Captopril inhibits thioacetamide-induced chronic liver injury associated with the suppression of inflammation / hypoxia- inducible factor 1-alpha / profibrogenic axis-mediated hepatotoxicity in rats / Captopril inhibe la lesión hepática crónica inducida por tioacetamida asociada con la supresión de la inflamación/factor 1-alfa inducible por hipoxia/hepatotoxicidad mediada por el eje profibrogénico en rata

SUMMARY: Liver transplantation is the only available method to treat liver failure induced by chronic liver injury. We sought to determine whether the angiotensin-converting enzyme inhibitor, captopril, can inhibit the development of chronic liver injury induced by the hepatotoxic agent thioacetamide (TAA) in association with the suppression of inflammation (hsCRP, TNF-α, and IL-6) / hypoxia- inducible factor 1-alpha (HIF-1α) / profibrosis (TIMP-1, MMP-9, and α-SMA) axis that mediates liver injury. Therefore, the model group of rats was injected for eight weeks with 200 mg/kg TAA starting at week two. The protective group was pretreated with 150 mg/ kg captopril daily for two weeks prior to TAA injections and continued receiving both capropril and TAA agents until being humanely scrificed at week 10. We observed a substantial damage to liver tissue in the model group as demonstrated by a significant (p<0.0001) increase in blood and hepatic tissue levels of high sensitivity C-reactive protein (hsCRP), tumor necrosis factor-a (TNF-α), interleukin- 6 (L-6), HIF-1α, tissue inhibitor of metalloproteinases-1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), alpha-smooth muscle actin (α-SMA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). All these parameters were significantly (p<0.0244) protected by captopril. Also, a significant (p<0.0001) positive correlation was observed between a-SMA (profibrosis) and the serum and tissue levels of hsCRP, TNF-α, HIF-1α, TIMP-1, MMP-9, and ALT. Thus, these findings suggest that the induction of chronic liver injury by the hepatotoxic compound, TAA is associated with the upregulation of inflammation/HIF-1α/profibrosis, with captopril exhibiting beneficial hepatic pleotropic effects.

El trasplante de hígado es el único método disponible para tratar la insuficiencia hepática inducida por una lesión hepática crónica. Buscamos determinar si el inhibidor de la enzima convertidora de angiotensina, captopril, puede inhibir el desarrollo de lesión hepática crónica inducida por el agente hepatotóxico tioacetamida (TAA) en asociación con la supresión de la inflamación (hsCRP, TNF-α e IL-6) / factor inducible por hipoxia 1-alfa (HIF-1α) / profibrosis (TIMP-1, MMP-9 y α- SMA) eje que media la lesión hepática. Por lo tanto, al grupo modelo de ratas se le inyectó durante ocho semanas 200 mg/kg de TAA a partir de la semana dos. El grupo protector fue pretratado con 150 mg/kg de captopril al día durante dos semanas antes de las inyecciones de TAA y continuó recibiendo capropril y agentes TAA hasta que fue sacrificado en la semana 10. Observamos un daño sustancial en el tejido hepático en el grupo modelo, como lo demuestra un aumento significativo (p<0,0001) de los niveles en sangre y tejido hepático de proteína C reactiva de alta sensibilidad (hsCRP), factor de necrosis tumoral-α (TNF-a), interleucina-6 (L-6), HIF-1α, inhibidor tisular de metaloproteinasas-1 (TIMP-1), metaloproteinasa de matriz-9 (MMP-9), actina de músculo liso alfa (α-SMA), alanina aminotransferasa (ALT) y aspartato aminotransferasa (AST). Todos estos parámetros estaban significativamente (p<0,0244) protegidos por captopril. Además, se observó una correlación positiva significativa (p<0,0001) entre α-SMA (profibrosis) y los niveles séricos y tisulares de hsCRP, TNF-α, HIF-1α, TIMP- 1, MMP-9 y ALT. Por lo tanto, estos hallazgos sugieren que la inducción de daño hepático crónico por el compuesto hepatotóxico, TAA, está asociada con la regulación al alza de la inflamación/HIF-1α/profibrosis, con captopril exhibiendo efectos pleotrópicos hepáticos beneficiosos.

Gender-based incidence, recovery period, and mortality rate of COVID-19 among the population of district Attock, Pakistan / Incidência baseada em gênero, período de recuperação e taxa de mortalidade de COVID-19 entre a população do distrito de Attock, Paquistão

Abstract COVID-19 is reported as an extremely contagious disease with common symptoms of fever, dry cough, sore throat, and tiredness. The published literature on incidence and gender-wise prevalence of COVID-19 is scarce in Pakistan. Therefore, the present study was designed to compare the distribution, incubation period and mortality rate of COVID-19 among the male and female population of district Attock. The data were collected between 01 April 2020 and 07 December 2020 from the population of district Attock, Pakistan. A total of 22,962 individuals were screened and 843 were found positive for RT-qPCR for SARS-CoV-2. The confirmed positive cases were monitored carefully. Among the positive cases, the incidence of COVID-19 was 61.7% among males and 38.2% among females. The average recovery period of males was 18.89±7.75 days and females were 19±8.40 days from SARS-CoV-2. The overall mortality rate was 8.06%. The death rate of male patients was significantly higher (P<0.05) compared to female patients. Also, the mortality rate was higher (P<0.05) in male patients of 40-60 years of age compared to female patients of the same age group. Moreover, the mortality rate significantly increased (P<0.05) with the increase of age irrespective of gender. In conclusion, the incidence and mortality rate of COVID-19 is higher in males compared to the female population. Moreover, irrespective of gender the mortality rate was significantly lower among patients aged <40 years.

Resumo Covid-19 é relatada como uma doença extremamente contagiosa com sintomas comuns de febre, tosse seca, dor de garganta e cansaço. A literatura publicada sobre incidência e prevalência de Covid-19 com base no gênero é escassa no Paquistão. Portanto, o presente estudo teve como objetivo comparar a distribuição, o período de incubação e a taxa de mortalidade de Covid-19 entre a população masculina e feminina do distrito de Attock. Os dados foram coletados entre 1 de abril de 2020 e 7 de dezembro de 2020 da população do distrito de Attock, Paquistão. Um total de 22.962 indivíduos foi selecionado, e 843 foram considerados positivos para RT-qPCR para SARS-CoV-2. Os casos positivos confirmados foram monitorados cuidadosamente. Entre os casos positivos, a incidência de Covid-19 foi de 61,7% no sexo masculino e 38,2% no feminino. O período médio de recuperação dos homens foi de 18,89 ± 7,75 dias e das mulheres 19 ± 8,40 dias do SARS-CoV-2. A mortalidade geral foi de 8,06%. A taxa de mortalidade de pacientes do sexo masculino foi significativamente maior (P < 0,05) em comparação com pacientes do sexo feminino. Além disso, a taxa de mortalidade foi maior (P < 0,05) em pacientes do sexo masculino com 40-60 anos de idade em comparação com pacientes do sexo feminino da mesma faixa etária. Além disso, a taxa de mortalidade aumentou significativamente (P < 0,05) com o aumento da idade, independentemente do sexo. Em conclusão, a incidência e a taxa de mortalidade de Covid-19 são maiores no sexo masculino em comparação com a população feminina. E também, independentemente do sexo, a taxa de mortalidade foi significativamente menor entre os pacientes com idade < 40 anos.

