Simultaneous detection of infectious bronchitis virus and avian metapneumovirus genotypes A, B, and C by multiplex RT-qPCR assay in chicken tracheal samples in Ecuador.

Respiratory RNA viruses such as Infectious bronchitis virus (IBV) and Avian metapneumovirus (aMPV), which are characterized by generating both respiratory damage and adverse effects on reproductive organs, affect poultry production economically due to high mortality rate and decrease in egg production and quality. Particularly, aMPV has three genotypes that have been reported with greater frequency in chickens: aMPV-A, aMPV-B, and aMPV-C. The present study proposes the design of a multiplex RT-qPCR assay for the simultaneous diagnosis of the 3 genotypes of interest of aMPV and IBV, followed by testing of 200 tracheal samples of vaccinated chickens with respiratory symptoms and finally a phylogenetic analysis of the sequences found. The assay detected up to 1 copy of each viral genome. The standard curves showed an efficiency between 90 and 100% in the multiplex assay and inter- and intra-assay coefficients of variation of 0.363 and 0.459, respectively and inter- and intra-assay coefficients of variation of 0.363 and 0.459, respectively. 69.5% of samples were found positive alone or in coinfection. 114 samples were positive for IBV, 13 for aMPV-A and 25 for aMPV-B. RNA of aMPV-C was no detected. The most commonly found combination was aMPV-B and IBV within 6 samples, and the least common was aMPV-A and aMPV-B in coinfection in 2 samples. The assay was specific for amplification of the genomes of the studied respiratory viruses (IBV, aMPV-A, aMPV-B, aMPV-C) as no amplification was shown from other viral genomes (ChPV, CAstV, ANV, and FAdV) or from the negative controls. Partial genomic Sanger sequencing enabled to identify circulating vaccine-derived and wild-type strains of IBV and vaccine and vaccine-derived strains of aMPV-B. In conclusion, this newly developed multiplex RT-qPCR was shown to be able to detect individual infections as well as co-infections among the respiratory viruses investigated. It was demonstrated to be a reliable and efficient tool for rapidly and safely diagnosing these infections. Furthermore, this study represents the first report of aMPV strains in Ecuadorian poultry and demonstrates the circulation of aMPV-A, aMPV-B, and GI-13 IBV strains in unvaccinated chicken populations in the country. Thus, it highlights the importance of simultaneously identifying these pathogens in greater detail and on a regular basis in Ecuador.

Population-Specific Distribution of TPMT Deficiency Variants and Ancestry Proportions in Ecuadorian Ethnic Groups: Towards Personalized Medicine.

Purpose: Thiopurine S-methyltransferase (TPMT) is an enzyme that metabolizes purine analogs, agents used in the treatment of acute lymphoblastic leukemia. Improper drug metabolism leads to toxicity in chemotherapy patients and reduces treatment effectiveness. TPMT variants associated with reduced enzymatic activity vary across populations. Therefore, studying these variants in heterogeneous populations, such as Ecuadorians, can help identify molecular causes of deficiency for this enzyme. Methods: We sequenced the entire TPMT coding region in 550 Ecuadorian individuals from Afro-Ecuadorian, Indigenous, Mestizo, and Montubio ethnicities. Moreover, we conducted an ancestry analysis using 46 informative ancestry markers. Results: We identified 8 single nucleotide variants in the coding region of TPMT. The most prevalent alleles were TPMT*3A, TPMT*3B, and TPMT*3C, with frequencies of 0.055, 0.012, and 0.015, respectively. Additionally, we found rare alleles TPMT*4 and TPMT*8 with frequencies of 0.005 and 0.003. Correlating the ancestry proportions with TPMT-deficient genotypes, we observed that the Native American ancestry proportion influenced the distribution of the TPMT*1/TPMT*3A genotype (OR = 5.977, p = 0.002), while the contribution of African ancestral populations was associated with the TPMT*1/TPMT*3C genotype (OR = 9.769, p = 0.003). The rates of TPMT-deficient genotypes observed in Mestizo (f = 0.121) and Indigenous (f = 0.273) groups provide evidence for the influence of Native American ancestry and the prevalence of the TPMT*3A allele. In contrast, although Afro-Ecuadorian groups demonstrate similar deficiency rates (f = 0.160), the genetic factors involved are associated with contributions from African ancestral populations, specifically the prevalent TPMT*3C allele. Conclusion: The distribution of TPMT-deficient variants offers valuable insights into the populations under study, underscoring the necessity for genetic screening strategies to prevent thiopurine toxicity events among Latin American minority groups.

Fast, cheap and sensitive: Homogenizer-based RNA extraction free method for SARS-CoV-2 detection by RT-qPCR.

Background: The SARS-CoV-2 gold standard detection method is an RT-qPCR with a previous step of viral RNA extraction from the patient sample either by using commercial automatized or manual extraction kits. This RNA extraction step is expensive and time demanding. Objective: The aim of our study was to evaluate the clinical performance of a simple SARS-CoV-2 detection protocol based on a fast and intense sample homogenization followed by direct RT-qPCR. Results: 388 nasopharyngeal swabs were analyzed in this study. 222 of them tested positive for SARS-CoV-2 by the gold standard RNA extraction and RT-qPCR method, while 166 tested negative. 197 of those 222 positive samples were also positive for the homogenization protocol, yielding a sensitivity of 88.74% (95% IC; 83.83 - 92.58). 166 of those negative samples were also negative for the homogenization protocol, so the specificity obtained was 97% (95% IC; 93.11 - 99.01). For Ct values below 30, meaning a viral load of 103 copies/uL, only 4 SARS-CoV-2 positive samples failed for the RNA extraction free method; for that limit of detection, the homogenizer-based method had a sensitivity of 97.92% (95% CI; 96.01 - 99.83). Conclusions: Our results show that this fast and cheap homogenization method for the SARS-CoV-2 detection by RT-qPCR is a reliable alternative of high sensitivity for potentially infectious SARS-CoV-2 positive patients. This RNA extraction free protocol would help to reduce diagnosis time and cost, and to overcome the RNA extraction kits shortage experienced during COVID-19 pandemic.

Effect of thermal liquefaction on quality, chemical composition and antibiofilm activity against multiresistant human pathogens of crystallized eucalyptus honey.

Thermal liquefaction is a conventional method used by beekeepers to liquefy crystallized honey. However, an abusive use of heat may affect its quality, chemical composition and bioactivity. The purpose of this study was to investigate the effect of thermal liquefaction on the quality, chemical composition and antibiofilm properties of eucalyptus honey. Thermal liquefaction (at 45 and 60 °C) did not affect the honey's quality; however, a significant reduction in the reducing capacity, total phenolic content and hydrogen peroxide content was observed. At 60 °C, a significant reduction in the honey's ability to inhibit biofilm formation was observed in Pseudomonas aeruginosa, as well as a reduction in its ability to remove preformed biofilms in both Staphylococcus aureus and Pseudomonas aeruginosa. Structural changes in biofilm architecture caused by honey were not affected by thermal treatment. Therefore, we recommend liquefaction at 45 °C as the most convenient for honey liquefaction without affecting its characteristics.