TGFß signaling pathway in salivary gland tumors.

OBJECTIVE: Pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are the most prevalent salivary gland tumors. Their pathogenesis has been recently associated with complex molecular cascades, including the TGFß signaling pathway. The aim of this study was to evaluate the expression of genes associated with the TGFß signaling pathway (TGFB1, ITGB6, SMAD2, SMAD4, FBN1, LTBP1, and c-MYC) to map possible downstream alterations in the TGFß cascade. DESIGN: Thirteen PA, 17 MEC, 13 ACC, and 10 non-neoplastic salivary gland samples were analyzed by real-time RT-PCR. RESULTS: Cases of PA presented increased TGFB1, LTPB1, c-MYC, and FBN1 expressions, whereas SMAD2 expression was decreased when compared to non-neoplastic tissue. MEC patients displayed increased expressions of TGFB1, ITGB6, FBN1, and c-MYC and decreased expressions of SMAD2 and SMAD4. ACC cases exhibited elevated expressions of the investigated genes except TGFB1. The present results suggest that decreased expression of SMAD2 and SMAD4 does not impede the transcriptional regulation of c-MYC, especially in PA and MEC. Increased expressions of ITGB6, TGFB1, LTBP1, and FBN1 appear to be related to the regulation of the TGFß signaling pathway in these tumors. Additionally, we observed a higher expression of SMAD4 in ACC and a raised expression of ITGB6 and lowered expression of SMAD2 in MEC. CONCLUSIONS: Our study demonstrated the differential expression of TGFß cascade members in salivary gland tumors such as SMAD2/SMAD4 and c-MYC as well as the participation of ITGB6, TGFB1, LTBP1, and FBN1, contributing to the understanding of the mechanisms involved in tumor progression.

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.

High-Throughput Sequencing of Grapevine in Mexico Reveals a High Incidence of Viruses including a New Member of the Genus Enamovirus.

This is the first viral metagenomic analysis of grapevine conducted in Mexico. During the summer of 2021, 48 plants displaying virus-like symptoms were sampled in Queretaro, an important grapevine-producing area of Mexico, and analyzed for the presence of viruses via high-throughput sequencing (HTS). The results of HTS were verified by real-time RT-PCR following a standardized testing scheme (Protocol 2010). Fourteen different viruses were identified, including grapevine asteroid mosaic-associated virus (GAMaV), grapevine Cabernet Sauvignon reovirus (GCSV), grapevine fanleaf virus (GFLV), grapevine fleck virus (GFkV), grapevine Pinot gris virus (GPGV), grapevine red globe virus (GRGV), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus 1 (GSyV-1), grapevine virus B (GVB), and grapevine leafroll-associated viruses 1, 2, 3, 4 (GLRaV1, 2, 3, 4). Additionally, divergent variants of GLRaV4 and GFkV, and a novel Enamovirus-like virus were discovered. This is the first report of GAMaV, GCSV, GLRaV4, GPGV, GRGV, GRVFV, and GSyV-1 infecting grapevines in Mexico; the impact of these pathogens on production is unknown.

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

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 acceptable for their use in adverse contexts, decentralization, and different epidemiological scenarios, for rapid and accurate SARS-CoV-2 detection.

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.

Positivity of dengue, chikungunya, and Zika infections in women in Northeast Brazil post-Zika epidemic.

The state of Ceará, in the Northeast Region of Brazil, presents the simultaneous circulation of Zika (ZIKV), dengue (DENV) and chikungunya (CHIKV) viruses. In 2017 there were a high number of cases of these three arboviruses, especially CHIKV. Here, we detected the presence of arboviruses ZIKV, DENV and CHIKV and their coinfections in women in endemic regions of the city of Fortaleza, Ceará in a post-Zika epidemic year. Sociodemographic and environmental characteristics associated with arbovirus positivity were also analyzed. Women (n = 1289) between 15 and 39 years old were included. RT-qPCR was performed for virus detection and IgM antibody positivity was also analyzed. One hundred and six (8.3%) participants were positive for one or more arboviruses. Monoinfections (76; 5.9%) were distributed between 22 (1.7%) for ZIKV, 39 (3.1%) for DENV and 15 (1.2%) for CHIKV. Co-infections were detected in 30 (2.3%) of the positive participants and one case with triple infection was found. IgM positivity was found in 2.4% of ZIKV RT-qPCR, 9.6% of DENV and 16.3% of CHIKV. RT-qPCR positivity for arboviruses was associated with low socioeconomic class and presence of a water box sealing in the household. A higher positivity to the three viruses occurred in the month with the lowest wind velocity, which was also preceded by the highest peak of rain and humidity. We identified the simultaneous circulation and co-infection of ZIKV, DENV and CHIKV in Fortaleza in a post-Zika epidemic year. We also highlight the need for continuous epidemiological surveillance combined with molecular diagnostic tools.