Comparative study on the distribution and expression of Neuroglobin and Hypoxia-inducible factor-1α in the telencephalon of yak and cattle / Estudo comparativo da distribuição e expressão de fator-1α indutível por neuroglobina e hipóxia no telencéfalo de iaques e bovinos

The telencephalon refers to the most highly developed and anterior part of the forebrain, consisting mainly of the cerebral hemispheres. The study determined Neuroglobin (Ngb) and Hypoxia-inducible factor (HIF-1α) expression in the telencephalon of yak and cattle, and compare the expression and distribution pattern of Ngb and HIF-1α in the two animals. Immunohistochemistry (IHC), quantitative real-time Polymerase Chain Reaction (qRT-PCR), and Western blot (WB) were employed to investigate Ngb and Hif-1α expression in the telencephalon of yak and cattle. mRNA and protein expressions of Ngb and HIF-1α showed positive in different tissues of the yak and cattle telencephalon. Ngb expression in tissues of the yak recorded higher as compare to cattle while HIF-1α expression was found higher in cattle than yak. The HIF-1α expression in some tissues of yak telencephalon was consistent with the cattle. The results documented that HIF-1α may have a direct or indirect synergistic effect on Ngb expression in the yak telencephalon to improve hypoxia adaptation. It is suggested that yak may need more Ngb expression for adaptation, but the expression of HIF-1α seems to be down-regulated during long-term adaptation, and the specific causes of this phenomenon needs to be further verified.

O telencéfalo refere-se à parte anterior e mais desenvolvida do prosencéfalo, consistindo principalmente dos hemisférios cerebrais. O estudo determinou a expressão de neuroglobina (Ngb) e fator indutível por hipóxia (HIF-1α) no telencéfalo de iaques e bovinos e comparou a expressão e o padrão de distribuição de Ngb e HIF-1α nos dois animais. Imuno-histoquímica (IHC), reação em cadeia da polimerase quantitativa em tempo real (qRT-PCR) e Western blot (WB) foram empregados para investigar a expressão de Ngb e Hif-1α no telencéfalo de iaques e bovinos. As expressões de mRNA e proteínas de Ngb e HIF-1α mostraram-se positivas em diferentes tecidos do telencéfalo de iaque e bovino. A expressão de Ngb nos tecidos do iaque foi registrada mais alta em comparação com o gado, enquanto a expressão do HIF-1α foi encontrada mais alta no gado do que no iaque. A expressão de HIF-1α em alguns tecidos do telencéfalo de iaque foi consistente com o gado. Os resultados documentaram que o HIF-1α pode ter um efeito sinérgico direto ou indireto na expressão de Ngb no telencéfalo de iaque para melhorar a adaptação à hipóxia. É sugerido que o iaque pode precisar de mais expressão de Ngb para adaptação, mas a expressão de HIF-1α parece ser regulada para baixo durante a adaptação de longo prazo, e as causas específicas desse fenômeno precisam ser verificadas.

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.

A review of the recent advances on Lassa fever with special reference to molecular epidemiology and progress in vaccine development

Lassa fever, a viral hemorrhagic fever caused by the Lassa virus (LASV), is endemic in West Africa and is associated with high morbidity and mortality. At least three of the four proposed seven lineages of LASV are found in Nigeria, where the multimammate rat, Mastomys natalensis, serves as the primary reservoir. Endemic countries report approximately 200,000 infections and 5,000 deaths annually, with Nigeria experiencing thousands of infections and hundreds of deaths including healthcare workers. The aim of this review is to provide scientific information for better understanding of the evolutionary biology, molecular epidemiology, pathogenesis, diagnosis, and prevention of Lassa fever in Nigeria and other endemic regions worldwide, which can lead to improved control efforts and reduce morbidity and mortality from recurrent epidemics. To achieve this aim, observational studies such as case series, cross-sectional and cohort studies published between December 2017 and September 2022 were searched for on various online databases including Google Scholar, Africa Journals Online (AJOL), Research Gates, PubMed, PMIC, NCDC, and WHO websites. Although the origin and evolutionary history, and the transmission dynamics of Lassa virus have been revealed through recent molecular epidemiological studies, the factors that drive the evolution of the virus remain unclear. Genetic changes in the viral genome may have enabled the virus to adapt to humans. Diagnosis of Lassa fever has also advanced from basic serological tests to more sophisticated methods such as quantitative real time polymerase chain reaction (qRT-PCR) and sequencing, which are particularly useful for identifying outbreak strains. Several vaccines, including recombinant vesicular stomatitis virus (rVSV), virus-like particle (VLP), and DNA-based vaccines, have shown promise in animal models and some have progressed to phase 2 clinical trials. Preventing and controlling Lassa fever is critical to safeguard the health and well-being of affected communities. Effective measures such as rodent control, improved sanitation, and early detection and isolation of infected individuals are essential for reducing transmission. Ongoing research into the genetic and ecological factors that drive the evolution of Lassa virus is necessary to reduce the impacts of Lassa fever

La fièvre de Lassa, une fièvre hémorragique virale causée par le virus de Lassa (LASV), est endémique en Afrique de l'Ouest et est associée à une morbidité et une mortalité élevées. Au moins trois des quatre lignées proposées de LASV se trouvent au Nigeria, où le rat multimammaire, Mastomys natalensis, sert de réservoir principal. Les pays endémiques signalent environ 200,000 infections et 5,000 décès par an, le Nigéria connaissant des milliers d'infections et des centaines de décès, y compris des travailleurs de la santé. L'objectif de cette revue est de fournir des informations scientifiques pour une meilleure compréhension de la biologie évolutive, de l'épidémiologie moléculaire, de la pathogenèse, du diagnostic et de la prévention de la fièvre de Lassa au Nigeria et dans d'autres régions endémiques du monde, ce qui peut conduire à des efforts de contrôle améliorés et réduire la morbidité et la mortalité des épidémies récurrentes. Pour atteindre cet objectif, des études observationnelles telles que des séries de cas, des études transversales et de cohorte publiées entre décembre 2017 et septembre 2022 ont été recherchées sur diverses bases de données en ligne, notamment Google Scholar, Africa Journals Online (AJOL), Research Gate, PubMed, PMIC, Sites Web du NCDC et de l'OMS. Bien que l'origine et l'histoire évolutive, ainsi que la dynamique de transmission du virus de Lassa aient été révélées par des études épidémiologiques moléculaires récentes, les facteurs qui déterminent l'évolution du virus restent flous. Des modifications génétiques du génome viral pourraient avoir permis au virus de s'adapter à l'homme. Le diagnostic de la fièvre de Lassa est également passé des tests sérologiques de base à des méthodes plus sophistiquées telles que la réaction quantitative en chaîne par polymérase en temps réel (qRTPCR) et le séquençage, qui sont particulièrement utiles pour identifier les souches épidémiques. Plusieurs vaccins, y compris le virus recombinant de la stomatite vésiculeuse (rVSV), les particules pseudo-virales (VLP) et les vaccins à base d'ADN, se sont révélés prometteurs dans des modèles animaux et certains ont progressé vers des essais cliniques de phase 2. La prévention et le contrôle de la fièvre de Lassa sont essentiels pour préserver la santé et le bien-être des communautés touchées. Des mesures efficaces telles que le contrôle des rongeurs, l'amélioration de l'assainissement et la détection et l'isolement précoces des personnes infectées sont essentielles pour réduire la transmission. Des recherches continues sur les facteurs génétiques et écologiques qui déterminent l'évolution du virus de Lassa sont nécessaires pour réduire les impacts de la fièvre de Lassa.

