Efficacy of clarithromycin-based triple therapy for treating Helicobacter pylori in Thai non-ulcer dyspeptic patients with clarithromycin-resistant strains.

BACKGROUND: Antibiotic resistance of H. pylori is problematic because it reduces the efficacy of eradication therapy. The objective of the present study was to assess the eradication rates of triple therapy against clarithromycin-sensitive and clarithromycin-resistant strains of H. pylori in Thai non-ulcer dyspeptic patients. MATERIAL AND METHOD: Patients who underwent upper gastrointestinal endoscopy at King Chulalongkorn Memorial Hospital between September 2002 and December 2003 were included. The patients who had positive urease test and culture were enrolled for antimicrobial resistance. Isolates were considered resistant when the MIC was more than 1 mcg/ml for clarithromycin. The patients received a combination of pantoprazole 40 mg BID, clarithromycin MR 1 gm OD, and amoxicillin 1 gm BID, for 7 days. Urea [14C] breath test was performed for evaluation of H. pylori eradication at least 1 month after treatment. RESULTS: Of the 470 patients, H. pylori were identified by positive rapid urease test in 282 patients (69.0%). Of these, cultures for H. pylori were achieved in 113 patients (54.6%) and E-tests for clarithromycin were successfully placed in 69 isolations. There were 29 males and 40 females, mean age was 38.7 +/- 13.3 years. Primary H. pylori resistance to clarithromycin was observed in 16 of 69 patients (23.2%). The eradication rates were 90.6% (48/53) and 56.3% (9/16) in patients with clarithromycin sensitive and clarithromycin resistant H. pylori strains, respectively (p = 0.002). CONCLUSION: The authors reported a high rate of clarithromycin resistant H.pylori isolates in Thailand. Pretreatment resistance to clarithromycin has a significant impact on treatment failure with clarithromycin-based regimen.

Searching for virulence Burkholderia pseudomallei genes by immunoscreening the lambda ZAPII expressed genomic library.

The lambdaZAP II expressed genomic library of B. pseudomallei was screened with pooled melioidosis serum preabsorbed with E. coli host cell. The positive clones were detected by using protein A-CDP-star chemiluminescence. All of 14 positive clones reacted with only the pooled absorbed melioidosis serum and not the pooled absorbed normal serum when tested with the plaque dot blot analysis. The expressed genes were detected by using a combination of immunoscreening, bioinformatics and molecular biology. At least six in vivo expressed genes were identified by this approach. Two were well known virulent genes, gmhA (a capsule biosynthetic gene) and bipD (type III secretion protein gene). Another two were genes coded for conserved hypothetical protein. The last two isolated genes were groEL (a chaperonine protein gene), and a gene encoding transmembrane protein.

Clinical spectrum of hepatic tuberculosis: comparison between immunocompetent and immunocompromised hosts.

