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.

The Immune Response in Adipocytes and Their Susceptibility to Infection: A Possible Relationship with Infectobesity.

The current obesity pandemic has been expanding in both developing and developed countries. This suggests that the factors contributing to this condition need to be reconsidered since some new factors are arising as etiological causes of this disease. Moreover, recent clinical and experimental findings have shown an association between the progress of obesity and some infections, and the functions of adipose tissues, which involve cell metabolism and adipokine release, among others. Furthermore, it has recently been reported that adipocytes could either be reservoirs for these pathogens or play an active role in this process. In addition, there is abundant evidence indicating that during obesity, the immune system is exacerbated, suggesting an increased susceptibility of the patient to the development of several forms of illness or death. Thus, there could be a relationship between infection as a trigger for an increase in adipose cells and the impact on the metabolism that contributes to the development of obesity. In this review, we describe the findings concerning the role of adipose tissue as a mediator in the immune response as well as the possible role of adipocytes as infection targets, with both roles constituting a possible cause of obesity.

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.

Cervical lymph node tuberculosis and TNF, IL8, IL10, IL12B and IFNG polymorphisms.

Cervical lymph node tuberculosis (LNTB) is the most common manifestation of extrapulmonary tuberculosis, resulting from the interaction of environmental and genetic factors. The immune response against TB is regulated by several cytokines, which have single nucleotide polymorphisms (SNPs), leading to different levels of expression. The aim of this study was to evaluate the association of LNTB with the TNF, IL8, IL10, IL12B and IFNG gene polymorphisms in Mexican patients. We investigated the association of ten SNPs in 14 patients with LNTB and 138 healthy controls. Significant differences were found for the allele TNF-238A (P=0.03) and the genotypes TNF-238GA (P=0.03), IL8+396GG (P=0.01) and IL12B+1188CC (P=0.04). Allele IL8+781C showed some association trend (P=0.08). Haplotypes TNF-AA and IL10-GTA were of susceptibility, whereas haplotype IL8-ATT was of protection. No association was found with IFNG. The association of these polymorphisms with extrapulmonary TB was compared in different populations. Our results suggest that these cytokine SNPs may influence the manifestation of LNTB in Mexican patients; however, we are aware of the limitations of our study, so it is necessary to make a replica using a larger sample of patients, as well as an increased number of cytokines with SNPs.

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.

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.

The amniotic band syndrome in the rat is associated with the activation of transforming growth factor-ß.

Amniocentesis in rats is associated with different malformations, such as cleft palate and limb deformation, resembling the human congenital amniotic band syndrome (ABS). Despite many human cases reported in the literature, little is known about the mechanisms involved in ABS. This study addressed if the activation of the transforming growth factor-ß1 (TGF-ß1) pathway is, in part, associated with amniotic band formation and growth restriction induced in rats by amniocentesis, as by a previously published model. For this purpose, quantification of TGF-ß1, α-smooth muscle actin, and collagen type I mRNA and protein levels were determined by quantitative PCR and Western blot analysis, respectively, in the fetus, its amniotic membrane, and the uterus of experimental and control rats. We found that TGF-ß1 mRNA levels are increased in the fetus and the amniotic membrane at 6 hours, whereas α-smooth muscle actin, phosphorylated Smad3, and collagen type I increased at 48 hours, suggesting that a fibrotic response is induced after the amniotic sac puncture. Furthermore, fetuses had hemorrhages, syndactyly, and amputation of limbs, similar to human ABS.

Leptin receptor gene polymorphisms and morbid obesity in Mexican patients.