Expression of microRNAs miR-126, miR-133b and miR-221 associated with apoptosis in rats submitted to focal cerebral ischemia and physical exercise / Expresión de microRNAs miR-126, miR-133b y miR-221 asociados a apoptosis en ratas sometidas a isquemia cerebral focal y ejercicio físico

SUMMARY: Stroke is one of the main causes of death and disability worldwide. The great impact on the quality of life of the population and on the health system justifies that we seek relevant alternatives to reduce the incidence and improve the treatment and recovery of patients affected by this disease. Physical exercise appears as an important tool in this scenario, being already pointed out as a possible therapeutic approach for the prevention of non-contagious chronic diseases. In this context, biomarkers such as miRNAs that respond to physical exercise and are directly related to several epigenetic mechanisms appear. Therefore, explaining the molecular mechanisms involved during physical exercise will lead to a better understanding of each stimulus and the dose to be used to better respond to each situation, thus being a promising approach for the evolution of prescription and control of training and processes recovery from various diseases, including stroke. Forty-eight Wistar rats were used, divided into four experimental groups: control group, ischemia group, physical exercise group and exercise + ischemia group. Real-time PCR methodology was used to analyze the expression of miRNAs: miR-126, miR-133b and miR-221. In our study we observed a significant difference in the expression of miR- 221 between the control group and the others groups. However, microRNAs: miR-126 and miR-133b do not show significant differences in expression between groups.

El ictus es una de las principales causas de muerte y discapacidad en todo el mundo. El gran impacto en la calidad de vida de la población y en el sistema de salud justifica buscar alternativas pertinentes para reducir la incidencia y mejorar el tratamiento y recuperación de los pacientes afectados por esta enfermedad. El ejercicio físico aparece como una herramienta importante en este escenario, siendo ya señalado como un posible abordaje terapéutico para la prevención de enfermedades crónicas no contagiosas. En este contexto, aparecen biomarcadores como los miRNAs que responden al ejercicio físico y están directamente relacionados con varios mecanismos epigenéticos. Por lo tanto, explicar los mecanismos moleculares involucrados durante el ejercicio físico conducirá a una mejor comprensión de cada estímulo y la dosis a utilizar para responder mejor a cada situación, siendo así un enfoque prometedor para la evolución de la prescripción, el control del entrenamiento y los procesos de recuperación de diversas enfermedades, incluido el accidente cerebrovascular. Se utilizaron cuarenta y ocho ratas Wistar, divididas en cuatro grupos experimentales: grupo control, grupo isquemia, grupo ejercicio físico y grupo ejercicio + isquemia. Se utilizó la metodología de PCR en tiempo real para analizar la expresión de miRNAs: miR-126, miR-133b y miR-221. En nuestro estudio observamos una diferencia significativa en la expresión de miR-221 entre el grupo control y los demás grupos. Sin embargo, los microARN: miR-126 y miR-133b no mostraron diferencias significativas en la expresión entre grupos.

Comparación del test de antígeno y la reacción en cadena de polimerasa para SARS-CoV-2 en niños menores de 12 años / Comparison of antigen test and polymerase chain reaction for SARS-CoV-2 in children younger than 12 years

Frenar la propagación de la enfermedad por el coronavirus 2019 (COVID-19, por su sigla en inglés) es fundamental, y se puede realizar mediante técnicas de detección rápidas y efectivas. El objetivo fue comparar la precisión diagnóstica de un test rápido de antígeno (TRAg,) con la reacción en cadena de polimerasa con retrotranscripción (RT-qPCR, por su sigla en inglés) y describir los umbrales de amplificación (Ct, por su sigla en inglés). Participaron niños de 1 mes a 11 años que tuvieran menos de 7 días de síntomas, sin resultado detectable en los últimos 90 días, e inmunocompetentes. Se incluyeron 1855 pacientes con una prevalencia de COVID-19 del 4,7 %. La sensibilidad global del TRAg fue del 60,2 % y su especificidad, del 99,8 %; en niños mayores de 5 años los valores fueron de 69,8 % y 99,8 %, respectivamente. Los valores de Ct de las muestras discordantes fueron más altos. En conclusión, la precisión diagnóstica muestra que TRAg tiene una especificidad similar a la RT-qPCR, pero una sensibilidad considerablemente menor, sobre todo en niños de menos de 5 años.

Stopping the spread of coronavirus disease 2019 (COVID-19) is critical and can be achieved through rapid and effective detection techniques. Our objective was to compare the diagnostic accuracy of rapid antigen tests (RAgT) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) and to describe amplification cycle thresholds (Cts). Participants were children aged 1 month to 11 years with symptoms for less than 7 days, who did not have a detectable result in the past 90 days, and were immunocompetent. A total of 1855 patients were included; the prevalence of COVID-19 was 4.7%. For the RAgT, overall sensitivity was 60.2% and specificity, 99.8%; in children older than 5 years, values were 69.8% and 99.8%, respectively. Ct values for discordant samples were higher. To conclude, the diagnostic accuracy indicated that the specificity of RAgT is similar to that of RT-qPCR, but its sensitivity is notably lower,especially in children younger than 5 years.

