Genomic and phenotypic studies among Clostridioides difficile isolates show a high prevalence of clade 2 and great diversity in clinical isolates from Mexican adults and children with healthcare-associated diarrhea.

Clostridioides difficile (C. difficile) is widely distributed in the intestinal tract of humans, animals, and in the environment. It is the most common cause of diarrhea associated with the use of antimicrobials in humans and among the most common healthcare-associated infections worldwide. Its pathogenesis is mainly due to the production of toxin A (TcdA), toxin B (TcdB), and a binary toxin (CDT), whose genetic variants may be associated with disease severity. We studied genetic diversity in 39 C. difficile isolates from adults and children attended at two Mexican hospitals, using different gene and genome typing methods and investigated their association with in vitro expression of toxins. Whole-genome sequencing in 39 toxigenic C. difficile isolates were used for multilocus sequence typing, tcdA, and tcdB typing sequence type, and phylogenetic analysis. Strains were grown in broth media, and expression of toxin genes was measured by real-time PCR and cytotoxicity in cell-culture assays. Clustering of strains by genome-wide phylogeny matched clade classification, forming different subclusters within each clade. The toxin profile tcdA+/tcdB+/cdt+ and clade 2/ST1 were the most prevalent among isolates from children and adults. Isolates presented two TcdA and three TcdB subtypes, of which TcdA2 and TcdB2 were more prevalent. Prevalent clades and toxin subtypes in strains from children differed from those in adult strains. Toxin gene expression or cytotoxicity was not associated with genotyping or toxin subtypes. In conclusion, genomic and phenotypic analysis shows high diversity among C. difficile isolates from patients with healthcare-associated diarrhea. IMPORTANCE: Clostridioides difficile is a toxin-producing bacterial pathogen recognized as the most common cause of diarrhea acquired primarily in healthcare settings. This bacterial species is diverse; its global population has been divided into five different clades using multilocus sequence typing, and strains may express different toxin subtypes that may be related to the clades and, importantly, to the severity and progression of disease. Genotyping of children strains differed from adults suggesting toxins might present a reduced toxicity. We studied extensively cytotoxicity, expression of toxins, whole genome phylogeny, and toxin typing in clinical C. difficile isolates. Most isolates presented a tcdA+/ tcdB+/cdt+ pattern, with high diversity in cytotoxicity and clade 2/ST1 was the most prevalent. However, they all had the same TcdA2/TcdB2 toxin subtype. Advances in genomics and bioinformatics tools offer the opportunity to understand the virulence of C. difficile better and find markers for better clinical use.

Case­control study of Epstein­Barr virus and Helicobacter pylori serology in Latin American patients with gastric disease.

BACKGROUND: Chronic tissue damage induced by Helicobacter pylori (HP)-driven inflammation is considered the main risk of gastric carcinoma (GC). Epstein­Barr virus (EBV) infection has also been associated with GC. In this study, we aim to address the role of EBV in inflammatory GC precursor lesions and its added risk to HP infection. METHODS: Antibodies against EBV, HP and the bacterial virulence factor CagA were measured in sera from 525 Mexican and Paraguayan patients with gastric disease. Gastric samples were characterised according to the updated Sydney classification and associations were estimated between antibody responses and severity of both tissue damage and inflammation. RESULTS: We found significant associations (odd ratios and trends) between EBV and HP copositivity and premalignant lesions and intestinal-type GC. The EBV and HP coinfection was also significantly associated with increased infiltration of immune cells. No association was found between EBV and the less inflammation-driven diffuse-type GC. CONCLUSIONS: Our study suggests that EBV co-participates with HP to induce severe inflammation, increasing the risk of progression to intestinal-type GC.

The utility of serologic tests as biomarkers for Helicobacter pylori-associated precancerous lesions and gastric cancer varies between Latin American countries.

