In vitro effects of medium tonicity, nutrient concentration, and free chlorine content on Acanthamoeba.

BACKGROUND: The environment preferred by Acanthamoeba trophozoites and the mechanism by which the amebae enters the cornea are not yet fully understood. A better understanding of the pathogenesis of this disease may help with prevention and treatment. PURPOSE: To define the preferred environments for Acanthamoeba survival and proliferation in vitro by examining the effect of tonicity, nutrient concentration, and free chlorine content on Acanthamoeba. MATERIALS AND METHODS: Human corneal isolates of Acanthamoeba castellanii and Acanthamoeba polyphaga trophozoites were cultured at 22°C (room temperature) in PYG (peptone-yeast extract-glucose) medium. The effect of tonicity on amebae was determined by incubating trophozoites in sodium chloride solutions in concentrations ranging from 0% to 10% for 19 days. Two different sets of media were prepared-one with and the other without added nutrients. The tonicity varied from 50 to 3438 mOsm/L while the pH was maintained at 6.7-6.8. Aliquots were recovered to determine the number and morphologic type of the amebae. To test the effect of chlorine, Acanthamoeba trophozoites were incubated for 7 days in buffered solutions with free chlorine concentrations varying from 0 to 5 mg/L free chlorine at 22°C. The pH was maintained at 7.2 and the tonicity varied from 88 to 92 mOsm/L. Trophozoites were enumerated by hemocytometer. RESULTS: Low tonicity solutions (<300 mOsm/L) favored the trophozoite stage, but elevating tonicity encouraged encystment. Only 3.3-3.9% of the trophozoites remained in 10% NaCl, while 46-58% of the trophozoites were present in distilled water. Increasing osmolality yielded a smaller number of Acanthamoeba with a greater proportion of cysts. Nutrients improved the replication rate at lower concentrations, increased the number of trophozoites and reduced the percentage of cysts. Chlorine completely inhibited both species of Acanthamoeba at free chlorine levels of 5mg/L, while lesser concentrations were less inhibitory. CONCLUSIONS: Acanthamoeba prefer hypotonic environments. Nutrients merely slowed the conversion of trophozoites to cysts at higher tonicity levels. Chlorine concentrations less than 5 mg/L, ocular irritation level, did not effectively convert trophozoites into cysts. We conclude that contact lens patients should avoid hypotonic ocular exposures, especially tap water and stagnant media such as lake water, and water from poorly maintained swimming pools and hot-tubs.

Addition of gentamicin to endoscopic retrograde cholangiopancreatography (ERCP) contrast medium towards reducing the frequency of septic complications of ERCP.

OBJECTIVE: Bacteremia and sepsis are serious complications of endoscopic retrograde cholangiopancreatography (ERCP) and occur in between 0.5 and 3% of cases. Patients with obstructed bile ducts are at highest risk of developing septic complications. The purpose of this investigation was to determine whether the addition of gentamicin to the ERCP contrast medium prevents or reduces the growth of Pseudomonas aeruginosa in vitro. METHODS: Artificial bile ducts were fashioned out of dialysis tubing and suspended in flasks containing brain heart infusion (BHI) broth. The tubing contained BHI broth alone, with or without contrast medium or with contrast medium plus gentamicin. The artificial ducts were inoculated with gentamicin-sensitive or gentamicin-resistant P. aeruginosa and quantitative cultures were performed. RESULTS: The contrast medium alone was bacteriostatic to both sensitive and resistant P. aeruginosa isolates. The addition of gentamicin to the contrast medium eliminated the sensitive strain after 2 h and resulted in a reduction in the number of gentamicin-resistant P. aeruginosa after 4 h. Incubation of the resistant isolate in the presence of contrast and gentamicin for an additional 4 h led to a further reduction in viable bacteria but did not completely eliminate the organisms. CONCLUSION: These results support the use of gentamicin in the contrast medium injected into the biliary system as an ancillary method to prevent post-ERCP sepsis.

Disinfection of endoscopes from Helicobacter pylori-positive subjects: evaluation of the effectiveness of the Chinese Calijing disinfection kit.

