another 28 patients (4.7%) were repositive on day 7 after discharge, although none showed any clinical symptomatic recurrence. Both repositives and non­repositives have similar patterns of IgG and IgM. Notably, the length of hospitalization of non-repositive patients was longer than that of 24-h repositive patients and 7-day repositive patients. In addition, the length of hospitalization of 24-h repositive patients was shorter than that of 7-day repositive patients, indicating that the length of hospitalization was also a determinant of viral shedding. Conclusion: Our study provides further information for improving the management of recovered and discharged patients, and further studies should be performed to elucidate the infectiveness of individuals with prolonged or RNA repositivity.

The value of anal swab RT-PCR for COVID-19 diagnosis in adult Indonesian patients.

OBJECTIVE: This study will test the performance of the anal swab PCR test when compared with the nasopharyngeal swab PCR test as a diagnostic tool for COVID-19. DESIGN: An observational descriptive study which included hospitalised suspected, or probable cases of hopitalised COVID-19 patients, conducted in Dr. Cipto Mangunkusumo National Hospital, Ciputra Hospital, Mitra Keluarga Depok Hospital and Mitra Keluarga Kelapa Gading Hospital, Indonesia. Epidemiological, clinical, laboratory and radiology data were obtained. Nasopharyngeal and anal swabs specimens were collected for SARS-CoV-2 RNA detection. RESULTS: We analysed 136 subjects as part of this study. The clinical spectrum of COVID-19 manifesation in this study was typical of hospitalised patients, with 25% classified as mild cases, 14.7% in severe condition and 12.5% of subjects classified as having acute respiratory distress syndrome. When compared with nasopharyngeal swab as the standard specimen for reverse transcription polymerase chain reaction (RT-PCR) detection of SARS-CoV-2 antigen, the sensitivity and specificity of the anal swab was 36.7% and 93.8%, respectively. The positive and negative predictive value were 97.8% and 16.5 %, respectively. The performance of the anal swab remained similar when only the subgroup of patients with gastrointestinal symptoms (n=92, 67.6%) was analysed (sensitivity 40% and specificity 91.7%). Out of all the subjects included in analysis, 67.6% had gastrointestinal symptoms. Similarly, 73.3% of patients in the anal swab-positive group had gastrointestinal symptoms. The two most common gastrointestinal symptoms in the subjects' population were nausea and anorexia. CONCLUSION: Anal swab specimen has low sensitivity (36.7%) but high specificity (93.8%) for detecting SARS-CoV-2 antigen by RT-PCR. Only one additional positive result was found by anal swab among the nasopharyngeal swab-negative group. Anal swab may not be needed as an additional test at the beginning of a patient's diagnostic investigation and nasopharyngeal swab RT-PCR remains as the standard diagnostic test for COVID-19.

Expanding molecular diagnostic capacity for COVID-19 in Ethiopia: operational implications, challenges and lessons learnt.

Efforts towards slowing down coronavirus (COVID-19) transmission and reducing mortality have focused on timely case detection, isolation and treatment. Availability of laboratory COVID-19 testing capacity using reverse-transcriptase polymerase chain reaction (RT-PCR) was essential for case detection. Hence, it was critical to establish and expand this capacity to test for COVID-19 in Ethiopia. To this end, using a three-phrased approach, potential public and private laboratories with RT-PCR technology were assessed, capacitated with trained human resource and equipped as required. These laboratories were verified to conduct COVID-19 testing with quality assurance checks regularly conducted. Within a 10-month period, COVID-19 testing laboratories increased from zero to 65 in all Regional States with the capacity to conduct 18,454 tests per day. The success of this rapid countrywide expansion of laboratory testing capacity for COVID-19 depended on some key operational implications: the strong laboratory coordination network within the country, the use of non-virologic laboratories, investment in capacity building, digitalization of the data for better information management and establishing quality assurance checks. A weak supply chain for laboratory reagents and consumables, differences in the brands of COVID-19 test kits, frequent breakdowns of the PCR machines and inadequate number of laboratory personnel following the adaption of a 24/7 work schedule were some of the challenges experienced during the process of laboratory expansion. Overall, we learn that multisectoral involvement of laboratories from non-health sectors, an effective supply chain system with an insight into the promotion of local production of laboratory supplies were critical during the laboratory expansion for COVID-19 testing. The consistent support from WHO and other implementing partners to Member States is needed in building the capacity of laboratories across different diagnostic capabilities in line with International Health Regulations. This will enable efficient adaptation to respond to future public health emergencies.

