Development of quantitative multiplex RT-qPCR one step assay for detection of hepatitis delta virus.

Hepatitis Delta is a disease caused by exposure to hepatitis B (HBV) and hepatitis D (HDV) viruses, usually with a more severe clinical outcome when compared to an HBV monoinfection. To date, the real prevalence of HDV infection is underestimated and detection methods are poorly available, especially in more endemic regions. Therefore, a one-step RT-qPCR method for quantification of HDV-RNA was developed. Biological samples were selected between 2017 and 2023 from patients at the Ambulatório Especializado em Hepatites Virais of the Centro de Pesquisa em Medicina Tropical de Rondônia and Serviço de Assistência Especializada and underwent the test developed by this study and a second quantitative RT-qPCR assay. The slope of the initial quantitative assay was - 3.321 with an efficiency of 100.04% and amplification factor equal to 2. Analysis of the repeatability data revealed a Limit of Quantification of 5 copies/reaction and Limit of Detection (95%) of 2.83 copies per reaction. In the diagnostic sensitivity tests, there was an accuracy of 97.37% when compared to the reference test. This assay proved to be highly efficient and reproducible, making it a valuable tool to monitor hepatitis Delta patients and assess the risk of disease progression, as well as the effectiveness of treatment.

Multiplex qPCR assay to determine Leishmania infantum load in Lutzomyia longipalpis sandfly samples.

The study aimed to develop a multiplex qPCR to detect Leishmania infantum load in different sandfly sample settings using Leishmania kDNA and sandfly vacuolar ATPase (VATP) subunit C as internal control gene. The amplification of Lutzomyia longipalpis VATP gene was evaluated together with Leishmania infantum kDNA in a multiplex reaction. The concentration of VATP gene oligonucleotides was adjusted until no statistically significant difference was observed between all multiplex standard curves and singleplex curves, that is, only kDNA amplification. Limit of detection (LoD) was measured using a probit model and a cut-off defined by receiver operating characteristic analysis. Limit of quantification (LoQ) was assessed by a linear model using the coefficient of variation threshold of 25%. After assuring VATP gene amplification, its primer-probe concentrations were best at 100 nM/10 nM, respectively. The cut-off Cq value for L. infantum kDNA was defined as 35.46 with 100% of sensitivity and specificity. A total of 95% LoD was determined to be of 0.162 parasites while LoQ was 5.858. Our VATP/kDNA multiplex qPCR assay shows that it can be used to evaluate both DNA integrity and determine L. infantum load in L. longipalpis even for low yielded samples, that is, individual midguts.

SARS-CoV-2 genomic surveillance in Rondônia, Brazilian Western Amazon.

SARS-CoV-2 has spread rapidly around the world, with Brazil currently considered an epicenter of the pandemic. The Northern region has the second highest incidence coefficient, as well as the third highest mortality rate in the country. This study aimed to investigate information about the evolutionary history of epidemic spread and genetic aspects of strains isolated on the Western Amazon, in the State of Rondônia, Brazil. It was possible to detect a total of 22 mutations. Some of these alterations may possibly be related to effects on transmissibility, the fidelity of RNA replication, the ability of cancer patients to respond to infection, beyond a mutation that emerged after the introduction of SARS-CoV-2 in Rondônia. At least two events of introduction were detected, corresponding to the B.1 and B.1.1 European lineages. An introduction was observed possibly through Argentina, where strains originated that circulated in the Minas Gerais and Ceará Brazilian states, prior to Rondônia (B.1.), as well as through the Minas Gerais state and the Federal District, which gave rise to strains that spread to Rondônia, from the capital to more rural parts of the state (B.1.1.). The findings show the need to monitor the genetic epidemiology of COVID-19, in order to surveil the virus's evolution, dispersion and diversity.

External Control Viral-Like Particle Construction for Detection of Emergent Arboviruses by Real-Time Reverse-Transcription PCR.

Arboviruses have been emerging and reemerging worldwide, predominantly in tropical and subtropical areas. As many arbovirus infections, including dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV), have similar signs and symptoms, clinical diagnosis of arbovirus infections is challenging. Therefore, reliable laboratory tests are necessary to improve the clinical management of patients with suspected arbovirus infections. Real-time reverse-transcription PCR (RT-qPCR) is among the more effective methods to distinguish these viruses. The aim of this study was to construct a unique positive external control derived from a unique plasmid using genetic engineering for specific use in RT-qPCR assays to detect Zika, dengue (1-4), and chikungunya. An external control derived from the MS2 bacteriophage was constructed using sequences from arbovirus and human genomes. Laboratories were asked to test the control in the ZDC Biomol kit, a RT-qPCR kit which is able to detect Zika, dengue serotypes 1-4, chikungunya, and an internal human control. RNA extracted from the external control was able to be amplified and detected in RT-qPCR assays for each virus detected by using the ZDC Biomol kit. The external control, samples from viral culture, and infected patient samples display similar amplification using this assay. The pET47b(+)MS2-ZDC vector is a viable expression system for the production of external control viral-like particles (MS2-ZDC). The RNA from the recombinant particles can be easily extracted and can function as a tool to validate all steps of process from the extraction to the amplification of all targets in specific reaction. Thus, the MS2-ZDC particles are laboratory-safe in order to avoid risk for operators, and the phages are effective as positive control for use in the ZDC Biomol kit amplifying all kit targets making them effective for commercial profile.

Surveillance of donated blood during the 2016 arbovirus outbreak in Brazil.

Reports of arboviral transmission via blood transfusion may be a cause of concern among asymptomatic infected donors. This study evaluated the presence of arboviruses in donated blood products during the 2016 outbreak in Vitória da Conquista (Bahia-Brazil). Serum samples (n = 676) were screened for ZIKV, CHIKV, and the four DENV serotypes using a one-step reverse transcriptase-based Real-Time Polymerase Chain Reaction (RT-PCR). No samples tested positive for any of the targets, whereas positive controls performed as expected. The results suggest a low risk of arboviral transmission via transfusion of blood products in the endemic area studied during the 2016 outbreak.