Molecular diagnosis of Helicobacter pylori.

H. pylori infection can be diagnosed with many different tests. If the patient is undergoing endoscopy with gastric biopsies, culture and histology remain the diagnostic methods of choice. Indirect tests include rapid urease tests, urea breath tests, and serology. Molecular methods such as PCR offer marginal improvements when done on biopsy material, but has the advantage of being able to accurately identify H. pylori in areas outside the stomach where cultures usually fail. PCR can detect low numbers of organisms in gastric juice, bile, stool and oral secretions. Because of its high sensitivity it can also be used for epidemiologic investigations of environmental sources. However, the largest role for PCR may be in molecular fingerprinting. Arbitrary Primer PCR (RAPD) on the whole bacterial genome can reliably and accurately distinguish between isolates. PCR-based RFLP analysis can separate isolates based on restriction fragment sizes in a smaller amplified genome segment. REP-PCR can group isolates into clusters that appear to have different clinical expressions. These methods promise to shed new light on the transmission and pathogenicity of H. pylori.

PCR for the Detection of H. pylori in Gastric Juice Aspirates and Environmental Water Samples.

Helicobacter pylori is a curved Gram-negative bacillus that infects the human gastric mucosa. Most methods used to diagnose H. pylori require the collection of gastric samples by biopsy during gastroduodenoscopy. A positive culture or visualization of the organism by histologic examination of biopsy material can give a definitive diagnosis (1). To make a diagnosis without gastroduodenoscopy, indirect tests, such as ELISA or urea breath tests, must be relied on (2). The polymerase chain reaction (PCR) can be used to make a highly specific diagnosis from gastric juice aspirates or environmental samples.

Metronidazole and clarithromycin resistance in Helicobacter pylori determined by measuring MICs of antimicrobial agents in color indicator egg yolk agar in a miniwell format. The Gastrointestinal Physiology Working Group of Universidad Peruana Cayetano Heredia and the Johns Hopkins University.

Resistance of Helicobacter pylori to metronidazole often causes failure of commonly used combination drug treatment regimens. We determined the MICs of metronidazole and clarithromycin against 18 H. pylori strains from Peru using tetrazolium egg yolk (TEY) agar. The MIC results obtained by agar dilution with petri dishes were compared with the results found through a miniwell format. The results of the two protocols for measuring drug susceptibility differed by no more than 1 dilution in all cases. On TEY agar, bright-red H. pylori colonies were easy to identify against a yellow background. Sixty-one percent (11 of 18) of the strains were resistant to metronidazole (MIC, > or = 4 micrograms/ml) and 50% (9 of 18) were resistant to clarithromycin (MIC, > or = 0.125 micrograms/ml), whereas none (0 of 5) of the strains tested were resistant to tetracycline (MIC, > or = 1 micrograms/ml). Thus, the prevalence of metronidazole and clarithromycin resistance in Peru is higher than that in developed regions of the world. The miniwell plate with TEY agar allows easy H. pylori colony identification, requires about one-third less of the costly medium necessary for petri dish assaying, conserves space, and yields MICs equivalent to those with agar dilution in petri dishes.

Evaluation of three commercially available blood culture systems for cultivation of Helicobacter pylori.

Helicobacter pylori is unable to grow in regular blood culture systems, including the BACTEC (Johnston Laboratories), Septi-Chek (Hoffman-La Roche), and Bacto (Difco) systems. We tested three blood culture systems used for fastidious organisms: brucella broth with SPS and CO2 (Becton Dickinson), biphasic brain heart infusion agar or broth (Becton Dickinson), and supplemented peptone broth (Vacutainer). Blood culture bottles were inoculated with H. pylori and human blood and were then incubated by routine diagnostic laboratory procedures. All three blood culture systems were able to sustain the growth of H. pylori, but brucella broth had the highest CFU per milliliter after 72 h. We conclude that a diagnostic laboratory should be able to detect H. pylori bacteremia in a majority of cases by using brucella blood culture bottles.

Pathological significance and molecular characterization of the vacuolating toxin gene of Helicobacter pylori.