BACKGROUND: Human obesity is due to a complex interaction among environmental, behavioral, developmental and genetic factors, including the interaction of leptin (LEP) and leptin receptor (LEPR). Several LEPR mutations and polymorphisms have been described in patients with early onset severe obesity and hyperphagic eating behavior; however, some contradictory findings have also been reported. In the present study we explored the association of six LEPR gene polymorphisms in patients with morbid obesity. FINDINGS: Twenty eight patients with morbid obesity and 56 non-obese Mexican Mestizo individuals were included. Typing of rs1137100, rs1137101, rs1805134, Ser492Thr, rs1805094 and rs1805096 LEPR polymorphisms was performed by PCR and allele specific hybridization. The LEPR Ser492Thr polymorphism was monomorphic with the presence of only the Ser492Thr-G allele. Allele C and genotype T/C for rs1805134 polymorphism were associated with susceptibility to morbid obesity (p = 0.02 and p = 0.03, respectively). No association was observed with any haplotype. Linkage disequilibrium (LD) showed that five polymorphisms (rs1137100, rs1137101, rs1805134, rs1805094 and rs1805096) were in absolute (D' = 1) but none in perfect (r2 = 1) LD. CONCLUSIONS: Our results suggest that rs1805134 polymorphism could be involved in the development of morbid obesity, whilst none of the alleles of the LEPR gene, rs1137100, rs1137101, rs1805094 and rs1805096 were associated as risk factors. However, more studies are necessary to confirm or reject this hypothesis.

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.

Genetic Variation among the Partial Gene Sequences of the Ribosomal Protein Large-Two, the Internal Transcribed Spacer, and the Small Ribosomal Subunit of Blastocystis sp. from Human Fecal Samples.

In the present study, we compared the genetic variability of fragments from the internal transcribed spacer region (ITS) and the small subunit ribosomal DNA (SSUrDNA) as nuclear markers, in contrast with the ribosomal protein large two (rpl2) loci, placed in the mitochondrion-related organelles (MROs) within and among human fecal samples with Blastocystis. Samples were analyzed using polymerase chain reaction (PCR)-sequencing, phylogenies, and genetics of population structure analyses were performed. In total, 96 sequences were analyzed, i.e., 33 of SSUrDNA, 35 of rpl2, and 28 of ITS. Only three subtypes (STs) were identified, i.e., ST1 (11.4%), ST2 (28.6%), and ST3 (60%); in all cases, kappa indexes were 1, meaning a perfect agreement among ST assignations. The topologies of phylogenetic inferences were similar among them, clustering to each ST in its specific cluster; discrepancies between phylogeny and assignment of STs were not observed. The STRUCTURE v2.3.4 software assigned three subpopulations corresponding to the STs 1-3, respectively. The population indices were consistent with those previously reported by other groups. Our results suggest the potential use of the ITS and rpl2 genes as molecular markers for Blastocystis subtyping as an alternative approach for the study of the genetic diversity observed within and between human isolates of this microorganism.

New tetratrichomonas species in two patients with pleural empyema.

Two unusual occurrences of pleural trichomonosis due to a new Tetratrichomonas species previously reported but not named were confirmed. In one patient, Trichomonas tenax and a Tetratrichomonas species were also detected in the oral cavity by molecular methods. We suggest that this new Tetratrichomonas species be named Tetratrichomonas empyemagena.

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.

Interleukin-8 and -10 gene polymorphisms in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is one of the most common gastrointestinal diagnoses seen by primary care providers and gastroenterologists. Proinflammatory cytokines interleukin (IL)-6 and IL-8 have been found increased in IBS patients. Cytokine gene single nucleotide polymorphisms (SNPs) of IL-8 and IL-10 have not been assessed in Mexican IBS patients. DNA was extracted from peripheral blood leucocytes of 45 IBS unrelated patients and 137 controls. Allele, genotype, and haplotype frequencies were determined by analyzing SNPs of IL-8 and IL-10 genes. IL-8 + 396 G allele (P = 0.02), IL-8 + 396(G/G) and IL-8 + 781(C/T) genotypes (P < 0.001), IL-10 - 1082A allele and IL-10 - 1082(A/A) genotype (P < 0.001) were significantly increased in the IBS group. Haplotypes IL-8 ATCC (P = 0.03) and IL-10 ACC (P < 0.001) were associated with susceptibility to develop IBS. An association of certain polymorphisms of IL-8 and IL-10 in IBS patients compared to controls was demonstrated, suggesting a role of these cytokine SNPs in the pathophysiology of IBS.

Immunological mechanisms involved in the protection against intestinal taeniosis elicited by oral immunization with Taenia solium calreticulin.