Diagnóstico molecular del SARS-CoV-2 en los Laboratorios LIORAD-AICA+: experiencia de un año de trabajo / Molecular diagnosis of SARS-CoV-2 at LIORAD-AICA+ laboratories, a one-year work experience

Introducción: El aumento de los casos de COVID-19 en Cuba requirió el desarrollo de nuevas capacidades para el diagnóstico molecular de la infección. En la Unidad Empresarial de Base Laboratorios LIORAD-AICA+, de La Habana, se estableció un Laboratorio de Biología Molecular para el diagnóstico molecular de la enfermedad. Objetivo: Analizar la experiencia de un año de trabajo, en el diagnóstico molecular de la COVID-19, del Laboratorio de Biología Molecular de la UEB LIORAD. Métodos: Para iniciar el diagnóstico molecular del SARS-CoV-2 en la UEB Laboratorios LIORAD se llevó a cabo un conjunto de acciones que estuvieron dirigidas a la evaluación de los riesgos, establecimiento de las áreas y el flujo de trabajo, y formación de equipos de trabajo. El personal se capacitó, se modificaron y elaboraron procedimientos e instructivas. Resultados: La evaluación de los riesgos permitió detectar un conjunto de riesgos asociados a la actividad de diagnóstico y se establecieron las medidas para mitigarlos. El personal del laboratorio recibió un total de 23 capacitaciones, se elaboró un total de ocho procedimientos e instructivas y dos registros. El laboratorio procesó en un año un total de 125 154 muestras. Conclusiones: Durante el año de trabajo el Laboratorio de Biología Molecular de la UEB LIORAD se realizó el diagnóstico certero de la enfermedad. Esto evidencia la importancia de la capacitación del personal y el cumplimiento de las buenas prácticas y medidas de bioseguridad en el trabajo con muestras potencialmente infecciosas(AU)

Introduction: The increase in the number of cases of COVID-19 in Cuba demanded of new capacities for the molecular diagnosis of the infection. A Laboratory of Molecular Biology for the molecular diagnosis of this disease was installed at the Base Business Unit LIORAD-AICA+ Laboratories in Havana. Objective: To analyze a one-year work experience in the molecular diagnosis of COVID-19 at the Laboratory of Molecular Biology, LIORAD-AICA+. Methods: To begin with the molecular diagnosis of SARS-CoV-2 at LIORAD-AICA+, a group of actions were carried out aimed at evaluating the risks, establishing the working areas and flow, and training the work team. Personnel were trained, and procedures and guidelines were drawn up and modified. Results: Risk assessment allowed identifying several risks associated with the diagnostic activity, and measures were established to mitigate them. The laboratory personnel received 23 training sessions; and eight procedures and guidelines, and two registers were drawn up. The laboratory processed a total of 125 154 samples in a year. Conclusions: During the work year, the accurate diagnosis of the disease was conducted at the Laboratory of Molecular Biology, LIORAD-AICA+. This evidences the importance of personnel training and the compliance with good practices and biosafety measures when working with potentially infectious samples(AU)

Tracking SARS-CoV-2 Variants Using a Rapid Typification Strategy: A Key Tool for Early Detection and Spread Investigation of Omicron in Argentina.

