Jaime Cok

Helicobacter pylori from Peruvian Amerindians: Traces of human migrations in strains from remote Amazon, and genome sequence of an Amerind strain

Background The gastric pathogen Helicobacter pylori is extraordinary in its genetic diversity, the differences between strains from well-separated human populations, and the range of diseases that infection promotes. Principal Findings Housekeeping gene sequences from H. pylori from residents of an Amerindian village in the Peruvian Amazon, Shimaa, were related to, but not intermingled with, those from Asia. This suggests descent of Shimaa strains from H. pylori that had infected the people who migrated from Asia into The Americas some 15,000+ years ago. In contrast, European type sequences predominated in strains from Amerindian Lima shantytown residents, but with some 12% Amerindian or East Asian-like admixture, which indicates displacement of ancestral purely Amerindian strains by those of hybrid or European ancestry. The genome of one Shimaa village strain, Shi470, was sequenced completely. Its SNP pattern was more Asian- than European-like genome-wide, indicating a purely Amerind ancestry. Among its unusual features were two cagA virulence genes, each distinct from those known from elsewhere; and a novel allele of gene hp0519, whose encoded protein is postulated to interact with host tissue. More generally, however, the Shi470 genome is similar in gene content and organization to those of strains from industrialized countries. Conclusions Our data indicate that Shimaa village H. pylori descend from Asian strains brought to The Americas many millennia ago; and that Amerind strains are less fit than, and were substantially displaced by, hybrid or European strains in less isolated communities. Genome comparisons of H. pylori from Amerindian and other communities should help elucidate evolutionary forces that have shaped pathogen populations in The Americas and worldwide.

Optimization and Evaluation of a PCR Assay for Detecting Toxoplasmic Encephalitis in Patients with AIDS

ABSTRACT Toxoplasma gondii is a common life-threatening opportunistic infection. We used experimental murine T. gondii infection to optimize the PCR for diagnostic use, define its sensitivity, and characterize the time course and tissue distribution of experimental toxoplasmosis. PCR conditions were adjusted until the assay reliably detected quantities of DNA derived from less than a single parasite. Forty-two mice were inoculated intraperitoneally with T. gondii tachyzoites and sacrificed from 6 to 72 h later. Examination of tissues with PCR and histology revealed progression of infection from blood to lung, heart, liver, and brain, with PCR consistently detecting parasites earlier than microscopy and with no false-positive results. We then evaluated the diagnostic value of this PCR assay in human patients. We studied cerebrospinal fluid and serum samples from 12 patients with AIDS and confirmed toxoplasmic encephalitis (defined as positive mouse inoculation and/or all of the Centers for Disease Control clinical diagnostic criteria), 12 human immunodeficiency virus-infected patients with suspected cerebral toxoplasmosis who had neither CDC diagnostic criteria nor positive mouse inoculation, 26 human immunodeficiency virus-infected patients with other opportunistic infections and no signs of cerebral toxoplasmosis, and 18 immunocompetent patients with neurocysticercosis. Eleven of the 12 patients with confirmed toxoplasmosis had positive PCR results in either blood or cerebrospinal fluid samples (6 of 9 blood samples and 8 of 12 cerebrospinal fluid samples). All samples from control patients were negative. This study demonstrates the high sensitivity, specificity, and clinical utility of PCR in the diagnosis of toxoplasmic encephalitis in a resource-poor setting.

Attenuated CagA Oncoprotein in Helicobacter pylori from Amerindians in Peruvian Amazon

Population genetic analyses of bacterial genes whose products interact with host tissues can give new understanding of infection and disease processes. Here we show that strains of the genetically diverse gastric pathogen Helicobacter pylori from Amerindians from the remote Peruvian Amazon contain novel alleles of cagA, a major virulence gene, and reveal distinctive properties of their encoded CagA proteins. CagA is injected into the gastric epithelium where it hijacks pleiotropic signaling pathways, helps Hp exploit its special gastric mucosal niche, and affects the risk that infection will result in overt gastroduodenal diseases including gastric cancer. The Amerindian CagA proteins contain unusual but functional tyrosine phosphorylation motifs and attenuated CRPIA motifs, which affect gastric epithelial proliferation, inflammation, and bacterial pathogenesis. Amerindian CagA proteins induced less production of IL-8 and cancer-associated Mucin 2 than did those of prototype Western or East Asian strains and behaved as dominant negative inhibitors of action of prototype CagA during mixed infection of Mongolian gerbils. We suggest that Amerindian cagA is of relatively low virulence, that this may have been selected in ancestral strains during infection of the people who migrated from Asia into the Americas many thousands of years ago, and that such attenuated CagA proteins could be useful therapeutically.

