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.

An Overview on Research in a University Hospital, Using a Payback Framework Categorization Approach.

BACKGROUND AND OBJECTIVE: Little information is available on how to assess the impact of research studies conducted in government hospitals in Latin America and specifically in Mexico. We aimed to determine the returns on investment of the research projects that were carried out in the Hospital General "Dr. Manuel Gea Gonzalez" (HGMGG), a general university hospital located in Mexico City, using a categorization model. METHODS: We conducted a study including bibliometric analyses of publications associated with all research studies performed during the period 2016-2019 in the HGMGG and investigator interviews, according to the payback framework categorization model. RESULTS: All studies analyzed had a positive impact based on outcomes in 5 "payback categories": (1) knowledge; (2) research targeting, capacity building, and absorption; (3) policy and product development; (4) health benefits; and (5) broader economic benefits. CONCLUSIONS: To date, it has not been possible to establish a set of indicators that show the results of the investigations carried out by medical specialists in training, who carry out the bulk of medical care in general hospitals and in the National Institutes of Health in Mexico. We identified, in the 5 categories of the payback framework model, different areas of opportunity to improve the benefits of the hospital's medical services through the development of scientific research projects.

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.

Elevated Levels of Cytotoxicity, Cytokines, and Anti-SARS-CoV-2 Antibodies in Mild Cases of COVID-19.

Current evidence shows higher production of cytokines and antibodies against severe acute respiratory coronavirus 2 (SARS-CoV-2) in severe and critical cases of Coronavirus Disease 2019 (COVID-19) in comparison with patients with moderate or mild disease. A recent hypothesis proposes an important role of genotoxicity and cytotoxicity in the induction of the cytokine storm observed in some patients at later stages of the disease. Interestingly, in this study, we report significantly higher levels of interleukin (IL)-1ß, IL-6, MCP-1, and IL-4 cytokines in mild COVID-19 patients versus severe cases, as well as a high frequency of karyorrhexis (median [Me] = 364 vs. 20 cells) and karyolysis (Me = 266 vs. 52 cells) in the mucosal epithelial cells of both groups of patients compared with uninfected individuals. Although we observed higher levels of anti-SARS-CoV-2 IgM and IgG antibodies in COVID-19 patients, IgM antibodies were significantly higher only in mild cases, for the N and the S viral antigens. High levels of IgG antibodies were observed in both mild and severe cases. Our results showed elevated concentrations of proinflammatory and anti-inflammatory cytokines in mild cases, which may reflect an active innate immune response and could be related to the higher IgM and IgG antibody levels found in those patients. In addition, we found that SARS-CoV-2 infection induces cytotoxic damage in the oral mucosa, highlighting the importance of studying the genotoxic and cytotoxic events induced by infection and its role in the pathophysiology of COVID-19.

Presence of human adenovirus 36 in visceral fat tissue, viral load, and analysis of its genetic variability.

It has been proposed that infection by adipogenic viruses constitutes a "low risk" factor for obesity. Here, we report the presence of adenovirus 36 (Ad36) and its viral load copy number in fat tissue of participants with obesity and normal weight; phylogenetic analysis was performed to describe their relationship and genetic variability among viral haplotypes. Adipose tissue obtained from 105 adult patients with obesity (cases) and 26 normal-weight adult participants as controls were analyzed by quantitative polymerase chain reaction (qPCR) amplifying the partial Ad36 E1a gene. The amplicons were examined by melting curves and submitted to sequencing. Then, genetic diversity and phylogenetic inferences were performed. Ad36 was identified at rates of 82% and 46% in the case and control groups, respectively (p = 1.1 × 10-4 , odds ratio = 5.28); viral load copies were also significantly different between both groups, being 25% higher in the case group. Melting curve analysis showed clear amplification among positive samples. Phylogenetic inferences and genetic diversity analyses showed that the Ad36 E1a gene exhibits low genetic variability and differentiation with strong gene flow due to an expanding process. Our results suggest that the phenomenon of infectobesity by Ad36 might not be a low-risk factor, as has been previously argued by other authors.