SARS-CoV-2 variants of concern (VOC) and interest (VOI) present mutations in reference to the original virus, being more transmissible. We implemented a rapid strategy for the screening of SARS-CoV-2 VOC/VOIs using real time RT-PCR and performed monitoring and surveillance of the variants in our region. Consecutive real-time RT-PCRs for detection of the relevant mutations/deletions present in the Spike protein in VOC/VOIs (TaqMan™ SARS-CoV-2 Mutation Panel, Applied Biosystems) were implemented. A total of 6,640 SARS-CoV-2 RNA samples (Cts < 30) from infected individuals in Central Argentina during 2021 were analyzed using different algorithms that were gradually adapted to the changing scenarios of local variant circulation. The strategy developed allowed the early detection and the identification of VOC/VOIs that circulated through the year, with a 100% of concordance with the WGS. The analyses of the samples showed introductions of VOCs Alpha and Gamma in February and March 2021, respectively. Gamma showed an exponential increase, with a peak of detection in July (72%), being responsible of the second wave of COVID19 in Argentina. Since VOC Delta entered into the region, it increased gradually, together with VOI Lambda, replacing VOC Gamma, until being the main variant (84.9%) on November. By December, these variants were replaced by the emergent VOC Omicron in a term of 2 weeks, producing the third wave. We report a useful tool for VOC/VOI detection, capable to quickly and cost-effectively monitor currently recognized variants in resource-limited settings, which allowed to track the recent expansion of Omicron in our region, and contributed to the implementation of public health measures to control the disease spread.

SARS-CoV-2 Variants in Paraguay: Detection and Surveillance with an Economical and Scalable Molecular Protocol.

SARS-CoV-2 variant detection relies on resource-intensive whole-genome sequencing methods. We sought to develop a scalable protocol for variant detection and surveillance in Paraguay, pairing rRT-PCR for spike mutations with Nanopore sequencing. A total of 201 acute-phase nasopharyngeal samples were included. Samples were positive for the SARS-CoV-2 N2 target and tested with the Spike SNP assay to detect mutations associated with the following variants: alpha (501Y), beta/gamma (417variant/484K/501Y), delta (452R/478K), and lambda (452Q/490S). Spike SNP calls were confirmed using amplicon (Sanger) sequencing and whole-genome (Nanopore) sequencing on a subset of samples with confirmed variant lineages. Samples had a mean N2 Ct of 20.8 (SD 5.6); 198/201 samples (98.5%) tested positive in the Spike SNP assay. The most common genotype was 417variant/484K/501Y, detected in 102/198 samples (51.5%), which was consistent with the P.1 lineage (gamma variant) in Paraguay. No mutations (K417 only) were found in 64/198 (32.3%), and K417/484K was identified in 22/198 (11.1%), consistent with P.2 (zeta). Seven samples (3.5%) tested positive for 452R without 478K, and one sample with genotype K417/501Y was confirmed as B.1.1.7 (alpha). The results were confirmed using Sanger sequencing in 181/181 samples, and variant calls were consistent with Nanopore sequencing in 29/29 samples. The Spike SNP assay could improve population-level surveillance for mutations associated with SARS-CoV-2 variants and inform the judicious use of sequencing resources.

SARS-COV-2 detection in saliva and nasopharyngeal swabs using RT-PCR was similar

Abstract The World Health Organization has declared the widespread spread of SARS-CoV-2 and its associated disease (COVID-19) a public health emergency. The standard gold test for detecting the virus is the RT-PCR, performed from nasopharyngeal swab (NPS) samples. However, this test may be uncomfortable for the patient and requires specific training and attire from the health professional responsible for collecting the sample. Therefore, the search for alternative ways to collect samples that may be used in the diagnosis of COVID-19 is relevant. This study aimed to compare the results obtained from NPS and saliva samples. NPS and saliva samples were collected from 189 symptomatic outpatients suspected of COVID-19, who came to Piquet Carneiro Polyclinic. RNA extraction was performed using the Bio-Gene DNA/RNA Viral Extraction kit (Bioclin®). Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) reactions used the Molecular SARS-CoV-2 (E / RP) kit (Bio-Manguinhos). The results indicated that 142 showed a non-detectable result (ND), while 47 showed a detectable result (D). Among the 142 "ND", 137 (94.4%) saliva samples obtained the same result, while 5 samples (3.4%) were "D". Among the 47 "D" swab samples, 35 (74.4%) showed the same result in the saliva samples. The sensitivity of the saliva test was 0.74 and the specificity was 0.97. The positive predictive value was 0.88 while the negative predictive value was 0.92. The results showed that detection of Sars-CoV-2 using saliva samples showed high sensitivity and specificity compared to nasopharyngeal swabs.