PURPOSE: Currently, studies on serologic diagnosis of Helicobacter pylori-associated gastric cancer (GC) in Latin America are scarce. The aim of the present study was to evaluate the association between H. pylori serology tests in patients with early precancerous lesions or GC, when compared with non-atrophic gastritis in Colombia, Paraguay, and Mexico, three countries in Latin America with a high prevalence of H. pylori infection but contrasting rates of GC mortality. METHODS: Gastric biopsies and blood samples were obtained from patients attending the gastroenterology or oncology services of hospitals in the three participating countries. IgG antibodies against H. pylori whole-cell antigens and CagA were tested in 1,117 sera using an enzyme-linked immunoabsorbent assay. RESULTS: Positive and significant associations were shown for H. pylori seropositivity and preneoplastic lesions in Mexico (OR 2.0; 95 % CI 1.1-3.4) but not in Colombia (OR 1.2; 95 % CI 0.6-2.1) or Paraguay (OR 1.5; 95 % CI 0.6-3.2); no significant associations were shown for GC in any country. CagA seropositivity was associated with preneoplasic lesions in all three countries (ORs = 2.1, 3.0, and 3.1 for Mexico, Colombia, and Paraguay, respectively), and with GC only in Colombia (OR 4.3; 95 % CI 2.1-9.2). CONCLUSIONS: In countries of Latin America, the IgG CagA test might be a useful biomarker for patients with gastric preneoplastic lesions and for those at risk of developing gastric cancer.

Topographical localisation of cagA positive and cagA negative Helicobacter pylori strains in the gastric mucosa; an in situ hybridisation study.

BACKGROUND: The cagA gene is a marker for the presence of the cag pathogenicity island, and the presence of cagA positive strains of Helicobacter pylori can identify individuals with a higher risk of developing gastrointestinal diseases. AIMS: To study the interaction between H. pylori cagA(+) and cagA(-) strains and the gastric mucosa. METHODS: Patients with H. pylori associated gastritis and peptic ulcers were studied. Biopsies were obtained from the antrum, corpus, fundus, and incisura for H pylori culture, and for in situ hybridisation studies. From each biopsy, multiple single H. pylori colonies were isolated and propagated for DNA isolation, and cagA was detected by the polymerase chain reaction (PCR). For in situ detection of H. pylori an oligonucleotide specific for an H. pylori common antigen and an oligonucleotide specific for cagA were used as probes. Biotinylated probes were incubated with biopsy sections, developed with streptavidin-horseradish peroxidase, and amplified with the tyramide system. RESULTS: PCR results for cagA in isolated colonies confirmed the in situ hydridisation studies. In situ hybridisation identified cagA(+) bacteria in patients with cagA(+) isolates; cagA(-) bacteria in patients with cagA(-) isolates, and cagA(+) and cagA (-) bacteria in patients with both cagA(+) and cagA(-) isolates. CagA(-) bacteria usually colonised the mucous gel or the apical epithelial surface, whereas cagA(+) bacteria colonised the immediate vicinity of epithelial cells or the intercellular spaces. CONCLUSIONS: These results document a different in vivo interaction between H. pylori cagA(+) or cagA(-) strains and the gastric mucosa.

The association of intestinal parasitosis and H. pylori infection in children and adults from a Mexican community with high prevalence of parasitosis.

BACKGROUND: Experimental evidences have suggested that a Th1 response is unable to eliminate H. pylori colonization; whereas a Th2 response, like the one induced by vaccination, reduces H. pylori infection in animal models. Some parasitic infections induce a polarized Th2 response, which theoretically would favor a reduced H. pylori prevalence. The aim of this work was to study the possible association between parasitic infections and H. pylori prevalence. MATERIALS AND METHODS: The study population included 120 children and 188 adults from a low socioeconomic level village. H. pylori prevalence was determined in serum by ELISA; parasitic infections were identified in feces by microscopic examination; and total serum IgE levels, as an indirect indicator of some parasitic infections, were determined by ELISA. RESULTS: In children, H. pylori prevalence was no different between those with and without intestinal parasitic infection. By contrast, adults with intestinal parasitic infection had a significantly lower H. pylori prevalence than adults without parasites (62.6% compared with 80.4%; p = 0.006, OR 2.45). Also in adults, but not in children, total IgE levels were significantly higher in those without H. pylori infection than in those with H. pylori infection (p < 0.001). CONCLUSIONS: Intestinal parasitic infections and serum IgE levels showed an age-dependent association with H. pylori prevalence. In adults, but not in children, intestinal parasitic infections and increased IgE levels where associated with a reduced H. pylori prevalence.