Some strains of Helicobacter pylori are known to produce an extracellular cytotoxin that causes vacuolization in various mammalian cells. In this study, we found that concentrated culture supernatants from four Helicobacter strains isolated from patients infected with the bacterium, but having normal gastric mucosa, lacked cytotoxic activity. We also show that a higher percentage of strains isolated from patients with polymorphonuclear leukocyte infiltration of gastric mucosa are toxin positive (78%) versus those isolated from patients lacking such infiltration (33%). In addition to examining the relationship between pathology and cytotoxic activity, we used the previously published N-terminal sequence of the protein to clone and characterize vacA, the structural gene encoding the cytotoxin. Briefly, three oligonucleotides capable of encoding the first nine amino acids corresponding to the sense strand and four oligonucleotides corresponding to the noncoding strand of the last seven known amino acids of the cytotoxin protein were made. They were used in all 12 possible combinations in 12 different PCR reactions, with DNA from a cytotoxin-positive strain as template. In four combinations, the expected 69-bp fragment was seen. The sequence of this 69-bp fragment confirmed that it encoded the known N-terminal sequence of the cytotoxin. This gene is capable of encoding a 136-kDa protein with a 33-amino-acid signal peptide, whereas the purified cytotoxin is only 87 kDa, suggesting processing in the C-terminal region of the protein. A single copy of the vacA gene encodes the cytotoxin in H. pylori. Consequently, the insertion of a kanamycin resistance marker in the vacA gene produced an isogenic mutant lacking the cytotoxic activity. This mutant provides genetic evidence that vacA encodes the cytotoxin. Sequence analysis of the DNA adjacent to the vacA gene demonstrated that this gene is next to a putative cysteinyl tRNA synthetase gene. From the sequence arrangement, we predict that there are no other genes transcribed together with vacA. We also show that five of seven cytotoxin-negative strains examined still carry the sequences encoding it whereas the other two have suffered a deletion of the vacA gene. We further show that in at least one cytotoxin-negative but vacA-positive strain (MO19), there are variations in the length of the vacA gene that could explain the cytotoxin-negative phenotype in this strain.

Safety and immunogenicity of an inactivated hepatitis A vaccine: effect of dose and vaccination schedule.

The safety and immunogenicity of three doses (360 ELISA units [EU], 720 EU, and 1440 EU) and four immunization schedules (0, 1, and 2; 0, 1, and 6; 0, 1, and 12; 0, 2, and 4 months) of an inactivated hepatitis A vaccine were investigated in 80 healthy adult volunteers. Adverse effects were mild and comparable to those in a control group given hepatitis B vaccine. Antibody levels presumed to be protective (> or = 20 mIU/mL) were achieved after one injection in 90% of vaccinees given 360 and 720 EU and in 100% given 1440 EU. After two injections, 100% of vaccinees seroconverted regardless of the dose of vaccine given. All vaccine schedules were highly immunogenic. The accelerated and high-dose schedule could be useful for people who need to be rapidly immunized. The 12-month schedule induced very high levels of anti-hepatitis A virus but had problems with compliance. The 0-, 2-, and 4-month schedule produced higher antibody levels than the conventional 0-, 1-, and 6-month schedule (P = .016). This suggests that the hepatitis A vaccine could be incorporated into existing pediatric vaccine schedules.

Delayed treatment of pulmonary blastomycosis causing vertebral osteomyelitis, paraspinal abscess, and spinal cord compression.

A 36-year-old woman with gallbladder disease had an incidental finding of asymptomatic cavitary lung infection with Blastomyces dermatitidis. No treatment was given initially, and 2 months later she presented with vertebral osteomyelitis, paraspinal abscess, and spinal cord compression due to dissemination of the fungus. The patient recovered following surgical debridement and treatment with 1 g of amphotericin B, followed by itraconazole 400 mg QD for 6 months. In spite of previous reports of the self limiting nature of primary pulmonary blastomycosis in the normal host, antifungal therapy may be needed in cases that do not resolve spontaneously within a short period of time, or if transient immunosuppression may be anticipated as may occur following surgery or after acquisition of other infections.

In vitro susceptibility of Helicobacter pylori to trospectomycin, pirlimycin (U-57930E), mirincamycin (U-24729A) and N-demethyl clindamycin (U-26767A).