Resumo A Organização Mundial da Saúde declarou a disseminação generalizada do SARS-CoV-2 e sua doença associada (COVID-19) uma emergência de saúde pública. O teste padrão ouro para detecção do vírus é o RT-PCR, realizado a partir de amostras de swab nasofaríngeo (NPS). No entanto, esse exame pode ser desconfortável para o paciente e requer treinamento específico e vestimenta do profissional de saúde responsável pela coleta da amostra. Portanto, a busca por formas alternativas de coleta de amostras que possam ser utilizadas no diagnóstico de COVID-19 é relevante. O objetivo deste estudo foi comparar os resultados obtidos em amostras de NPS e saliva. Amostras de NPS e saliva foram coletadas de 189 pacientes ambulatoriais sintomáticos com suspeita de COVID-19, que procuraram a Policlínica Piquet Carneiro. A extração de RNA foi realizada com o kit Bio-Gene DNA / RNA Viral Extraction (Bioclin®) e as reações em tempo real da reação em cadeia da polimerase-transcriptase reversa (RT-PCR) usaram o kit Molecular SARS-CoV-2 (E / RP) (Bio-Manguinhos). Os resultados indicaram que 142 apresentaram resultado não detectável (ND), enquanto 47 apresentaram resultado detectável (D). Entre os 142 "ND", 137 (94,4%) amostras de saliva obtiveram o mesmo resultado, enquanto 5 amostras (3,4%) foram "D". Dentre as 47 amostras de swab "D", 35 (74,4%) apresentaram o mesmo resultado nas amostras de saliva. A sensibilidade do teste de saliva foi de 0,74 e a especificidade foi de 0,97. O valor preditivo positivo foi de 0,88, enquanto o valor preditivo negativo foi de 0,92. Os resultados mostraram que a detecção de Sars-CoV-2 em amostras de saliva apresentou alta sensibilidade e especificidade quando comparada com swabs nasofaríngeos.

Evaluation of the Efficiency of Commercial Vaccines Against Infectious Bronchitis Virus (IBV) Belonging to the GI-16 Lineage Isolated in an Argentinean Outbreak.

In this study we evaluated the effectiveness of adding serotype 793B vaccine to an immunization program in order to control the infectious bronchitis virus (IBV) GI-16 lineage. Therefore, two different experiments were performed. First, a virus cross-neutralization test was carried out, which indicated that neither the Massachusetts (Mass) nor 793B serotypes are antigenically related to the field isolate A13 (GI-16). We also performed a challenge trial to evaluate if the Mass/793B combination is more efficient than Mass/Connecticut (Conn) to protect chickens against the Argentinian variant A13. Thus, 40 chickens were organized in four groups. Chickens in Group A were vaccinated at 1 day of age with Mass serotype and then at 14 days old with Mass plus Conn serotypes. Chickens in Group B received Mass and 793B serotypes at 1 and 14 days old, respectively. Groups C and D remained unvaccinated. At 28 days of age, Groups A, B, and C were challenged with the A13 isolate, while Group D remained as the negative control. The statistical analysis of the ciliostasis evaluation, performed at 7 days postchallenge (dpch), indicated that the difference between Mass/793B and Mass/Conn was not significant (p > 0.05). However, the comparison against the negative control showed that only Group A was significantly different, suggesting a slightly better performance on blocking ciliostasis for the Mass/793B combination. On the other hand, no significant differences were observed in the viral load, quantified by reverse-transcription quantitative real-time PCR (RT-qPCR) in tracheal swabs and kidneys (at 3 and 7 dpch, respectively) between vaccinated groups. Furthermore, some amounts of the viral genome were found in both vaccinated groups that could indicate that neither the Mass/793B nor the Mass/Conn combinations totally inhibited the viral replication. Such viral replication in vaccinated chickens should seriously be taken into consideration because it could promote the selection of new variants in the future.