Zika virus RNA detection in blood donors in São Paulo, Brazil

ABSTRACT Introduction: The Zika Virus (ZIKV) is a single-stranded RNA genome virus, belonging to the family Flaviviridae, genus Flavivirus. Outbreaks around the world have demonstrated that the presence of asymptomatic viremic blood donors provides an increase in the risk of transfusion transmission (TT) and nucleic acid test (NAT) screening has been proposed to ensure the blood safety. This study implemented an "in-house" method to detect ZIKV RNA in blood sample donations. Methods: Primary plasma tubes are submitted to nucleic acid extraction on an automated platform. After extraction, the NAT set-up is performed in the robotic pipettor, in which an amplification mixture containing primers and probes for ZIKV and Polio vaccine virus (PV) are added in duplex as an internal control. The real-time polymerase chain reaction is then performed in a thermocycler, using the protocol established by the supplier. Results: From May 2016 to May 2018, 3,369 samples were collected from 3,221 blood donors (confidence coefficient 95%), of which 31 were considered false positive (0.92%), as they did not confirm initial reactivity when repeated in duplicates and 14 (0.42%) had their results invalid due to repeat failure in the internal control, 4 (0.12%), due to insufficient sample volume and 2 (0.05%), due to automatic pipettor failures. No Zika RNA reactive sample was identified. Conclusion: The test showed feasible to be incorporated into the blood screening routine. Our data do not indicate the need to screen for ZIKV RNA in São Paulo during the evaluated period. However, a generic NAT system covering a group of flaviviruses which are circulating in the region, such as DENV and YFV, among others, could be a useful tool.

Amplificación directa de ADN de Bordetella pertussis purificado de hisopados nasofaríngeos por un método de bajo costo, rápido (60-segundos) y libre de equipos / Direct amplification of Bordetella pertussis DNA purified from nasopharyngeal swabs by a low-cost, fast (60-second), and equipment-free method

RESUMEN Objetivo. Desarrollar y evaluar un método de bajo costo basado en celulosa para la purificación rápida y amplificación directa de ADN de Bordetella pertussis de hisopados nasofaríngeos. Materiales y métodos. Se prepararon discos de celulosa y se evaluaron diferentes parámetros (buffers de lisis/lavado, número de discos y elución de ADN). El método se acopló a una amplificación directa por PCR en tiempo real (qPCR) y se estimó el rendimiento utilizando hisopados nasofaríngeos que fueron positivos (n=100) y negativos (n=50) para ADN B. pertussis por qPCR, comparado con el método basado en columnas de sílice. Se calculó el grado de concordancia, sensibilidad, especificidad, valor predictivo positivo (VPP) y valor predictivo negativo (VPN). Se evaluó la factibilidad del método rápido para ser acoplado a un ensayo colorimétrico de amplificación isotérmica mediada por lazo (LAMP). Resultados. El método rápido con un disco de celulosa y buffer de lisis y lavado conteniendo PVP-40 y Tween 20, respectivamente, mostró una mayor capacidad para purificar ADN amplificable de B. pertussis. El método tuvo una sensibilidad de 89,0% (IC95%, 80,2%-94,9%) y una especificidad de 98,5% (IC95%, 92,1%-100,0%), con un buen grado de concordancia (Kappa=0,867; IC95% 0,788 - 0,946), respecto al método referencial. Los VPP y VPN fueron 98,6% (IC95%, 92,7,2%-100,0%) y 88,2% (IC95%, 78,7%-94,4%), respectivamente. Se evidenció una amplificación exitosa por LAMP, y se obtuvieron resultados comparables con el método por columnas de sílice. Conclusión. El método desarrollado es simple, de bajo costo y libre de equipos para la obtención rápida (60 segundos) de ADN en el punto de atención, y puede ser implementado en diversas técnicas moleculares orientados al diagnóstico oportuno y al estudio epidemiológico de tos ferina.

ABSTRACT Objective. To develop and evaluate a low-cost cellulose-based method for rapid purification and direct amplification of Bordetella pertussis DNA from nasopharyngeal swabs. Materials and methods. We prepared cellulose discs and evaluated different parameters (lysis/wash buffers, number of discs and DNA elution). The method was coupled to a direct real-time PCR (qPCR) amplification and the performance was estimated using nasopharyngeal swabs that were positive (n=100) and negative (n=50) for B. pertussis DNA by qPCR, compared to the silica column-based method. We calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) and the degree of agreement. The feasibility of the rapid method to be coupled to a loop-mediated isothermal amplification colorimetric assay (LAMP) was evaluated. Results. The rapid method, with a cellulose disk and lysis and wash buffer containing PVP-40 and Tween 20, respectively, showed a greater capacity to purify amplifiable DNA from B. pertussis. The method had a sensitivity of 89.0% (95%CI: 80.2%-94.9%) and a specificity of 98.5% (95%CI: 92.1%-100.0%), with a good degree of agreement (Kappa=0.867; 95%CI: 0.788 - 0.946), compared to the reference method. The PPV and NPV were 98.6% (95%CI: 92.7.2%-100.0%) and 88.2% (95%CI: 78.7%-94.4%), respectively. Successful amplification by LAMP was evident, and comparable results were obtained with the silica column method. Conclusion. The developed method is simple, low-cost and equipment-free for rapid (60 seconds) DNA collection at the point of care, and can be implemented in various molecular techniques aimed at the timely diagnosis and epidemiological study of pertussis.

Low-dose cisplatin exposure and SNAIL, Vimentin, E-cadherin expression in HEPG2 cell line / Exposición a dosis bajas de cisplatino y expresión de SNAIL, Vimentina, E-cadherina en línea celular HEPG2

Cisplatin, the first platinum compound approved for cancer treatment, is widely used in the treatment of various cancers including hepatocellular carcinoma (HCC). HCC incidence rates rise globally. Epithelial mesenchymal transition (EMT) is implicated in cancer invasion and metastasis, which are associated with increased mortality. Cisplatin dose might influence cancer invasion and metastatic behavior of the cells. The aim of the study was to investigate the effect of low-dose cisplatin treatment on EMT- related changes in HepG2 cells. Following treatment with 4 µM cisplatin, HepG2 cells were evaluated morphologically. Gene expression of E-cadherin, Vimentin, Snail1 was assessed by quantitative PCR. Immunofluorescence analyses of NA-K ATPase were performed. Although the low-dose cisplatin treated cells exhibited a more stretched morphology, no statistical difference was detected in gene expression of E-cadherin, Vimentin, Snail1 and immunofluorescence of NA-K ATPase. Findings on low-dose cisplatin effects in HepG2 might contribute to the knowledge of antineoplastic inefficacy by further understanding the molecular mechanisms of drug action.