BACKGROUND: Hepatic tuberculosis has been reported in normal and immunocompromised hosts. However, no published comparisons between these two groups of subjects with hepatic tuberculosis have been found. The aim of this study was to compare the clinical manifestations, biochemical tests, radiologic features and pathological findings of hepatic tuberculosis in immunocompromised and immunocompetent patients. METHOD: The authors reviewed retrospectively 20 patients with hepatic tuberculosis admitted between January 1993 and October 2000 to Chulalongkorn University Hospital, Thailand. There were 12 immunocompromised patients (10 HIV-infected males, 1 systemic lupus erythematosus (SLE) male, 1 SLE female) and 8 immunocompetent patients (6 males, 2 females). The clinical manifestations, biochemical tests, radiologic features and pathological findings were compared between these 2 groups. The diagnosis of Mycobacterium tuberculosis (M. tb) was the combination of a demonstrated organism in hemo- or specimen culture, histopathology (positive acid fast bacilli) and rapid identification of M. tb from nested polymerase chain reaction (nPCR) assay based on amplification of the IS 6110 insertion sequences. RESULTS: The clinical features were similar in both groups with fever, weight loss and hepatomegaly as the main manifestations. The biochemical findings were also similar but the alkaline phosphatase (ALP) was significantly higher in the immunocompromised group (p < 0.001). Hepatomegaly and diffuse increased echogenicity were common in both groups. Ascitis and calcifications were found more commonly in the immunocompetent subjects, although the differences were not statistically significant. Non-caseating granuloma without detection of acid fast bacilli was a common finding in both groups. The nested PCR assay increased the sensitivity from 49 per cent to 86 per cent compared to the regular PCR assay but specificity was 100 per cent in both techniques. The mortality was significantly higher in immunocompetent patients (p < 0.05) due to the extreme age and severe coexisting diseases. CONCLUSION: Fever, weight loss, hepatomegaly, disproportionate elevation of ALP and reverse A/G ratio were common in hepatic tuberculosis. A disproportionate elevation of ALP was significantly higher in the immunocompromised hosts. Nested PCR assay showed good sensitivity and specificity in the diagnosis of this disease.

Pattern of drug resistant Helicobacter pylori in dyspeptic patients in Thailand.

Emergence of drug resistant Helicobacter pylori (H. pylori) has occurred in various countries and could compromise the efficacy of current treatment regimens. The aim of the study was to identify the pattern of antibiotic resistant H. pylori in Thailand and evaluate various factors associated with drug resistance. Between June 2001 and December 2002, a total of 560 dyspeptic patients who underwent upper gastrointestinal endoscopy at King Chulalongkorn Memorial Hospital were included in this study. Antral gastric biopsies were obtained for H. pylori cultures and susceptibility tests using Epsilometer test (E-test). The value of antibiotic resistant breakpoints were amoxicillin 0.5 microg/ml, clarithromycin 1.0 microg/ml, metronidazole 8 microg/ml, and tetracycline 4 microg/ml, respectively. H. pylori were detected in 315 patients using the rapid urease test (56.25%). Cultures for H. pylori were positive in 172 patients. E-test for all four antibiotics was successfully placed in 79 isolations. The prevalence of antibiotic resistant H. pylori were amoxicillin 13.9 per cent (11/79), clarithromycin 19.0 per cent (15/79), metronidazole 30.4 per cent (24/79), tetracycline 5.1 per cent (4/79), and multi-drugs 16.5 per cent (13/79), respectively. However, age, sex, or endoscopic findings did not differ between the patients with H. pylori resistant strains and sensitive strains. The emergence of antibiotic and multi-drug resistant H. pylori in Thailand were relatively high and these could compromise the efficacy of current treatment regimens. The factors associated with drug resistant H. pylori could not be demonstrated in the present study. Further study in a larger number of patients might be necessary to identify factors associated with resistant H. pylori.

Inhibitory effect of Lactobacillus acidophilus on Helicobacter pylori in peptic ulcer patients: in vitro study.

