Newborn screening blood spot analysis in the UK: influence of spot size, punch location and haematocrit.

OBJECTIVE: In dried blood spot analysis, punch location and variations in applied sample volume and haematocrit can produce different measured concentrations of analytes. We investigated the magnitude of these effects in newborn screening in the UK. METHODS: Heparinized blood spiked with thyroid stimulating hormone (TSH), phenylalanine, tyrosine, leucine, methionine, octanoyl carnitine (C8), and immunoreactive trypsinogen (IRT) was spotted onto filter paper: (i) at a constant haematocrit of 50% at various volumes, and (ii) at a range of haematocrits using a constant volume. Subpunches (3.2 mm) of the dried blood spots were then analysed. RESULTS: Compared with a central punch from a 50 µL blood spot with 50% haematocrit, 10 µL spots can have significantly lower measured concentrations of all analytes, with decreases of 15% or more observed for leucine, methionine, phenylalanine, and tyrosine. Punching at the edge of a spot can increase measured concentrations up to 35%. Higher haematocrit decreased measured TSH and C8 yet increased amino acids and IRT by 15% compared with 50% haematocrit. Lower haematocrits had the opposite effect, but only with higher concentrations of some analytes. CONCLUSIONS: Differences in blood spot size, haematocrit and punch location substantially affect measured concentrations for analytes used in the UK newborn screening programme, and this could affect false positive and negative rates. To minimize analytical bias, these variables should be controlled or adjusted for where possible.

Significantly higher frequency of Helicobacter suis in patients with idiopathic parkinsonism than in control patients.

BACKGROUND: There is increased proportional mortality from Parkinson's disease amongst livestock farmers. The hypokinesia of Parkinson's disease has been linked to Helicobacter pylori. H. suis is the most common zoonotic helicobacter in man. AIM: To compare the frequency of H. suis, relative to H. pylori, in gastric biopsies of patients with idiopathic parkinsonism (IP) and controls from gastroenterology services. METHODS: DNA extracts, archived at a Helicobacter Reference Laboratory, from IP patient and gastroenterology service biopsies were examined anonymously for H. suis, using species-specific RT-PCR. RESULTS: Relative risk of having H. suis in 60 IP patients compared with 256 controls was 10 times greater than that of having H. pylori. In patients with IP and controls, respectively, frequencies of H. suis were 27 (exact binomial 95% C.I. 15, 38) and 2 (0, 3)%, and of H. pylori, 28 (17, 40) and 16 (12, 21)%. Excess of H. suis in IP held when only the antral or corporal biopsy was considered. Of 16 IP patients with H. suis, 11 were from 19 with proven H. pylori eradication, 3 from 17 pre-H. pylori eradication, 2 from 24 H. pylori culture/PCR-negative. Frequency was different between groups (P = 0.001), greatest where H. pylori had been eradicated. Even without known exposure to anti-H. pylori therapy, H. suis was more frequent in IP patients (5/41) than in controls (1/155) (P = 0.002). Partial multilocus sequence typing confirmed that strains from IP patients (6) and control (1) differed from RT-PCR standard strain. CONCLUSIONS: Greater frequency of H. suis in idiopathic parkinsonism appears exaggerated following H. pylori eradication. Multilocus sequence testing comparison with porcine strains may clarify whether transmission is from pigs/porcine products or of human-adapted, H. suis-like, bacteria.

Percutaneous transhepatic self-expanding metal stents for palliation of malignant biliary obstruction.