El cisplatino, el primer compuesto de platino aprobado para el tratamiento del cáncer, es ampliamente utilizado en el tratamiento de varios tipos de cáncer, incluido el carcinoma hepatocelular (CHC). Las tasas de incidencia de CHC aumentan a nivel mundial. La transición mesenquimal epitelial (EMT) está implicada en la invasión del cáncer y la metástasis, que se asocian con un aumento de la mortalidad. La dosis de cisplatino podría influir en la invasión del cáncer y el comportamiento metastásico de las células. El objetivo del estudio fue investigar el efecto del tratamiento con dosis bajas de cisplatino en los cambios relacionados con la EMT en las células HepG2. Tras el tratamiento con cisplatino de 4 µM, se evaluaron morfológicamente las células HepG2. La expresión génica de E-cadherina, vimentina, caracol1 se evaluó mediante PCR cuantitativa. Se realizaron análisis de inmunofluorescencia de NA-K ATPasa . Aunque las células tratadas con cisplatino en dosis bajas exhibieron una morfología más estirada, no se detectaron diferencias estadísticas en la expresión génica de E-cadherina, vimentina, Snail1 e inmunofluorescencia de NA-K ATPasa. Los hallazgos sobre los efectos del cisplatino en dosis bajas en HepG2 podrían contribuir al conocimiento de la ineficacia antineoplásica al comprender mejor los mecanismos moleculares de la acción del fármaco.

mRNA expression of myosin heavy chain isoforms in the sphenomandibularis portion of the temporalis muscle / Expresión de ARNm de las isoformas de la cadena pesada de la miosina en la porción esfenomandibular del músculo temporal

SUMMARY: The main objective of this study was to analyze by real-time quantitative polymerase chain reaction (RT-qPCR) the expression patterns of the myosin heavy chain (MHC) isoforms (MHC-I, MHC-IIa, MHC-IIx) in the sphenomandibularis portion of the temporalis muscle. We expected to find differences between the sphenomandibularis and the other portions of the temporalis that could be related to the functional characteristics of the sphenomandibularis identified by electromyography. We dissected the right temporalis muscle of ten adult human individuals (five men and five women). Samples of the anterior and posterior temporalis and of the sphenomandibularis portion were obtained from each dissected muscle. These samples were analyzed by RT-qPCR to determine the percentages of expression of the MHC-I, MHC-IIa and MHC-IIx isoforms. No significant differences were identified between the anterior and the posterior temporalis in the expression patterns of the MHC-I, MHC-IIa and MHC-IIx isoforms. However, there were significant differences between the sphenomandibularis and the anterior temporalis. Specifically, the sphenomandibularis portion had a higher percentage of expression of the MHC-I isoform (P=0.04) and a lower percentage of expression of the MHC-IIx isoform (P=0.003). The pattern of expression that we observed in the sphenomandibularis reflects a greater resistance to fatigue, a lower contraction speed, and a lower capacity of force generation in the sphenomandibularis compared to the anterior temporalis. These characteristics are consistent with electromyographic findings on the functional differences between these two portions.

RESUMEN: El principal objetivo de este estudio fue analizar mediante real-time quantitative polymerase chain reaction (RT-qPCR) los patrones de expresión de las isoformas de la cadena pesada de la miosina (MHC-I, MHC-IIa y MHC-IIx) en la porción esfenomandibular del músculo temporal. Se esperó encontrar diferencias entre el esfenomandibular y las otras porciones del músculo temporal que se pudieran relacionar con las características funcionales del esfenomandibular, identificadas mediante electromiografía. Para obtener estos resultados, se diseccionó el músculo temporal derecho en diez humanos adultos (cinco hombres y cinco mujeres) y se obtuvieron muestras de la porción anterior y posterior del músculo temporal y de su porción esfenomandibular. Estas muestras fueron analizadas mediante RT-qPCR para determinar los porcentajes de expresión de las isoformas MHC-I, MHC- IIa y MHC-IIx. No se identificaron diferencias significativas de los patrones de expresión entre la porción anterior y la porción posterior del músculo temporal, pero sí que se observaron diferencias significativas entre la porción anterior del músculo temporal y su porción esfenomandibular. Concretamente, la porción esfenomandibular presentó un mayor porcentaje de expresión de la isoforma MHC-I (P=0.04) y un menor porcentaje de expresión de la isoforma MHC-IIx (P=0.003). El patrón de expresión que hemos observado en la porción esfenomandibular del músculo temporal refleja una mayor resistencia a la fatiga, una velocidad de contracción más lenta y una menor capacidad de generar fuerza si se compara esta porción con la porción anterior del músclo temporal. Estas características son consistentes con las diferencias funcionales que presentan estas dos porciones, que han sido descritas mediante electromiografía.

Results of a sample-to-cutoff ratio using Abbott Architect rHTLV-I/II assay allow to predict detection of HTLV-1 and HTLV-2 proviral DNA by real-time PCR / Resultados da razão sinal da amostra sobre o cutoff da reação no ensaio Abbott Architect rHTLV-I/II permite prever a detecção de DNA proviral por PCR em tempo real

The present study aims to correlate the sample-to-cutoff ratios (S/CO) distributions of reactive results for HTLV-1/2 antibodies with the detection of proviral DNA in a population of blood donor candidates. It was carried out a retrospective data search of 632 HTLV-1/2 reactive samples, submitted to confirmatory testing from January 2015 to December 2019. Serological screening was performed by chemiluminescent microparticle immunoassay Architect rHTLV-I/II, whereas confirmatory testing was performed by in-house real-time polymerase chain reaction method. 496 out of 632 samples (78%) had undetectable HTLV-1/2 proviral DNA and 136 (22%) had detectable proviral DNA. HTLV infection was not confirmed in any individual for whom the S/CO ratio value was <4, and proviral DNA detection rates gradually escalated as S/CO ratio values increased. The sensitivity and predictive positive value found for the Architect rHTLV-I/II was 100% and 22%, respectively. The receiver operating characteristic (ROC) curve analysis showed that the optimal S/CO ratio value for predicting the presence of HTLV-1/2 was 18.11. High S/CO ratios were more associated with the detection of proviral DNA. The S/CO ratio value <4 suggests excluding true HTLV infection and the risk of blood transmission (AU).

O estudo tem como objetivo correlacionar às distribuições das razões sample-to-cutoff (S/CO) de resultados reagentes para anticorpos HTLV-1/2 com a detecção de DNA proviral em uma população de candidatos à doação de sangue. Realizou-se uma busca retrospectiva de dados de 632 amostras reagentes para HTLV-1/2 submetidas à testagem confirmatória entre janeiro de 2015 a dezembro de 2019. A triagem sorológica foi realizada pelo imunoensaio quimioluminescente de micropartículas Architect rHTLV-I/II, enquanto o teste confirmatório foi realizado pelo método de PCR em tempo real in-house. 496 de 632 amostras (78%) apresentaram DNA proviral indetectável e 136 (22%) apresentaram DNA proviral detectável. A infecção por HTLV não foi confirmada em nenhum indivíduo com valor de S/CO <4 e as taxas de detecção de DNA proviral escalonaram gradualmente à medida que as razões S/CO aumentaram. A sensibilidade e valor preditivo positivo encontrados para o Architect rHTLV-I/II foram 100% e 22%, respectivamente. Utilizando análise de curva ROC, o valor de razão S/CO ideal para predizer a presença de DNA proviral foi de 18,11. Razões S/CO elevadas foram mais associadas à detecção de DNA proviral. Em suma, o valor de S/CO <4 sugere a exclusão de infecção por HTLV e o risco de transmissão pelo sangue (AU).