Lassa fever outcomes and prognostic factors in Nigeria (LASCOPE): a prospective cohort study.

BACKGROUND: Lassa fever is a viral haemorrhagic fever endemic in parts of west Africa. New treatments are needed to decrease mortality, but pretrial reference data on the disease characteristics are scarce. We aimed to document baseline characteristics and outcomes for patients hospitalised with Lassa fever in Nigeria. METHODS: We did a prospective cohort study (LASCOPE) at the Federal Medical Centre in Owo, Nigeria. All patients admitted with confirmed Lassa fever were invited to participate and asked to give informed consent. Patients of all ages, including newborn infants, were eligible for inclusion, as were pregnant women. All participants received standard supportive care and intravenous ribavirin according to Nigeria Centre for Disease Control guidelines and underwent systematic biological monitoring for 30 days. Patients' characteristics, care received, mortality, and associated factors were recorded using standard WHO forms. We used univariable and multivariable logistic regression models to investigate an association between baseline characteristics and mortality at day 30. FINDINGS: Between April 5, 2018, and March 15, 2020, 534 patients with confirmed Lassa fever were admitted to hospital, of whom 510 (96%) gave consent and were included in the analysis. The cohort included 258 (51%) male patients, 252 (49%) female patients, 426 (84%) adults, and 84 (16%) children (younger than 18 years). The median time between first symptoms and hospital admission was 8 days (IQR 7-13). At baseline, 176 (38%) of 466 patients had a Lassa fever RT-PCR cycle threshold (Ct) lower than 30. From admission to end of follow-up, 120 (25%) of 484 reached a National Early Warning Score (second version; NEWS2) of 7 or higher, 67 (14%) of 495 reached a Kidney Disease-Improving Global Outcome (KDIGO) stage of 2 or higher, and 41 (8%) of 510 underwent dialysis. All patients received ribavirin for a median of 10 days (IQR 9-13). 62 (12%) patients died (57 [13%] adults and five [6%] children). The median time to death was 3 days (1-6). The baseline factors independently associated with mortality were the following: age 45 years or older (adjusted odds ratio 16·30, 95% CI 5·31-50·30), NEWS2 of 7 or higher (4·79, 1·75-13·10), KDIGO grade 2 or higher (7·52, 2·66-21·20), plasma alanine aminotransferase 3 or more times the upper limit of normal (4·96, 1·69-14·60), and Lassa fever RT-PCR Ct value lower than 30 (4·65, 1·50-14·50). INTERPRETATION: Our findings comprehensively document clinical and biological characteristics of patients with Lassa fever and their relationship with mortality, providing prospective estimates that could be useful for designing future therapeutic trials. Such trials comparing new Lassa fever treatments to a standard of care should take no more than 15% as the reference mortality rate and consider adopting a combination of mortality and need for dialysis as the primary endpoint. FUNDING: Institut National de la Santé et de la Recherche Médicale, University of Oxford, EU, UK Department for International Development, Wellcome Trust, French Ministry of Foreign Affairs, Agence Nationale de Recherches sur le SIDA et les hépatites virales, French National Research Institute for Sustainable Development.

Variability of cycle threshold values in an external quality assessment scheme for detection of the SARS-CoV-2 virus genome by RT-PCR.

