Molecular detection of SARS-CoV-2 in Argentina: Evaluation of alternative diagnostic tools for the decentralization of the diagnosis

Abstract The rocketing number of COVID-19 cases highlighted the critical role that diagnostic tests play in medical and public health decision-making to contain and mitigate the SARS-CoV-2 pandemic. This study reports the evaluation and implementation of different tests for the molecular detection of SARS-CoV-2 in the central region of Argentina. We evaluated 3 real time RT-PCR kits (GeneFinder COVID-19 Plus RealAmp Kit, DisCoVery SARS-CoV-2 RT-PCR Detection Kit and WGene SARS-CoV-2 RT Detection), 2 nucleic acid extraction methods [MagaBio plus Virus DNA/RNA Purification Kit II (BioFlux), 35-min vs. 9-min, a pre-analytical reagent (FlashPrep®) and 2 isothermal amplification tests (Neokit Plus and ELA CHEMSTRIP®). The order according to the best performance of the 3 real-time RT-PCR kits evaluated was: DisCoVery > GeneFinderTM> WGene. The 2 RNA extraction methods showed similar good results: MagaBio plus Virus RNA Purification Kit II (BioFlux) 9-min was selected due to its faster performance. FlashPrep® reagent showed excellent results to perform direct RNA detection. Isothermal amplification assays showed acceptable sensitivity and specificity values (>80%), except in samples with Ct> 30. Our data show optimal real time RT-PCR kits and alternative molecular methods for SARS-CoV-2 diagnostic. These alternative assays proved to be aceptable.

Resumen La explosión de casos de COVID-19 resaltó el papel fundamental que desempeñan las pruebas de diagnóstico en la toma de decisiones médicas y de salud pública para contener y mitigar la pandemia de SARS-CoV-2. Este estudio reporta la evaluación y la implementación de diferentes test para la detección molecular de SARS-CoV-2 en la región central de Argentina. Evaluamos tres kits de RT-PCR en tiempo real (GeneFinder COVID-19 Plus RealAmp Kit, DisCoVery SARS-CoV-2 RT-PCR Detection Kit y WGene SARS-CoV-2 RT Detection), dos métodos de extracción de ácidos nucleicos (MagaBio plus Virus DNA/RNA Purification Kit II [BioFlux, 35-min vs. 9-min), un reactivo pre-analítico (FlashPrep®) y dos test de amplificación isotérmica (Neokit Plus and ELA CHEMSTRIP®). El orden de rendimiento de los tres kits de RT-PCR en tiempo real evaluados fue el siguiente: DisCoVery GeneFinder™ WGene. Los dos métodos de extracción de RNA mostraron buenos y similares resultados; se seleccionó MagaBio plus Virus RNA Purification Kit II (BioFlux) 9-min debido a su rápido tiempo de procesamiento. El reactivo FlashPrep® mostró excelentes resultados para realizar detección directa de RNA. Los ensayos de amplificación isotérmica mostraron valores de sensibilidad y de especificidad aceptables (80%), excepto en muestras con Ct 30. Nuestros resultados muestran kits de RT-PCR en tiempo real óptimos, como así también métodos moleculares alternativos para el diagnóstico de SARS-CoV-2 que resultan aceptables para su uso en contextos adversos, de descentralización y en diferentes escenarios epidemiológicos, para la detección rápida y precisa del SARS-CoV-2.

Material genético do SARS-CoV-2 em superfícies de equipamentos utilizados na prática de atividades físicas / Genetic material of SARS-CoV-2 on the surface of equipment used in the practice of physical activities

A humanidade foi impactada por uma Pandemia que expôs a população ao contato com um vírus de elevado contágio e com o índice de letalidade alarmante. Este estudo objetivou avaliar a possibilidade da persistência de material genético do SARS-CoV-2 na superfície dos equipamentos de estabelecimentos de prática de atividades físicas indoor e outdoor. Foram coleta das amostras de equipamentos utilizados para a prática de exercícios físicos em cinco academias, cinco praças e entre os frequentadores desses ambientes. Aplicou-se a técnica RT-PCR para a detecção doRNA do SARS-CoV-2 e posterior análise dos resultadose foi detectada a existência de partículas de RNA viral do SARS-CoV-2 em 48,57% das amostras coletadas dos equipamentos das academias e 12,85% das amostras coletadas nas praças, evidenciando uma incidência menor em equipamentos utilizados em locais abertos em todas as áreas comparadas.Além disso, constatou-se que 35,7% dos participantes do estudo testaram positivo para COVID-19.Os casos positivos para COVID-19 detectados apresentaram sintomas classificados como levesa moderados e uma recuperação rápida.A presença de material genético nos equipamentos,por sua vez, leva-nos a perceber a importância da higienização adequada das superfícies, como forma de prevenção.

Humanity was impacted by a Pandemic that exposed the population to contact with a highly contagious virus with an alarming lethality rate. The present study aimed to evaluate the possibility of the persistence of genetic material from SARS-CoV-2 on the surface of equipment used to practice indoor and outdoor physical activities. A sample of equipment used for the practice of physical activity was collected in five gyms and five squares and among the regulars of these environments. The RT-PCR technique was applied to detect the RNA of SARS-CoV-2 and subsequent analysis of the results. The existence of SARS-CoV-2 viral RNA particles was detected in 48.57% of the samples collected from gym equipment and 12.85% of the samples collected in squares, evidencing a lower incidence in equipment used in open spaces in all areas compared and it was found that 35.7% of the study participants tested positive for COVID-19. The positive cases for COVID-19 detected, had symptoms classified as mild to moderate and a quick recovery. The presence of genetic material in the equipment, in turn, leads us to realize the importance of proper cleaning of surfaces, as a form of prevention.