Identification of Blastocystis sp. in school children from a rural Mexican village: subtypes and risk factors analysis.

Blastocystis sp. is a common eukaryotic microorganism that colonizes the intestinal tract of several animals, including humans, although its role as a pathogen is still unclear. In the present study, we report the prevalence and risk factors associated with Blastocystis infection in scholars from a rural community in Mexico. A cross-sectional observational study was carried out on schoolchildren aged 3 to 15 years old; fecal samples were analyzed by culture, Faust technique, and molecular analysis. In addition, a structured questionnaire was applied to identify possible risk factors. Of the 177 samples obtained, Blastocystis sp. was the microorganism that presented the highest frequency (n=78, 44%), and included the following subtypes (STs): ST1 (n=43, 56.5%), ST2 (n=18, 23.6%), and ST3 (n=15, 19.7%); Blastocystis STs were not identified in two cases. No associating factors were found between Blastocystis infection or among STs vs. symptoms. During bivariate analysis, no statistically significant risk factors were found, except for the variable of "eating sweets, snacks, and handmade food on the way home" (p=0.04). Therefore, it is plausible to conclude that schoolchildren become infected with Blastocystis sp. mainly outside their homes, perhaps by eating contaminated handmade food on their way to or from school; however, this variable should be evaluated in detail in future studies.

Long-term infection passaging of Human Adenovirus 36 in monkey kidney cells.

Human Adenovirus 36 (HAdV-36) has been related to diverse effects on metabolism and may attenuate the lipid accumulation in kidneys with increased adiposity. Some of these effects would be related to viral persistence. However, until now, a model of persistent in vitro infection by HAdV-36 is unknown. In this study, we examined the cells of the Vero lineage to explore their permissiveness to long-term HAdV-36 infection. HAdV-36 was productively replicated in Vero cells and maintained long-term infection for up to 35 cell passages. A subculture was obtained from the cells that survived the primary infection at a low MOI (0.5). The production of the extracellular infectious virus with titers ranging from 104 to 106 TCID50/mL and DNA-bearing cells was detected. In long-term infected cells, the intracellular distribution of viral antigen was demonstrated by performing immunolocalization (IFI) and expression of cell-viral antigen in 50% of cells by flow cytometry, using anti-HAdV-36 hyperimmune rabbit serum. Furthermore, E1a and E4orf1 genes in long-term infected passages showed a decreasing trend. Our preliminary results reveal that renal epithelial monkey cells are permissive for the productive infection of HAdV-36. Vero cell culture long-term infection might be a promising model for addressing the fundamental aspects of the HAdV-36 biology that cannot reveal broadly-used cultures, which do not maintain long-term infection in primary or transformed cells.

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.

Identification of α-L-fucosidase (ALFuc) of Blastocystis sp. subtypes ST1, ST2 and ST3.

Blastocystis sp. is a common intestinal microorganism. The α-L-fucosidase (ALFuc) is an enzyme long associated with the colonization of the gut microbiota. However, this enzyme has not been experimentally identified in Blastocystis cultures. The objective of the present study was to identify ALFuc in supernatants of axenic cultures of Blastocystis subtype (ST)1 ATCC-50177 and ATCC-50610 and to compare predicted ALFuc proteins of alfuc genes in sequenced STs1-3 isolates in human Blastocystis carriers. Excretion/secretion (Es/p) and cell lysate proteins were obtained by processing Blastocystis ATCC cultures and submitting them to SDS-PAGE and immunoblotting. In addition, 18 fecal samples from symptomatic Blastocystis human carriers were analyzed by sequencing of amplification products for subtyping. A complete identification of the alfuc gene and phylogenetic analysis were performed. Immunoblotting showed that the amplified band corresponding to ALFuc (~51 kDa) was recognized only in the ES/p. Furthermore, prediction analysis of ALFuc 3D structures revealed that the domain α-L-fucosidase and the GH29 family's catalytic sites were conserved; interestingly, the galactose-binding domain was recognized only in ST1 and ST2. The phylogenetic inferences of ALFuc showed that STs1-3 were clearly identifiable and grouped into specific clusters. Our results show, for the first time through experimental data that ALFuc is a secretion product of Blastocystis sp., which could have a relevant role during intestinal colonization; however, further studies are required to clarify this condition. Furthermore, the alfuc gene is a promising candidate for a phylogenetic marker, as it shows a conserved classification with the SSU-rDNA gene.

