Identification of SARS-CoV-2 in urban rodents from Southern Mexico City at the beginning of the COVID-19 pandemic.

Currently, there are some concerns about the situation and, in particular, about the future of the COVID-19 pandemic and the new emerging variants of SARS-CoV-2. Rodents are an example of synanthropic animals in urban environments that harbor important zoonoses. Although the molecular identification of SARS-CoV-2 in Rattus norvegicus from New York City had been reported, in other studies, urban wild rodents infected with this virus have not been found. This study aimed to molecularly identify the presence of SARS-CoV-2 in urban wild rodents from Mexico City, trapped along a water channel of a public park as part of a pest control program, at the beginning of the COVID-19 pandemic, during the fall and winter of 2020. Up to 33 Mus musculus and 52 R. norvegicus were captured and euthanized, large intestine samples with feces from the animals were obtained. RNAs were obtained and subjected to qRT-PCR for SARS-CoV-2 identification and threshold cycle (Ct) values were obtained. Four mice (12.1%) and three rats (5.8%) were positive, three rodents exhibited Ct<30. Our results on the frequency of SARS-CoV-2 in urban rats are in line with other previous reports. Thus, similar to other authors, we suggest that surveillance for the detection of SARS-CoV-2 in urban wild rodents, as sentinel animals, should be maintained.

Identification of SARS-CoV-2 in urban rodents from Southern Mexico City at the beginning of the COVID-19 pandemic

ABSTRACT Currently, there are some concerns about the situation and, in particular, about the future of the COVID-19 pandemic and the new emerging variants of SARS-CoV-2. Rodents are an example of synanthropic animals in urban environments that harbor important zoonoses. Although the molecular identification of SARS-CoV-2 in Rattus norvegicus from New York City had been reported, in other studies, urban wild rodents infected with this virus have not been found. This study aimed to molecularly identify the presence of SARS-CoV-2 in urban wild rodents from Mexico City, trapped along a water channel of a public park as part of a pest control program, at the beginning of the COVID-19 pandemic, during the fall and winter of 2020. Up to 33 Mus musculus and 52 R. norvegicus were captured and euthanized, large intestine samples with feces from the animals were obtained. RNAs were obtained and subjected to qRT-PCR for SARS-CoV-2 identification and threshold cycle (Ct) values were obtained. Four mice (12.1%) and three rats (5.8%) were positive, three rodents exhibited Ct<30. Our results on the frequency of SARS-CoV-2 in urban rats are in line with other previous reports. Thus, similar to other authors, we suggest that surveillance for the detection of SARS-CoV-2 in urban wild rodents, as sentinel animals, should be maintained.

Genetic Variability of the Internal Transcribed Spacer and Pyruvate:Ferredoxin Oxidoreductase Partial Gene of Trichomonas vaginalis from Female Patients.

In the present study, we evaluated the genetic variability of the internal transcribed spacer (ITS) region and the pyruvate:ferredoxin oxidoreductase (pfor) A gene of Trichomonas vaginalis from female patients and its possible implications in the host-parasite relationship. Phylogenetic and genetics of populations analyses were performed by analyzing sequences of the ITS region and partial pfor A gene of clinical samples with T. vaginalis, as previously documented. Alignments of protein sequences and prediction of three-dimensional structure were also performed. Although no correlation between the main clinical characteristics of the samples and the results of phylogeny was found, a median-joining analysis of ITS haplotypes showed two main clusters. Also, pfor A, due to its phylogenetic divergence, could be used as a marker to confirm the genus and species of trichomonads. Alignment of protein sequences and prediction of three-dimensional structure showed that PFOR A had a highly conserved structure with two synonymous mutations in the PFOR domain, substituting a V for a G or a S for a P. Our results suggest that the role of genetic variability of PFOR and ITS may not be significant in the symptomatology of this pathogen; however, their utility as genus and species markers in trichomonads is promising.

Unexpected Presence of Blastocystis Subtype 1-3 DNA in Human Vaginal and Sperm Samples Coinfected with Trichomonas vaginalis.

There have been few reports on extra-enteric infections by Blastocystis STs and none have been molecularly identified in samples from human reproductive organs. We report for the first time the identification of 3 different subtypes of Blastocystis (ST1-3) in vaginal and sperm samples, from patients infected with Trichomonas vaginalis. Blastocystis STs were identified by PCR-sequencing and by phylogenetic inferences using 28 vaginal swab samples and 7 sperm samples from patients trichomoniasis. Blastocystis STs were identified in 6 of 28 vaginal swabs (21.4%) and in 3 of 7 sperm samples (42.8%). In both biological samples, STs 1-3 were found; one vaginal sample showed subtype co-infection with ST1 and ST3. High genetic variation was observed in the sequences obtained and no specific clustering in the phylogenetic trees was detected. Most of the haplotypes identified were placed far from the main dispersal centers. Our finding suggested that incorrect cleaning of the genital area or a contamination by combination of anal and vaginal intercourse.

