Comparison of three PCR methods for detection of Helicobacter pylori DNA and detection of cagA gene in gastric biopsy specimens

AIM:To comparatively evaluate PCR and other diagnostic methods (the rapid urease test and/or culture) in order to determine which of the three PCR methods (ureA,glmM and 26-kDa,SSA gene) was most appropriate in the diagnosis of Helicobacterpylori(Hpylori) infection and also to evaluate the detection of a putative virulence marker of H pylori,the cage,gene,by PCR in biopsy specimens. METHODS:One hundred and eighty-nine biopsy specimens were collected from 63 patients (three biopsies each) undergoing upper gastroduodenal endoscopy for various dyspeptic symptoms.The PCR methods used to detect H pylori DNA directly from biopsies were the glmM,26-kDa, ureA and then cagA was used to compare the culture technique and CLO for urease with the culture technique being used as the gold standard. RESULTS:Thirty-five percent of the biopsies were positive for H pylori DNA using the 3 PCR methods,while 68% of these were positive for the cagA gene.Twenty-four percent of the biopsies were negative for H pylori DNA in all PCR methods screened.The remaining 41% were either positive for ureA gene only,glmM only,26-kDa only,or ureA+glmM, ureA+26-kDa,glmM+26-kDa.Out of the 35% positive biopsies,41% and 82% were positive by culture and CLO respectively,while all negative biopsies were also negative by culture and cagA.Cag A+ infection was also predominantly found in H pylori DNA of the biopsies irrespective of the clinical diagnosis. CONCLUSION:This method is useful for correctly identifying infections caused by H pylori and can be easily applied in our laboratory for diagnostic purposes.

Assessment of co-segregated TLR4 genotypes among Nigerian children with asymptomatic and clinical malaria

Objective: To assess the occurrence and pattern of Toll-like receptor 4(TLR4) cosegregated genotypes among children with Plasmodium falciparum malaria in Nigeria.Methods: In this case-control study, a total of 79 Plasmodium falciparum infected children aged 2–7 years and 105 age-matched uninfected controls of Yoruba descents in Lagos were studied. The extracted DNA samples were used for TLR4 genotyping at codons 299(Asp > Gly) and 399(Thr > Ile) by PCR-restriction fragment length polymorphism. Malaria infection was diagnosed by blood smear microscopy and infected children were stratified into asymptomatic, uncomplicated and severe malaria sub-groups.Malnutrition was determined by measuring the mid upper arm circumference and anemia was defined as hemoglobin < 11 g/dL.Results: The proportions of children with acute malnutrition and severe anemia were12.0% and 3.2%, respectively. Parasitemia and malnutrition were not correlated and four distinct patterns of TLR4 genotypes were found in the study population: Asp299Asp/Thr399Thr(90.2%), Asp299Gly/Thr399Thr(4.3%), Gly299Gly/Thr399Thr(3.8%) and Asp299Gly/Thr399Ile(1.6%). These genotypes did not differ significantly(P > 0.05) in frequency between infected and non-infected children. However, low and high occurrences of the TLR4 Asp299Asp/Thr399 Thr and Asp299Gly/Thr399 Thr genotypes were observed in the severe malaria subgroup.Conclusions: This study reveals a protective role for TLR4 Asp299Gly/Thr399 Ile and Asp299Asp/Thr399 Thr genotypes against severe malaria in Nigerian children.