Identification and Molecular Characterization of Four New Blastocystis Subtypes Designated ST35-ST38.

Three recent studies of Blastocystis epidemiology in mammalian hosts identified four novel sequences that appeared to share B. lapemi as the most similar sequence. However, full-length ssu rRNA gene sequences were not available to confirm the validity of these new subtypes. In the present study, Nanopore MinION sequencing was used to obtain full-length reference sequences for each of the new subtypes. Additionally, phylogenetic analyses and pairwise distance comparisons were performed to confirm the validity of each of these new subtypes. We propose that the novel sequences described in this study should be assigned the subtype designations ST35-ST38. The full-length reference sequences of ST35-ST38 will assist in accurate sequence descriptions in future studies of Blastocystis epidemiology and subtype diversity.

Long-term infection passaging of Human Adenovirus 36 in monkey kidney cells

ABSTRACT Human Adenovirus 36 (HAdV-36) has been related to diverse effects on metabolism and may attenuate the lipid accumulation in kidneys with increased adiposity. Some of these effects would be related to viral persistence. However, until now, a model of persistent in vitro infection by HAdV-36 is unknown. In this study, we examined the cells of the Vero lineage to explore their permissiveness to long-term HAdV-36 infection. HAdV-36 was productively replicated in Vero cells and maintained long-term infection for up to 35 cell passages. A subculture was obtained from the cells that survived the primary infection at a low MOI (0.5). The production of the extracellular infectious virus with titers ranging from 104 to 106 TCID50/mL and DNA-bearing cells was detected. In long-term infected cells, the intracellular distribution of viral antigen was demonstrated by performing immunolocalization (IFI) and expression of cell-viral antigen in 50% of cells by flow cytometry, using anti-HAdV-36 hyperimmune rabbit serum. Furthermore, E1a and E4orf1 genes in long-term infected passages showed a decreasing trend. Our preliminary results reveal that renal epithelial monkey cells are permissive for the productive infection of HAdV-36. Vero cell culture long-term infection might be a promising model for addressing the fundamental aspects of the HAdV-36 biology that cannot reveal broadly-used cultures, which do not maintain long-term infection in primary or transformed cells.

Identification of α-L-fucosidase (ALFuc) of Blastocystis sp. subtypes ST1, ST2 and ST3

ABSTRACT Blastocystis sp. is a common intestinal microorganism. The α-L-fucosidase (ALFuc) is an enzyme long associated with the colonization of the gut microbiota. However, this enzyme has not been experimentally identified in Blastocystis cultures. The objective of the present study was to identify ALFuc in supernatants of axenic cultures of Blastocystis subtype (ST)1 ATCC-50177 and ATCC-50610 and to compare predicted ALFuc proteins of alfuc genes in sequenced STs1-3 isolates in human Blastocystis carriers. Excretion/secretion (Es/p) and cell lysate proteins were obtained by processing Blastocystis ATCC cultures and submitting them to SDS-PAGE and immunoblotting. In addition, 18 fecal samples from symptomatic Blastocystis human carriers were analyzed by sequencing of amplification products for subtyping. A complete identification of the alfuc gene and phylogenetic analysis were performed. Immunoblotting showed that the amplified band corresponding to ALFuc (~51 kDa) was recognized only in the ES/p. Furthermore, prediction analysis of ALFuc 3D structures revealed that the domain α-L-fucosidase and the GH29 family's catalytic sites were conserved; interestingly, the galactose-binding domain was recognized only in ST1 and ST2. The phylogenetic inferences of ALFuc showed that STs1-3 were clearly identifiable and grouped into specific clusters. Our results show, for the first time through experimental data that ALFuc is a secretion product of Blastocystis sp., which could have a relevant role during intestinal colonization; however, further studies are required to clarify this condition. Furthermore, the alfuc gene is a promising candidate for a phylogenetic marker, as it shows a conserved classification with the SSU-rDNA gene.

