The prevalence of Chlamydia pneumoniae in atherosclerotic and nonatherosclerotic blood vessels of patients attending for redo and first time coronary artery bypass graft surgery

The current genetic strategies used to identify Tropheryma whippelii, the putative agent of Whipple's disease, are based on PCR-mediated amplification of a part of its 16S rRNA gene (16S rDNA). Because there is very little intraspecies variation in these molecules, they are not suitable as targets for epidemiologic investigations. However, the intergenic spacer region between the 16S and 23S rDNAs is usually much more variable and has repeatedly been used for epidemiologic purposes. We have therefore amplified the spacer region of T. whippelii directly from clinical specimens from nine independent Swiss patients with Whipple's disease by PCR with primers complementary to the 3' and 5' ends of the 16S and 23S rDNAs, respectively. The amplicons were directly sequenced and the sequences were compared to the T. whippelii reference sequence in GenBank/EMBL (accession no. X99636). Complete sequence homogeneity was found between the samples from our nine patients; the spacer sequence was also identical to the reference sequence. However, the sequences corresponding to the 3' and 5' ends of the 16S and the 23S rDNAs of T. whippelii, respectively, differed from the respective sequences in GenBank/EMBL. The same sequence found in our patients was then found in a sample from the German patient from which the published sequence had been derived. We conclude that the 16S-23S rDNA spacer region seems to be very conserved in T. whippelii and that the respective reference entry in public databases should be revised.     

Spondylodiscitis caused by Tropheryma whippelii

We describe the first case of spondylodiscitis caused by Tropheryma whippelii in which this so far unculturable organism was shown to be present at the site of infection in a patient without significant gastrointestinal symptoms. The methods used included broad-range PCR amplification with universal primers complementary to constant sequences of the gene coding for 16S rRNA, direct sequencing of the amplified fragment, and comparison of the sequence determined with those deposited in sequence databases. In addition to demonstrating the presence of this organism in the affected vertebral body, we found in our patient that the specific PCR is more sensitive than histology for detecting Whipple's bacilli in bowel biopsy specimens. Because histology of small bowel biopsies from the duodenum were-in contrast to PCR from the same site-not diagnostic for Whipple's disease in our patient, we recommend PCR whenever Whipple's disease has to be excluded.     

Estimation of the relative abundance of different Bacteroides and Prevotella ribotypes in gut samples by restriction enzyme profiling of PCR-amplified 16S rRNA gene sequences

We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.     

Whipple's syndrome (uveitis, B27-negative spondylarthropathy, meningitis, and lymphadenopathy) associated with Arthrobacter sp. infection

OBJECTIVE: To report an unusual case of Whipple's disease, including uveitis, seronegative spondylarthropathy, meningitis, and lymphadenopathy, associated with an Arthrobacter sp. infection. DESIGN: Interventional case report. PATIENT AND INTERVENTION: A 60-year-old white man presenting with severe chronic uveitis and systemic inflammatory manifestations was treated efficiently for Whipple's disease after histopathologic analysis of vitreous and inguinal adenopathy biopsy specimens. The authors performed a retrospective, laboratory-based evaluation of stored tissue specimens. MEASUREMENTS: Molecular analysis based on 16S ribosomal RNA gene amplification was applied to pretreatment biopsy specimens of inguinal lymph node to identify a causative bacterial agent. RESULTS: Tropheryma whippelii genome was not detected in these specimens. However, an amplification product was obtained after the first polymerase chain reaction run and subsequently was sequenced. It corresponded to an Arthrobacter sp., a gram-positive agent presenting diagnostic patterns and therapeutic management similar to those of Whipple's disease caused by T. whippelii. CONCLUSION: The absence of T. whippelii identification by molecular amplification during a clinically and histologically oriented Whipple's syndrome should not rule out the diagnosis. Arthrobacter infection may represent a new bacterial etiology of systemic inflammatory disorders involving the eye and associated with periodic acid-Schiff-positive inclusions.     

Diagnosis of meningococcal meningitis by broad-range bacterial PCR with cerebrospinal fluid

We used broad-range bacterial PCR combined with DNA sequencing to examine prospectively cerebrospinal fluid (CSF) samples from patients with suspected meningitis. Fifty-six CSF samples from 46 patients were studied during the year 1995. Genes coding for bacterial 16S and/or 23S rRNA genes could be amplified from the CSF samples from five patients with a clinical picture consistent with acute bacterial meningitis. For these patients, the sequenced PCR product shared 98.3 to 100% homology with the Neisseria meningitidis sequence. For one patient, the diagnosis was initially made by PCR alone. Of the remaining 51 CSF samples, for 50 (98.0%) samples the negative PCR findings were in accordance with the negative findings by bacterial culture and Gram staining, as well as with the eventual clinical diagnosis for the patient. However, the PCR test failed to detect the bacterial rRNA gene in one CSF sample, the culture of which yielded Listeria monocytogenes. These results invite new research efforts to be focused on the application of PCR with broad-range bacterial primers to improve the etiologic diagnosis of bacterial meningitis. In a clinical setting, Gram staining and bacterial culture still remain the cornerstones of diagnosis.     

Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms

Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.     

Investigation of film curing stages by dielectric analysis and physical characterization

A previously published sequence of the 23S rRNA gene of Coxiella burnetii has been reported to contain an intervening sequence of 444 base pairs (bp). The sequence information on the intervening sequence and the 23S rRNA gene was exploited to develop a specific PCR-based assay for C. burnetii. A primer set was designed that amplified a 477-bp fragment encompassing part of the intervening sequence and part of the 23S rDNA. From all of nine C. burnetii strains tested, a fragment of the expected size was amplified. As predicted from the published sequence, restriction endonuclease digestion of the PCR product from the Coxiella strains with RsaI produced two distinct fragments approximately 210- and 270-bp in size. The PCR-based method showed a detection limit of 10(2) bacteria as determined by visualization of the amplicon on an agarose gel. When experimentally infected blood was analyzed, the detection limit was 10(3) bacteria. No visible amplicons were observed when 41 bacterial strains, representing 29 species other than C. burnetii, were tested. The presence of the DNA in all bacterial samples was confirmed by amplification of a 350-bp fragment of the 16S rDNA using two universal primers. The described method proved to be specific for C. burnetii and may become a rapid and sensitive diagnostic assay for C. burnetii. The results also demonstrate that the intervening sequence within the 23S rRNA gene is generally found among isolates of C. burnetii.     

Identification and analysis of PCB dechlorinating anaerobic enrichments by amplification: accuracy of community structure based on restriction analysis and partial sequencing of 16S rRNA genes

The composition of polychlorinated biphenyl (PCB) dechlorinating mixed communities was analysed by restriction fragment length polymorphism of PCR amplified rDNAs (ARDRA) and partial sequencing of 16S rRNA genes amplified from PCB degrading enrichments. Restriction analysis confirms that the 16S rRNA genes amplified from PCB dechlorinating communities vary depending on the PCB congener dechlorinated. Comparison of 16S rRNA sequences to published ribosomal databases indicates that the two most abundant Operational Taxonomic Units (OTUs) appear to be species of the genus Clostridium. The amount that the amplification procedure contributed to this result was determined by varying the amplification procedure and by creating an artificial template mixture. Varying the amount of template by sixfold in the amplification did not affect the distribution of OTUs but the number of OTUs observed decreased with decreasing template concentration. Comparison of products amplified from mixtures of 16S rDNA clones indicates that the more abundant Clostridium OTU did not amplify more efficiently than those of less abundant OTUs. Hybridization to a probe designed to detect the most abundant OTUs indicates that two other OTUs are closely related to this Clostridium species.     

Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria

The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different Clostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.     

PCR analysis of T. whippelii DNA in a case of Whipple's disease: effect of antibiotics and correlation with histology

A 58-yr-old man developed severe weight loss, arthralgias, and diarrhea. Endoscopic examination of the stomach and duodenum revealed thickened folds of duodenal mucosa. Biopsy of the gastric mucosa was negative, whereas duodenal biopsy revealed blunted epithelial villi and PAS-positive foamy macrophages within the lamina propria. Bacilli typical of those associated with Whipple's disease were found by electron microscopy. The diagnosis was confirmed by polymerase chain reaction (PCR) assay, which detected a portion of the 16S ribosomal RNA gene sequence corresponding to the Whipple bacillus (Tropheryma whippelii) in duodenum, stomach, and liver biopsies before therapy. T. whippelii DNA was eliminated from all tissues tested within 3 months of starting antibiotic treatment, but the histological improvement lagged behind the clinical and molecular evidence of improvement.     

16S rRNA gene sequence of Rubrobacter radiotolerans and its phylogenetic alignment with members of the genus Arthrobacter, gram-positive bacteria, and members of the family Deinococcaceae

The nearly complete sequence of the 16S rRNA gene of an extremely highly radiotolerant bacterium, Rubrobacter radiotolerans (reclassified from Arthrobacter radiotolerans based on chemical characteristics), was determined by PCR amplification of the genomic DNA followed by cloning of the amplified gene and sequencing by the dideoxynucleotide method. The sequence was aligned with the sequences of members of the genus Arthrobacter and also with the sequences of representatives of the gram-positive bacteria having high G + C contents and the family Deinococcaceae (radioresistant micrococci and their relatives). The results of our phylogenetic analysis confirmed that R. radiotolerans is not a member of the Arthrobacter group and thus supported the previous reclassification. Moreover, although it is radioresistant and has a high G+C content, R. radiotolerans is more closely related to the gram-positive bacteria with high G+C contents than to the radioresistant members of the Deinococcaceae.     