Association between cycle threshold (Ct ) values and clinical and laboratory data in inpatients with COVID-19 and asymptomatic health workers.

In-house assays for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), are feasible alternatives, particularly in developing countries. Cycle threshold (Ct ) values obtained by qRT-PCR were compared with clinical and laboratory data from saliva of inpatients with COVID-19 and asymptomatic health workers (AHW) were studied. Saliva specimens from 58 inpatients confirmed by qRT-PCR for SARS-CoV-2 using nasopharyngeal specimens, and 105 AHW were studied by qRT-PCR using three sets of primers for the N (N1, N2, and N3) gene of SARS-CoV-2, according to the CDC Diagnostic Panel protocol, showing a positivity of 88% for inpatients and 8% for AHW. Bivariate analysis revealed an association between Ct < 38.0 values for N2 and mechanical ventilation assistance among patients (p = .013). In addition, values of aspartate-transaminase, lactate dehydrogenase, and ferritin showed significant correlations with Ct values of N1 and N3 genes in inpatients. Therefore, our results show that Ct values correlate with some relevant clinical data for inpatients with COVID-19.

Is the genetic variability of Cathepsin B important in the pathogenesis of Blastocystis spp.?

The potential role of Blastocystis as a pathogen is controversial because it is found in both symptomatic and asymptomatic carriers. Since Cathepsin B has been identified as a main virulence factor that contributes to the pathogenesis of this parasite, the purpose of this study was to analyze the genetic polymorphisms of cathepsin B from Blastocystis from patients with irritable bowel syndrome and from asymptomatic carriers. DNA from fecal samples of both groups, which were previously genotyped by 18S sequencing, was used to amplify a fragment of the cathepsin B gene. Phylogenetic reconstructions were performed and some genetic population indexes were obtained. Amplicons of 27 samples (15 cases, 10 controls, and two commercial ATCC strains) were obtained and analyzed. Phylogenetic reconstructions using nucleotides or inferred amino acid sequences did not separate between cases or controls or among subtypes. Regarding the values of genetic variability, we found that the haplotype and nucleotide diversity indexes of cathepsin B from cases and controls were similar to the values of 18S from controls. By contrast, 18S from cases showed low variability, suggesting that the genetic variability of cathepsin B was not related to the symptomatology of Blastocystis carriers. However, since no polymorphisms related to cases or controls were found, it is logical to assume that the potential damage caused by Blastocystis in situ may be due to unclear mechanisms of Cathepsin B regulation and expression that should be studied in future studies.

Can the pyruvate: ferredoxin oxidoreductase (PFOR) gene be used as an additional marker to discriminate among Blastocystis strains or subtypes?

BACKGROUND: Blastocystis spp. are the most prevalent intestinal eukaryotes identified in humans, with at least 17 genetic subtypes (ST) based on genes coding for the small-subunit ribosomal RNA (18S). It has been argued that the 18S gene should not be the marker of choice to discriminate between STs of these strains because this marker exhibits high intra-genomic polymorphism. By contrast, pyruvate:ferredoxin oxidoreductase (PFOR) is a relevant enzyme involved in the core energy metabolism of many anaerobic microorganisms such as Blastocystis, which, in other protozoa, shows more polymorphisms than the 18S gene and thus may offer finer discrimination when trying to identify Blastocystis ST. Therefore, the objective of the present study was to assess the suitability of the PFOR gene as an additional marker to discriminate among Blastocystis strains or subtypes from symptomatic carrier children. METHODS: Faecal samples from 192 children with gastrointestinal symptoms from the State of Mexico were submitted for coprological study. Twenty-one of these samples were positive only for Blastocystis spp.; these samples were analysed by PCR sequencing of regions of the 18S and PFOR genes. The amplicons were purified and sequenced; afterwards, both markers were assessed for genetic diversity. RESULTS: The 18S analysis showed the following frequencies of Blastocystis subtypes: ST3 = 43%; ST1 = 38%; ST2 = 14%; and ST7 = 5%. Additionally, using subtype-specific primer sets, two samples showed mixed Blastocystis ST1 and ST2 infection. For PFOR, Bayesian inference revealed the presence of three clades (I-III); two of them grouped different ST samples, and one grouped six samples of ST3 (III). Nucleotide diversity (π) and haplotype polymorphism (θ) for the 18S analysis were similar for ST1 and ST2 (π = ~0.025 and θ = ~0.036); remarkably, ST3 showed almost 10-fold lower values. For PFOR, a similar trend was found: clade I and II had π = ~0.05 and θ = ~0.05, whereas for clade III, the values were almost 6-fold lower. CONCLUSIONS: Although the fragment of the PFOR gene analysed in the present study did not allow discrimination between Blastocystis STs, this marker grouped the samples in three clades with strengthened support, suggesting that PFOR may be under different selective pressures and evolutionary histories than the 18S gene. Interestingly, the ST3 sequences showed lower variability with probable purifying selection in both markers, meaning that evolutionary forces drive differential processes among Blastocystis STs.