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.

An Unusual Case of Extra-Enteric Blastocystosis in the Uterine Cervix.

Extra-enteric infections by Blastocystis spp. have rarely been documented. Here, we report a case of extra-enteric blastocystosis in a patient with minimal cervicitis symptoms. A 47-year-old Hispanic female patient was attended in a primary health centre in Michoacan state, Mexico, for her routine gynaecological medical examination. As only symptom, she referred to a slight vaginal itching. The presence of several vacuolar-stages of Blastocystis spp. were identified by Papanicolaou staining; molecular identification was attempted by culture-PCR sequencing of a region of 18S gene from cervical and faecal samples obtained 2 months after cytological examination, even when patient declared that she tried self-medicating with vaginal ovules. Blastocystis ST1 was identified only in the faecal sample. The presence of Blastocystis spp. in the cervix of a patient with scarce symptomatology, demonstrates the extraordinary flexibility of this microorganism to adapt to new environments and niches.

Interaction between human mucins and parasite glycoproteins: the role of lectins and glycosidases in colonization by intestinal protozoa.

Intestinal mucins are the first line of defense against microorganisms. Although knowledge about the mechanisms involved in the establishment of intestinal protozoa is limited, there is evidence that these parasites produce lectin-like molecules and glycosidases, that exert both, constitutive and secretory functions, promoting the establishment of these microorganisms. In the present review, we analyse the main interactions between mucins of the host intestine and the four main protozoan parasites in humans and their implications in intestinal colonization. There are lectin-like molecules that contain complex oligosaccharide structures and N-acetylglucosamine (GlcNAc), mannose and sialic acid as main components, which are excreted/secreted by Giardia intestinalis, and recognized by the host using mannose-binding lectins (MBL). Entamoeba histolytica and Cryptosporidium spp. express the lectin galactose/N-acetyl-D-galactosamine, which facilitates their adhesion to cells. In Cryptosporidium, the glycoproteins gp30, gp40/15 and gp900 and the glycoprotein lectin CpClec are involved in protozoan adhesion to intestinal cells, forming an adhesion-attack complex. G. intestinalis and E. histolytica can also produce glycosidases such as ß-N-acetyl-D-glucosaminidase, α-d-glucosidase, ß-d-galactosidase, ß-l-fucosidase, α-N-acetyl-d-galactosaminidase and ß-mannosidase. In Blastocystis, α-D-mannose, α-D-glucose, GlcNAc, α-D-fucose, chitin and sialic acid that have been identified on their surface. Fucosidases, hexosaminidases and polygalacturonases, which may be involved in the mucin degradation process, have also been described in the Blastocystis secretoma. Similarly, symbiotic coexistence with the intestinal microbiota promotes the survival of parasites facilitating cell invasion and nutrients obtention. Furthermore, it is necessary to identify and characterize more glycosidases, which have been only partially described by in silico analyses of the parasite genome.

An Unexpected Case of Lagochilascariasis: Interdisciplinary Management and Use of 12S and 18S rDNA Analysis.