Development and evaluation of a PCR-based assay for detection of Haemobartonella felis in cats and differentiation of H. felis from related bacteria by restriction fragment length polymorphism analysis

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats.     

Lacrimal canalicular obstruction associated with topical ocular medication

An uncultured bacterium of the Verrucomicrobiales cluster was identified by its 16S rDNA sequence as a major bacterium in Dutch Drentse A grassland soils. Potential metabolic activity of the according organism was estimated by applying direct ribosome isolation from soil and partial amplification of the 16S rRNA via RT-PCR using bacteria-specific primers. Temperature gradient gel electrophoresis separated the amplicons sequence specifically into reproducible fingerprints. One of the fingerprint bands matched with the signal of clone DA101. Southern blot hybridization with a DA101-specific V6 probe confirmed sequence identity. It is the first time that an organism of the Verrucomicrobiales cluster has been indicated as a potential major metabolizer in environmental microbial communities.     

Nested PCR assay for detection of granulocytic ehrlichiae

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.     

PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples

A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks species-specific sequences within the genes coding for 16S rRNA. The PCR product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3), M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples.     

Pharmacological determinants of the antitumour activity of mitomycin C

Raspberries were epidemiologically associated with cyclosporiasis outbreaks during 1996 and 1997. The 18S rRNA genes of Cyclospora cayetanensis and several species of a closely related genus, Eimeria, were sequenced and primers for a nested PCR developed in a previous study. The ability to distinguish amplified products of Cyclospora sp. from those of Eimeria spp. is important for testing food and environmental samples. Therefore, an RFLP analysis of amplified products was used to differentiate Cyclospora cayetanensis from Eimeria spp. PCR inhibitors and the low levels of Cyclospora oocysts present in raspberries make template preparation for PCR challenging. Several approaches for PCR template preparation from raspberry samples were evaluated. Template preparation methods using various washing and concentration steps, oocyst disruption protocols, resin matrix treatment, DNA precipitation, and/or the addition of nonfat dried milk solution to a PCR using modified primers were evaluated first with oocysts of Eimeria tenella then refined with oocysts of C. cayetanensis. Approximately 10 E. tenella oocysts per PCR or approximately 19 C. cayetanensis oocysts per PCR were detected with the optimized template preparation method. The addition of 20 microliters of raspberry wash sediment extract and nonfat dried milk solution did not inhibit the amplification of DNA from as few as 10 E. tenella and 25 C. cayetanensis oocysts in a 100-microliter PCR. The nucleotide sequences of C. cayetanensis and the Eimeria spp. are 94 to 96% similar in the amplified region, but the amplification products from the two genera were distinguished using an RFLP analysis with the restriction enzyme MnlI.     

Molecular identification of Gemella species from three patients with endocarditis

Gemella morbillorum and Gemella haemolysans are opportunistic pathogens which cause endocarditis and other severe infections. We report on three patients with endocarditis, one with endocarditis caused by G. haemolysans and two with endocarditis caused by G. morbillorum. The paucity of reports concerning these bacteria is probably related to the difficulties associated with their identification. For example, one of the strains reported in this study was originally sent to our laboratory with a preliminary characterization as a short "gram-negative" coccobacillus, highlighting the specific problem associated with Gram staining of these bacteria. The usefulness of 16S rRNA gene amplification, partial sequencing, and comparison of the nucleotide sequence to those in databases when standard phenotypic identification schemes are not helpful is emphasized. We also suggest that the use of simple tests, such as testing susceptibility to vancomycin for gram-negative bacteria and colistin for gram-positive bacteria, could prevent misinterpretation of Gram staining in gram-variable bacteria such as Gemella spp.     