OBJECTIVES: The qualitative results of SARS-CoV-2 specific real-time reverse transcription (RT) PCR are used for initial diagnosis and follow-up of Covid-19 patients and asymptomatic virus carriers. However, clinical decision-making and health management policies often are based additionally on cycle threshold (Ct) values (i.e., quantitative results) to guide patient care, segregation and discharge management of individuals testing positive. Therefore, an analysis of inter-protocol variability is needed to assess the comparability of the quantitative results. METHODS: Ct values reported in a SARS-CoV-2 virus genome detection external quality assessment challenge were analyzed. Three positive and two negative samples were distributed to participating test laboratories. Qualitative results (positive/negative) and quantitative results (Ct values) were assessed. RESULTS: A total of 66 laboratories participated, contributing results from 101 distinct test systems and reporting Ct values for a total of 92 different protocols. In all three positive samples, the means of the Ct values for the E-, N-, S-, RdRp-, and ORF1ab-genes varied by less than two cycles. However, 7.7% of reported results deviated by more than ±4.0 (maximum 18.0) cycles from the respective individual means. These larger deviations appear to be systematic errors. CONCLUSIONS: In an attempt to use PCR diagnostics beyond the identification of infected individuals, laboratories are frequently requested to report Ct values along with a qualitative result. This study highlights the limitations of interpreting Ct values from the various SARS-CoV genome detection protocols and suggests that standardization is necessary in the reporting of Ct values with respect to the target gene.

Thoracic imaging tests for the diagnosis of COVID-19.

