Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection.

The prompt and accurate identification of the etiological agents of viral respiratory infections is a critical measure in mitigating outbreaks. In this study, we developed and clinically evaluated a novel melting-curve-based multiplex real-time PCR (M-m-qPCR) assay targeting the RNA-dependent RNA polymerase (RdRp) and nucleocapsid phosphoprotein N of SARS-CoV-2, the Matrix protein 2 of the Influenza A virus, the RdRp domain of the L protein from the Human Respiratory Syncytial Virus, and the polyprotein from Rhinovirus B genes. The analytical performance of the M-m-qPCR underwent assessment using in silico analysis and a panel of reference and clinical strains, encompassing viral, bacterial, and fungal pathogens, exhibiting 100% specificity. Moreover, the assay showed a detection limit of 10 copies per reaction for all targeted pathogens using the positive controls. To validate its applicability, the assay was further tested in simulated nasal fluid spiked with the viruses mentioned above, followed by validation on nasopharyngeal swabs collected from 811 individuals. Among them, 13.4% (109/811) tested positive for SARS-CoV-2, and 1.1% (9/811) tested positive for Influenza A. Notably, these results showed 100% concordance with those obtained using a commercial kit. Therefore, the M-m-qPCR exhibits great potential for the routine screening of these respiratory viral pathogens.

Viral Coinfection of Children Hospitalized with Severe Acute Respiratory Infections during COVID-19 Pandemic.

The main pathogens of severe respiratory infection in children are respiratory viruses, and the current molecular technology allows for a rapid and simultaneous detection of a wide spectrum of these viral pathogens, facilitating the diagnosis and evaluation of viral coinfection. METHODS: This study was conducted between March 2020 and December 2021. All children admitted to the ICU with a diagnosis of SARI and who were tested by polymerase chain reaction on nasopharyngeal swabs for SARS-CoV-2 and other common respiratory viral pathogens were included in the study. RESULTS: The result of the viral panel identified 446 children, with one infected with a single virus and 160 co-infected with two or more viruses. This study employed descriptive analyses, where a total of twenty-two coinfections among SARI-causing viruses were identified. Thus, the five most frequent coinfections that were selected for the study are: hRV/SARS-CoV-2 (17.91%), hRV/RSV (14.18%), RSV/SARS-CoV-2 (12.69%), hRV/BoV (10.45%), and hRV/AdV (8.21%). The most significant age group was 38.1%, representing patients aged between 24 and 59 months (61 individuals). Patients older than 59 months represented a total of 27.5%, comprising forty-four patients. The use of oxygen therapy was statistically significant in coinfections with Bocavirus, other CoVs, Metapneumovirus, and RSV. Coinfections with SARS-CoV-2 and the other different coinfections presented a similar time of use of oxygen therapy with a value of (p > 0.05). In the year 2020, hRV/BoV was more frequent in relation to other types of coinfections, representing a total of 35.1%. The year 2021 presented a divergent profile, with hRV/SARS-CoV-2 coinfection being the most frequent (30.8%), followed by hRV/RSV (28.2%). Additionally, 25.6% and 15.4% represented coinfections between RSV/SARS-CoV-2 and hRV/AdV, respectively. We saw that two of the patients coinfected with hRV/SARS-CoV-2 died, representing 9.52% of all deaths in the study. In addition, both hRV/hBoV and hRV/RSV had death records for each case, representing 8.33% and 6.67% of all deaths, respectively. CONCLUSION: Coinfections with respiratory viruses, such as RSV and hBoV, can increase the severity of the disease in children with SARI who are admitted to the ICU, and children infected with SARS-CoV-2 have their clinical condition worsened when they have comorbidities.

Development of a melting-curve based multiplex real-time PCR assay for simultaneous detection of Streptococcus agalactiae and genes encoding resistance to macrolides and lincosamides.

BACKGROUND: Streptococcus agalactiae or Group B Streptococcus (GBS) remains the leading cause of infections in newborns worldwilde. Prenatal GBS screening of pregnant women for vaginal-rectal colonization is recommended in many countries to manage appropriate intrapartum antimicrobial prophylaxis for those identified as carriers. In this study, a novel melting-curve based multiplex real-time PCR assay for the simultaneous detection of GBS and macrolide and lincosamide resistance markers was developed. The usefulness of the assay was evaluated for rapid and accurate prenatal GBS screening. METHODS: One hundred two pregnant women who were at 35-37 weeks of gestation were enrolled in this study. The analytical performance of the multiplex real-time PCR was first tested using a panel of reference and clinical bacterial and fungal strains. To test the clinical performance, vaginal-rectal swabs were obtained from pregnant women who were seen at the teaching hospital for regular prenatal care. The results of real-time were compared with those obtained from microbiological analyses. RESULTS: The real-time PCR assay showed 100% specificity and a limit of detection of 104 colony forming units equivalent per reaction. The prevalence of GBS colonization among the population studied was 15.7% (16/102) based on a positive culture and the real-time PCR results. Agreement between the two assays was found for 11 (68.75%) GBS colonized women. Using the culture-based results as a reference, the multiplex real-time PCR had a sensitivity of 91.7% (11/12, CI 59.7-99.6%), a specificity of 95.5% (86/90, CI 89.8-98.7%), a positive predictive value of 73.3% (11/15, CI 44.8-91.1%) and a negative predictive value of 98.9% (86/87, CI 92.9-99.9%). CONCLUSION: The multiplex real-time PCR is a rapid, affordable and sensitive assay for direct detection of GBS in vaginal-rectal swabs.

Análise genômica funcional do Trypanosoma cruzi submetido a diferentes tipos de estresse / Functional genomic analysis of the Trypanosoma cruzi submitted the different types of stress

A diferenciação dos epimastigotas (formas replicativas e não-infectivas) em tripomastigotas metacíclicos (formas não-replicativa e infectivas) é o processo denominado metaciclogênese. Um evento essencial para a metaciclogênese, que pode ser mimetizado in vitro, é o estresse nutricional que os parasitas enfrentam nas porções posteriores do tubo digestivo do inseto vetor. Importantes mudanças no programa de expressão gênica ocorrem durante o estresse e desempenham um papel fundamental nas mudanças morfológicas e fisiológicas observadas durante a metaciclogênese. No entanto, poucas informações estão disponíveis em relação à capacidade e diferentes condições de estresse em disparar a metaciclogênese e, também, sobre os genes envolvidos na regulação dessas respostas. Para tanto, foi realizada uma análise comparativa das diferentes condições estresse (aumento temperatura, diminuição do pH e o estresse nutricional), em experimentos de metaciclogênese in vitro a fim de avaliar a capacidade de diferenciação do parasita diante de tais condições. Foi observado que todos os parasitas submetidos às condições de estresses são capazes de se diferenciar, entretanto, os parasitas provenientes dos estresses de pH e temperatura sofrem um retardo no disparo da metaciclogênese, porém esta diferença é minimizada até o final do processo. Para comparar as propriedades biológicas dos tripomastigotas metacíclicos originados de cada condição de estresse, foram feitos ensaios de infecção em células Vero e camundongos Swiss. Foi possível constatar que todos os ripomastigotas foram capazes de infectar as células Vero, assim como, infectar os camundongos. Para investigar a expressão dos genes foi utilizada a tecnologia de microarranjos. Foi selecionado um conjunto de 32 genes especificamente expressos nos tipos de estresse estudados. Para os genes selecionados foram realizadas análises quantitativas pela técnica de PCR em tempo real (RT-PCR) que validaram os dados de...epimastigotas.