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.

Differential Expressions of Selected Activating and Inhibitory Receptors on K562-Stimulated Natural Killer (NK) Cells in HIV-1 and HIV-2 Infections

Context: The functional activity of NK cells depends on the balance between the engagement of activating and inhibitory receptors on the cell surface with their ligands, which enables them to kill infected cells. Objectives: The aim of this study was to evaluate and compare expressions of selected activating and inhibitory receptors on stimulated NK cells in HIV-1 and HIV-2 infections. Methods: PBMCs were analysed for activating (NKp30, NKp44, NKp46) and inhibitory (CD158a, CD158b, p70) receptor expressions in 30 HIV-1, 30 HIV-2 and 30 HIV uninfected healthy control (HC) subjects by flow cytometry after stimulating with K562 cells. Results: There was an expression of other receptors following an already in vitro engagement of NK cells with K562 cells. Higher expression of the activating receptors, NKp44 (p = 0.029) and NKp46 (p = 0.032) on NK cells from HIV-2 compared to HIV-1 infected individuals but similar NKp30 expression (p = 0.980). The levels of expression of inhibitory receptor CD158a were similar between HIV-1 and HIV-2 infected subjects (p = 0.309) but there was significant up-regulation of inhibitory receptors p70 (p = 0.010) and CD158b (p = 0.05) in HIV-1 compared to HIV-2 subjects. Conclusion: Despite the in vitro engagement of NK cells with stimulating K562 cells, our data showed differential expressions of other selected activating and inhibitory receptors in HIV-1 and HIV-2 infected subjects.

Higher Type 1 Interferon Levels in Plasma of Asymptomatic HIV-2 than in HIV-1 Individuals

A number of cytokines are secreted during HIV infection that enhances both innate and adaptive immune responses. Interferon-α/β/γ, IL-12, IL-15 and IL-18 have been found to contribute to the development, maturation, differentiation and function of NK and other immune cells. The levels of IFN-α/β/γ, IL-12, IL-15 and IL-18 were compared in the plasma of 90 HIV-1 infected and 90 HIV-2 infected subjects by ELISA or Cytometric Beads Array assays. The HIV-infected subjects were stratified according to CD4+ T cell counts into three groups: >500, 200 - 500 and <200 cells/ul, with 30 subjects in each group. Cytokine levels were also determined in the plasma of 50 HIV uninfected blood bank donors. Among the cytokines tested, IFN-α was found to be significantly increased in HIV-2 infected compared to HIV-1 infected subjects at high CD4+ T cell counts (p = 0.001). The levels of IFN-β were seen to differ between the two infections in patients from the category of medium CD4+ T cell counts: this was significantly increased in HIV-2 infected patients (p < 0.001) as well as compared to uninfected controls (p = 0.001). The levels of IFN-γ were similar at all the CD4+ T cell categories except for an increase in HIV-2 infected patients at low CD4+ T cell counts (p = 0.02). The levels of these cytokines were similar in all HIV-1 subjects. Also, the level of IL-12p70 was similar between the two infections but significantly higher in HIV-2 at low compared to medium CD4+ T cells categories (p = 0.047). Similar to IFN-γ and IL-12p70, the levels of both IL-18 and IL-15 were found to be significantly higher in HIV-2 infected patients compared to HIV-1 at low CD4+ T cell counts (p < 0.05). These data show that there is variability in the levels of innate cytokines at different stages of HIV infection but the finding of increased IFN-α in HIV-2 infected asymptomatic subjects is consistent with the high innate NK responses previously noted at this stage of infection.