Gamma interferon augments macrophage activation by lipopolysaccharide by two distinct mechanisms, at the signal transduction level and via an autocrine mechanism involving tumor necrosis factor alpha and interleukin-1

Acanthamoebae are ubiquitous soil and water bactivores which may serve as amplification vehicles for a variety of pathogenic facultative bacteria and as hosts to other, presently uncultured bacterial endosymbionts. The spectrum of uncultured endosymbionts includes gram-negative rods and gram-variable cocci, the latter recently shown to be members of the Chlamydiales. We report here the isolation from corneal scrapings of two Acanthamoeba strains that harbor gram-negative rod endosymbionts that could not be cultured by standard techniques. These bacteria were phylogenetically characterized following amplification and sequencing of the near-full-length 16S rRNA gene. We used two fluorescently labelled oligonucleotide probes targeting signature regions within the retrieved sequences to detect these organisms in situ. Phylogenetic analyses demonstrated that they displayed 99.6% sequence similarity and formed an independent and well-separated lineage within the Rickettsiales branch of the alpha subdivision of the Proteobacteria. Nearest relatives included members of the genus Rickettsia, with sequence similarities of approximately 85 to 86%, suggesting that these symbionts are representatives of a new genus and, perhaps, family. Distance matrix, parsimony, and maximum-likelihood tree-generating methods all consistently supported deep branching of the 16S rDNA sequences within the Rickettsiales. The oligonucleotide probes displayed at least three mismatches to all other available 16S rDNA sequences, and they both readily permitted the unambiguous detection of rod-shaped bacteria within intact acanthamoebae by confocal laser-scanning microscopy. Considering the long-standing relationship of most Rickettsiales with arthropods, the finding of a related lineage of endosymbionts in protozoan hosts was unexpected and may have implications for the preadaptation and/or recruitment of rickettsia-like bacteria to metazoan hosts.     

Development of rRNA-targeted PCR and in situ hybridization with fluorescently labelled oligonucleotides for detection of Yersinia species

In this report, we present details of two rapid molecular detection techniques based on 16S and 23S rRNA sequence data to identify and differentiate Yersinia species from clinical and environmental sources. Near-full-length 16S rRNA gene (rDNA) sequences for three different Yersinia species and partial 23S rDNA sequences for three Y. pestis and three Y. pseudotuberculosis strains were determined. While 16S rDNA sequences of Y. pestis and Y. pseudotuberculosis were found to be identical, one base difference was identified within a highly variable region of 23S rDNA. The rDNA sequences were used to develop primers and fluorescently tagged oligonucleotide probes suitable for differential detection of Yersinia species by PCR and in situ hybridization, respectively. As few as 10(2) Yersinia cells per ml could be detected by PCR with a seminested approach. Amplification with a subgenus-specific primer pair followed by a second PCR allowed differentiation of Y. enterocolitica biogroup 1B from biogroups 2 to 5 or from other pathogenic Yersinia species. Moreover, a set of oligonucleotide probes suitable for rapid (3-h) in situ detection and differentiation of the three pathogenic Yersinia species (in particular Y. pestis and Y. pseudotuberculosis) was developed. The applicability of this technique was demonstrated by detection of Y. pestis and Y. pseudotuberculosis in spiked throat and stool samples, respectively. These probes were also capable of identifying Y. enterocolitica within cryosections of experimentally infected mouse tissue by the use of confocal laser scanning microscopy.     

Identification of Helicobacter in gastric biopsies by PCR based on 16S rDNA sequences: a matter of little significance for the prediction of H. pylori-associated gastritis?

The aim of the present study was to correlate molecular evidence of the presence of Helicobacter pylori in gastric biopsy samples, based on analysis of 16S rDNA, vacuolating toxin (vacA), urease A (ureA) and cagA genes, with the clinical, histological and serological findings in patients with H. pylori-associated gastritis. Fresh biopsy samples were collected from the gastric antrum and corpus of 22 asymptomatic volunteers with or without H. pylori-associated gastritis. Total DNA was extracted from the biopsy material and subjected to 16S rDNA PCR amplification, Southern blotting and 16S rDNA sequence analysis of the PCR products. The vacA, ureA and cagA genes were characterised by PCR amplification and Southern blot analysis. Based on partial 16S rDNA sequence analysis, DNA belonging to the genus Helicobacter was detected in gastric biopsy samples from 20 of 22 subjects, including seven of nine histologically and serologically normal controls. Six of 20 partial 16S rDNA sequences revealed variations within variable regions V3 and V4 that deviated from those of the H. pylori type strain ATCC 4350T and, therefore, possibly represented other species of Helicobacter. VacA genes identical with those of the type strain were found predominantly in the subjects with H. pylori gastritis, and all the patients except one were found to be cagA-positive. There was no evidence of false positive PCR reactions. In conclusion, the PCR-based molecular typing methods used here were apparently too sensitive when applied to the detection of H. pylori in human gastric tissues. The lack of quantitative analysis makes them inappropriate as clinical tools for the diagnosis of H. pylori-associated gastritis, despite the fact that they provide a qualitative and sensitive tool for the detection and characterisation of H. pylori in the gastrointestinal tract.