Nota de investigación­Evaluación de la eficacia de vacunas comerciales contra el virus de la bronquitis infecciosa (IBV) perteneciente al linaje GI-16 aislado durante un brote argentino. En este estudio se evaluó la efectividad de agregar la vacuna del serotipo 793B a un programa de inmunización para controlar al virus de la bronquitis infecciosa (con las siglas en inglés IBV) linaje GI-16. Por tanto, se realizaron dos experimentos diferentes. Primeramente, se llevó a cabo una prueba de neutralización cruzada de virus, que indicó que ni los serotipos Massachusetts (Mass) ni 793B están antigénicamente relacionados con el aislado de campo A13 (GI-16). También se realizó una prueba de desafío para evaluar si la combinación Massachussets/793B era más eficiente que Massachussets/Connecticut (Conn) para proteger a los pollos contra la variante argentina A13. De esta forma, 40 pollos se organizaron en cuatro grupos. Los pollos del Grupo A se vacunaron al día de edad con el serotipo Massachussets y luego a los 14 días con los serotipos Massachussets más Connecticut. Los pollos del Grupo B recibieron los serotipos Massachussets y 793B a los 1 y 14 días de edad, respectivamente. Los grupos C y D permanecieron sin vacunar. A los 28 días de edad, los Grupos A, B y C fueron desafiados con el aislado A13, mientras que el Grupo D permaneció como control negativo. El análisis estadístico de la evaluación de la ciliostasis, realizada a los 7 días después del desafío (dpch), indicó que la diferencia entre el tratamiento Massachussets/793B y Massachussets/Connecticut no fue significativa (P> 0.05). Sin embargo, la comparación con el control negativo mostró que solo el Grupo A fue significativamente diferente, lo que sugiere un desempeño ligeramente mejor en el bloqueo de la ciliostasis para la combinación Massachussets/793B. Por otro lado, no se observaron diferencias significativas (P> 0.05) en la carga viral, cuantificada mediante transcripción reversa y PCR cuantitativa en tiempo real de hisopos traqueales y riñones (a 3 y 7 días después del desafío, respectivamente) entre los grupos vacunados. Además, se encontraron algunas cantidades del genoma viral en ambos grupos vacunados que podrían indicar que ni las combinaciones Massachussets/793B ni Massachussets/Connecticut inhibieron totalmente la replicación viral. Esta replicación viral en pollos vacunados debe tenerse muy en cuenta porque podría promover la selección de nuevas variantes en el futuro.

Alternative RNA extraction-free techniques for the real-time RT-PCR detection of SARS-CoV-2 in nasopharyngeal swab and sputum samples.

Standard diagnoses of SARS-CoV-2 infections are done by RNA extraction and real-time RT-PCR (rRT-PCR). However, the need for RNA extraction complicates testing due to increased processing time, high cost, and limited availability of commercial kits. Therefore, alternative methods for rRT-PCR detection of SARS-CoV-2 without RNA extraction were investigated. Nasopharyngeal and sputum samples were used to compare the sensitivity of three techniques: Trizol RNA extraction, thermal shock, and the direct use of samples with an RNase inhibitor. Direct, extraction-free use of primary samples plus the RNase inhibitor produced diagnostic values of 100 % sensitivity and specificity compared to standard protocols, and these findings were validated in a second, independent laboratory.

PREX2 gene's expression in gastric antral epithelial cells of patients with H. pylori infection / Expressão do gene PREX2 em células epiteliais antrais gástricas em pacientes com infecção por H. pylori

ABSTRACT BACKGROUND: The Prex2 protein is a member of the Rac family proteins that belongs to small G proteins with a critical role in cell migration, cell proliferation, and apoptosis through its effects on PI3K cell signaling pathway and phosphatase activity of PTEN protein. The effect of PREX2 gene expression has been shown in some cancer cells. A survey of PREX2 gene expression in gastric antral epithelial cells of gastric cancer patients with Helicobacter pylori various genotypes infection can conduct to better understanding H. pylori infection's carcinogenesis. METHODS: In a case-control study, PREX2 gene expression was evaluated in gastric antral biopsy samples on four groups of patients referred to Sanandaj hospitals, including gastritis with (n=23) and without (n=27) H. pylori infection and gastric cancer with (n=21) and without (n=32) H. pylori infection. Each gastric biopsy sample's total RNA was extracted and cDNA synthesized by using Kits (Takara Company). The PREX2 gene expression was measured using the relative quantitative real-time RT-PCR method and ΔΔCt formula. RESULTS: The PREX2 gene expression increased in gastric antral biopsy samples of gastritis and gastric cancer patients with H. pylori infection (case groups) than patients without H. pylori infection (control groups) 2.38 and 2.27 times, respectively. The patients with H. pylori vacA s1m1 and sabB genotypes infection showed a significant increase of PREX2 gene expression in gastric cancer antral epithelial cells. CONCLUSION: H. pylori vacA s1m1 and sabB genotypes have the positive correlations with PREX2 gene expression in gastric antral epithelial cells of gastritis and gastric cancer patients.

