Prospective multicentre clinical study on inter- and intrapatient genetic variability for antimicrobial resistance of Helicobacter pylori.

OBJECTIVES: We report on a large prospective, multicentre clinical investigation on inter- and intrapatient genetic variability for antimicrobial resistance of Helicobacter pylori. METHODS: Therapy-naive patients (n = 2004) who had undergone routine diagnostic gastroscopy were prospectively included from all geographic regions of Austria. Gastric biopsy samples were collected separately from antrum and corpus. Samples were analysed by histopathology and real-time PCR for genotypic resistance to clarithromycin and quinolones. Clinical and demographic information was analysed in relation to resistance patterns. RESULTS: H. pylori infection was detected in 514 (26%) of 2004 patients by histopathology and confirmed in 465 (90%) of 514 patients by real-time PCR. PCR results were discordant for antrum and corpus in 27 (5%) of 514 patients, indicating inhomogeneous infections. Clarithromycin resistance rates were 17% (77/448) and 19% (84/455), and quinolone resistance rates were 12% (37/310) and 10% (32/334) in antrum and corpus samples, respectively. Combination of test results per patient yielded resistance rates of 21% (98/465) and 13% (50/383) for clarithromycin and quinolones, respectively. Overall, infection with both sensitive and resistant H. pylori was detected in 65 (14%) of 465 patients. CONCLUSIONS: Anatomically inhomogeneous infection with different, multiple H. pylori strains is common. Prospective clinical study design, collection of samples from multiple sites and microbiologic methods that allow the detection of coinfections are mandatory for collection of reliable data on antimicrobial resistance patterns in representative patient populations. (ClinicalTrials.gov identifier: NCT02925091).

Comprehensive evaluation of chemiluminescent immunoassays for the laboratory diagnosis of Clostridium difficile infection.

For the microbiological diagnosis of a Clostridium (C.) difficile infection (CDI), a two-test algorithm consisting of a C. difficile glutamate dehydrogenase (GDH)-immunoassay followed by a toxin-immunoassay in positive cases is widely used. In this study, two chemiluminescent immunoassays (CLIAs), one for GDH and the other for the toxins A and B, have been evaluated systematically using appropriate reference methods. Three-hundred diarrhoeal stool specimens submitted for CDI diagnosis were analysed by the LIAISON CLIAs (DiaSorin). Toxigenic culture (TC) and cell cytotoxicity assay (CCTA) were used as "gold standard" reference methods. In addition, GDH and toxin A and B enzyme immunoassays (EIAs), C. diff Chek-60 and toxin A/B II (TechLab), and the Cepheid Xpert C. difficile polymerase chain reaction (PCR) were performed. C. difficile was grown in 42 (14%), TC was positive in 35 (11.7%) and CCTA in 25 (8.3%) cases. CLIAs were more sensitive but less specific than the respective EIAs. Using culture as reference, the sensitivity of the GDH CLIA was 100%. In comparison to CCTA sensitivity, specificity, positive predictive value and negative predictive value of the two-test algorithm were 88, 99.3, 91.7 and 98.9% by CLIAs and 72, 99.6, 94.7 and 97.5% by EIAs. Discrepant results by CLIAs were more frequent than that by EIAs (9% vs. 6.3%); in those cases, PCR allowed for the accurate detection of toxigenic strains. Due to performance characteristics and testing comfort, CLIAs in combination with PCR represent a favourable option for the rapid laboratory C. difficile diagnostics.

Prevalence and risk factor assessment of Tropheryma whipplei in a rural community in Gabon: a community-based cross-sectional study.