BACKGROUND: The aim of this study was to evaluate the effectiveness of the Calijing disinfection kit (an endoscope disinfection method used in Chinese hospitals) in eradicating Helicobacter pylori and assess whether use of the kit in 1994 during endoscopies in the Shandong Intervention Trial (SIT), Shandong, China, could have resulted in iatrogenic transmission of H pylori . METHODS: Bacterial culture studies at the Veterans Affairs Medical Center, Houston, Texas, using endoscopes and forceps from 49 H pylori -positive patients were performed on contaminated endoscopes before and after disinfection with the Calijing kit. RESULTS: At least 1 endoscope culture site was H pylori positive in 39 of 49 (79.6%) specimens predisinfection, whereas H pylori was not isolated from any endoscopic culture site postdisinfection. Non- H pylori bacteria and fungi were recovered from 22.6% of the postdisinfection cultures. CONCLUSION: Although no viable H pylori were recovered following the disinfection procedures, levels of H pylori below the detection threshold of the bacteriologic assay may have contributed to an increase in H pylori seroprevalence noted in the SIT. In addition, the kit was unable to provide disinfection against non- H pylori organisms, suggesting the need to adhere to internationally accepted disinfection procedures for endoscope reprocessing.

B-cell and T-cell immune responses to experimental Helicobacter pylori infection in humans.

The acute antibody and T-cell immune response to Helicobacter pylori infection in humans has not been studied systematically. Serum from H. pylori-naive volunteers challenged with H. pylori and cured after 4 or 12 weeks was tested by enzyme-linked immunosorbent assays for anti-H. pylori-specific immunoglobulin M (IgM) and IgA established using bacterial lysates from homologous (the infecting strain) and heterologous H. pylori. Proteins recognized by IgM antibody were identified by mass spectrometry of immunoreactive bands separated by two-dimensional gel electrophoresis. Mucosal T-cell subsets (CD4, CD8, CD3, and CD30 cells) were assessed by immunohistochemistry. All 18 infected volunteers developed H. pylori-specific IgM responses to both homologous or heterologous H. pylori antigens. H. pylori antigens reacted with IgM antibody at 4 weeks postinfection. IgM Western blotting showed immunoreactivity of postinfection serum samples to multiple H. pylori proteins with molecular weights ranging between 9,000 (9K) to 150K with homologous strains but only a 70K band using heterologous antigens. Two-dimensional electrophoresis demonstrated that production of H. pylori-specific IgM antibodies was elicited by H. pylori flagellins A and B, urease B, ABC transporter binding protein, heat shock protein 70 (DnaK), and alkyl hydroperoxide reductase. Mucosal CD3, CD4, and CD8 T-cell numbers increased following infection. IgM antibody responses were detected to a range of homologous H. pylori antigens 2 to 4 weeks postchallenge. The majority of H. pylori proteins were those involved in motility and colonization and may represent targets for vaccine development.

Eradication of Helicobacter pylori by 7-day triple-therapy regimens combining pantoprazole with clarithromycin, metronidazole, or amoxicillin in patients with peptic ulcer disease: results of two double-blind, randomized studies.

AIM: To compare the short-term (7-day) safety and efficacy of two triple-therapy regimens using pantoprazole with those of two dual-therapy regimens (one with pantoprazole and one without), for Helicobacter pylori eradication in patients with peptic ulcer disease. METHODS: H. pylori infection was identified by rapid urease (CLOtest), and confirmed by histology and culture. Patients were enrolled into one of two randomized, double-blind, multicenter, parallel-group studies. In study A, patients received oral pantoprazole 40 mg, clarithromycin 500 mg, and metronidazole 500 mg (PCM); pantoprazole, clarithromycin and amoxicillin 1000 mg (PCA); or pantoprazole and clarithromycin (PC). In study B, patients received PCM, PCA, PC, or clarithromycin and metronidazole without pantoprazole (CM). Treatments were given twice daily for 7 days. H. pylori status after therapy was assessed by histology and culture at 4 weeks after completing the course of study treatment. Modified intent-to-treat (MITT; each study: n = 424, n = 512) and per-protocol (PP; each study: n = 371, n = 454) populations were analyzed. The MITT population comprised all patients whose positive H. pylori status was confirmed by culture and histology; the PP population comprised patients who also complied with > or = 85% of study medication doses. RESULTS: A total of 1016 patients were enrolled. Cure rates among patients with clarithromycin-susceptible H. pylori strains were 82 and 86% for PCM, and 72 and 71% for PCA, in studies A and B, respectively. Cure rates among patients with metronidazole-susceptible H. pylori strains were 82 and 87% for PCM, and 71 and 69% for PCA, in studies A and B, respectively. The combined eradication rates observed with the PCM regimen were superior to those of all other regimens tested. Side-effects were infrequent and mild. CONCLUSIONS: PCM had the highest overall eradication rate in these two studies examining 7-day treatment regimens. All regimens were safe and well tolerated.