BACKGROUND: Malignant biliary obstruction is often inoperable at presentation and has a poor prognosis. Percutaneously placed self-expanding metal stents (SEMS) have been widely used for palliation of malignant biliary obstruction as an alternative to major bypass surgery or when endoscopic drainage is not technically feasible. The success rate, procedural complications and outcomes in patients who underwent placement of SEMS in a tertiary referral centre are presented. METHODS: All patients who had percutaneous transhepatic cholangiography (PTC) and SEMS for palliation of malignant biliary obstruction between May 2008 and July 2010 at Groote Schuur Hospital, Cape Town, were reviewed. A retrospective chart review was undertaken using multidisciplinary case notes of all patients. The data analysed included demographic information, diagnosis, level of biliary obstruction, number and type of procedures, efficacy and complications of SEMS insertion. Boston Scientific 69 mm by 10 mm Wallstent SEMS were used in all patients. RESULTS; Fifty patients (28 men, 22 women, mean age 61 years, range 48 - 80 years) underwent percutaneous SEMS placement. Twenty-one patients had biliary obstruction at the level of the hilum involving the hepatic duct bifurcation, 5 in the mid-common bile duct and 24 in the low common bile duct. In 20 patients (40%) SEMS were placed at the time of initial biliary drainage (one-stage procedure), while the remaining 30 patients underwent stent placement within 2 - 23 days of biliary drainage as a two-stage procedure because of difficult access through the lesion during the initial procedure. Five patients (10%) required bilateral SEMS insertion. Stent placement was successful in all patients and biliary obstruction was relieved in all. The mean serum bilirubin level decreased by a mean of 56% from 294 µmol/l to 129 µmol/l measured 5 days after stent insertion. Mean hospital stay after stent insertion was 4.1 days. The average length of hospital stay for patients who underwent a one-stage procedure was 3.2 days (range 1 - 11 days), and for patients who underwent a two-stage procedure 7.6 days (range 3 - 23 days). Nine patients (18%) developed a procedure-related complication, which included cholangitis after stent insertion (n=4), cholangitic liver abscesses (n=1), subphrenic liver collection (n=1), bile leakage (n=1) and cholecystitis (n=2). Three patients (6%) developed complications unrelated to SEMS insertion, which included myocardial ischaemia (n=2) and pneumonia (n=1). Stent occlusion occurred in 4 patients (8%) within a week as result of stent migration (n=3) or presumed biliary sludge (n=1); 2 (4%) stents occluded between 7 days and 1 month. Four patients (8%) died during hospital admission due to pre-existing biliary sepsis (n=3) and pneumonia (n=1). Nine patients developed duodenal obstruction due to disease progression and required endoscopic duodenal stenting. Four patients (8%) survived less than 1 month, 12 (24%) between 1 month and 3 months, 11 (22%) between 3 and 6 months, and 10 (20%) beyond 6 months. Follow-up was not possible for 9 patients (18%) from distant referral sites. CONCLUSION: These results demonstrate that percutaneously placed SEMS achieved satisfactory palliation with a low complication rate in a high-risk patient group with advanced malignant biliary obstruction.

Is Helicobacter pylori antibiotic resistance surveillance needed and how can it be delivered?

BACKGROUND: Most patients are prescribed Helicobacter pylori treatment without culture and antibiotic susceptibility testing, as current guidance recommends that patients with recurrent dyspepsia should be tested for H. pylori using a non-invasive breath or faecal antigen test. AIMS: To determine the prevalence of H. pylori antibiotic resistance in patients attending endoscopy in England and Wales, and the feasibility of an antibiotic resistance surveillance programme testing. METHODS: We tested the antibiotic susceptibility of H. pylori isolates from biopsy specimens from 2063 of 7791 (26%) patients attending for endoscopy in Gloucester and Bangor, and 339 biopsy specimens sent to the Helicobacter Reference Unit (HRU) in London. Culture and susceptibility testing was undertaken in line with National and European methods. RESULTS: Helicobacter pylori were cultured in 6.4% of 2063 patients attending Gloucester and Bangor hospitals. Resistance to amoxicillin, tetracycline and rifampicin/rifabutin was below 3% at all centres. Clarithromycin, metronidazole and quinolone resistance was significantly higher in HRU (68%, 88%, 17%) and Bangor isolates (18%, 43%, 13%) than Gloucester (3%, 22%, 1%). Each previous course of these antibiotics is associated with an increase in the risk of antibiotic resistance to that agent [clarithromycin: RR = 1.5 (P = 0.12); metronidazole RR = 1.6 (P = 0.002); quinolone RR = 1.8 (P = 0.01)]. CONCLUSIONS: Helicobacter pylori infection is now uncommon in dyspeptic patients at endoscopy. A surveillance system is feasible and necessary to inform dyspepsia management guidance. Clinicians should take a thorough antibiotic history before prescribing metronidazole, clarithromycin or levofloxacin for H. pylori.

Molecular epidemiology of human Campylobacter jejuni shows association between seasonal and international patterns of disease.