Lactobacillus acidophilus (L. acidophilus) have been introduced into many fermented dairy products. The presence of L. acidophilus appears to decrease Helicobacter pylori (H. pylori) density in the human stomach and could enhance antibiotic therapy for H. pylori eradication. This study was designed to determine the optimal density of L. acidophilus that has a maximum inhibitory effect on H. pylori in peptic ulcer patients. To determine whether L. acidophilus has an inhibitory effect on H. pylori isolated from peptic ulcer patients and to determine whether the optimal density of L. acidophilus has a maximum inhibitory effect on H. pylori isolated from peptic ulcer patients. H. pylori was isolated from gastric biopsy specimens of peptic ulcer patients. The suspension of pure H. pylori colonies were inoculated into the broth and adjusted to match the density of No.3 MacFarland standard (approximately 9x10(8) cells/ml). Forty microliters of the suspension were equally spread onto each quadrant of the plate and left to dry. L. acidophilus was prepared from LC-1 (Nestle Research Center, Switzerland). They were grown on blood agar and incubated overnight at 37 degrees C. The suspension of L. acidophilus was inoculated into the broth and adjusted to match the density of No.1, No.2, No.3 and No.4 MacFarland standard (approximately 3x10(8), 6x10(8), 9x10(8) and 12x10(8) cells/ml respectively). Ten microliters of each density of L. acidophilus was dropped onto each quadrant of a previously inoculated H. pylori plate and then the plate was kept under microaerophilic conditions for 72 hours. Inhibition clear halo zone of H. pylori around the colonies of L. acidophilus was interpreted as the inhibitory effect. H. pylori were isolated from gastric biopsy specimens of fifteen peptic ulcer patients (eleven patients with gastric ulcer and four patients with duodenal ulcer). A total of sixty tests of inhibitory effect of L. acidophilus on H. pylori were evaluated in the present study. L. acidophilus had inhibitory effect on H. pylori in 13/15 patients (86.67%) and L. acidophilus in the density of No.3 MacFarland standard had a significantly higher inhibitory effect on H. pylori in the density of No.3 MacFarland than that of No.1 (60% vs 20%; p<0.05), No.2 (60% vs 20%; p<0.05) and No.4 (60% vs 20%; p<0.05) MacFarland standard. In conclusion, L. acidophilus has an inhibitory effect on H. pylori isolated from peptic ulcer patients. Approximately an equal density of L. acidophilus on H. pylori has the most favorable effect. This optimal density of L. acidophilus should have maximum effect on H. pylori clearance and could enhance antibiotic therapy for H. pylori eradication in humans.

Amplification of P1 gene by polymerase chain reaction for detection of Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a causative agent of human respiratory tract infection of which the clinical features are not significantly different from those of infections caused by other respiratory pathogens. The diagnosis is based principally on laboratory tests. Since conventional methods such as culture and serological tests are time-consuming, insensitive, and non-specific, polymerase chain reaction (PCR) was employed for laboratory diagnostics. This study was aimed to develop PCR method to detect M. pneumoniae by designing primers to amplify fragment of the P1 adhesin gene. Two protocols, PCR-probe hybridization and nested PCR, were carried out. False-positive result due to amplicon carry over was prevented by using dUTP instead of dTTP and the addition of enzyme uracil DNA glycosylase (UDG). For nested PCR, UDG was added only in the first round reaction mixture. The sensitivity of PCR was 10 fg of M, pneumoniae DNA as detected by agarose gel electrophoresis and increased to be 1 fg as detected by either probe hybridization or nested PCR. The specificity of PCR was tested with DNAs from Mycoplasma spp, a variety of different bacterial genera and human leukocyte. All gave negative results. Considering of the speed, sensitivity, specificity and the prevention of amplicon carryover, the developed PCR-based protocols were suitable and reliable for the detection of M. pneumoniae in routine laboratory.

Duodenal Helicobacter pylori associated duodenal ulcer depend on gastric Helicobacter pylori status.