Socioeconomic and Nutritional Factors Account for the Association of Gastric Cancer with Amerindian Ancestry in a Latin American Admixed Population

Gastric cancer is one of the most lethal types of cancer and its incidence varies worldwide, with the Andean region of South America showing high incidence rates. We evaluated the genetic structure of the population from Lima (Peru) and performed a case-control genetic association study to test the contribution of African, European, or Native American ancestry to risk for gastric cancer, controlling for the effect of non-genetic factors. A wide set of socioeconomic, dietary, and clinic information was collected for each participant in the study and ancestry was estimated based on 103 ancestry informative markers. Although the urban population from Lima is usually considered as mestizo (i.e., admixed from Africans, Europeans, and Native Americans), we observed a high fraction of Native American ancestry (78.4% for the cases and 74.6% for the controls) and a very low African ancestry (<5%). We determined that higher Native American individual ancestry is associated with gastric cancer, but socioeconomic factors associated both with gastric cancer and Native American ethnicity account for this association. Therefore, the high incidence of gastric cancer in Peru does not seem to be related to susceptibility alleles common in this population. Instead, our result suggests a predominant role for ethnic-associated socioeconomic factors and disparities in access to health services. Since Native Americans are a neglected group in genomic studies, we suggest that the population from Lima and other large cities from Western South America with high Native American ancestry background may be convenient targets for epidemiological studies focused on this ethnic group.

Validation of String Test for Diagnosis of Helicobacter pylori Infections

ABSTRACT The method of recovering Helicobacter pylori DNA or viable cells absorbed on a string that a person has swallowed and that is retrieved an hour later (string test) should be a useful alternative to traditional analysis of cells or DNA obtained by endoscopy, which is invasive, uncomfortable, relatively costly, and ill-suited for community-based and pediatric studies. Here we assayed the sensitivity and validity of the string test versus conventional endoscopic biopsy for detecting and analyzing H. pylori infection. Forty-four people with gastric complaints were studied using both H. pylori culture and urease gene (ureB) PCR. H. pylori organisms cultured from strings and biopsy specimens from the same patients were fingerprinted by the randomly amplified polymorphic DNA (RAPD) method. Biopsy sections were also hematoxylin and eosin and silver stained for H. pylori detection. H. pylori was cultured from 80% of strings and detected by PCR from 91% of strings from participants whose biopsies had been H. pylori positive by culture, PCR, and/or histology. Strains recovered from strings and biopsy specimens yielded identical or closely related RAPD profiles in each of the 24 cases tested. We conclude that the string test is a useful method for H. pylori recovery and analysis when relatively noninvasive procedures are needed.

Detection of Soluble Antigen and DNA of Trypanosoma cruzi in Urine Is Independent of Renal Injury in the Guinea Pig Model

The diagnosis of Chagas disease in humans is generally limited to the detection of specific antibodies. Detection of T. cruzi antigens in urine has been reported previously, but is not used in the diagnosis. In this study, soluble T. cruzi antigens and DNA were detected in urine samples and were associated with kidney injury and systemic detection of the parasite. We used 72 guinea pigs infected with T. cruzi Y strain and 18 non-infected guinea pigs. Blood, kidney, heart and urine samples were collected during the acute phase and chronic phase. Urine samples were concentrated by ultrafiltration. Antigens were detected by Western Blot using a polyclonal antibody against trypomastigote excretory-secretory antigen (TESA). T. cruzi DNA was detected by PCR using primers 121/122 and TcZ1/TcZ2. Levels of T. cruzi DNA in blood, heart and kidney were determined by quantitative PCR. T. cruzi antigens (75 kDa, 80 kDa, 120 kDa, 150 kDa) were detected in the acute phase (67.5%) and the chronic phase (45%). Parasite DNA in urine was detected only in the acute phase (45%). Kidney injury was characterized by high levels of proteinuria, kidney injury molecule-1 (KIM-1) and urea, and some histopathological changes such as inflammation, necrosis, fibrosis and scarce parasites. The detection of antigens and DNA in urine was associated with the presence of parasite DNA in blood and heart and with high levels of parasite DNA in blood, but not with the presence of parasite in kidney or kidney injury. These results suggest that the detection of T. cruzi in urine could be improved to be a valuable method for the diagnosis of Chagas disease, particularly in congenital Chagas disease and in immunocompromised patients.