We sought to explain seasonality and other aspects of Campylobacter jejuni epidemiology by integrating population genetic and epidemiological analysis in a large 3-year longitudinal, two-centre, population-based study. Epidemiological information was collected for 1505 isolates, which were multilocus sequence-typed. Analyses compared pathogen population structure between areas, over time, and between clinical presentations. Pooled analysis was performed with published international datasets. Subtype association with virulence was not observed. UK sites had nearly identical C. jejuni populations. A clade formed by ST45 and ST283 clonal complexes showed a summer peak. This clade was common in a Finnish dataset but not in New Zealand and Australian collections, countries with less marked seasonality. The UK, New Zealand and Australian collections were otherwise similar. These findings map to known in-vitro differences of this clade. This identifies a target for studies to elucidate the drivers of the summer peak in human C. jejuni infection.

Genome sequence of the emerging pathogen Helicobacter canadensis.

We determined the genome sequence of the type strain of Helicobacter canadensis, an emerging human pathogen with diverse animal reservoirs. Potential virulence determinants carried by the genome include systems for N-linked glycosylation and capsular export. A protein-based phylogenetic analysis places H. canadensis close to Wolinella succinogenes.

Persistence of Campylobacter species, strain types, antibiotic resistance and mechanisms of tetracycline resistance in poultry flocks treated with chlortetracycline.

OBJECTIVES: The aim of this study was to investigate the persistence of Campylobacter species, strain types, antibiotic resistance and mechanisms of tetracycline resistance in poultry flocks treated with chlortetracycline. METHODS: Three commercially reared broiler flocks, naturally colonized with Campylobacter, were treated with chlortetracycline under experimental conditions. The numbers of Campylobacter isolated, and the species, flaA short variable region allele, and antimicrobial resistance of isolates were determined. RESULTS: For two of three flocks, tetracycline-resistant strains predominated prior to chlortetracycline exposure. Presence of the antibiotic had no discernible effect on the numbers or types of Campylobacter and the tetracycline-resistant strains persisted in numbers similar to those observed before treatment. With all flocks, some faecal samples were obtained that contained no Campylobacter, irrespective of exposure to chlortetracycline; this was more common as the birds grew older. For the third flock, tetracycline-resistant Campylobacter were in the minority of samples before and during exposure to chlortetracycline, but at sampling times after this, no resistant strains were found in the treated (or untreated) birds, irrespective of exposure to the antibiotic. All tetracycline-resistant isolates (MICs 16 to >128 mg/L) contained tet(O) and, for some isolates, this was transferable to Campylobacter jejuni 81116. The efflux pump inhibitor PAbetaN reduced the MICs of tetracycline for these isolates by 4-fold, suggesting that an intact efflux pump, presumably CmeABC, is required for high-level tetracycline resistance. CONCLUSIONS: Our data indicate that chlortetracycline treatment does not eradicate tetracycline-resistant Campylobacter spp. from poultry. However, if a low number of resistant isolates are present, then the antibiotic pressure appears insufficient to select such strains as the dominant population.

Serotypes, intimin subtypes, and antimicrobial resistance patterns of atypical enteropathogenic Escherichia coli isolated in England from 1993 to 1996.

The aim of this study was to characterise the atypical enteropathogenic Escherichia coli (EPEC) strains isolated during a study of intestinal infectious disease in the UK by serotyping, intimin subtyping, and antimicrobial resistance typing. Serotypes, intimin subtypes, and resistance patterns of strains from cases were then compared with those from the control group. A wide range of serotypes, intimin subtypes, and antimicrobial resistance patterns was identified in isolates from both cases and controls, with O70:H11 and O111:H- being the most frequently detected serotypes. The most common intimin types were gamma and gamma(2). Thirty-six percent of the EPEC isolates were resistant to at least one antimicrobial agent. No significant differences in the characteristics of EPEC strains isolated from patients with symptoms of gastrointestinal disease versus those isolated from healthy controls were detected, although strains harbouring the beta-intimin subtype were more commonly isolated from children under 5 years of age (p=0.002). The compilation of data on atypical EPEC strains presented here indicates the need for further study of their virulence and epidemiology in order to assess their significance as human pathogens.

Molecular characterisation of an outbreak strain of multiresistant Salmonella enterica serovar Typhimurium DT104 in the UK.

A major national outbreak of multiresistant Salmonella enterica serovar Typhimurium definitive phage type 104 (MR DT104) occurred in England and Wales in the summer of 2000. Isolates of MR DT104 were characterised by antimicrobial resistance type (R-type), pulsed-field gel electrophoresis (PFGE), plasmid profiling and fluorescent amplified fragment length polymorphism (fAFLP) analysis. Results of R-type, PFGE and fAFLP showed that summer 2000 outbreak-associated isolates were indistinguishable from most MR DT104 isolates collected in England and Wales during the 1980s and 1990s. However, outbreak-associated isolates all had an additional 2-MDa plasmid (PP D), and this distinct profile allowed outbreak cases to be distinguished from background MR DT104 infections, thereby facilitating the epidemiological investigation by improving the specificity of the case definition. The study demonstrated the highly clonal nature of MR DT104 and the importance of a hierarchical approach to molecular subtyping for outbreak investigations.