Correlación entre el umbral de ciclo de la RPC en tiempo real y la clasificación clínica de la COVID-19 / Correlation between real-time PCR cycle threshold and clinical classification of COVID-19

INTRODUCCIÓN: El umbral de ciclo (en inglés cycle threshold-Ct) de la reacción de polimerasa en cadena en tiempo real con transcripción reversa (RT-qPCR) indica la concentración relativa de una secuencia de ARN; este valor se ha relacionado con la expresión de cuadros clínicos en infecciones virales. OBJETIVO: Determinar la correlación entre el valor Ct y la clasificación clínica de la COVID-19. MÉTODO: Se realizó un estudio transeccional correlacional; los valores Ct se obtuvieron mediante RT-qPCR dirigida al gen N del SARS-CoV-2 agrupándolos mediante un estimador robusto central y relacionándose con la clasificación clínica de la COVID-19. RESULTADOS: De los 718 casos incluidos en el estudio; 77,7% (558) fueron leves; 21,3% (153) moderados y 1% (7) graves. El valor Ct se agrupó en niveles: Ct bajo 18,83 - 30,10 y Ct alto > 30,10. Existió correlación significativa inversa débil (p = 0,002; rho de Spearman = -0,117) entre el valor Ct y la clasificación clínica. Las características: sexo, edad menor a 65 años, fiebre, escalofrío, diarrea, anosmia y sobrepeso-obesidad estuvieron asociadas al valor de Ct. CONCLUSIÓN: A menor valor Ct se espera una clasificación de mayor gravedad de la COVID-19; no obstante, debido a que la correlación es débil, su utilidad como predictor de gravedad es limitada.

BACKGROUND: The cycle threshold (Ct) of real-time reverse transcription PCR (RT-qPCR) indicates the relative concentration of an RNA sequence, this value has been related to clinical profile in viral infections. AIM: To determine the correlation between the Ct value and the clinical classification of COVID-19. METHOD: A correlational cross-sectional study was carried out, the Ct values were obtained by RT-qPCR directed to the N gene of SARS-CoV-2, grouping them by means of a central robust estimator and related to the clinical classification of COVID-19. RESULTS: Of the 718 cases included in the study; 77.7% (558) were mild; 21.3% (153) moderate and 1% (7) severe. The Ct value was grouped into levels: low Ct 18.83-30.10 and high Ct> 30.10. There was a weak inverse significant correlation (p = 0.002; Spearman's rho = -0.117) between the Ct value and the clinical classification. The characteristics: sex, age under 65 years, fever, chills, diarrhea, anosmia, and overweightobesity were associated with the Ct value. CONCLUSION: The lower the Ct value, a classification of greater severity of COVID-19 is expected, however, because the correlation is weak, its usefulness as a severity predictor is limited.

Effects of nitric oxide on C2C12 cell inflammatory responses and notexin-induced muscle injury / Efectos del óxido nítrico en las respuestas inflamatorias de las células C2C12 y la lesión muscular inducida por notexina

SUMMARY: Skeletal muscle injury is an acute inflammatory condition caused by an inflammatory response. To reduce inflammatory cell infiltration and relieve skeletal muscle injury, efficient treatment is urgently needed. Nitric oxide is a free radical molecule reported to have anti-inflammatory effects. In this study, we showed that NO could inhibit the inflammatory response of C2C12 cells in vitro and protect rat skeletal muscle injury from notexin in vivo. NO synthase inhibitor (L-NG-Nitroarginine Methyl Este?L-NAME) and NO donor (sodium nitroprusside dehydrate ?SNP) were used to explore the vital role of lipopolysaccharides (LPSs) in LPS-stimulated C2C12 myoblasts.The expression of IL-18 and IL-1b was upregulated by L-NAME and downregulated by SNP, as indicated by the ELISA results. NO can reduce ASC, Caspase-1, and NLRP3 mRNA and protein levels. Furthermore, NO was detected in the rat model. The results of immunohistochemical staining showed that the production of DMD decreased. We conducted qRT-PCR and western blotting to detect the expression of Jo-1, Mi-2, TLR2, and TLR4 on day 6 post injury following treatment with L-NAME and SNP. The expression of Jo-1, Mi-2, TLR2, and TLR4 was upregulated by L-NAME and significantly reversed by SNP. NO can alleviate C2C12 cell inflammatory responses and protect rat skeletal muscle injury from notexin.

RESUMEN: La lesión del músculo esquelético es una afección inflamatoria aguda causada por una respuesta inflamatoria. Para reducir la infiltración de células inflamatorias y aliviar la lesión del músculo esquelético es necesario un tratamiento eficaz. El óxido nítrico es una molécula de radicales libres que tiene efectos antiinflamatorios. En este estudio, demostramos que el ON podría inhibir la respuesta inflamatoria de las células C2C12 in vitro y proteger la lesión del músculo esquelético de rata de la notexina in vivo. El inhibidor de ON sintasa (L-NG-nitroarginina metil este, L-NAME) y el donante de ON (nitroprusiato de sodio deshidratado, SNP) se utilizaron para explorar el papel vital de los lipopolisacáridos (LPS) en los mioblastos C2C12 estimulados por LPS. La expresión de IL- 18 e IL-1b fue regulada positivamente por L-NAME y regulada negativamente por SNP, como indican los resultados de ELISA. El ON puede reducir los niveles de proteína y ARNm de ASC, Caspasa-1 y NLRP3. Además, se detectó ON en el modelo de rata. Los resultados de la tinción inmunohistoquímica mostraron que disminuyó la producción de DMD. Realizamos qRT-PCR y transferencia Western para detectar la expresión de Jo-1, Mi-2, TLR2 y TLR4 el día 6 después de la lesión después del tratamiento con L-NAME y SNP. La expresión de Jo-1, Mi-2, TLR2 y TLR4 fue regulada positivamente por L- NAME y significativamente revertida por SNP. El ON puede aliviar las respuestas inflamatorias de las células C2C12 en ratas, y proteger la lesión del músculo esquelético de la notexina.