Estudio comparativo entre un test de antígeno y la prueba de la reacción en cadena de la polimerasa en tiempo real (RT-PCR) para el diagnóstico de infección por COVID-19 en pediatría / Comparative study between an antigen test and the reverse transcription-polymerase chain reaction (RT-PCR) test for the diagnosis of COVID-19 in pediatrics

Introducción. La pandemia por COVID-19 ha puesto de manifiesto la necesidad de pruebas diagnósticas rápidas. La prueba de referencia es la reacción en cadena de la polimerasa en tiempo real (RT-PCR). Requiere un equipo y personal capacitado, y su resultado puede llevar un tiempo de espera prolongado. El sistema BD Veritor® es el método rápido cromatográfico utilizado para la detección del antígeno del coronavirus de tipo 2 del síndrome respiratorio agudo grave, en individuos sintomáticos. El objetivo primario del siguiente trabajo es evaluar sensibilidad y especificidad del test de antígeno (TA) comparadas con la RT-PCR en población pediátrica. Población y métodos. Estudio prospectivo, de prueba diagnóstica. Se incluyó a todo menor de 17 años en los primeros 5 días de inicio de síntomas, que consultó desde julio de 2021 hasta febrero de 2022. Se calculó un mínimo de 300 muestras para lograr una precisión de ± 8,76 % y de ± 3,68 % para sensibilidad y especificidad respectivamente. Se analizaron en paralelo las muestras por ambas metodologías. Resultados. De 316 muestras pareadas, 33 fueron positivas por ambos métodos; 6 fueron positivas solo por RT-PCR. La especificidad del TA fue del 100 %; la sensibilidad, del 84,6 %, con un valor predictivo positivo y negativo del 100 % y del 98 % respectivamente. Conclusiones. El TA demostró ser útil en el diagnóstico de pacientes pediátricos con COVID-19 en los primeros 5 días de inicio de síntomas, aunque aquellos con TA negativo y alta sospecha clínica deberían confirmar su resultado con la RT-PCR.

Introduction. The COVID-19 pandemic has brought to light the need for rapid diagnostic tests. The gold standard test is reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR requires equipment and trained personnel, and results may take a long waiting time. The BD Veritor® System is a rapid chromatographic method used for the detection of severe acute respiratory syndrome coronavirus 2 antigen in symptomatic individuals. The primary objective of this study is to assess the sensitivity and specificity of the antigen test (AT) compared to the RT-PCR in the pediatric population. Population and methods. Prospective study with a diagnostic test. All children younger than 17 years in the first 5 days of symptom onset, who consulted between July 2021 and February 2022, were included. A minimum of 300 specimens was estimated to achieve an accuracy of ±8.76% and ±3.68% for sensitivity and specificity, respectively. Specimens were analyzed in parallel using both methodologies. Results. Of 316 paired samples, 33 were positive by both methods; 6 were positive only by RT-PCR. The specificity of the AT was 100%; sensitivity was 84.6%, with a positive and negative predictive value of 100% and 98%, respectively. Conclusions. The AT proved to be useful in the diagnosis of pediatric patients with COVID-19 in the first 5 days of symptom onset, although those with a negative AT and high clinical suspicion should confirm their result with a RT-PCR.

ASSOCIATION OF SERUM FERRITIN IN POST COVID-19 ANAEMIC PATIENTS TREATED WITH UMBILICAL CORD BLOOD AND HAEMATINICS.

COVID-19 patients commonly present with lower respiratory symptoms with other systemic involvement. Haematological manifestation such as low haemoglobin, thrombocytopenia, lymphocytopenia also common in COVID19 patients. In this study, we investigated prevalence, association with serum ferritin in post COVID-19 anaemic patients, after human umbilical cord blood transfusion in relation to control group. Among 155 COVID-19 RT-PCR positive patients 36 (23%) was anaemic. In our study 18 patients was transfused human umbilical cord blood, 12 patients were treated with haematinics and 6 patients denied taking any of the above. In most cases anaemia was moderate to severe that may be due to inflammation or due to pre-existing iron deficiency.Umbilical cord blood transfusion to post COVID -19 patients for the treatment of anaemia because of the unique composition of UCB. Haematological analysis and serum ferritin estimation reflecting the treatment out come in post COVID-19 anaemic patients. There was a difference between the dependent variable's serum ferritin (p <.001) in anaemic COVID-19 patients. In conclusion, our result highlight serum ferritin is widely used in diagnosis and monitoring of COVID-19 disease.