RESUMO CONTEXTO: A proteína Prex2 é membro das proteínas da família Rac que pertencem a pequenas proteínas G com um papel crítico na migração celular, na proliferação celular e na apoptose através de seus efeitos na via de sinalização celular PI3K e atividade fosfatase da proteína PTEN. O efeito da expressão genética PREX2 tem sido mostrada em algumas células cancerosas. Um levantamento da expressão genética PREX2 em células epiteliais antrais gástricas de pacientes infectados com vários genótipos de Helicobacter pylori pode conduzir a um melhor entendimento da carcinogênese da infecção por H. pylori. MÉTODOS: Em estudo de caso-controle, a expressão genética PREX2 foi avaliada em amostras de biópsia antral gástrica em quatro grupos de pacientes encaminhados aos hospitais de Sanandaj, incluindo gastrite com (n=23) e sem (n=27) infecção por H. pylori e de câncer gástrico com (n=21) e sem (n=32) infecção por H. pylori. O RNA total de cada amostra de biópsia gástrica foi extraído e cDNA sintetizado por meio de kits (Takara Company). A expressão genética PREX2 foi medida utilizando-se o método RT-PCR em tempo real quantitativo relativo e a fórmula ΔΔCt. RESULTADOS: A expressão genética PREX2 aumentou em amostras de biópsia antral gástrica de pacientes com gastrite e câncer gástrico com infecção por H. pylori (grupos de casos) em relação aos sem infecção por H. pylori (grupos de controle) 2,38 e 2,27 vezes, respectivamente. Os pacientes com infecção por genótipos H. pylori vacA s1m1 e sabB apresentaram um aumento significativo da expressão genética PREX2 em células epiteliais antrais de câncer gástrico. CONCLUSÃO: Os genótipos H. pylori vacA s1m1 e sabB têm correlações positivas com a expressão genética PREX2 em células epiteliais antrais gástricas de pacientes com câncer gástrico e gastrites.

Analytical and clinical performance of molecular assay used by the Brazilian public laboratory network to detect and discriminate Zika, Dengue and Chikungunya viruses in blood.

In response to the Zika epidemics in Brazil, the ZDC molecular assay (Bio-Manguinhos) was developed and registered at the Brazilian Regulatory Agency of Health Surveillance - ANVISA. The circulation of Zika (ZIKV) Dengue (DENV) and Chikungunya (CHIKV) viruses and their clinical similarities are challenges to correctly diagnose these viruses. The simultaneous detection of ZIKV, DENV and CHIKV is an important tool for diagnosis and surveillance. Here, we present the analytical and clinical performance evaluation of ZDC molecular assay (Bio-Manguinhos) at the public health laboratories three years after its registration at ANVISA. The clinical performance demonstrates the ZDC molecular assay (Bio-Manguinhos) has 100% sensitivity and 100% specificity to detect and discriminate ZIKV, CHIKV, and DENV from clinical plasma samples. The ZDC molecular assay (Bio-Manguinhos) results were highly reproducible and no cross-reactivity was seen during testing with a panel of other infectious agents. In conclusion, the ZDC molecular assay (Bio-Manguinhos) is an accurate and reliable tool to monitor Zika, dengue and chikungunya infections in countries like Brazil with simultaneous circulation of the three viruses.

Cetacean morbillivirus in Humpback whales' exhaled breath.