BACKGROUND: The respiratory illness caused by SARS-CoV-2 infection continues to present diagnostic challenges. Early research showed thoracic (chest) imaging to be sensitive but not specific in the diagnosis of coronavirus disease 2019 (COVID-19). However, this is a rapidly developing field and these findings need to be re-evaluated in the light of new research. This is the first update of this 'living systematic review'. This update focuses on people suspected of having COVID-19 and excludes studies with only confirmed COVID-19 participants. OBJECTIVES: To evaluate the diagnostic accuracy of thoracic imaging (computed tomography (CT), X-ray and ultrasound) in people with suspected COVID-19. SEARCH METHODS: We searched the COVID-19 Living Evidence Database from the University of Bern, the Cochrane COVID-19 Study Register, The Stephen B. Thacker CDC Library, and repositories of COVID-19 publications through to 22 June 2020. We did not apply any language restrictions. SELECTION CRITERIA: We included studies of all designs that recruited participants of any age group suspected to have COVID-19, and which reported estimates of test accuracy, or provided data from which estimates could be computed. When studies used a variety of reference standards, we retained the classification of participants as COVID-19 positive or negative as used in the study. DATA COLLECTION AND ANALYSIS: We screened studies, extracted data, and assessed the risk of bias and applicability concerns using the QUADAS-2 domain-list independently, in duplicate. We categorised included studies into three groups based on classification of index test results: studies that reported specific criteria for index test positivity (group 1); studies that did not report specific criteria, but had the test reader(s) explicitly classify the imaging test result as either COVID-19 positive or negative (group 2); and studies that reported an overview of index test findings, without explicitly classifying the imaging test as either COVID-19 positive or negative (group 3). We presented the results of estimated sensitivity and specificity using paired forest plots, and summarised in tables. We used a bivariate meta-analysis model where appropriate. We presented uncertainty of the accuracy estimates using 95% confidence intervals (CIs). MAIN RESULTS: We included 34 studies: 30 were cross-sectional studies with 8491 participants suspected of COVID-19, of which 4575 (54%) had a final diagnosis of COVID-19; four were case-control studies with 848 cases and controls in total, of which 464 (55%) had a final diagnosis of COVID-19. Chest CT was evaluated in 31 studies (8014 participants, 4224 (53%) cases), chest X-ray in three studies (1243 participants, 784 (63%) cases), and ultrasound of the lungs in one study (100 participants, 31 (31%) cases). Twenty-six per cent (9/34) of all studies were available only as preprints. Nineteen studies were conducted in Asia, 10 in Europe, four in North America and one in Australia. Sixteen studies included only adults, 15 studies included both adults and children and one included only children. Two studies did not report the ages of participants. Twenty-four studies included inpatients, four studies included outpatients, while the remaining six studies were conducted in unclear settings. The majority of included studies had a high or unclear risk of bias with respect to participant selection, index test, reference standard, and participant flow. For chest CT in suspected COVID-19 participants (31 studies, 8014 participants, 4224 (53%) cases) the sensitivity ranged from 57.4% to 100%, and specificity ranged from 0% to 96.0%. The pooled sensitivity of chest CT in suspected COVID-19 participants was 89.9% (95% CI 85.7 to 92.9) and the pooled specificity was 61.1% (95% CI 42.3 to 77.1). Sensitivity analyses showed that when the studies from China were excluded, the studies from other countries demonstrated higher specificity compared to the overall included studies. When studies that did not classify index tests as positive or negative for COVID-19 (group 3) were excluded, the remaining studies (groups 1 and 2) demonstrated higher specificity compared to the overall included studies. Sensitivity analyses limited to cross-sectional studies, or studies where at least two reverse transcriptase polymerase chain reaction (RT-PCR) tests were conducted if the first was negative, did not substantively alter the accuracy estimates. We did not identify publication status as a source of heterogeneity. For chest X-ray in suspected COVID-19 participants (3 studies, 1243 participants, 784 (63%) cases) the sensitivity ranged from 56.9% to 89.0% and specificity from 11.1% to 88.9%. The sensitivity and specificity of ultrasound of the lungs in suspected COVID-19 participants (1 study, 100 participants, 31 (31%) cases) were 96.8% and 62.3%, respectively. We could not perform a meta-analysis for chest X-ray or ultrasound due to the limited number of included studies. AUTHORS' CONCLUSIONS: Our findings indicate that chest CT is sensitive and moderately specific for the diagnosis of COVID-19 in suspected patients, meaning that CT may have limited capability in differentiating SARS-CoV-2 infection from other causes of respiratory illness. However, we are limited in our confidence in these results due to the poor study quality and the heterogeneity of included studies. Because of limited data, accuracy estimates of chest X-ray and ultrasound of the lungs for the diagnosis of suspected COVID-19 cases should be carefully interpreted. Future diagnostic accuracy studies should pre-define positive imaging findings, include direct comparisons of the various modalities of interest on the same participant population, and implement improved reporting practices. Planned updates of this review will aim to: increase precision around the accuracy estimates for chest CT (ideally with low risk of bias studies); obtain further data to inform accuracy of chest X-rays and ultrasound; and obtain data to further fulfil secondary objectives (e.g. 'threshold' effects, comparing accuracy estimates across different imaging modalities) to inform the utility of imaging along different diagnostic pathways.

[Prediction of end of lockdown post-peak of cases in first wave of the COVID-19 pandemic in Chile]. / Predicción del fin del confinamiento después del máximo de casos en la primera ola de la pandemia COVID-19 en Chile.

INTRODUCTION: The results of mandatory confinement have been detrimental in several respects. Nonetheless, they have resulted in reducing the number of active cases of COVID-19. Chile has begun the de-escalation and needs to know the best time to end the restrictions. OBJECTIVE: We discuss the best conditions and guarantees for the end of compulsory confinement. METHODS: This study is based on a trend model with prediction estimation. The data of the variables of interest were subjected to linear regression studies to determine the curve that best explained the data. The coefficient of determination, the standard deviation of y in x, and the confidence interval of the observed curve were estimated. The trend curve was chosen in accordance with the regression estimates. OUTCOMES: It was found that all dependent variables tended to decrease over time in a quadratic fashion, except for the new cases variable. In general, the R2 and MAPE estimates are satisfactory, except for the variable number of PCR tests per day. CONCLUSIONS: Gradual and cautious steps should be taken before ending mandatory confinement. In the current de-escalator, daily PCR tests should be increased, maintaining vigilance on indicators of incidence, prevalence, and positivity of PCR tests. Evidence suggests with some degree of confidence that mandatory confinement could be safely lifted as of August 30, 2020. Long-term preparations must be made to contain future waves of new cases.