Tropheryma whipplei is the causative agent of Whipple's disease and has been detected in stools of asymptomatic carriers. Colonization has been associated with precarious hygienic conditions. There is a lack of knowledge about the epidemiology and transmission characteristics on a population level, so the aim of this study was to determine the overall and age-specific prevalence of T. whipplei and to identify risk factors for colonization. This molecular epidemiological survey was designed as a cross-sectional study in a rural community in Central African Gabon and inhabitants of the entire community were invited to participate. Overall prevalence assessed by real-time PCR and sequencing was 19.6% (95% CI 16-23.2%, n=91) in 465 stool samples provided by the study participants. Younger age groups showed a significantly higher prevalence of T. whipplei colonization ranging from 40.0% (95% CI 27.8-52.2) among the 0-4 year olds to 36.4% (95% CI 26.1-46.6) among children aged 5-10 years. Prevalence decreased in older age groups (p<0.001) from 12.6% (95% CI 5.8-19.4%; 11-20 years) to 9.7% (95% CI 5.7-13.6) among those older than 20. Risk factor analysis revealed young age, male sex, and number of people sharing a bed as factors associated with an increased risk for T. whipplei carriage. These results demonstrate that T. whipplei carriage is highly prevalent in this part of Africa. The high prevalence in early life and the analysis of risk factors suggest that transmission may peak during childhood facilitated through close person-to-person contacts.

A novel fluorescence in situ hybridization test for rapid pathogen identification in positive blood cultures.

A novel molecular beacon-based fluorescence in situ hybridization (FISH) test allowing for the identification of a wide range of bacterial pathogens directly in positive blood cultures (BCs) was evaluated with positive BCs of 152 patients. Depending on the Gram stain, either a Gram-negative or a Gram-positive panel was used. The time to result was 30 min, and the hands-on time was only 10 min. Seven per cent of the cultured microorganisms were not included in the FISH panels; the identification rate of those included was 95.2%. Overall, the FISH test enabled accurate pathogen identification in 88.2% of all cases analysed.

Evaluation of a novel internally controlled real-time PCR assay targeting the 16S rRNA gene for confirmation of Neisseria gonorrhoeae infections.

A novel HybProbe real-time LightCycler PCR assay was developed for confirmation of Neisseria gonorrhoeae in samples positive according to the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR assay. The new assay amplifies 375 bp of the N. gonorrhoeae 16S rRNA gene and includes an internal amplification control introduced during DNA purification. The assay had 100% specificity because of the high specificity of the HybProbes and primers. Other Neisseria spp. failed to generate positive crossing-point values and melting peaks. The analytical sensitivity for N. gonorrhoeae DNA was 0.5 fg/PCR, corresponding to 0.3 CFU/PCR. Sensitivity was not impaired in the presence of higher DNA concentrations (>or=1000-fold) from Neisseria spp. other than N. gonorrhoeae. The sensitivity was similar to that reported for the COBAS AMPLICOR assay with cervical swab samples. To assess its clinical applicability as a confirmatory test, 38 (2.9%) of 1313 swabs that were positive according to the COBAS AMPLICOR assay were tested using the new in-house assay and the commercially available GenFlow Neisseria test. Twenty-one samples negative according to COBAS AMPLICOR also underwent confirmatory testing. Both confirmatory tests yielded identical results; the 21 negative samples remained negative, and only 11 (28.9%) of the samples positive according to COBAS AMPLICOR were positive after retesting, suggesting a low prevalence (0.84%) of N. gonorrhoeae infection in the study population. These data suggest that the novel real-time PCR assay is an excellent and easy to interpret confirmatory test for the existing COBAS AMPLICOR assay for N. gonorrhoeae.

Evaluation of a protocol for molecular broad-range diagnosis of culture-negative bacterial infections in clinical routine diagnosis.

AIM: Introduction of a protocol for broad-range diagnosis of bacterial infections, which remain negative in culture. METHODS AND RESULTS: The new TaqMan real-time PCR assay amplifies part of the 16S rRNA gene. Species are identified by subsequent sequencing and phylogenetic blast analysis. The analytical sensitivity showed to be 50 fg DNA per PCR. The lowest detectable bacterial cell concentration in blood was 1000 CFU per 200 mul EDTA-blood. The utility in clinical routine diagnosis was evaluated by testing 136 clinical specimens. Bacterial pathogens were detected in 33 samples (24.3%) either by culture or molecular diagnosis. In 10 culture negative cases, pathogens such as Mycoplasma timone/orale, Ureaplasma parvum/urealyticum, Treponema pallidum, different streptococci and staphylococci were identified by molecular diagnosis only. CONCLUSIONS: The introduced broad-range real-time PCR protocol showed to be useful in the clinical routine in cases where bacterial infection was highly anticipated but culture remained negative. However, the obtained data have to be always interpreted with caution and in conjunction with the clinical data, crossing-point values and with the Blast result of both the sample and the controls. SIGNIFICANCE AND IMPACT OF THE STUDY: This work introduces a new and well-evaluated broad-range real-time PCR protocol for diagnosis of bacterial infections.