Optimización del control interno de calidad de RT-PCR en tiempo real para detección cualitativa de SARS-CoV-2

Objetivo: Optimizar el control interno de calidad de RT-PCR en tiempo real para detección cualitativa de SARS-CoV-2, utilizando los valores Cq de controles negativos y positivos. Material y método: Estudio prospectivo-longitudinal. La muestra estuvo constituida por 143 valores Cq para los controles negativos de alicuotado y extracción, así como para el control positivo. Se analizó la distribución normal de los valores Cq mediante la prueba de Anderson-Darling (AD) y se aplicaron pruebas de aleatoriedad. Se calculó límites de control a partir de 51 valores Cq, para luego, mediante gráficas de control, monitorizar 92 valores Cq obtenidos desde noviembre del 2020 hasta marzo del 2021. Se evaluó aceptación de lote e índices Cpk como indicadores de optimización. Los cálculos se hicieron con el programa Minitab. Resultados: Se aceptaron los lotes de valores Cq y se obtuvieron índices Cpk superiores a 1.33 para los tres tipos de control. Discusión: No existen estudios publicados que apliquen control estadístico de calidad a la detección cualitativa de SARS-CoV-2. Conclusiones: Es posible utilizar los valores Cq de los controles para optimizar el control interno de calidad de RT-PCR en tiempo real para detección cualitativa de SARS-CoV-2, como si se tratara de una técnica de tipo cuantitativo.

Objective: To optimize the internal quality control of real-time RT-PCR for the qualitative detection of SARS-CoV-2, using the Cq values ​​of negative and positive controls. Material and method : Prospective-longitudinal study. The sample consisted of 143 Cq values for the negative aliquot and extraction controls, as well as for the positive control. The normal distribution of Cq values ​​was analyzed using the Anderson-Darling (AD) test and randomness tests were applied. Control limits were calculated from 51 Cq values, and then, using control charts, to monitor 92 Cq values ​​obtained from November 2020 to March 2021. Lot acceptance and Cpk indices were evaluated as optimization indicators. The calculations were made with the Minitab program. Results: The batches of Cq values ​​were accepted and Cpk indices higher than 1.33 were obtained for the three types of control. Discussion : There are no published studies that apply statistical quality control to the qualitative detection of SARS-CoV-2. Conclusions : It is possible to use the Cq values ​​of the controls to optimize the internal quality control of real-time RT-PCR for qualitative detection of SARS-CoV-2, as if it were a quantitative technique.

SARS-CoV-2 antigen detection in deceased bodies: implications for infection prevention

Background & objectives: High transmissibility of the SARS-CoV-2 has significant implications on healthcare workers’ safety, preservation, handling, transportation and disposal of the deceased bodies. The objective of this study was to detect SARS-CoV-2 antigen in nasopharyngeal samples and its implications in handling and care of COVID-19 deceased bodies. Methods: A study was conducted at a dedicated COVID-19 centre on deceased individuals from April to December 2020. Rapid antigen test (RAT) and reverse transcription (RT)-PCR was compared on all the SARS-CoV-2 positive cadavers recruited in the study. Results: A total of 115 deceased individuals were included in the study. Of these, 79 (68.7%) were male and 36 (31.3%) were female and majority were in the age group of 51-60 yr [31 (27%)]. SARS-CoV-2 antigen test was positive in 32 (27.8%) and negative in 83 (72.1%) individuals. The mean time interval between deaths to the sample collection was 13.2 h with interquartile range of eight to 20 h. Reverse transcription (RT)-PCR was used as the reference test and 24 (20.9%) cases were true positive; 93.6 per cent [95% confidence interval (CI) 88.8-98.4%] sensitivity, 45.2 per cent (95% CI 35.5-55%) specificity, 60.2 per cent (95% CI 50.6-69.8%) positive predictive value and 88.8 per cent (95% CI 82.7-95%) negative predictive value of antigen test was computed. Interpretation & conclusions: SARS-CoV-2 antigen test was positive beyond 19 h in COVID-19 deceased individuals. Antigen test was found to be highly sensitive in the deceased. Patients, suspected of having died due to COVID-19, can be screened by this method. As infectiousness of the virus in the deceased bodies cannot be directly concluded from either the antigen or RT-PCR test, yet possible transmission cannot be completely ruled out. Strict infection control measures need to be followed during the handling and clearance of COVID-19 cadavers.

Accuracy of a raw saliva-based COVID-19 RT-LAMP diagnostic assay

ABSTRACT The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic demanded rapid diagnosis to isolate new COVID-19 cases and prevent disease transmission. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) rapidly became the gold standard for diagnosis. However, due to the high cost and delay of the results, other types of diagnosis were implemented, such as COVID-19 Ag Rapid Tests and Reverse Transcription Technique followed by Loop-Mediated isothermal Amplification (RT-LAMP). In this work, we validated the use of RT-LAMP in saliva samples rather than nasopharyngeal swabs, as the collection is more comfortable. First, we selected 5 primer sets based on the limit of detection for SARS-CoV-2 RNA, then validated their sensitivity and specificity in patient samples. A total of 117 samples were analyzed by fluorometric RT-LAMP and compared with qRT-PCR results. Our results show that the use of a high-sensitive primer ORF1-a, together with a low-sensitive primer set Gene E (time to threshold of 22.9 and 36.4 minutes, respectively, using 200 copies of viral RNA), achieved sensitivity in purified RNA from saliva samples of 95.2% (95% CI 76.1-99.8) with 90.5% specificity (95% CI 69.6-98.8) (n = 42).As RNA purification increases the turnaround time, we tested the outcome of RT-LAMP utilizing raw saliva samples without purification. The test achieved a sensitivity of 81.8% (95% CI 59.7-94.8) and a specificity of 90.9% (95% CI 70.8-98.8). As a result, the accuracy of 92.9% (95% CI 80.5-98.5) in purified RNA-saliva samples was lowered to an acceptable level of 86.4% (95% CI 72.6-94.8) in raw saliva. Although mass vaccination has been implemented, new strains and low vaccination progress helped to spread COVID-19. This study shows that it is feasible to track new COVID-19 cases in a large population with the use of raw saliva as sample in RT-LAMP assay which yields accurate results and offers a less invasive test.