Gastric Helicobacter pylori (H. pylon) plays an important role in the pathogenesis of duodenal ulcer (DU), although not all H. pylori infected persons will develop disease. Duodenal H. pylori was supposed to be one of the factors related with DU. The aim of this study was to investigate whether H. pylori in the duodenum of patients with DU plays a critical role in the pathogenesis of DU regarding the gastric H. pylori status. Furthermore, it was to determine the prevalence of duodenal H. pylori infection in Thailand. Ninety three patients were included in the study. They underwent gastroscopic evaluation for dyspeptic symptoms and none of them had previous H. pylori eradication therapy. An upper gastrointestinal endoscopy was performed and two specimens were collected each form the antrum, midcorpus and duodenal bulb in order to diagnose H. pylori infection. The gold standard for H. pylori detection is a positive specimen culture or polymerase chain reaction (PCR) assay for the vac A gene or positive urease test plus H. pylori seen in the pathology. Ninety three dyspeptic patients (43 males and 50 females; mean age 48.2 years; range 22 to 79 years) were included in the study. Duodenal H. pylori was detected in 31/93 (33.33%) patients which included 15 (48.38%) patients with duodenal ulcer, 2 (6.45%) patients with gastric ulcer and 14 (45.16%) patients with NUD. Five of thirty one (16.21%) patients with duodenal H. pylori infection had negative gastric H. pylori. These five patients included 1 with DU, 1 with DU and 3 with NUD. Duodenal H. pylori was associated with DU dependent of the presence of gastric H. pylori (p<0.05) and there was no association between duodenal H. pylori with negative gastric H. pylori and duodenal ulcer (p>0.05). Duodenal H. pylori is associated with duodenal ulcer dependent on the presence of gastric H. pylori. These results suggest that transmission of gastric H. pylori to the duodenum was prerequisited for the formation of DU.

Gastric juice urease test and brushing urease test for Helicobacter pylori detection.

The aim of the study was to demonstrate the sensitivity, specificity and accuracy of gastric juice urease test and brushing-urease test compared to the biopsy-urease test for Helicobacter pylori (H. pylori) detection. For each patient, two milliliters of gastric juice was collected and one milliliter in the supernatant was tested for rapid urease reactions. One gastric mucus brushing and two biopsies were taken from the body and the antrum. The brushing specimens were tested for rapid urease reaction by shaking the brush into the urea broth. The gold standards for diagnosing of H. pylori are positive H. pylori upon specimen culture or positive identification of H. pylori from polymerase chain reaction (PCR) assay using primer for vac A gene. Forty patients were enrolled in the study including ten patients with gastric ulcer, six patients with duodenal ulcer and twenty four patients with non-ulcer dyspepsia. Brushing-urease test and biopsy-urease test were not different sensitivity (87.50% vs 93.20%), specificity (100% vs 100%) and accuracy (90.25% vs 95.50%). The gastric juice urease test had a sensitivity of 65.25 per cent, specificity of 100 per cent and accuracy of 75 per cent for detecting of H. pylori infection. In conclusion, gastric juice urease test had low sensitivity in the diagnosis of H. pylori infection. Brushing-urease test is as accurate as biopsy-urease test in detecting H. pylori infection. However, the brushing method had lower gastric tissue injury than the biopsy and so should be used for detecting H. pylori infection in patients with coagulopathy.

A comparative study of diagnostic tests for tuberculous lymphadenitis: polymerase chain reaction vs histopathology and clinical diagnosis.

To compare diagnostic tests for tuberculous lymphadenitis by polymerase chain reaction (PCR) with histopathology and clinical diagnosis in sensitivity, specificity and predictive value. This retrospective analytic, single blind study was done at King Chulalongkorn Memorial Hospital. Paraffin-embedded specimens were classified into 2 groups. The study group contained 30 proved AFB positive paraffin-embedded specimens from patients who also had clinical diagnosis of tuberculosis and improved by antituberculous treatment. The control group contained 30 formalin-fixed, paraffin-embedded specimens of lymph node hyperplasia proved by histopathological and clinical review. All 60 specimens were slided, and systematically labeled and sent to PCR lab. Polymerase Chain Reaction method had sensitivity = 43.33 per cent, specificity = 100 per cent, positive predictive value = 100 per cent and negative predictive value = 63.83 per cent. The present findings revealed that the PCR results were related to the age of the paraffin-embedded tissues. No positive results were obtained from tissues kept since 1996. Positive results were obtained in 3/7 cases (42.86%), 2/3 (66.67%) and 8/10 cases (80%) from tissue of 1997, 1998 and 1999 respectively. Conclusion: Polymerase chain reaction has sufficient reliability best as a confirmatory diagnostic test for tuberculous lymphadenitis; however, it is not appropriate as a screening test.