The in vitro activity of trospectomycin, pirlimycin, mirincamycin and N-demethyl clindamycin was measured against 46 clinical isolates of Helicobacter pylori using an agar dilution technique. The MIC50 and MIC90 were 4 and 64 micrograms/ml for pirlimycin and N-demethyl clindamycin, and 32 and 128 micrograms/ml for mirincamycin, respectively. All 46 strains were sensitive to trospectomycin with an MIC50 of 8 micrograms/ml and an MIC90 of 16 micrograms/ml. Of seven strains with the highest trospectomycin MICs (8 or 16 micrograms/ml) 100% were found to be resistant to metronidazole. Among ten strains with low trospectomycin MICs (2 micrograms/ml or less) 100% were sensitive to metronidazole. Possible explantations for the apparent correlation between the MICs of the two drugs are discussed. Since all metronidazole resistant strains were sensitive to trospectomycin, this drug may be useful in treating infection with metronidazole resistant Helicobacter pylori.

Evaluation of QuickVue, a rapid enzyme immunoassay test for the detection of serum antibodies to Helicobacter pylori.

QuickVue is an enzyme immunoassay test for qualitative detection of serum immunoglobulin-G antibodies to Helicobacter pylori. We evaluated its ability to predict infection by H. pylori in 100 adult and 49 pediatric patients referred for gastric endoscopy. A patient was defined as infected with H. pylori if either culture or histology was positive. Of the 100 adult patients, 64 had H. pylori infection and QuickVue correctly identified 59 of the 64. Of 36 H. pylori-negative patients, 20 were correctly identified as negative by the test. In this sample of patients, QuickVue had a sensitivity of 92% and a specificity of 56%. In the 49 pediatric patients, QuickVue correctly identified nine of 11 infected cases and 34 of 38 noninfected patients. In this group, the sensitivity was 82% and the specificity was 89%. Overall the test had a sensitivity of 91% and a specificity of 73%. The positive predictive value was 77% and the negative predictive value was 89%.

Diagnosis of Helicobacter pylori infection by means of a polymerase chain reaction assay for gastric juice aspirates.

A polymerase chain reaction (PCR) assay for Helicobacter pylori was developed with use of primer sequences from the ureA structural gene coding for the small subunit of urease. The PCR amplification was 100% specific for H. pylori in tests with 40 stock isolates of this species and with 30 control organisms, including two species of urease-producing Helicobacter. Thirty-four dyspeptic patients were evaluated by culture and histologic assessment of antral biopsy samples as well as by PCR of gastric juice aspirates. In 26 of the 34 patients, infection with H. pylori was diagnosed by culture and histology. PCR correctly identified 25 of these 26 patients. All eight patients with negative cultures and histologic findings also had negative PCR results. In this group of patients, therefore, PCR had a sensitivity of 96% and a specificity of 100%. Thus PCR of gastric juice aspirates can be used to diagnose H. pylori infection. This information is important since gastric juice can be aspirated through a nasogastric tube without gastroduodenoscopy. In addition, since clinical samples can be collected at one institution and mailed to a laboratory at another without compromising the outcome of the test, diagnostic PCR is accessible even to those clinicians at whose institutions the technology required for the procedure is not available.

An in vitro adherence assay reveals that Helicobacter pylori exhibits cell lineage-specific tropism in the human gastric epithelium.

Helicobacter pylori is a microaerophilic bacterium found in the stomach of asymptomatic humans as well as patients with acid peptic disease and gastric adenocarcinoma. We have developed an in situ adherence assay to examine the cell lineage-specific nature of binding of this organism and to characterize the nature of cell surface receptors that recognize its adhesin. Fluorescein isothiocyanate-labeled H. pylori strains were bound to surface mucous cells present in the pit region of human and rat gastric units but not to mucous neck, parietal, or chief cell lineages present in the glandular domains of these units. Binding was abolished by proteinase K treatment of tissue sections and by pretreatment of the bacteria with bovine submaxillary gland mucin, a rich source of fucosylated and sialylated carbohydrates. Several lines of evidence suggest that binding to surface mucous cells is not dependent upon terminal nonsubstituted alpha 2,3- and alpha 2,6-linked sialic acids in the adhesin receptor: (i) binding was not inhibited by incubating H. pylori strains with sialylated glycoconjugates such as fetuin and free sialyllactose; (ii) immunohistochemical stainings using the sialic acid-specific Sambucus nigra and Maackia amurensis lectins and the cholera toxin B subunit did not detect any sialylated glycoconjugates in these epithelial cells; and (iii) binding was not sensitive to metaperiodate under conditions that selectively cleaved carbons 8 and 9 of terminal nonmodified sialic acids. A role for fucosylated epitopes in the glycoprotein(s) that mediate binding of H. pylori to surface mucous cells was suggested by the facts that this lineage coexpresses the adhesin receptor and major fucosylated histo-blood group antigens, that monoclonal antibodies specific for histo-blood group antigens H, B, and Leb block binding, and that the lectin Ulex europaeus type 1 agglutinin, which is specific for alpha-L-fucose, also bound to the same cells that bound the bacteria. Furthermore, human colostrum secretory IgA inhibited adhesion in a metaperiodate- and alpha-L-fucosidase-sensitive but neuraminidase-independent fashion. The in situ adherence assay should be useful in further characterizing the H. pylori adhesin and its receptor and for identifying therapeutically useful compounds that inhibit strain-specific and cell lineage-specific binding of this human pathogen.