Subtyping intimin genes from enteropathogenic Escherichia coli associated with outbreaks and sporadic cases in the United Kingdom and Eire.

PCR-RFLP methods for subtyping the intimin gene from strains of typical and atypical enteropathogenic Escherichia coli (EPEC) and Verocytotoxin-producing E. coli (VTEC) were compared. A novel HhaI PCR-RFLP method was developed that was rapid, easy to use and amplified an 1852 bp fragment of the intimin gene from all isolates examined. This method was used to investigate the intimin sub-types of EPEC strains associated with 14 outbreaks of diarrhoeal disease between 1967 and 2001, and 20 sporadic cases between January and December 2000, in the UK and Eire. In this study, genes encoding alpha, beta, gamma, delta and zeta-intimin were detected in the EPEC strains associated with outbreaks and beta, gamma, epsilon, theta and zeta-intimin genes were identified in isolates from sporadic cases. The beta-intimin gene was the most frequently detected sub-type in both the outbreak and sporadic strains.

A national outbreak of multi-resistant Salmonella enterica serovar Typhimurium definitive phage type (DT) 104 associated with consumption of lettuce.

Between 1 August and 15 September 2000, 361 cases of Salmonella enterica serotype Typhimurium definitive phage type (DT) 104, resistant to ampicillin, chloramphenicol, streptomycin, sulphonamides, spectinomycin and tetracycline (R-type ACSSuSpT), were identified in England and Wales residents. Molecular typing of 258 isolates of S. Typhimurium DT104 R-type ACSSuSpT showed that, although isolates were indistinguishable by pulsed-field gel electrophoresis, 67% (174/258) were characterized by a particular plasmid profile. A statistically significant association between illness and consumption of lettuce away from home was demonstrated (OR = 7.28; 95% CI=2.25-23.57; P=0.0006) in an unmatched case-control study. Environmental investigations revealed that a number of food outlets implicated in the outbreak had common suppliers of salad vegetables. No implicated foods were available for microbiological testing. An environmental audit of three farms that might have supplied salad vegetables to the implicated outlets did not reveal any unsafe agricultural practices. The complexity of the food supply chain and the lack of identifying markers on salad stuffs made tracking salad vegetables back to their origin extremely difficult in most instances. This has implications for public health since food hazard warnings and product withdrawal are contingent on accurate identification of the suspect product.

Detection of campylobacter species: a comparison of culture and polymerase chain reaction based methods.

AIMS: To investigate the optimal method for the detection of campylobacters from stool samples by comparing selective culture with membrane filtration and the polymerase chain reaction (PCR). METHODS: Three hundred and forty three stool samples were investigated by each of the three methods mentioned above. Selective culture was performed with charcoal cefoperazone desoxycholate agar plates. Membrane filtration was performed using cellulose triacetate membranes with 0.45 micro m pores placed on blood agar plates. Enteropathogenic campylobacters were detected using a PCR identification algorithm, consisting of screening PCRs and species identification using a PCR enzyme linked immunosorbent assay (PCR-ELISA), both based on the 16S rRNA gene. RESULTS: Of the 343 samples tested, 23 were positive by one or more method. Of these, 17 were positive by selective culture, 12 by membrane filtration, and 20 by the PCR identification algorithm. A total of 18 of 23 positives were identified as C jejuni and/or C coli by the PCR identification algorithm, compared with 14 identified to the genus level by selective culture, and 10 by membrane filtration. Among the remaining five positive samples, one C hyointestinalis was detected only by the PCR identification algorithm; one C upsaliensis was detected only by the PCR identification algorithm; one Campylobacter sp was detected by membrane filtration and selective culture and later identified as C concisus; one Campylobacter sp was detected by membrane filtration alone and later identified as Arcobacter sp; and one Campylobacter sp detected only by selective culture was lost to study and therefore not speciated. There was no significant difference between detection by selective culture and the other two methods. However, detection by PCR was significantly better than by membrane filtration (0.05 > p > 0.02). CONCLUSION: The PCR identification algorithm can detect and identify Campylobacter spp to the species level and the result is obtained on the same day. However, PCR is expensive, labour intensive, and does not provide an isolate for further identification or typing. Selective culture is as good as the PCR identification algorithm for the detection of the two most common species, C jejuni and C coli, and it is cheap and practical. However, it does miss the less common species, results take 48 hours, and identification is only to the genus level. Membrane filtration showed a low sensitivity compared with the other methods and is not appropriate for the diagnostic laboratory, although it was the only method to detect the Arcobacter sp. The optimum method for the detection of campylobacters from stool samples in the diagnostic laboratory remains selective culture.