Enterococcus gastritis.

Helicobacter pylori infection is the most common cause of gastritis with its associated sequelae. Gastritis secondary to other bacteria is rare. This report describes Enterococcus-associated gastritis in a 59-year-old diabetic man. Nine months after receiving treatment for H. pylori-associated gastritis, he underwent endoscopy to confirm H. pylori eradication and to evaluate the status of previously seen ulcers. Mucosal biopsy specimens revealed severe active but focal gastritis adjacent to gram-positive coccobacilli in short to long chains with no H. pylori. Culture grew an Enterococcus similar to E. hirae and E. durans. No treatment was given, and endoscopy done 2 months later showed complete resolution of the gastritis and absence of H. pylori or enterococci. Our patient's gastritis represents a previously undescribed manifestation of Enterococcus infection. It is possible that the presence of NSAID gastric mucosal injury and diabetes predisposed this individual to the development of transient Enterococcus gastritis.

Studies regarding the mechanism of false negative urea breath tests with proton pump inhibitors.

OBJECTIVE: The mechanism of false negative urea breath tests (UBTs) results among proton pump inhibitor (PPI) users is unknown. We studied the time course of PPI-associated negative UBT, the relation to Helicobacter pylori density, and whether gastric acidification would prevent false negative UBT results. METHOD: In the UBT experiment, H. pylori-infected volunteers received omeprazole 20 mg b.i.d. for 13.5 days. UBTs with citric acid were done before, after 6.5 days of PPI, and 1, 2, 4, 7, and 14 days after therapy. In the culture and histology experiment, after a wash-out of >5 months, nine of the original subjects were rechallenged with omeprazole for 6.5 days. Antral and corpus biopsies for histology and culture were done before and 1 day after PPI administration. RESULTS: Thirty subjects (mean age 42 yr) were enrolled. UBTs were significantly reduced on day 6.5 (p = 0.031); 10 subjects (33%) developed transient negative UBTs. The UBT recovered in all but one subject by the fourth day post-PPI and in all subjects by day 14. In the culture and histology experiment, upon PPI rechallenge, three of nine subjects (33%) had negative UBTs. H. pylori density, whether measured by culture or histology, decreased with PPI therapy; antral biopsies became histologically negative in five subjects and corpus biopsies in three subjects. CONCLUSION: PPI-induced negative UBT results were related to the anti-H. pylori effect of the PPI. Acidification of the stomach did not prevent false negative UBT results. Three days is likely the minimum delay from stopping PPI until one should perform a test for active infection. A delay of 14 days is preferred.

Bismuth-based quadruple therapy using a single capsule of bismuth biskalcitrate, metronidazole, and tetracycline given with omeprazole versus omeprazole, amoxicillin, and clarithromycin for eradication of Helicobacter pylori in duodenal ulcer patients: a prospective, randomized, multicenter, North American trial.

OBJECTIVES: This multicenter, randomized, active-controlled trial assessed efficacy of bismuth-based quadruple therapy with omeprazole, bismuth biskalcitrate, metronidazole, and tetracycline (OBMT) using a single-triple capsule of BMT compared with triple therapy with omeprazole, amoxicillin, and clarithromycin (OAC) in treatment of patients with Helicobacter pylori infection and duodenal ulcers. METHODS: Patients with active duodenal ulcer or diagnosed within the past 5 yr and with infection documented by (13)C-urea breath test plus histology or culture were randomly assigned to 10-day course of OBMT using a single-triple capsule containing bismuth biskalcitrate 140 mg, metronidazole 125 mg, and tetracycline 125 mg given as three capsules q.i.d. with omeprazole 20 mg b.i.d., or a 10-day course of OAC, omeprazole 20 mg plus amoxicillin 1 g plus clarithromycin 500 mg, all b.i.d. Eradication was confirmed by two negative urea breath tests at >1 month and >2 months after therapy. RESULTS: One hundred thirty-eight patients received OBMT and 137 OAC. Modified intent-to-treat eradication rates were 87.7% for OBMT and 83.2% for OAC (95% CI = -3.9%-12.8%; p = 0.29). OBMT eradicated 91.7% metronidazole-sensitive and 80.4% metronidazole-resistant strains (p = 0.06). OAC eradicated 92.1% clarithromycin sensitive and 21.4% clarithromycin-resistant strains (p < 0.001). Adverse events occurred in 58.5% of OBMT patients and 59.0% of OAC patients. CONCLUSIONS: OBMT regimen using the single-triple capsule is as efficacious and well-tolerated as the widely used OAC regimen for H. pylori eradication. This OBMT therapy largely overcomes H. pylori metronidazole resistance, present in 40% of patients in this study.