Paracrystalline inclusions of a novel ferritin containing nonheme iron, produced by the human gastric pathogen Helicobacter pylori: evidence for a third class of ferritins.

An abundant 19.3-kDa Helicobacter pylori protein has been cloned, and the sequence is homologous with a ferritin-like protein produced by Escherichia coli K-12. Homologies are also present with a number of eucaryotic ferritins, as well as with the heme group-containing bacterioferritins. All amino acids involved in chelation of inorganic iron by ferritins from humans and other higher species are conserved in the H. pylori protein. Consistent with the structural data indicating an iron-binding function, E. coli overexpressing the H. pylori ferritin-like protein accumulates almost 10 times more nonheme iron than vector controls, and the iron-binding activity copurifies with the 19.3-kDa protein. Immunoelectron microscopy of H. pylori, as well as of E. coli overexpressing the H. pylori gene, demonstrates that the gene product has a cytoplasmic location where it forms paracrystalline inclusions. On the basis of these structural and functional data, we propose that the H. pylori gene product (termed Pfr) forms the basis for a second class of bacterial ferritins designed to store nonheme iron.

PCR-based RFLP analysis of DNA sequence diversity in the gastric pathogen Helicobacter pylori.

DNA sequence diversity among 60 independent isolates of the gastric pathogen Helicobacter pylori was assessed by testing for restriction fragment length polymorphisms (RFLPs) in several PCR-amplified gene segments. 18 Mbol and 27 HaeIII RFLPs were found in the 2.4 kb ureA-ureB (urease) segment from the 60 strains; this identified 44 separate groups, with each group containing one to four isolates. With one exception, each isolate not distinguished from the others by RFLPs in ureA-ureB was distinguished by Mbol digestion of the neighboring 1.7 kb ureC-ureD segment. The 1.5 kb flaA (flagellin) gene, which is not close to ure gene cluster, was also highly polymorphic. In contrast, isolates from initial and followup biopsies yielded identical restriction patterns in each of the three cases tested. The potential of this method for detecting population heterogeneity was tested by mixing DNAs from different strains before amplification: the arrays of restriction fragments obtained indicated co-amplification from both genomes in each of the five pairwise combinations tested. These results show that H. pylori is a very diverse species, that indicate PCR-based RFLP tests are almost as sensitive as arbitrary primer PCR (RAPD) tests, and suggest that such RFLP tests will be useful for direct analysis of H. pylori in biopsy and gastric juice specimens.

DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting.

The RAPD (or AP-PCR) DNA fingerprinting method was used to distinguish among clinical isolates of Helicobacter pylori, a bacterium whose long term carriage is associated with gastritis, peptic ulcers and gastric carcinomas. This method uses arbitrarily chosen oligonucleotides to prime DNA synthesis from genomic sites to which they are fortuitously matched, or almost matched. Most 10-nt primers with > or = 60% G + C yielded strain-specific arrays of up to 15 prominent fragments, as did most longer (> or = 17-nt) primers, whereas most 10-nt primers with 50% G+C did not. Each of 64 independent H. pylori isolates, 60 of which were from patients in the same hospital, was distinguishable with a single RAPD primer, which suggests a high level of DNA sequence diversity within this species. In contrast, isolates from initial and followup biopsies were indistinguishable in each of three cases tested.