The humpback whale (HW; Megaptera novaeangliae) population that seasonally resides along the Brazilian coast concentrates in the Abrolhos Bank (Bahia and Espírito Santo states) for breeding during austral winter and spring. Cetacean morbillivirus (CeMV, Paramyxoviridae family) is currently one of the most significant biological threats to cetaceans worldwide with high infection and mortality rates. CeMV is pleiotropic yet it has special tropism for the respiratory, lymphoid and nervous system and is primarily transmitted by the aerogenous route. A new lineage of CeMV, the Guiana dolphin morbillivirus (GDMV), is known to affect cetaceans off Brazil. GDMV was first detected in a Guiana dolphin (Sotalia guianensis) stranded in the Abrolhos Bank region, in 2010. In addition to pathologic examinations on stranded HW, pathogen survey of free-ranging HW may provide valuable insight into the epidemiology of diseases. We hypothesized that HW in the Brazilian breeding ground could be exposed to CeMV. Thus, in the present study, we investigated the presence of CeMV in exhaled breath condensates (EBC) of HW in the Abrolhos Bank. Overall, 73 samples of EBC from 48 groups of HW were collected during the breeding seasons of 2011 (n = 16) and 2012 (n = 57). One sample failed to have the reference gene amplified and was excluded from the study. CeMV was detected by a RT-qPCR method in 2 EBC samples, representing 2 whale groups. Phylogenetic analysis of partial morbillivirus phosphoprotein gene showed 100% homology to GDMV. Our results show that HW in Brazil are infected by CeMV with a relative prevalence of 4.3% (2/47) and demonstrate the suitability of using EBC and RT-qPCR as a non-invasive tool for CeMV survey in free-ranging whales. This pioneer study provides scientific basis for non-invasive CeMV monitoring of HW, suggests HW may play a role in the dynamics of CeMV and raises concern for potential conservation implications for this species.

Analytical and clinical performance of molecular assay used by the Brazilian public laboratory network to detect and discriminate Zika, Dengue and Chikungunya viruses in blood

ABSTRACT In response to the Zika epidemics in Brazil, the ZDC molecular assay (Bio-Manguinhos) was developed and registered at the Brazilian Regulatory Agency of Health Surveillance - ANVISA. The circulation of Zika (ZIKV) Dengue (DENV) and Chikungunya (CHIKV) viruses and their clinical similarities are challenges to correctly diagnose these viruses. The simultaneous detection of ZIKV, DENV and CHIKV is an important tool for diagnosis and surveillance. Here, we present the analytical and clinical performance evaluation of ZDC molecular assay (Bio-Manguinhos) at the public health laboratories three years after its registration at ANVISA. The clinical performance demonstrates the ZDC molecular assay (Bio-Manguinhos) has 100% sensitivity and 100% specificity to detect and discriminate ZIKV, CHIKV, and DENV from clinical plasma samples. The ZDC molecular assay (Bio-Manguinhos) results were highly reproducible and no cross-reactivity was seen during testing with a panel of other infectious agents. In conclusion, the ZDC molecular assay (Bio-Manguinhos) is an accurate and reliable tool to monitor Zika, dengue and chikungunya infections in countries like Brazil with simultaneous circulation of the three viruses.

Evaluation of Two Real-Time, TaqMan Reverse Transcription-PCR Assays for Detection of Rabies Virus in Circulating Variants from Argentina: Influence of Sequence Variation.

In rabies diagnosis, it is essential to count on a rapid test to give a quick response. The combined sensitivity and robustness of the TaqMan RT-PCR assays (qRT-PCR) have made these methods a valuable alternative for rabies virus (RABV) detection. We conducted a study to compare the applicability of two widely used qRT-PCR assays targeting the nucleoprotein gene (LysGT1 assay) and leader sequences (LN34 qRT-PCR assay) of RABV genomes, in all variants circulating in Argentina. A total of 44 samples obtained from bats, dogs, cattle, and horses, that were previously tested for rabies by FAT and conventional RT-PCR, were used in the study. All variants were successfully detected by the pan-lyssavirus LN34 qRT-PCR assay. The LysGT1 assay failed to detect three bat-related variants. We further sequenced the region targeted by LysGT1 and demonstrated that the presence of three or more mismatches with respect to the primers and probe sequences precludes viral detection. We conclude that the LysGT1 assay is prone to yield variant-dependent false-negative test results, and in consequence, the LN34 assay would ensure more effective detection of RABV in Argentina.

A novel real-time PCR to detect Cetacean morbillivirus in Atlantic cetaceans.