Screening of reference genes for quantitative real-time PCR in Artemisia argyi / 中国中药杂志

Artemisia Argyi Folium, a traditional Chinese medicine of important medicinal and economic value, sees increasing demand in medicinal and moxibustion product market. Screening stable and reliable reference genes for quantitative real-time PCR(qRT-PCR) is a prerequisite for the analysis of gene expression in Artemisia argyi. In this study, eight commonly used reference genes, Actin, 18s, EF-1α, GAPDH, SAND, PAL, TUA, and TUB, from the transcriptome of A. argyi, were selected as candidate genes. The expression of each gene in different tissues(roots, stems, and leaves) of A. argyi and in leaves of A. argyi after treatment with methyl jasmonate(MeJA) for different time(0, 4, 8, 12 h) was detected by qRT-PCR. Then, geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder were employed to evaluate their expression stability. The results demonstrated that Actin was the most stable reference gene in different tissues and in leaves treated with MeJA, and coming in the second was SAND. Furthermore, the expression of DXS and MCT which are involved in terpenoid backbone biosynthesis was detected in different tissues and after MeJA treatment. The results showed that the expression patterns of DXS and MCT in different tissues and under MeJA treatment calculated with Actin and SAND as internal reference genes were consistent, which validated the screening results. In conclusion, Actin is the most suitable reference gene for the analysis of gene expression in different tissues of A. argyi and after MeJA treatment. This study provides valuable information for gene expression analysis in A. argyi and lays a foundation for further research on molecular mechanism of quality formation of Artemisia Argyi Folium.

Single-copy Loss of Rho Guanine Nucleotide Exchange Factor 10 ( arhgef10) Causes Locomotor Abnormalities in Zebrafish Larvae / 生物医学与环境科学（英文）

OBJECTIVE@#To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation.@*METHODS@#Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 -/-, arhgef10 +/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively.@*RESULTS@#WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 -/- zebrafish had more severe symptoms than arhgef10 +/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis.@*CONCLUSION@#Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.

Application of two RT-PCR methods for detection of norovirus in market-sold oysters and norovirus genetic characteristic analysis, a survey conducted in Beijing / 中华流行病学杂志

Objective: To evaluate the application of real-time RT-PCR and semi-nested RT-PCR in the detection of norovirus in oysters and analyzing the genetic characteristics of the isolates. Methods: Real-time fluorescent RT-PCR and semi-nested RT-PCR were used to detect norovirus GⅠ/GⅡ in fresh oysters collected from the markets in Beijing from November 2014 to October 2015. The detection rate of the parallel test was also analyzed. In addition, the reliability of semi-nested RT-PCR was evaluated by agreement rate and consistency test (Kappa value). The positive products of norovirus GⅠ/GⅡ capsid protein region gene by semi-nested RT-PCR were sequenced. Software BioEdit 7.0.9.0 was used for sequence alignment, and software Mega 6.0 was used to construct the evolutionary tree. Results: In 72 samples, the detection rate of norovirus was 31.94% (23/72) by real-time RT-PCR, 38.89% (28/72) by semi-nested RT-PCR and 48.61% (35/72) by parallel test. The coincidence rate of the two methods was 73.61%, a moderate degree (Kappa value =0.43). A total of 13 norovirus strains were successfully sequenced, and 11 strains (7 GⅡ.17 strains, 2 GⅡ. 4 Sydney_ 2012 strains, 1 GⅡ. 1 strain and 1 GⅡ. 21 strain) were obtained from norovirus positive samples by two RT-PCR methods, two strains (1 GⅡ. 17 strain and 1 GⅡ. 3 strain) were obtained from real-time RT-PCR negative samples which were positive for norovirus by semi-nested RT-PCR. The similarity between these strains and reference strains from diarrhea patients, environmental sewage, and shellfish products were 84.4% - 100.0%. Conclusions: The parallel test of norovirus in oysters by two RT-PCR methods can improve the detection rate and detect more genotypes. Norovirus strains in oysters were highly homologous with reference strains from diarrheal patients, environmental sewage, and shellfish products. Therefore, surveillance, prevention and control for norovirus should be carried out in people who have frequent contacts with oysters and related environments.

Accurate quantification of 3'-terminal 2'-O-methylated small RNAs by utilizing oxidative deep sequencing and stem-loop RT-qPCR / 医学前沿

The continuing discoveries of novel classes of RNA modifications in various organisms have raised the need for improving sensitive, convenient, and reliable methods for quantifying RNA modifications. In particular, a subset of small RNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), are modified at their 3'-terminal nucleotides via 2'-O-methylation. However, quantifying the levels of these small RNAs is difficult because 2'-O-methylation at the RNA 3'-terminus inhibits the activity of polyadenylate polymerase and T4 RNA ligase. These two enzymes are indispensable for RNA labeling or ligation in conventional miRNA quantification assays. In this study, we profiled 3'-terminal 2'-O-methyl plant miRNAs in the livers of rice-fed mice by oxidative deep sequencing and detected increasing amounts of plant miRNAs with prolonged oxidation treatment. We further compared the efficiency of stem-loop and poly(A)-tailed RT-qPCR in quantifying plant miRNAs in animal tissues and identified stem-loop RT-qPCR as the only suitable approach. Likewise, stem-loop RT-qPCR was superior to poly(A)-tailed RT-qPCR in quantifying 3'-terminal 2'-O-methyl piRNAs in human seminal plasma. In summary, this study established a standard procedure for quantifying the levels of 3'-terminal 2'-O-methyl miRNAs in plants and piRNAs. Accurate measurement of the 3'-terminal 2'-O-methylation of small RNAs has profound implications for understanding their pathophysiologic roles in biological systems.

Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples / Validação da nested PCR em tempo real em um tubo para detecção de Cryptosporidium spp. em amostras fecais de aves

Abstract The aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36/443 (8.1%) samples were positive for Cryptosporidium spp. by conventional nested PCR. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. An evaluation of analytical specificity did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability analysis showed the same result in 27 out of 30 samples (90%). As for the reproducibility of one-tube nested real-time PCR, 24 of the 30 samples examined (80%) showed the same result. All the 90 samples amplified by one-tube real-time nested PCR were successfully sequenced, leading to the identification of C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium avian genotype I. Genetic sequencing of conventional nested PCR amplicons was successful in 10/36 (27.8%) of positive samples.

Resumo O objetivo deste trabalho foi validar um protocolo de nested PCR em tempo real em um tubo (nPCR-TR-1T) seguida de sequenciamento genético para detectar e caracterizar as espécies e genótipos de Cryptosporidium em aves. Um total de 443 amostras de DNA genômico, extraído de amostras fecais de aves, foi analisado pela nPCR-TR-1T e pela nested PCR convencional. Pela nPCR-TR-1T, foi observada positividade para Cryptosporidium spp. de 20,3% (90/443), em contraste com a nested PCR convencional, que apresentou positividade de 8,1% (36/443). O teste de sensibilidade analítica mostrou que a nPCR-TR-1T detecta aproximadamente 0,5 oocisto (2 esporozoítos) por reação. A avaliação da especificidade analítica não revelou amplificação de microrganismos que comumente apresentam amplificação inespecífica com primers utilizados para o diagnóstico de Cryptosporidium spp. O cálculo da repetibilidade evidenciou o mesmo resultado em 27 de 30 amostras (90%). Em relação à reprodutibilidade da nPCR-TR-1T, foi observado o mesmo resultado em 80% (24/30) das amostras examinadas. Foi possível realizar o sequenciamento em todas as 90 amostras amplificadas pela nPCR-TR-1T, com identificação de C. baileyi, C. galli, C. meleagridis, C. proventriculi e Cryptosporidium genótipo I em aves. O sequenciamento dos fragmentos amplificados pela nested PCR convencional foi possível em 10/36 (27,8%) das amostras positivas.