INTRODUCCIÓN: Los resultados del confinamiento obligatorio han sido perjudiciales en varios aspectos. No obstante, han surtido efecto en lograr el descenso de casos activos de COVID-19. Chile ha comenzado la desescalada y precisa conocer el mejor momento para poner fin a las restricciones. OBJETIVOS: Discutir las mejores condiciones y garantías para el fin del confinamiento obligatorio sobre la base de los casos nuevos, casos activos y positividad de exámenes de reacción en cadena de la polimerasa. MÉTODOS: Estudio basado en un modelo de tendencia con estimación de predicciones. Los datos de las variables de interés fueron sometidas a estudios de regresión lineal, con el objeto de determinar la curva que mejor explicaba los datos. Se estimó el coeficiente de determinación, la desviación estándar de y en x y el intervalo de confianza de la curva observada. Posteriormente, fue escogida la curva de tendencia en concordancia con las estimaciones de regresión. RESULTADOS: Se encontró que todas las variables dependientes tendían a disminuir con el tiempo de forma cuadrática, con excepción de la variable casos nuevos. En general, las estimaciones de coeficiente de determinación (R2) y error porcentual absoluto medio son satisfactorias, con excepción de la variable: número de exámenes de reacción en cadena de la polimerasa por día. CONCLUSIONES: Se deben tomar medidas graduales y cautelosas antes de poner fin al confinamiento obligatorio. En la actual desescalada, se deben aumentar los exámenes de reacción en cadena de la polimerasa diarios y mantener vigilancia en los indicadores de incidencia, prevalencia y positividad de dichos exámenes. La evidencia sugiere con cierto grado de confiabilidad que el confinamiento obligatorio podría levantarse de forma segura a contar del día 30 de agosto de 2020. Se deben hacer preparativos a largo plazo en contención de las futuras olas, es decir, una nueva alza de casos nuevos y activos luego del descenso.

Predicción del fin del confinamiento después del máximo de casos en la primera ola de la pandemia COVID-19 en Chile / Prediction of end of lockdown post-peak of cases in first wave of the COVID-19 pandemic in Chile

Introducción Los resultados del confinamiento obligatorio han sido perjudiciales en varios aspectos. No obstante, han surtido efecto en lograr el descenso de casos activos de COVID-19. Chile ha comenzado la desescalada y precisa conocer el mejor momento para poner fin a las restricciones. Objetivos Discutir las mejores condiciones y garantías para el fin del confinamiento obligatorio sobre la base de los casos nuevos, casos activos y positividad de exámenes de reacción en cadena de la polimerasa. Métodos Estudio basado en un modelo de tendencia con estimación de predicciones. Los datos de las variables de interés fueron sometidas a estudios de regresión lineal, con el objeto de determinar la curva que mejor explicaba los datos. Se estimó el coeficiente de determinación, la desviación estándar de y en x y el intervalo de confianza de la curva observada. Posteriormente, fue escogida la curva de tendencia en concordancia con las estimaciones de regresión. Resultados Se encontró que todas las variables dependientes tendían a disminuir con el tiempo de forma cuadrática, con excepción de la variable casos nuevos. En general, las estimaciones de coeficiente de determinación (R2) y error porcentual absoluto medio son satisfactorias, con excepción de la variable: número de exámenes de reacción en cadena de la polimerasa por día. Conclusiones Se deben tomar medidas graduales y cautelosas antes de poner fin al confinamiento obligatorio. En la actual desescalada, se deben aumentar los exámenes de reacción en cadena de la polimerasa diarios y mantener vigilancia en los indicadores de incidencia, prevalencia y positividad de dichos exámenes. La evidencia sugiere con cierto grado de confiabilidad que el confinamiento obligatorio podría levantarse de forma segura a contar del día 30 de agosto de 2020. Se deben hacer preparativos a largo plazo en contención de las futuras olas, es decir, una nueva alza de casos nuevos y activos luego del descenso.