New serological assay for detection of putative Helicobacter pylori virulence factors.

We evaluated a new immunoblot assay (Helicoblot 2.1) for Helicobacter pylori infection and CagA and VacA status by using serum samples from 222 patients. The test accurately detected H. pylori infection and VacA status, but improvements in the interpretation criteria are required before it can be recommended for determination of CagA status.

Helicobacter pylori infection in Kazakhstan: effect of water source and household hygiene.

The epidemiology of Helicobacter pylori infection and risk factors associated with its transmission are not well understood. Kazakhstan is country with two ethnic groups, Asian (Kazakhs) and Western (Russians), living under similar socioeconomic conditions. The aim of this study was to examine the seroepidemiologic pattern of H. pylori and hepatitis A among the same individuals from both ethnic groups, with emphasis on water source and household sanitation practices. This was a cross-sectional seroepidemiologic study conducted among unrelated healthy individuals in Kazakhstan. From May through August 1999, individuals between the ages of 10 and 60 years from Almaty, Kazakhstan, were invited to participate. Demographic information, socioeconomic factors, living conditions, and various aspects of the local household environment including access to water were collected. A clean water index (CWI) was created based on combined factors, consistency of boiling water before drinking, frequency of storing and reusing water, and frequency of bathing and showering. H. pylori and hepatitis A antibodies were assessed by enzyme-linked immunosorbent assay. Two hundred eighty-eight individuals between the ages of 10 and 60 years participated. The prevalence of H. pylori infection was almost identical among the two ethnic groups (Russians 79% and Kazakhs 80%). H. pylori infection was inversely correlated with the CWI (i.e., 56%, 79%, and 95% for high, middle, and low, respectively (P < .05). Drinking river water had highest risk of H. pylori infection (OR = 13.6, 95% CI = 1.8-102.4; P < .01, compared with tap water). Crowding showed no significant effect on H. pylori prevalence. Anti-HAV antibodies were found in 86% of the population, 90% among the Russians versus 82% among the Kazakhs (OR = 1.8, 95% CI = 1.1-3.8, P = .05). Although the two infections were highly correlated (P < .001), antibody to both infections were present simultaneously in only 74%. The prevalence of H. pylori infection in Kazakhstan is very high. The data suggest that transmission of H. pylori can be water borne, related to poor sanitary practices, or both. The high prevalence of antibodies to H. pylori and HAV among this population is a marker for poor sanitation and hygienic practices. Reducing the rate of H. pylori transmission will require improvements in overall sanitation including clean water, waste disposal, as well as in household hygienic practices.

Real-time quantitative PCR for detection of Helicobacter pylori.

Helicobacter pylori is one of the most common chronic infections in humans, in whom it is a key etiological factor in peptic ulcer disease, gastric mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma. Humans are the bacterium's only host. Here we report the development of a real-time quantitative (Q) PCR-based assay to measure ureC gene copy number to detect H. pylori, based on the fact that there is only one copy of the ureC gene per bacterium. Upon optimization of LightCycler Q-PCR conditions, we obtained a standard curve with a linear range (correlation coefficient = 1) across six logs of DNA concentration. We were able to accurately quantify as few as 1,000 bacteria in our assay. Analysis of variance on 15 randomly selected clinical samples showed good reproducibility of this assay. Comparison of Q-PCR results with bacterial culture and histopathological results from an additional 85 clinical biopsy samples showed a significant difference for the presence of H. pylori. Many samples that were negative for H. pylori by culture and histopathology were positive by Q-PCR. Contamination of PCR by H. pylori or H. pylori genetic material could not be ruled out. In summary, we developed a rapid, sensitive, and real-time Q-PCR method for detecting H. pylori. This technique offers a significant improvement over other available methods for detecting H. pylori in clinical and research samples.