Avaliação da infecção por Parvovírus Humano B19 em diferentes grupos populacionais: diagnóstico diferencial e aspectos imunopatogênicos da infecção aguda e persistente / Evaluation of Human Parvovirus B19 infection in different population groups: differential diagnosis and immunopathogenic aspects of acute and persistent infection

A infecção pelo Parvovírus B19 (B19V) pode ocorrer em indivíduos imunocompetentes e imunocomprometidos, de todas as faixas etárias, e se caracteriza por ser aguda e autolimitada, podendo levar a quadros de doença exantemática (DE), doença febril aguda (DFA), doença renal crônica (DRC) e falência hepática aguda (FHA). O diagnóstico diferencial de B19V nessas populações, muitas vezes, não ocorre e estudos sobre a prevalência do B19V são antigos e escassos, não refletindo a atualidade. Marcadores da infecção podem ser detectados na circulação e em diferentes tipos de tecidos, inclusive em tecidos não eritroides, por meses ou anos. A infecção pode levar a manifestações clínicas graves, que requer tratamento hospitalar, e a doenças inflamatórias atípicas, como: cardiomiopatia, artrite reumatoide, hepatite e vasculite. No entanto, a detecção de B19V DNA não implica necessariamente na presença de vírions infecciosos e na associação do B19V com essas manifestações atípicas. Dessa forma, o objetivo do trabalho foi otimizar técnicas de PCR em tempo real para quantificação do B19V DNA e de detecção de partículas virais infecciosas, a fim de realizar o diagnóstico diferencial da infecção pelo B19V em pacientes com DE, DFA, DRC e FHA. Para o diagnóstico da infecção, amostras de diferentes populações foram testadas: DE (n=54), DFA (n=60), DRC (n=221), e FHA (n=30). Amostras de soro (e de tecido hepático para FHA) foram submetidas a avaliação de marcadores sorológicos (IgM e IgG anti-B19V) e moleculares do B19V, a fim de determinar a fase da infecção em que o paciente se encontrava. Para a avaliação de marcadores moleculares, a metodologia de PCR quantitativo e em tempo real foi otimizada e permitiu um diagnóstico sensível e específico do B19V DNA. Além disso, a presença de vírions em amostras de pacientes com B19V (n=10) e de macacos cynomolgus (n=4) infectados experimentalmente foram avaliadas por meio da técnica de pré-tratamento das amostras com uma enzima endonuclease. O teste molecular (qPCR) otimizado durante o estudo, apresentou sensibilidade e especificidade de 100%. O ensaio com a endonuclease revelou que a maioria das amostras de soro humano tornou-se B19V DNA negativa após o pré-tratamento, indicando que não eram infecciosas. Foi observado prevalências do B19V DNA em 5,5% dos pacientes com DE; 6,6% em DFA; 65,6% em DRC, e 23,3% em FHA. Como conclusão a técnica de qPCR otimizada no presente estudo foi efetiva para o esclarecimento de casos da infecção por B19V e é adequada para diagnóstico diferencial. Além disso, o teste laboratorial baseado em endonuclease possibilitou a discriminação do B19V DNA (se encapsidado em vírions ou não). Portanto, estes testes podem ser utilizados para esclarecer o papel do B19V como agente etiológico associado a diversas manifestações clínicas. As prevalências encontradas nesse estudo indicam que o B19V está circulando entre os diversos grupos populacionais estudados e deve ser feita uma melhor vigilância da infecção, pois está presente tanto em indivíduos imunocompetentes como em imunocomprometidos. Além disso, os resultados sugerem a importância da inclusão de B19V no diagnóstico laboratorial diferencial, não apenas para fins epidemiológicos, mas também para o manejo adequado do paciente.

Parvovirus B19 (B19V) infection can occur in immunocompetent and immunocompromised individuals of all group ages and is characterized as acute and self limiting, which can lead to rash disease (RD), acute febrile illness (AFI), chronic kidney disease (CKD), and acute liver failure (ALF). Differential diagnosis of B19V in these populations often does not occur and studies on the prevalence of B19V are scarce, outdated, and do not reflect the current situation. B19V markers of acute infection can be detected in the circulation and in different tissue types, including non-erythroid tissues, for months to years and may lead to severe clinical manifestations, requiring hospital treatment, and to atypical inflammatory diseases, such as cardiomyopathy, rheumatoid arthritis, hepatitis, and vasculitis. However, the detection of B19V DNA does not necessarily imply the presence of infectious virions and the causal relation between B19V and atypical manifestations could not be proved yet. Thus, the aim of this study was to standardize the real-time PCR for quantification of B19V DNA and detection of infectious viral particles in order to perform the differential diagnosis of the B19V infection in RD, AFI, CKD, and ALF patients. For the diagnosis of the infection, samples from different populations were tested: RD (n=54), AFI (n=60), CKD (n=221), and ALF (n=30). Serum samples (and hepatic tissue for ALF) were submitted to the evaluation of B19V serological status (anti-B19V IgM and IgG antibodies) and molecular markers, in order to determine the stage of infection in which the patient is. For the evaluation of molecular markers, a quantitative real-time PCR methodology was optimized and allowed a sensitive and specific diagnosis of B19V DNA. In addition, the presence of virions in samples from patients with B19V (n=10) and from cynomolgus monkeys (n=4) experimentally infected were evaluated by endonuclease enzyme pretreatment. The molecular test optimized during the study showed 100% sensitivity and specificity. The endonuclease treatment assay revealed that most human serum samples became negative after pretreatment, as indicative of non-infective particles. Concerning the prevalence of B19V DNA: 5.5% were obtained in patients with RD; 6.6% in AFI; 65.6% in CKD, and 23.3% in ALF. In conclusion, the qPCR technique optimized in the present study was effective for clarifying cases of B19V infection and is suitable for differential diagnosis. In addition, the endonuclease-based laboratory test made it possible to discriminate B19V DNA (whether encapsidated in virions or not). Therefore, these tests can be used to clarify the role of B19V as an etiologic agent associated with several clinical manifestations. The prevalence found in this study indicate that B19V is circulating among the different populational groups that have been studied and better surveillance of the infection should be carried out, as it is present in both immunocompetent and immunocompromised individuals. In addition, the results suggest the importance of including B19V in the differential laboratory diagnosis, not only for epidemiological purposes but also for the proper management of the patient.