Mapping of SARS-CoV-2 in Waldeyer's lymphatic ring and visceral biopsies: the age and the illness duration's impact

Abstract Objective: To study the impact of age and the interval between disease diagnosis and death on the organotropism of SARS-CoV-2. Method: Patients underwent post-mortem biopsies from lungs, Waldeyer ring, heart, liver, kidneys and bone marrow between 2020-2021. SARS-CoV-2 organotropism was mapped by using molecular RT-PCR analyses for SARS-CoV2 targeting the Envelope gene (E), the RNA Polymerase Gene (RdRp), and the Nucleocapsid gene (N). Statistical and linear regression analysis was performed to study the impact of age and illness duration in SARS-CoV-2 organotropism. Results: We performed 158 postmortem biopsies in 21 patients, with a mean age of 76 years old. The mean interval between the diagnosis of the infection to the death was 23 days. The RNA of the SARS-CoV-2 was detected in 100% of lung biopsies, 76%-82% of Waldeyer's ring biopsies, 55% of heart biopsies, 40% of kidney biopsies, 33% of liver and 25% of bone marrow biopsies. Patients who died before the day 9, presented extensive visceral dissemination of SARS-CoV-2 RNA. Most of the patients older than 80 years (90%) presented visceral dissemination of SARS-CoV-2 RNA, while among younger patients, only 3/11 patients presented visceral dissemination of the virus. The relationship between "age" and "illness duration" and multitropism of the virus was statistically significant (p< 0.001). Conclusion: Disease interval and age were factors that were significantly associated with RT-PCR positive results in multiple organs. Critical COVID-19 patients have multiorganic viral dissemination in early stages. In the critical older patients, multiorganic viral dissemination is the rule. Level of evidence: 4. Case Series.

Risque de transmission de certains arbovirus à travers le don de sang à Bamako / Risk of transmission of certain arbovirus through blood donation in the city of Bamako

Introduction. Au Mali, le dépistage de certains virus tels que la dengue, Zika et la fièvre de la vallée du Rift n'est pas systématique au centre national de transfusion sanguine (CNTS). Le risque peut être considérable en raison de leurs courtes périodes de virémie asymptomatique dans la population dont l'incidence est variable et parfois extrêmement élevée. Cette étude avait pour objectif d'explorer la possibilité de transmission de certains arbovirus à travers le don de sang au CNTS de Bamako. Méthodes. Il s'agissait d'une étude transversale, de juillet 2019 à juin 2020 à Bamako. Au total deux cents (200) donneurs de sang du CNTS ont été inclus. Les examens ont été réalisés au Centre d'Infectiologie Charles Mérieux (CICM) de Bamako avec le dépistage du génome des virus responsables de la Dengue, de la fièvre de la Vallée du Rift, et du Zika à l'aide de la technique de la RT-PCR en temps réel. Le Test de Dépistage Rapide (TDR) a été utilisé pour la détection des anticorps IgG et IgM spécifiques de la Dengue. Résultats. Le sexe masculin représente 84% (168/200). Le TDR a détecté 4,5% (9/200) de Dengue IgG positifs et aucun cas de Dengue IgM positif. La technique de RT-PCR n'a détecté aucun des trois virus. Conclusion. Cette étude prouve que le risque de transmission de certains arbovirus à travers le don de sang existe, mais il semble être minime au CNTS de Bamako

Background. In Mali, screening for certain viruses such as dengue, Zika, and Rift Valley fever is not systematic at the national blood transfusion center (CNTS). The risk can be considerable due to their short periods of asymptomatic viremia in the population with variable and sometimes extremely high incidence. The objective of this study was to explore the possibility of transmission of certain arboviruses through blood donation at the CNTS of Bamako. Methods. This was a cross-sectional study, from July 2019 to June 2020 in Bamako. A total of two hundred (200) blood donors from the CNTS were included. The examinations were performed at the Centre d'Infectiologie Charles Mérieux (CICM) in Bamako with the screening of the genome of viruses responsible for Dengue, Rift Valley fever, and Zika using the real-time RT-PCR technique. The Rapid Screening Test (RST) was used for the detection of Dengue-specific IgG and IgM antibodies. Results. Male sex represented 84% (168/200). The RDT detected 4.5% (9/200) of IgG positive Dengue and no IgM positive Dengue cases. The RT-PCR technique did not detect any of the three viruses. Conclusion. This study proves that the risk of transmission of certain arboviruses through blood donation exists, but it seems to be minimal at the CNTS of Bamako.