Increasing multidrug resistance in Helicobacter pylori strains isolated from children and adults in Mexico.

The susceptibilities to three antimicrobials of 195 Helicobacter pylori strains isolated from Mexican patients is reported; 80% of the strains were resistant to metronidazole, 24% were resistant to clarithromycin, and 18% presented a transient resistance to amoxicillin. Resistance to two or more antimicrobials increased significantly from 1995 to 1997.

A community-based seroepidemiologic study of Helicobacter pylori infection in Mexico.

A nationwide community-based survey for Helicobacter pylori infection had not been done. This study sought to determine the seroprevalence of infection in Mexico, and the socioeconomic and demographic variables that are risk factors for infection. The survey assessed 11,605 sera from a sample population representing persons ages 1-90 years from all socioeconomic and demographic levels and from all regions of Mexico. Antibodies against H. pylori were studied by ELISA using whole cell antigen. Among the findings were that 66% of the population was infected and that age was the strongest risk factor for infection. By age 1 year, 20% were infected and by age 10 years, 50% were infected. Crowding (odds ratio [OR], 1.4), low educational level (OR, 2.42), and low socioeconomic level (OR, 1.43) were risk factors for infection. Prevalence was similar in urban and in rural communities (OR, 0.95). This study is the largest community-based seroepidemiologic study of H. pylori to date.

Validation of a serologic test for the diagnosis of Helicobacter pylori infection and the immune response to urease and CagA in children.

OBJECTIVE: Little is known about Helicobacter pylori infections and the immune response to urease and CagA in pediatric populations. Our aims were: 1) to validate serological assays for antibodies against whole cell extract, CagA, and urease of H. pylori; 2) to examine their role in diagnosis of infection in children with recurrent abdominal pain (RAP); and 3) to examine the antibody responses to CagA and urease in children. METHODS: An enzyme-linked immunosorbent assay (ELISA) for diagnosis of H. pylori infection using whole cell extracts was validated in 50 children with biopsy-confirmed infection. The IgG and IgA antibody responses against recombinant CagA and urease were compared by ELISA in 82 children with RAP and in 246 age- and sex-matched healthy children. RESULTS: The whole-cell extract ELISA had a sensitivity of 85 % and specificity of 87%. Children with RAP were more infected with H. pylori than were healthy control subjects; however, IgG and IgA CagA seropositivity was lower among those with RAP than among asymptomatic children (34% and 23% vs 76% and 55%, respectively; p < 0.0001). In both groups of children, the immune response to urease was low. CONCLUSION: A serodiagnosis of H. pylori infection using native strains was developed. The difference in the immune response between children with RAP and control subjects suggests that RAP occurs during the acute phase of H. pylori infection. Our results also suggest that urease is a poor immunogen.

Antisecretory activity in a lectin fraction of plasma from patients with acute diarrhea.

The present study describes the first attempt to detect antisecretory activity in a lectin fraction of plasma from patients with acute diarrhea. The plasma antisecretory protein (ASP) was purified by affinity chromatography in agarose, and its antisecretory activity tested in rats subjected to intestinal challenge with cholera toxin. During the first 24 h of the diarrheal episode, antisecretory activity in patients (median 0, range 0-25%) was lower than that seen in the asymptomatic group (median 10, range 0-30%); 3 days later, when diarrhea ceased in most of the patients, the ASP activity increased significantly (median 30, range 0-75%). However, 5 days later the activity decreased again (median 0, range 0-55%). No differences in ASP levels were found between cases associated with an enteropathogen and those where no pathogen was identified. These findings reveal an inverse relationship between the increase in ASP and the patient's intestinal secretion; suggesting that ASP plays a role in the compensatory mechanisms that occur in diarrhea in humans.