Cetacean morbillivirus (CeMV, family Paramyxoviridae) is a re-emergent pathogen associated with severe epizootic outbreaks causing high mortality among cetaceans worldwide. Recently, CeMV caused an unusual mortality event of Guiana dolphins (Sotalia guianensis) in Brazil. Partial sequence of the viral phosphoprotein (P) gene showed that the Guiana dolphin morbillivirus (GDMV) might represent a new lineage of CeMV. This study aimed to develop a molecular technique to detect the most common CeMV strains known to circulate in the Atlantic Ocean: GDMV, Dolphin morbillivirus (DMV) and Pilot-whale morbillivirus (PWMV). A sensible real-time reverse transcription polymerase chain reaction (RT-qPCR) method based on intercalating dye, targeting the P gene was described. This assay successfully detected GDMV, PWMV and DMV from field samples. Its performance was compared to a RT-qPCR method that specifically detects GDMV. Both assays had high sensibility and excellent intra- and inter-assay reproducibility. A total of 109 field samples from 32 Guiana dolphins were screened for CeMV by conventional RT-PCR in parallel with the RT-qPCR assay. The detection rate increased from 32% to 60% by use of the novel RT-qPCR. The RT-qPCR assay described herein allows rapid and sensitive detection of Atlantic CeMV strains, and is potentially suitable for screening of CeMV globally.

Real-time RT-PCR assay to detect Granada virus and the related Massilia and Arrabida phleboviruses.

BACKGROUND: Granada virus belongs to the genus Phlebovirus within the Naples serocomplex and was detected for the first time in sand flies from Spain in 2003. Seroprevalence studies have revealed that Granada virus may infect humans with most cases being asymptomatic. Moreover, recent studies in vector samples revealed that the related Massilia and Arrabida phleboviruses could be also circulating in Spain. The objective of this study was to develop and assess a new sensitive real-time RT-PCR assay for Granada virus diagnosis able to detect the related phleboviruses Massilia and Arrabida. METHODS: Two specific primers and one unique probe to detect Granada, Massilia and Arrabida viruses, without differentiating between them, were designed targeting the conserved L-segment of their genome. Sensitivity was assessed using 10-fold serial dilutions of quantified in vitro DNA samples. Specificity was evaluated by testing different genomic RNA extracted from other representative phleboviruses. The new assay was used for virus detection in sand flies collected in 2012 from the Balearic Archipelago, a touristic hotspot in the Mediterranean. RESULTS: The real-time RT-PCR assay exhibited a sensitivity per reaction of 19 copies for Granada and Arrabida, and 16 copies for Massilia. No other related phleboviruses were detected. From the 37 pools of sand fly samples studied from four different Balearic Islands, we detected one positive in the island of Cabrera. CONCLUSIONS: To our knowledge, the method described here is the first real-time RT-PCR designed to detect Granada virus and the related Massilia and Arrabida phleboviruses. The study demonstrated that this is a rapid, robust and reliable assay for the accurate diagnosis of human infections as well as for virus surveillance in vectors.

Molecular and biological characterization of the immunological potency of Newcastle disease virus oil emulsion-inactivated vaccines prepared from field isolate obtained from vaccinated chickens outbreak.

This study was conducted to characterize the immunological parameters of chickens vaccinated with two formulated inactivated vaccines, water in oil (WO) and water in oil in water (WOW), prepared from velogenic Newcastle disease virus (vNDV) genotype VIIj isolated from outbreak among vaccinated chickens. Six groups (G1-G6) of commercial broiler chickens were established (n = 20). The G1-G3 were received homologous (WO and WOW) and heterologous (LaSota) inactivated vaccines, respectively. The G4 was vaccinated with live heterologous (LaSota) vaccine, while G5 and G6 were kept as control positive and control negative non-vaccinated groups. The antibody titers were measured against vNDV and LaSota antigens using hemagglutination inhibition (HI) test, the cytokine gene expressions of IFNγ, IL1ß, IL4, IL6, IL8, and IL18 were quantified using real-time RT-PCR, and the virus shedding was titrated on chicken embryo fibroblast cells after challenging by vNDV. The classical clinical signs and 100% mortality were observed only in G5 after vNDV challenging. The highest HI titers were detected in G1, G2, and G3 using NDV/168 antigen with no significant differences among them. These groups showed higher HI titer than G4 (2-4log2). Cytokine gene expression of IFNγ, IL1, IL6, IL8, and IL18 were significantly downregulated in vaccinated chickens with upregulation of IL4 than non-vaccinated challenge group. Viral shedding titers were significantly (0.0001, p ≤ 0.001) reduced in all samples form vaccinated chickens. In conclusion, the prepared vaccines produced highly efficient immunological responses and could be used for controlling the NDV infection.