Clinical presentation in relation to diversity within the Helicobacter pylori cag pathogenicity island.

OBJECTIVE: This study investigated the genetic diversity of the cag pathogenicity island (PAI) in Helicobacter pylori (H. pylori) in relation to clinical outcome and interleukin (IL)-8 production. METHODS: Seven genes in the cag PAI (cagA, cagE, cagG, cagM, cagT, open reading frame 13 and 10) were examined by polymerase chain reaction and Southern blot hybridization using H. pylori from 120 patients with different presentations (duodenal ulcer, gastric cancer, gastritis alone). IL-8 production from AGS cells (gastric cancer cell line) cocultured with H. pylori was measured by ELISA. RESULTS: An intact cag PAI was present in 104 (87%) isolates, and five (4%) had deletions within the cag PAI; 11 (9%) lacked the entire cag PAI. Clinical isolates containing the complete cag PAI induced a greater secretion of IL-8 as compared with those without the cag PAI (3048 +/- 263 vs 480 +/- 28 pg/ml, p < 0.001). Deletion of only cagG reduced IL-8 secretion by two thirds. Deletions of more than one locus reduced IL-8 secretion to background. A similar proportion of H. pylori from patients with gastritis, duodenal ulcer, or gastric cancer had intact cag PAI (88%, 88%, and 85%, respectively). Although the presence of cagG was a better predictor of the presence of an intact cag PAI than cagA or cagE, the presence or absence of any of these genes had no association with clinical presentation. CONCLUSION: Although the cag PAI plays an important role in IL-8 production, clinical presentation cannot be predicted by the presence of an intact cag PAI or any of these seven cag PAI genes.

Importance of Helicobacter pylori oipA in clinical presentation, gastric inflammation, and mucosal interleukin 8 production.

BACKGROUND & AIMS: Disease-associated virulence factors of Helicobacter pylori may not be independent of one another. The aim was to determine which H. pylori virulence factor(s) was the most important predictor of severity of gastric inflammation or clinical outcome. METHODS: cag Pathogenicity island (PAI), vacA babA2, and iceA status were determined by polymerase chain reaction (PCR). oipA functionality was based on switch status determined by PCR-based sequencing. A backward stepwise multiple regression analysis was performed to determine which factor(s) was the most discriminating for clinical outcome as well as the relationship to mucosal histology (H. pylori density, neutrophil infiltration, intestinal metaplasia, and gastric atrophy) and mucosal interleukin 8 (IL-8) production. RESULTS: H. pylori were obtained from 247 patients (86 with gastritis, 86 with duodenal ulcer, and 75 with gastric carcinoma). Although oipA status was closely linked to specific cag PAI, vacA, and babA2 genotypes, only oipA status remained in the final model to discriminate duodenal ulcer from gastritis (adjusted odds ratio [OR] = 5 and 95% confidence interval [CI] = 2.1-11.9). Among the factors, only a functional oipA was significantly associated with high H. pylori density, severe neutrophil infiltration, and high mucosal IL-8 levels (P < 0.001). oipA status had no relationship to gastric atrophic changes. CONCLUSIONS: oipA functional status was related to clinical presentation, H. pylori density, and gastric inflammation. cag PAI, babA2, or vacA status appear important only as surrogate markers for a functional oipA gene.

Helicobacter pylori in North and South America before Columbus.

We present a molecular epidemiologic study, based on an analysis of vacA, cagA and cag right end junction genotypes from 1042 Helicobacter pylori isolates, suggesting that H. pylori was present in the New World before Columbus. Eight Native Colombian and Alaskan strains possessed novel vacA and/or cagA gene structures and were more closely related to East Asian than to non-Asian H. pylori. Some Native Alaskan strains appear to have originated in Central Asia and to have arrived after strains found in South America suggesting that H. pylori crossed the Bering Strait from Asia to the New World at different times.

Discrimination between cases of duodenal ulcer and gastritis on the basis of putative virulence factors of Helicobacter pylori.

The BabA, cagA, and vacA statuses of 827 Helicobacter pylori isolates were used in logistic regression models to discriminate duodenal ulcer from gastritis. Only BabA was a candidate for a universal virulence factor, but the low c statistic value (0.581) indicates that none of these factors were helpful in predicting the clinical presentation.