Interleukin-1 beta single-nucleotide polymorphism's C allele is associated with elevated risk of gastric cancer in helicobacter pylori-infected Peruvians

Particular alleles of the interleukin-1B (IL-1B) gene have been correlated with increased risk of atrophic gastritis and gastric cancer in the populations of East Asia and Europe. No such data exist from Peru, a developing country with a population genotypically different from others studied and with a high prevalence of Helicobacter pylori infection and gastric cancer. We conducted a case-control study comparing 334 hospitalized patients with atrophic gastritis or gastric cancer with 158 nonatrophic gastritis patients (controls). Conditional logistic regression analysis revealed that an increased risk of atrophic gastritis (odds ratio, 5.60) and gastric cancer (odds ratio, 2.36) was associated with the IL-1B-511 C allele. Our study is the first to establish this allele as a risk for these conditions. Given the high prevalence of H. pylori and recurrence rate after treatment, IL-1B-511 single-nucleotide polymorphism analysis may identify those individuals who would benefit most from robust H. pylori eradication efforts in Peru.

Soil-Transmitted Helminth Infections Are Associated With an Increase in Human Papillomavirus Prevalence and a T-Helper Type 2 Cytokine Signature in Cervical Fluids

BACKGROUND An ecological correlation between invasive cervical cancer incidence and burden of soil-transmitted helminths (STH) is hypothesized to explain the excess in detectable human papillomavirus (HPV) infection in Latin America, via a global T-helper type 2 (Th2)-biased mucosal immune response secondary to STH infection. METHODS The association between current STH infection and HPV prevalence was compared in regions of Peru where STH is or is not endemic. Adjusted prevalence ratios (PRs) with robust variance were estimated as an effect measure of STH infection on HPV prevalence in each study site. Soluble immune marker profiles in STH-infected and STH-uninfected women were compared using Spearman rank correlation with the Sidak correction. RESULTS Among women in the helminth-endemic region of the Peruvian Amazon, those with STH infection women had a 60% higher prevalence of HPV, compared with those without STH infection (PR, 1.6; 95% confidence interval, 1.0-2.7). Non-STH parasitic/protozoal infections in the non-STH-endemic population of Peru were not associated with HPV prevalence. In Iquitos, A Th2 immune profile was observed in cervical fluid from helminth-infected women but not helminth-uninfected women. CONCLUSIONS A proportion of the increased HPV prevalence at older ages observed in Latin America may be due to a population-level difference in the efficiency of immunological control of HPV across the lifespan due to endemic STH infection.

Early detection of gastric cancer using global, genome-wide and IRF4, ELMO1, CLIP4 and MSC DNA methylation in endoscopic biopsies

Clinically useful molecular tools to triage gastric cancer patients are not currently available. We aimed to develop a molecular tool to predict gastric cancer risk in endoscopy-driven biopsies obtained from high-risk gastric cancer clinics in low resource settings. We discovered and validated a DNA methylation biomarker panel in endoscopic samples obtained from 362 patients seen between 2004 and 2009 in three high-risk gastric cancer clinics in Lima, Perú, and validated it in 306 samples from the Cancer Genome Atlas project (“TCGA”). Global, epigenome wide and gene-specific DNA methylation analyses were used in a Phase I Biomarker Development Trial to identify a continuous biomarker panel that combines a Global DNA Methylation Index (GDMI) and promoter DNA methylation levels of IRF4, ELMO1, CLIP4 and MSC. We observed an inverse association between the GDMI and histological progression to gastric cancer, when comparing gastritis patients without metaplasia (mean = 5.74, 95% CI, 4.97−6.50), gastritis patients with metaplasia (mean = 4.81, 95% CI, 3.77−5.84), and gastric cancer cases (mean = 3.38, 95% CI, 2.82−3.94), respectively (p < 0.0001). Promoter methylation of IRF4 (p < 0.0001), ELMO1 (p < 0.0001), CLIP4 (p < 0.0001), and MSC (p < 0.0001), is also associated with increasing severity from gastritis with no metaplasia to gastritis with metaplasia and gastric cancer. Our findings suggest that IRF4, ELMO1, CLIP4 and MSC promoter methylation coupled with a GDMI>4 are useful molecular tools for gastric cancer risk stratification in endoscopic biopsies.

Colonoscopic Findings in Peruvian Patients with Chronic Diarrhea

Objective To report the colonoscopic and pathological findings in patients with chronic diarrhea from a gastroenterology unit during approximately 3 years in a general teaching hospital located in Lima-Peru. Materials and Methods Patients with chronic diarrhea as the motive for colonoscopy from March 2008 to December 2010 were selected from the colonoscopy report computerized database. Colonoscopic findings were registered. Biopsies taken during the procedure were prospectively reviewed. Results 226 patients were included, of which 162 (71.7%) had a colon biopsy available. The average age of the patients was 53.6±16.36. 85.8% of patients were reported to have a normal colon. 14.8% of patients were found to have a normal colonic mucosa or mucosal edema, 35.8% of patients had lymphocytic colitis and 28.4% had paucicelular colitis. Conclusions The majority of colonoscopies were reported with unremarkable macroscopic findings. Lymphocytic colitis was unusually frequent compared to previous reports.