Sensitivity in culture of epithelial cells from rhesus monkey kidney and human colon carcinoma to toxins A and B from Clostridium difficile.

The effect of toxins A and B from Clostridium difficile on human colon carcinoma cells (HT-29, epithelial), rhesus monkey kidney cells (MA-104, epithelial) and green monkey kidney cells (VERO, fibroblast) was studied. Both toxins caused rounding of HT-29 cells and rounding with projections remaining attached to the substrate in MA-104 and VERO cells; however, the sensitivity to each toxin varies considerably. Toxin A was detected in ng by VERO, pg by HT-29 and fractions of pg by MA-104 cells; for toxin B, pg were detected by VERO, ng by MA-104 and micrograms by HT-29 cells. HT-29 cells were grown with galactose to allow their differentiation to enterocytes, and their sensitivity to the toxins during the process was studied. At early stages, the sensitivity to both toxins was similar, and as the differentiation proceeded, the response to both toxins decreased continuously, and after 16 days no evident morphological effect was observed, even with micrograms amounts of either toxin. In contrast to all cell lines reported to date, HT-29 and MA-104 epithelial cells are exquisitely sensitive to toxin A and less responsive to toxin B. The rounding of HT-29 by these toxins depends on the degree of differentiation of the cell.

Frequency of identification of cytotoxigenic strains of Escherichia coli in cases of diarrhea from rural and urban communities.

It has been suggested that strains of Escherichia coli producing Vero-Toxin (VTEC) may cause diarrhea or hemorrhagic colitis; however, there are not enough studies to support this hypothesis. We studied the frequency of isolation of VTEC strains in patients with acute diarrhea from rural and urban communities. A total of 1430 strains were analyzed, 361 coming from 118 patients from the rural community (Cadereyta, Qro.) and 1069 from the urban district (D.F.); 95 of these patients were asymptomatic, 213 suffered from watery diarrhea and 43 had bloody diarrhea. For production of toxins, strains were grown in tryptic soy broth for 24h and the culture supernatant was inoculated on HeLa cells; strains were considered cytotoxic when they caused lysis in at least 50% of the cells. In the rural community, VTEC strains were isolated in 20% of the asymptomatics, in 45% of the watery diarrhea patients and in 76% of patients with bloody diarrhea. Frequency of isolation was significantly higher in patients with diarrhea than in asymptomatics (P less than 0.05). The relative risk to present watery diarrhea was 3 and to present bloody diarrhea was 12. In the urban district, VTEC strains were isolated in 13, 7.9 and 4.5% from asymptomatics, watery diarrhea and bloody diarrhea patients, respectively; the relative risk for diarrhea was 1. Colonization by VTEC strains is significantly higher in patients from the rural community and these infected patients have an important risk to develop diarrhea.

Epidemiological aspects of Clostridium difficile in a pediatric hospital and its role in diarrheal disease.

The influence of antibiotics on the frequency of colonization by Clostridium difficile and the presence of its cytotoxin in infants and older children was examined to determine its role in diarrheal disease. Cytotoxin was more closely associated with cases of diarrhea, both in infants and in children than the microorganism, although not significantly. The isolates were typed by means of sensitivity to bacteriophages and bacteriocins and their cytotoxigenic potential was also determined. Less than 30% of the colonized patients had toxigenic strains. A study of strain variability over a four-year period in the same hospital showed that two bacteriophage-bacteriocin types and non-toxigenic strains predominated. The common presence of non-toxigenic strains could explain in part the lack of correlation between isolation of Clostridium difficile and diarrhea. Most of the non-toxigenic strains showed moderate resistance to tetracycline, a property which might explain their ability to persist for long periods in the hospital.