Oral immunization with functional recombinant Taenia solium calreticulin (rTsCRT) induces 37% reduction in tapeworm burden in the experimental model of intestinal taeniosis in hamsters. Furthermore, tapeworms recovered from vaccinated animals exhibit diminished length, being frequently found in more posterior parts of the small intestine. The aim of this study was to analyze the immunological mechanisms involved in protection in response to rTsCRT oral immunization. Hamsters were orally immunized with rTsCRT using cholera toxin (CT) as adjuvant, weekly for 4 weeks. Fifteen days after the last boost animals were challenged with four T. solium cysticerci. Reduction in the adult worm recovery and increased transcription of mRNA for IL-4 and IFN-γ in the mucosa of rTsCRT+CT immunized animals were observed. Immunization also induced goblet cell hyperplasia in the mucosa surrounding the implantation site of the parasite. Specific IgG and IgA antibodies in serum and fecal supernatants were detected after the second immunization, being more pronounced after challenge. Our data suggest that oral vaccination with rTsCRT+CT regulates a local expression of IL-4 and IFN-γ, stimulating secretion of IgA that, together with the increase of goblet cells and mucin production, could result in an unfavorable environment for T. solium promoting an impaired tapeworm development.

Findings related to IL-8 and IL-10 gene polymorphisms in a Mexican patient population with irritable bowel syndrome infected with Blastocystis.

The intestinal protozoan parasite Blastocystis is one of the most common parasites worldwide in humans and, although its ability to cause human disease has been questioned, some reports have demonstrated that this microorganism is associated to the development of irritable bowel syndrome (IBS) and to a proinflammatory response, in which the expression of some cytokines is unregulated. Since inflammatory cytokine gene polymorphisms might have a role in the pathophysiology of IBS, we assessed the role of single nucleotide polymorphisms (SNPs) for interleukin (IL)-8 and IL-10, in previously collected DNA samples from IBS patients and controls, with or without Blastocystis infection. IL-8+396(G) and IL-10-1082 (A) alleles (p=0.0437 and p=0.0267, respectively), as well as their homozygous (p<0.0001 and p=0.0039, respectively) and IL-8+781(CT) (p=0.0248) genotypes were significantly overrepresented in patients with IBS in comparison with controls. IL-8+396(GG) genotype was relevant because it was associated to IBS (p<0.0001), to Blastocystis (p=0.0025), and to IBS­Blastocystis (p=0.0272). In the latter binomial association, this genotype presented a high contribution (etiological fraction =0.452) and a risk >fourfold to develop IBS. IL-8+781 (T) and IL-10-592 (C) alleles were also associated to Blastocystis and to IBS­Blastocystis, respectively (p=0.0448 and p=0.0166). Our results suggest that some IL-8 and IL-10 SNPs could change individual susceptibility increasing the relative risk in the development of IBS in Blastocystis carriers.

Blastocystis infection is associated with irritable bowel syndrome in a Mexican patient population.

In recent times, some common "non-pathogenic" parasites, such as Blastocystis and Dientamoeba fragilis, have been associated to the aetiology of irritable bowel syndrome (IBS), while host pro-inflammatory cytokine gene polymorphisms might have a role in the pathophysiology of the disease. Therefore, Blastocystis subtypes (ST), D. fragilis and gene promoter single nucleotide polymorphisms of interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) in IBS patients and controls were studied. After giving written consent, 45 patients with symptoms of IBS according to the Rome III criteria and 45 controls were enrolled. DNA was extracted from peripheral blood for SNP analysis at position -174 for IL-6 as well as -238 and -308 for TNF-α. Blastocystis was more common in the IBS group (p = 0.043). Interestingly, D. fragilis was found more frequently in the control group (p = 0.002); Blastocystis ST1 and 3 were most frequent in both groups. Haploview analysis revealed linkage disequilibrium in TNF-α (p < 0.0001); however, none of the SNPs for IL-6 and TNF-α were found to be significantly related with IBS. The clinical and molecular approaches undertaken for the first time in Latin American IBS patients demonstrated an association with Blastocystis that supports a pathogenic role of this parasite in IBS Furthermore, co-infections with ST1 and ST3 were frequent; thus, the genetic diversity proposed within ST polymorphisms does not rule out that particular strains might be associated with disease. In addition, our results do not support a major contribution of IL-6 and TNF-α gene polymorphisms in the susceptibility to IBS.

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.