Prevalence of COVID-19 at the Wahgnion-Gold mining site in Burkina Faso and use of RT-PCR initial cycle threshold to monitor the dynamics of SARS-CoV-2 load

Background: To control the spread of coronavirus disease-19 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), it is necessary to adequately identify and isolate infectious patients particularly at the work place. Real time polymerase chain reaction (RT-PCR) assay is the recommended confirmatory method for the diagnosis of SARS-CoV-2 infection. The aim of this study was to determine the prevalence of SARSCoV-2 infection in Burkina Faso and to use the initial cycle threshold (Ct) values of RT-PCR as a tool to monitor the dynamics of the viral load. Methodology: Between September 2021 and February 2022, oropharyngeal and/or nasopharyngeal swab samples of consecutively selected COVID-19 symptomatic and apparently healthy workers from the Wahgnion mining site in the South-western Burkina Faso who consented to the study were collected according to the two weeks shift program and tested for SARS-CoV-2 using RT-PCR assay. Patients positive for the virus were followed-up weekly until tests were negative. Association of the initial RT-PCR Ct values with disease duration was assessed by adjusted linear regression approach. Two-sided p value < 0.05 was considered statistically significant. Results: A total of 1506 (92.9% males) participants were recruited into the study, with mean age and age range of 37.18.7 and 18-68 years respectively. The overall prevalence of SARS-CoV-2 infection was 14.3% (216/1506). Of the 82 patients included in the follow-up study, the longest duration of positive RT-PCR test, from the first positive to the first of the two negative RT-PCR tests, was 33 days (mean 11.6 days, median 10 days, interquartile range 8- 14 days). The initial Ct values significantly correlated with the duration of RT-PCR positivity (with ß=-0.54, standard error=0.09 for N gene, and ß=-0.44, standard error=0.09 for ORF1ab gene, p<0.001). Participants with higher Ct values corresponding to lower viral loads had shorter viral clearance time than those of lower Ct values or higher viral loads. Conclusion: Approximately 1 out of 7 tested miners had SARS-CoV-2 infection and the duration of their RT-PCR tests positivity independently correlated with the initial viral load measured by initial Ct values. As participants with lower initial Ct values tended to have longer disease duration, initial RT-PCR Ct values could be used to guide COVID-19 patient quarantine duration particularly at the work place.

Contexte: Pour contrôler la propagation de la maladie à coronavirus 19 (COVID-19) causée par le syndrome respiratoire aigu sévère coronavirus-2 (SRAS-CoV-2), il est nécessaire d'identifier et d'isoler de manière adéquate les patients infectieux, en particulier sur le lieu de travail. Le test de réaction en chaîne par polymérase en temps réel (RT-PCR) est la méthode de confirmation recommandée pour le diagnostic de l'infection par le SRAS-CoV-2. Le but de cette étude était de déterminer la prévalence de l'infection par le SRAS-CoV-2 au Burkina Faso et d'utiliser les valeurs du seuil initial du cycle (Ct) de la RT-PCR comme outil de suivi de la dynamique de la charge virale. Méthodologie: Entre septembre 2021 et février 2022, des écouvillonnages oropharyngés et/ou nasopharyngés de travailleurs symptomatiques COVID-19 et apparemment en bonne santé sélectionnés consécutivement du site minier de Wahgnion dans le sud-ouest du Burkina Faso qui ont consenti à l'étude ont été prélevés selon les deux programme de quart de semaines et testé pour le SRAS-CoV-2 à l'aide d'un test RT-PCR. Les patients positifs pour le virus ont été suivis chaque semaine jusqu'à ce que les tests soient négatifs. L'association des valeurs Ct initiales de la RT-PCR avec la durée de la maladie a été évaluée par une approche de régression linéaire ajustée. Une valeur p bilatérale < 0,05 a été considérée comme statistiquement significative. Résultats: Un total de 1506 participants (92,9% d'hommes) ont été recrutés dans l'étude, avec un âge moyen et une tranche d'âge de 37,1 à 8,7 ans et de 18 à 68 ans, respectivement. La prévalence globale de l'infection par le SRAS-CoV-2 était de 14,3% (216/1506). Sur les 82 patients inclus dans l'étude de suivi, la plus longue durée de test RT-PCR positif, du premier test positif au premier des deux tests RT-PCR négatifs, était de 33 jours (moyenne 11,6 jours, médiane 10 jours, intervalle interquartile 8-14 jours). Les valeurs Ct initiales étaient significativement corrélées à la durée de positivité de la RT-PCR (avec ß=-0,54, erreur standard=0,09 pour le gène N et ß=-0,44, erreur standard=0,09 pour le gène ORF1ab, p<0,001). Les participants avec des valeurs de Ct plus élevées correspondant à des charges virales plus faibles avaient un temps de clairance virale plus court que ceux avec des valeurs de Ct plus basses ou des charges virales plus élevées. Conclusion: Environ 1 mineur testé sur 7 était infecté par le SRAS-CoV-2 et la durée de la positivité de ses tests RTPCR était indépendamment corrélée à la charge virale initiale mesurée par les valeurs Ct initiales. Comme les participants avec des valeurs Ct initiales inférieures avaient tendance à avoir une durée de maladie plus longue, les valeurs Ct initiales de la RT-PCR pourraient être utilisées pour guider la durée de la quarantaine des patients COVID19, en particulier sur le lieu de travail.