Heterogeneity in expression of lipopolysaccharide by strains of Salmonella enterica serotype Typhimurium definitive phage type 104 and related phage types.

AIMS: To investigate lipopolysaccharide (LPS) expression in Salmonella enterica serotype Typhimurium definitive phage type 104 (Salmonella Typhimurium DT104) and related phage types. METHODS AND RESULTS: Isolates were examined for the expression of LPS by SDS-PAGE and silver staining and subtyped by Pulsed Field Gel Electrophoresis (PFGE). The 100 isolates expressed one of two LPS profiles designated A (72%) and B (28%). LPS profiling was able to discriminate between isolates of identical PFGE type. Among 10 groups of outbreak isolates examined, each group was of a single LPS profile: A, 8/10 and B, 2/10. All 10 outbreaks were identical by PFGE analysis. CONCLUSIONS: Isolates of Salmonella Typhimurium DT104 and related phage types expressed one of two distinct LPS profiles. The two LPS profiles appear similar but shifted and in phase with one another, suggesting that the heterogeneity is due to changes in the LPS core region rather than among the repeating oligosaccharide units of the long-chain LPS. SIGNIFICANCE AND IMPACT OF THE SUTDY: LPS profiling provides a useful adjunct to PFGE and other molecular methods for the subtyping of this group of bacteria in epidemiological investigations.

Campylobacter hominis sp. nov., from the human gastrointestinal tract.

Sequences of 16S rDNA of a novel campylobacter from faeces of healthy humans were previously shown to originate from a new taxon, 'Candidatus Campylobacter hominis', which could not be cultured. Since phylogenetic analysis suggested that anaerobic conditions might be required for growth, an isolation strategy was developed employing initial non-selective membrane filtration onto fastidious anaerobe agar. Campylobacters were then isolated from the resulting mixed microbial flora by a dilution strategy and/or by immunomagnetic separation with genus-specific polyclonal antibody. Isolates were identified by a genus and taxon-specific PCR assay, and 16S rDNA nucleotide sequence analysis was carried out. All isolates exhibited the typical Campylobacter characteristics of being non-fermentative, oxidase-positive, catalase-negative and Gram-negative. Unusually, however, they were straight rods lacking flagella. The 16S rDNA nucleotide sequence analysis, DNA and mol% G+C were consistent with a new Campylobacter species whose nearest phylogenetic neighbours were Campylobacter gracilis and Campylobacter sputorum. The unique species status of the isolates was further confirmed by taxonomic analysis of 47 phenotypic characteristics. The name Campylobacter hominis sp. nov. is proposed for the new species, the type strain of which is NCTC 13146T (= LMG 19568T).

Use of a LightCycler gyrA mutation assay for rapid identification of mutations conferring decreased susceptibility to ciprofloxacin in multiresistant Salmonella enterica serotype Typhimurium DT104 isolates.

A LightCycler-based PCR-hybridization gyrA mutation assay (GAMA) was developed to rapidly detect gyrA point mutations in multiresistant (MR) Salmonella enterica serotype Typhimurium DT104 with decreased susceptibility to ciprofloxacin (MIC, 0.25 to 1.0 mg/liter). Ninety-two isolates (49 human, 43 animal) were tested with three individual oligonucleotide probes directed against an Asp-87-to-Asn (GAC-->AAC) mutation, an Asp-87-to-Gly (GAC-->GGC) mutation, and a Ser-83-to-Phe (TCC-->TTC) mutation. Strains homologous to the probes could be distinguished from strains that had different mutations by their probe-target melting temperatures. Thirty-seven human and 30 animal isolates had an Asp-87-to-Asn substitution, 6 human and 6 animal isolates had a Ser-83-to-Phe substitution, and 5 human and 2 animal isolates had an Asp-87-to-Gly substitution. The remaining six strains all had mismatches with the three probes and therefore different gyrA mutations. The sequencing of gyrA from these six isolates showed that one human strain and two animal strains had an Asp-87-to-Tyr (GAC-->TAC) substitution and two animal strains had a Ser-83-to-Tyr (TCC-->TAC) substitution. One animal strain had no gyrA mutation, suggesting that this isolate had a different mechanism of resistance. Fifty-eight of the strains tested were indistinguishable by several different typing methods including antibiograms, pulsed-field gel gel electrophoresis, and plasmid profiling, although they could be further subdivided according to gyrA mutation. This study confirmed that MR DT104 with decreased susceptibility to ciprofloxacin from humans and food animals in England and Wales may have arisen independently against a background of clonal spread of MR DT104.