A Mexican 24-year-old male patient was referred to our hospital due to increased left retroauricular volume with skin fistulisation, resembling an infection by the uncommon worm Lagochilascaris minor. The patient was submitted to lateral skull base surgery. No adult worms or eggs were observed during light and scanning electron microscopy analysis, as well as by histopathologic examination of the small piece of removed tissue, only L3 stage larvae of Lagochilascaris spp. were identified. Polymerase chain reaction-sequencing assays were performed using primers for the mitochondrial 12S and the nuclear 18S rDNA gene. DNA of some L minor adults, previously identified, were used as control. The molecular analysis identified the worm as L minor. According to previous reports, lagochilascariasis is a complicated infection that requires an interdisciplinary management by different clinical specialists. This is the first time that 12S and 18S rDNA genes are reported as molecular markers for diagnosis of L minor.

The Short-Term Effects of Roux-en-Y Gastric Bypass on Renal Excretion of Sodium and Its Association with Blood Pressure.

INTRODUCTION: Bariatric surgery has been shown to be effective in reducing weight and has benefits, such as lowering blood pressure. An increase in urinary sodium excretion has been suggested as a possible mechanism. This study explored changes in sodium excretion and their correlation with blood pressure after Roux-en-Y gastric bypass. MATERIALS AND METHODS: This study was conducted on 28 obese participants with body mass index (BMI) of 44.54 ± 7.81 kg/m2 who underwent gastric bypass. Before surgery and at the third and sixth months after gastric bypass, blood pressure, urinary sodium concentration, 24-hour (24-h) urinary sodium excretion, and fractional excretion of sodium were evaluated. In addition, serum sodium and potassium levels were determined. Nonparametric tests were used to analyze the data. RESULTS: Blood pressure decreased after surgery and remained at low levels over the 3- and 6-month periods. The urinary sodium concentration increased at 3 months after surgery; however, the 24-h urinary sodium excretion and urine volume decreased. Interestingly, although some associations between variables were observed, significant correlations between the 24-h urinary sodium excretion and the systolic, diastolic, and mean blood pressures were found. In addition, the urine volume was higher in the sixth month than in the third month following surgery. CONCLUSIONS: In the months immediately following surgery, a low-salt and low-volume diet favors decreases in urine volume and 24-h urinary sodium excretion. In addition, in the sixth month after surgery, an association between blood pressure and 24-h urinary sodium excretion was observed.

Molecular genotyping of Blastocystis spp. in wild mammals from Mexico.

Blastocystis spp. are common intestinal parasites found worldwide in humans and a wide range of animals. They exhibit extensive genetic diversity; currently, 17 subtypes (STs) and some groups called non-mammalian and avian STs (NMASTs) have been proposed. In addition, a large variety of animals have been reported as hosts of the parasite, and new hosts and STs are still being described. In this study, Blastocystis infection of wild animals in two sylvatic areas of Mexico was surveyed. Of one hundred twenty-four fecal samples, six were positive for Blastocystis: specifically, one sample from an opossum, one sample from a bat, and four samples from different species of rodents. ST4, ST17, and nucleotide sequences similar to Blastocystis lapemi were identified based on SSU rDNA sequences. To our knowledge, this is the first report to investigate species poorly or not previously evaluated for Blastocystis infection. Mammals having different niches and geographical distribution were infected with similar genetic type of Blastocystis, so that we suggest that local water or food sources could play an important role in Blastocystis transmission and ST maintenance in wild animals. Additionally, there are STs with scarce genetic variation, suggesting that they could be highly adapted to their hosts. These data contribute to our understanding of the host range and genetic diversity of Blastocystis.

Myiasis caused by Dermatobia hominis in Mexico: morphological and molecular identification using the cytochrome oxidase I gene.

Myiasis caused by Dermatobia hominis , the human botfly, is frequent in the Americas, however, scarce morphological and molecular information exist regarding this dipteran. We describe three cases in urban areas of Mexico were D. hominis is not endemic. Morphological and genetic identification were performed using the cytochrome oxidase I as a molecular marker. The mitochondrial cytochrome oxidase I gene is useful for inferring the genetic divergence of D. hominis .