Delayed SARS-COV-2 clearance in infected persons in Ghana

Objective: This study aimed to determine the duration of SARS-CoV-2 clearance in persons in Ghana. The research question was whether the duration of virus clearance in Ghana matched the 14 days recommended by the World Health Organization (WHO); this had direct implications for transmission, which was key in managing the COVID-19 pandemic. Design: This was a retrospective analytical study. Setting: All facilities that submitted clinical specimens to Noguchi Memorial Institute for Medical Research (NMIMR) for SARS-CoV-2 diagnosis between March to June 2020 were included in the study. Interventions: Samples from 480 persons who tested positive for SARS-CoV-2 by RT-PCR from March to June 2020 at NMIMR and submitted at least two follow-up samples were retrospectively analysed. Individuals with two consecutive negative RT-PCR retesting results were considered to have cleared SARS-CoV-2. Results: The median time from the initial positive test to virus clearance was 20 days (IQR: 5-56 days). This was six days longer than the WHO-recommended 14 days, after which infected persons could be de-isolated. Sputum and nasopharyngeal swabs proved more sensitive for detecting viral RNA as the infection progressed. At a significance level of 0.05, age and sex did not seem to influence the time to SARS-CoV-2 clearance. Conclusions: The median time to SARS-CoV-2 clearance in this study was 20 days, suggesting that SARS-CoV-2 infected persons in Ghana take longer to clear the virus. This finding calls for further investigations into whether patients who remain PCR positive continue to be infectious and inform isolation practices in Ghana.

Clinical significance of LncRNA-MIAT as a non-invasive diagnostic marker of nonHodgkin's lymphoma associated with occult hepatitis C virus infection

Aims: the current research aimed to investigate LncRNA-MIAT in patients with nonHodgkin lymphoma (NHL) and to assess its correlation with clinicopathological features and treatment protocols of NHLs among Egyptian patients with Occult hepatitis C virus (HCV) infection (OCI). Patients & Methods: This study was conducted on 20 patients with NHL and 30 healthy subjects as the control group. All subjects were screened for HCV-RNA in both plasma and PBMCs. RT-PCR determined lncRNA-MIAT. Results: lncRNA-MIAT relative expression level was upregulated in NHL groups (2.73±0.86) compared to controls (1.06±0.07), P ˂0.001*. Among NHL, patients with OCI (3.2±0.63) had significantly higher levels of lncRNA-MIAT compared to HCV (2.6±1.08) and non-HCV (2.4±0.4), P ˂0.001*. Additionally, the relative expression levels of lncRNA-MIAT were significantly positively correlated with laboratory and clinicopathological features of NHL. Interestingly, concerning the treatment of DLBCLNHL, there were significantly higher levels of lncRNA-MIAT in no treatment subgroup (n=10, 3.31±0.95) compared to successfully treated subgroups [CHOP (n=7, 1.58±0.34) and R-CHOP (n=3, 11.16±0.21), P ˂0.001* Conclusions: lncRNA-MIAT level was upregulated in NHL patients, particularly patients with OCI. Thus, circulatory lncRNA-MIAT may serve as a promising non-invasive diagnostic marker for NHL associated with OCI

COVID-19 surveillance via wastewater:Introduction of wastewater-based epidemiology / 西安交通大学学报(医学版)

Since the outbreak of the COVID-19 global pandemic in 2019, monitoring COVID-19 infection status and trend through wastewater, known as wastewater-based epidemiology (WBE), has been widely used in many countries and regions. WBE consists of five steps:wastewater sample collection, viral concentration, viral nucleic acid extraction, quantification of virus using quantitative RT-PCR, and dissemination of the wastewater surveillance results. This method could be used for early warning of COVID-19 outbreak in a population, monitoring COVID-19 distributions and epidemic trend, prediction of COVID-19 prevalence rate, understanding of temporal trend of SARS-COV-2 variants, and simultaneous surveillance of multiple pathogens. WBE and clinical surveillance can be used concurrently and the former is a good complement to the latter.

Application and Modification of RT-LAMP for Rapid Detection of SARS-CoV-2 Viral Genome

@#COVID-19 outbreak caused by the newly discovered SARS-CoV-2 has become a major public health threat around the world and has create a tremendous effect on the global economy. Hence, there is a high demand for rapid and accurate diagnosis to contain the spread of the disease. The Reverse-Transcription Polymerase Chain Reaction (RTPCR), the current standard for diagnosis of COVID-19 however possesses certain drawbacks that limits its application to meet the high demand of the continually increasing COVID-19 cases. Conversely, Loop-Mediated Isothermal Amplification (LAMP) is another nucleic acid amplification method that shows a great potential as an alternative tool in rapid diagnosis of COVID-19 due to its simplicity and rapidity. This review summarized the recent published research articles related to the application and modification of RT-LAMP assay for the rapid detection of COVID-19 in comparison with other available diagnostic methods.

Development and verification of multiplex real-time RT-PCR assay for classing and identification of HPIV1,HPIV2 and HPIV3 / 中国生物制品学杂志