Campylobacter lanienae sp. nov., a new species isolated from workers in an abattoir.

Campylobacter-like organisms were isolated from the faeces of healthy individuals during a hygiene survey of abattoir workers. The strains, which exhibited characteristics of Campylobacter, being non-glucose-fermenting, oxidase- and catalase-positive, Gram-negative, motile rods, were identified to the genus level by a PCR assay. Nucleotide sequence analysis of the 16S rRNA gene, DNA homology experiments and determination of G + C content demonstrated that they constituted a previously undescribed species, whose nearest phylogenetic neighbours were Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter fetus and Campylobacter mucosalis. The name Campylobacter lanienae sp. nov. is proposed for this taxon and species-specific PCR primers were evaluated which will find use in the study of its epidemiology, prevalence and pathogenicity.

Large-scale survey of Campylobacter species in human gastroenteritis by PCR and PCR-enzyme-linked immunosorbent assay.

A PCR-based study of the incidence of enteropathogenic campylobacter infection in humans was done on the basis of a detection and identification algorithm consisting of screening PCRs and species identification by PCR-enzyme-linked immunosorbent assay. This was applied to DNA extracted from 3,738 fecal samples from patients with sporadic cases of acute gastroenteritis, submitted by seven regional Public Health Laboratories in England and Wales over a 2-year period. The sending laboratories had cultured "Campylobacter spp." from 464 samples. The PCR methodologies detected 492 Campylobacter-positive samples, and the combination of culture and PCR yielded 543 Campylobacter-positive samples. There was identity (overlap) for 413 samples, but 79 PCR-positive samples were culture negative, and 51 culture-positive samples were PCR negative. While there was no statistically significant difference between PCR and culture in detection of C. jejuni-C. coli (PCR, 478 samples; culture, 461 samples), PCR provided unique data about mixed infections and non-C. jejuni and non- C. coli campylobacters. Mixed infections with C. jejuni and C. coli were found in 19 samples, and mixed infection with C. jejuni and C. upsaliensis was found in one sample; this was not apparent from culture. Eleven cases of gastroenteritis were attributed to C. upsaliensis by PCR, three cases were attributed to C. hyointestinalis, and one case was attributed to C. lari. This represents the highest incidence of C. hyointestinalis yet reported from human gastroenteritis, while the low incidence of C. lari suggests that it is less important in this context.

The effect of omeprazole dosing on the isolation of Helicobacter pylori from gastric aspirates.

BACKGROUND: Animal experiments suggest that omeprazole dosing increases shedding of Helicobacter into the gastric lumen, and hence into gastric juice. AIM: To assess the effect of omeprazole dosing on the yield of H. pylori from gastric aspirates of infected volunteers. METHODS: Six serial nasogastric aspirates, three before and three during dosing with omeprazole 40 mg b.d., were obtained for culture from 10 H. pylori infected volunteers and one uninfected volunteer. To reduce contamination, samples were diluted 1:10 with Maximum Recovery Diluent (MRD; pH 7.0) or HCl-KCl buffer (pH 2.2) prior to culture on Columbia and Dent's agar. RESULTS: Undiluted gastric juice cultures were rapidly overgrown by upper respiratory tract flora. HCl-KCl dilution resulted in isolation of H. pylori from 77% of infected subject aspirates before, and 67% of aspirates during dosing with omeprazole. The yields were significantly lower with MRD dilution, 47% and 10%, respectively. Omeprazole dosing significantly decreased the yield after MRD dilution, but not after HCl-KCl dilution. CONCLUSIONS: Decreasing intragastric acidity, by dosing with omeprazole, decreases the isolation of H. pylori from routinely processed gastric aspirates. In vitro acidification of gastric aspirates, by HCl-KCl dilution, increases the isolation of H. pylori both before and during omeprazole dosing.