Introduction The results of mandatory confinement have been detrimental in several respects. Nonetheless, they have resulted in reducing the number of active cases of COVID-19. Chile has begun the de-escalation and needs to know the best time to end the restrictions. Objective We discuss the best conditions and guarantees for the end of compulsory confinement. Methods This study is based on a trend model with prediction estimation. The data of the variables of interest were subjected to linear regression studies to determine the curve that best explained the data. The coefficient of determination, the standard deviation of y in x, and the confidence interval of the observed curve were estimated. The trend curve was chosen in accordance with the regression estimates. Outcomes It was found that all dependent variables tended to decrease over time in a quadratic fashion, except for the new cases variable. In general, the R2 and MAPE estimates are satisfactory, except for the variable number of PCR tests per day. Conclusions Gradual and cautious steps should be taken before ending mandatory confinement. In the current de-escalator, daily PCR tests should be increased, maintaining vigilance on indicators of incidence, prevalence, and positivity of PCR tests. Evidence suggests with some degree of confidence that mandatory confinement could be safely lifted as of August 30, 2020. Long-term preparations must be made to contain future waves of new cases.

Comparison of Primer-Probe Sets among Different Master Mixes for Laboratory Screening of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

BACKGROUND: There is a shortage of chemical reagents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis and a surge of SARS-CoV-2 cases, especially in limited-resource settings. Therefore, the combination of an optimal assay kit is necessary. METHODS: We compared the ability to screen SARS-CoV-2 among three primer-probe sets in two different master mixes, Invitrogen™ SuperScript™ III One-Step RT-PCR and LightCycler Multiplex RNA Virus Master. RESULTS: The assay with TIB-Molbiol, IDT, and Phu Sa sets for LightCycler Multiplex RNA Virus Master or Invitrogen™ SuperScript™ III One-Step RT-PCR showed positive results from a single reaction of triplicate in the three days of 4.8 copies per reaction. R squared and amplification efficiency were 0.97 and ranged from 107 to 108%, respectively. CONCLUSIONS: Our findings indicated that TIB-Molbiol, IDT, and Phu Sa primer-probe sets could be beneficial for the laboratory screening of SARS-CoV-2 by RT-qPCR assay of E gene. There is a need to consider the combination of these reagent sets as a new strategy to increase the testing capacity of screening programs for COVID-19.

[Repetition of microbiological tests in suspect of SARS-CoV-2 infection: utility of a score based on clinical probability]. / Repetición de las pruebas microbiológicas en la sospecha de la infección por SARS-CoV-2: utilidad de un score basado en la probabilidad clínica.

OBJECTIVE: The diagnosis of SARS-CoV-2 infection presents some limitations. RT-PCR in nasopharyngeal swabs is considered the gold standard for the diagnosis, although it can have false negative results. We aimed to analyze the accuracy of repeating nasopharyngeal swabs based on different clinical probabilities. METHODS: Retrospective observational study of the first patients admitted to a two COVID Internal Medicine wards at the University Hospital Marqués de Valdecilla, Santander, from March to April 2020. RT-PCR targering E, N, RdRP and ORFab1 genes and antibody tests detecting IgG. RESULTS: A total of 145 hospitalized patients with suspected SARS-Cov2 infection were admitted and in 98 (67.5%) diagnosis was confirmed. The independent predictive variables for SARS-CoV-2 infection were: epidemiological contact, clinical presentation as pneumonia, absence of pneumonia in the last year, onset of symptoms > 7 days, two or more of the following symptoms -dyspnea, cough or fever- and serum lactate dehydrogenase levels >350 U/L (p<0.05). A score based on these variables yielded an AUC-ROC of 0.89 (CI95%, 0.831-0.946; p<0.001). The accuracy of the first nasopharyngeal swabs was 54.9%. Repeating nasopharyngeal swabs two or three times allows to detect an additional 16% of positive cases. The overall accuracy of successive RT-PCR tests in patients with low pre-test probability was <5%. CONCLUSIONS: We have defined a pre-test probability score based on epidemiological and clinical data with a high accuracy for diagnosis of SARS-CoV-2. Repeating nasopharyngeal swabs avoids sampling errors, but only in medium of high probability pre-test clinical scenarios.

Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is commonly diagnosed by reverse transcription polymerase chain reaction (RT-PCR) to detect viral RNA in patient samples, but RNA extraction constitutes a major bottleneck in current testing. Methodological simplification could increase diagnostic availability and efficiency, benefitting patient care and infection control. Here, we describe methods circumventing RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated or lysed samples. Our data, including benchmarking using 597 clinical patient samples and a standardised diagnostic system, demonstrate that direct RT-PCR is viable option to extraction-based tests. Using controlled amounts of active SARS-CoV-2, we confirm effectiveness of heat inactivation by plaque assay and evaluate various generic buffers as transport medium for direct RT-PCR. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. This could aid expansion of COVID-19 testing.

[Analysis of the demand for detection of SARSCoV-2 in a health area of Spain]. / Análisis de la demanda de detección de SARS-CoV-2 en un área de salud de España.

OBJECTIVE: Since the discovery of the SARS-CoV-2 virus, the polymerase chain reaction technique (RT-PCR) has become the fundamental method for diagnosing the disease in its acute phase. The objective is to describe the demand-based series of RT-PCR determinations received at a Microbiology Service at a third-level reference hospital for a health area for three months spanning from the onset of the epidemic by SARS-CoV-2. METHODS: A retrospective analysis of the total of the RT-PCR requested in the Microbiology Service analyzed from 02/25/2020 to 05/26/2020 (90 days) has been carried out. They have been grouped by epidemiological weeks and by the petitioner service. A descriptive analysis was carried out by age, gender and number of requests for each patient. In the tests carried out, a confidence level of 95% (p <0.05) was considered significant. RESULTS: A total of 27,106 requests was received corresponding to 22,037 patients. Median age 53.7 (RIC 40.9-71.7) years, women: 61.3%. Proportion of patients with any positive RT-PCR: 14%. Of the total requests for RT-PCR, positive 3,710. Week 13 had the highest diagnosis performance (39.0%). The primary care has been the service thar has made the most requests (15,953). Patients with 3 or more RT-PCR: 565, of them, 19 patients had a positive result after previously having a negative one. CONCLUSIONS: Requests have been increasing depending on the evolution of the epidemic. The RT-PCR has a high diagnostic performance in the phases of highest contagiousness and / or transmissibility of the virus.

Optimising triage procedures for patients with cancer needing active anticancer treatment in the COVID-19 era.

BACKGROUND: Immunosuppression induced by anticancer therapy in a COVID-19-positive asymptomatic patient with cancer may have a devastating effect and, eventually, be lethal. To identify asymptomatic cases among patients receiving active cancer treatment, the Federico II University Hospital in Naples performs rapid serological tests in addition to hospital standard clinical triage for COVID-19 infection. METHODS: From 6 to 17 April 2020, all candidates for chemotherapy, radiotherapy or target/immunotherapy, if negative at the standard clinical triage on the day scheduled for anticancer treatment, received a rapid serological test on peripheral blood for COVID-19 IgM and IgG detection. In case of COVID-19 IgM and/or IgG positivity, patients underwent a real-time PCR (RT-PCR) SARS-CoV-2 test to confirm infection, and active cancer treatment was delayed. RESULTS: Overall 466 patients, negative for COVID-19 symptoms, underwent serological testing in addition to standard clinical triage. The average age was 61 years (range 25-88 years). Most patients (190, 40.8%) had breast cancer, and chemotherapy with or without immunotherapy was administered in 323 (69.3%) patients. Overall 433 (92.9%) patients were IgG-negative and IgM-negative, and 33 (7.1%) were IgM-positive and/or IgG-positive. Among the latter patients, 18 (3.9%), 11 (2.4%) and 4 (0.9%) were IgM-negative/IgG-positive, IgM-positive/IgG-negative and IgM-positive/IgG-positive, respectively. All 33 patients with a positive serological test, tested negative for RT-PCR SARS-CoV-2 test. No patient in our cohort developed symptoms suggestive of active COVID-19 infection. CONCLUSION: Rapid serological testing at hospital admission failed to detect active asymptomatic COVID-19 infection. Moreover, it entailed additional economic and human resources, delayed therapy administrationand increased hospital accesses.