@#ObjectiveTo develop and verify a multiplex real-time RT-PCR assay for simultaneous identification of human parainfluenza virus type 1(HPIV1),type 2(HPIV2)and type 3(HPIV3).MethodsThe whole genome sequences of HPIV1,HPIV2 and HPIV3 were downloaded from the database for alignment analysis,and the conserved regions were selected. Specific primers and probes were designed for the three viruses respectively to develop a multiplex real-time RTPCR assay with human ribonuclease P(RNase P)as theinternal quality control. The method was verified for the sensitivity,specificity and precision and used to detect 192 clinical samples.ResultsAfter optimization,the multiplex real-time RTPCR reaction system was determined to be 30 μL,including 10 × NeoscriptRTase/UNG Multi mix 3 μL,5 × Neoscript RT Premix Multi Buffer 6 μL,upstream and downstream primers of HPIV1,HPIV2 and HPIV3(100 μmol/L)0. 1 μL respectively,HPIV1,HPIV2,HPIV3 probes(100 μmol/L)0. 05 μL respectively,RNase P upstream and downstream primers(50 μmol/L)0. 06 μL respectively,RNase P probe(50 μmol/L)0. 03 μL respectively,template 15 μL,and ddH2O supplemented to 30 μL. The reaction conditions were 50 ℃ 20 min,95 ℃ 3 min and 45 cycles of 95 ℃ 15 s and54 ℃ 30 s. Fluorescence signals were collected during annealing in each cycle. The minimum detection limits of HPIV1,HPIV2 and HPIV3 were all 500 copies/mL by the multiplex real-time RT-PCR assay;The method showed no cross-reaction with influenza A virus,influenza B virus,respiratory syncytial virus andnovel coronaviruses. The coefficients of variation(CVs)in intra-and inter-groups of the recombinant plasmid standard mixture with three different concentrations were all less than 3%. HPIV1,HPIV2 and HPIV3 were detected in 192 clinical samples,and the positive rates were7. 81%,0. 05% and 3. 1%,respectively.ConclusionThe multiplex real-time RT-PCR assay for detection of HPIV1,HPIV2 and HPIV3 developed in this study has good sensitivity,specificity and precision,which has a high clinical application prospect in the field of rapid diagnosis and identification of HPIV.

Establishment of duplex TaqMan RT-PCR method for detection of Entamoeba histolytica and Giardia lamblia in fecal sample / 中国热带医学

@#Abstract: Objective To establish the duplex TaqMan RT-PCR method for detection of Entamoeba histolytica and Giardia lamblia in fecal samples. Methods Primer pairs and probes for Entamoeba histolytica and Giardia lamblia were designed and duplex TaqMan RT-PCR amplification system was constructed. PCR products were inserted into the pUC57 plasmid, and the lower limit of detection of the method was determined. Clinical stool samples were tested in order to evaluated the efficacy of the method. Results The detection limits of duplex TaqMan RT-PCR were 31.6 copies/μL for Entamoeba histolytica and 32 copies/μL for Giardia lamblia, respectively. Of the total of 212 clinical stool samples tested, all 3 samples with E. histolytica-positive patients by microscopy were positive by PCR, while 1 from 209 samples with E. histolytica-negative patients by microscopy were positive by PCR, and the remaining samples were negative. For Giardia lamblia, all 8 samples positive by microscopy were positive by PCR, and 1 from 204 sample with a microscopy-negative patient was positive by PCR, and the remaining samples were negative. The amplification product sequencing and blast analysis were used to confirm that the amplified sequence in the specimen of a patient with negative microscopy but positive PCR belongs to the targeted pathogen, supported by clinical symptoms and laboratory test results. PCR results for other diarrhea-causing pathogens were negative, indicating no cross-reactivity. Conclusions The dual TaqMan RT-PCR method developed in this study can not only detect microscopy-positive samples of Entamoeba histolytica and Giardia lamblia but also can detect samples that were missed by microscopy, with higher sensitivity than the microscopy method. Further, this detection method does not cross-react with other diarrhea-causing pathogens, including cross-react with other diarrhea-causing pathogens including Iodamoeba butschlii, Blastocystis hominis, Plesiomonas, Aeromonas, Salmonella, Shigella, Sphaerozoum fuscum, and Entamoeba hartmani, thus has a good specificity.

Establishment of qRT-PCR for absolute quantitative detection of Chikungunya virus / 中国热带医学

@#Abstract: Objective　To develop a real-time fluorescent quantitative RT-PCR (qRT-PCR) method for qualitative and quantitative Chikungunya virus (CHIKV) analysis. Methods Based on the systematic analysis of the genomic sequences of Chikungunya and its related arboviruses, the specific nucleic acid sequences for Chikungunya virus were screened and identified, and then the primers and TaqMan probe were designed. Meanwhile, the human GAPDH gene was used as an internal reference. The reaction system for qRT-PCR was systematically optimized by L9(34) orthogonal design, and a rapid detection method for Chikungunya by qRT-PCR based on TaqMan probe methods was established. The sensitivity, specificity, reproducibility, and coverage of the established method were analyzed in detail. The standard curve was made, and the absolute quantitative method was established using the cloned nucleic acid fragments as positive samples. Results　A real-time fluorescent quantitative RT-PCR assay was developed for the qualitative and quantitative analysis of Chikungunya virus. The reaction system included Chikungunya virus and reference internal gene specific primers and probe, RT/Taq enzyme mixture, reaction buffer, and negative and positive reference. The established method obtained positive results with the ROSS strain of ECSA subtype, LR2006 strain of IOL branch, 181/25 strain of Asian type and Dongguan 2010 epidemic strains of Chikungunya virus, but there was no cross-reaction with other 18 arboviruses belonging to Flaviviruses, Alphaviruses and Bunyavirus. The minimum detection limit of the established method was 5.80 copies/mL, and a linear relationship was observed between the amount of input plasmid DNA and fluorescence signal value over a range of 5.80×102 copies/mL to 5.80×1010 copies/mL, and the correlation coefficient was 0.999 5. The qRT-PCR amplification efficiency was 91%, and the intra-assay variations and inter-assay variations were 0.01-0.07 and 0.03-0.11, respectively. Conclusions　The TaqMan qRT-PCR method developed in this study can qualitatively and quantitatively detect Chikungunya virus rapidly with specificity and sensitivity, providing a technical method for the prevention and control of this viral disease.

Differential expression of sigma factor of Mycobacterium tuberculosis under in vivo infection and in vitro culture conditions / 公共卫生与预防医学

Objective To investigate the gene expression of sigma factors in vivo, and to explore the sigma factors that may be closely related to the virulence of pathogenic Mycobacterium tuberculosis Methods Tuberculosis (TB) patients diagnosed in the outpatient department of Tianjin Tuberculosis Control Center from January to December 2018 were selected, and 20 sputum-positive specimens were randomly selected from TB patients confirmed with Xpert-positive for the present study. Two immediate sputum specimens were collected from each case of pulmonary tuberculosis before treatment, one for RNA extraction and one for in vitro culture. In vitro cultured strains in the logarithmic phase of growth were harvested for RNA extraction. The specific primers for 13 sigma factors were designed. The differential expression of the 13 sigma factors between sputum isolates and in vitro cultured strains was analyzed by fluorescence quantitative PCR. Taking ribosomal 16s as the reference gene, the transcription level of sigma factors was analyzed by 2ΔCt. Using the stably expressed sigA as the control reference, the expression differences of other sigma factors were analyzed by one-way ANOVA. Results Within 0 days, stress-associated sigma factors have a different expression profile in clinical isolate strains vs H37Rv or in vitro. All the sigma factors induced up regulation in sputum ,while no difference transcription between clinical isolate strains vs H37Rv（P＞0.05）. When compared to in vitro culture ,only sigM transcript highest in sputum(P＜0.05). Conclusion SigM plays an important role in the initial stages of bacterial infection, but its exact role is unclear.We assumed it could have a role in the interplay between the host immune defenses and the bacterial escape mechanisms.

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.

Detección molecular de Cucumber mosaic virus y Pepper severe mottle virus en arvenses asociadas al cultivo de ají (Capsicum spp.)

En los últimos años, el ají (Capsicum chinense, Capsicum frutescens y Capsicum annuum var. Acuminatum), cultivado en el Valle del Cauca, se ha visto afectado por enfermedades virales causadas por Cucumber mosaic virus (CMV-ají) y Pepper severe mottle virus (PepSMoV). Pese a que estos dos virus son limitantes para producción del cultivo de ají, en la actualidad, pocos estudios han identificado los hospederos alternos de CMV-ají y PepSMoV. En este trabajo, se evaluó la presencia de CMV-ají y PepSMoV, mediante RT-PCR, en muestras de tejido foliar, de 121 plantas arvenses, asociadas al cultivo de ají, en el Valle del Cauca, Colombia. El análisis molecular indicó la presencia de CMV-ají, en el 21,4 % de las plantas recolectadas y de PepSMoV, en el 20,6 %. Se identificaron las especies arvenses Amaranthus viridis, Parthenium hysterophorus, Hippobroma longiflora, Commelina diffusa, Clitoria ternatea, Crotalaria incana, Desmodium tortuosum, Desmodium intortum, Macroptilium lathyroides, Anoda acerifolia, Boerhavia erecta, Bougainvillea glabra, Rivina humilis, Browallia americana, Capsicum rhomboideum, Solanum americanum y Lantana camara, como hospederas de CMV-ají o PepSMoV. Se presentó infección mixta de CMV-ají y PepSMoV, en 57 % de las arvenses positivas a virus, las cuales, están distribuidas en zonas productores de ají, localizadas en seis municipios del Valle del Cauca. Estos resultados brindan información sobre la distribución de estos virus en el Valle del Cauca, contribuyen al conocimiento de la epidemiología viral y servirán para diseñar medidas de manejo, orientadas a prevenir las infecciones virales en los cultivos de ají.

In recent years, chili pepper (Capsicum chinense, Capsicum frutescens y Capsicum annuum var. Acuminatum) grown in Valle del Cauca has been affected by viral diseases caused by Cucumber mosaic virus (CMV-chili pepper) and Pepper severe mottle virus (PepSMoV). Although these two viruses are limiting to the production of the chili pepper crop, at present, few studies have identified the alternate hosts of CMV-chili pepper and PepSMoV. In this work, the presence of CMV-chili pepper and PepSMoV were evaluated by RT-PCR in leaf tissue samples from 121 weed plants associated with chili pepper cultivation in Valle del Cauca, Colombia. Molecular analysis indicated the presence of CMV-chili pepper in 21.4 % of the collected plants and PepSMoV in 20.6 %. Weed species Amaranthus viridis, Parthenium hysterophorus, Hippobroma longiflora, Commelina diffusa, Clitoria ternatea, Crotalaria incana, Desmodium tortuosum, Desmodium intortum, Macroptilium lathyroides, Anoda acerifolia, Boerhavia erecta, Bougainvillea glabra, Rivina humilis, Browallia americana, Capsicum rhomboideum, Solanum americanum and Lantana camara, as hosts of CMV-chili pepper or PepSMoV. Mixed infection of CMV-chili pepper and PepSMoV was present in 57 % of the weeds positive for viruses, which are distributed in chili pepper producing areas located in six municipalities of Valle del Cauca. These results provide information on the distribution of these viruses in Valle del Cauca. Contribute to the knowledge of viral epidemiology and will serve to design management measures aimed to prevent viral infections in chili pepper crops.