The clinical sensitivity of a single SARS-CoV-2 upper respiratory tract RT-PCR test for diagnosing COVID-19 using convalescent antibody as a comparator.

The clinical false negative rate of reverse transcriptase polymerase chain reaction (RT-PCR) testing for SARS-CoV-2 on a single upper respiratory tract sample was calculated using convalescent antibody testing as a comparator. The sensitivity in symptomatic individuals was 86.2% (25/29). Of the missed cases, one (3.5%) was detected by repeat RT-PCR, one by CT thorax and two (7.1%) by convalescent antibody. The clinical false negative rate of a single RT-PCR on an upper respiratory tract sample of 14% in symptomatic patients is reassuring when compared to early reports. This report supports a strategy of combining repeat swabbing, use of acute and convalescent antibody testing and CT thorax for COVID-19 diagnosis.

Chest CT in patients with a moderate or high pretest probability of COVID-19 and negative swab.

OBJECTIVES: We aimed to assess the diagnostic performance of CT in patients with a negative first RT-PCR testing and to identify typical features of COVID-19 pneumonia that can guide diagnosis in this case. METHODS: Patients suspected of COVID-19 with a negative first RT-PCR testing were retrospectively revalued after undergoing CT. CT was reviewed by two radiologists and classified as suspected COVID-19 pneumonia, non-COVID-19 pneumonia or negative. The performance of both first RT-PCR result and CT was evaluated by using sensitivity (SE), specificity (SP), positive predictive value (PPV), negative predictive value (NPV) and area under the curve (AUC) and by using the second RT-PCR test as the reference standard. CT findings for confirmed COVID-19 positive or negative were compared by using the Pearson chi-squared test (P values < 0.05) RESULTS: Totally, 337 patients suspected of COVID-19 underwent CT and nasopharyngeal swabs in March 2020. Eighty-seven out of 337 patients had a negative first RT-PCR result; of these, 68 repeated RT-PCR testing and were included in the study. The first RT-PCR test showed SE 0, SP = 100%, PPV = NaN, NPV = 70%, AUC = 50%, and CT showed SE = 70% SP = 79%, PPV = 86%, NPV = 76%, AUC = 75%. The most relevant CT variables were ground glass opacity more than 50% and peripheral and/or perihilar distribution. DISCUSSION: Negative RT-PCR test but positive CT features should be highly suggestive of COVID-19 in a cluster or community transmission scenarios, and the second RT-PCR test should be promptly requested to confirm the final diagnosis.

A reverse-transcription recombinase-aided amplification assay for the rapid detection of N gene of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).

The current outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China firstly. A rapid, highly sensitive, specific, and simple operational method was needed for the detection of SARS-CoV-2. Here, we established a real-time reverse-transcription recombinase-aided amplification assay (RT-RAA) to detect SARS-CoV-2 rapidly. The primers and probe were designed based on the nucleocapsid protein gene (N gene) sequence of SARS-CoV-2. The detection limit was 10 copies per reaction in this assay, which could be conducted within 15 min at a constant temperature (39 °C), without any cross-reactions with other respiratory tract pathogens, such as other coronaviruses. Furthermore, compared with commercial real-time RT-PCR assay, it showed a kappa value of 0.959 (p < 0.001) from 150 clinical specimens. These results indicated that this real-time RT-RAA assay may be a valuable tool for detecting SARS-CoV-2.