In vitro activity of fosfomycin alone and in combination with amoxicillin, clarithromycin and metronidazole against Helicobacter pylori compared with combined clarithromycin and metronidazole.

In order to evaluate the suitability of fosfomycin in combination with other agents for the treatment of Helicobacter pylori infections, the susceptibility profiles of 65 H. pylori strains were determined against multiple antimicrobial agents and combinations thereof using the agar dilution method. For fosfomycin alone, the range of minimum inhibitory concentration (MIC) results and the MICs at which 50% and 90% of strains were inhibited were 0.5-32 microg/ml and 2 and 4 microg/ml, respectively. For the combination of fosfomycin with amoxicillin, clarithromycin or metronidazole, the means calculated for the minimum and maximum fractional inhibitory concentration index were 0.70-1.17 and 1.15-2.03, respectively, suggesting partial synergy or indifference in the majority of strains. The combination of clarithromycin and metronidazole showed synergistic activity against 14 of 28 H. pylori strains tested. The in vitro activity results suggest the combination of fosfomycin with either amoxicillin or clarithromycin may be a promising alternative for the treatment of H. pylori infection. However, the clinical efficacy of these regimens remains to be investigated.

Antimicrobial susceptibility profiles of clinically relevant blood culture isolates from nine surgical intensive care units, 1996-2000.

In order to elucidate trends in the incidence and susceptibility profiles of causative agents of bacteremia/fungemia in nine surgical intensive care units, a total of 744 isolates obtained during a 5-year period (1996-2000) were studied. The isolates included 698 bacteria and 46 fungi obtained from 523 positive blood cultures, representing 317 episodes of bacteremia/fungemia. Methicillin-resistant Staphylococcus aureus accounted for 2.3 episodes per 1000 surgical ICU admissions in 1996, 1.6 in 1997, 0.3 in 1998, 0.6 in 1999, and 1.7 in 2000. One Enterococcus faecalis (VanA) isolate resistant to both vancomycin and teicoplanin was recovered in 1996. Ciprofloxacin resistance in Pseudomonas aeruginosa decreased from 36% in 1996 to 20% in 2000, and resistance to third-generation cephalosporins decreased from 40% in 1996 to 9% in 2000. In light of differences between these results and those found elsewhere, these findings might prove useful for making infection control policy decisions in intensive care units.

[Helicobacter pylori: virulence factors, resistance and diagnosis]. / Helicobacter pylori: Virulenzfaktoren, Resistenz und Diagnostik.

Helicobacter pylori disposes of various virulence factors such as urease, vacuolating cytotoxin and the cag-pathogenicity island which--though not alone, but possibly in conjunction with host-specific factors--may explain the varying course of the infection (asymptomatic, dyspepsia, ulcer, distal gastric carcinoma). Increasing resistance to macrolide antibiotics and the lack of new therapeutic approaches makes treatment of the infection increasingly difficult. The resultant call for a strict indication for treatment is in obvious contrast to the finding that Helicobacter pylori represents the major risk factor for gastric carcinoma and eradication would therefore be absolutely desirable. Increased use of culture and susceptibility testing would be desirable for the purpose of therapy optimization but also for reasons of resistance epidemiology. The indication for diagnostic screening should be guided by the treatment indications as proposed by the guidelines of the Maastricht Consensus Conference 2-2000. In addition--and regardless of clinical picture--therapeutic follow-up primarily relying on non-invasive tests (13C urea breath test, stool antigen test) should be a matter of course.

Endocarditis by Lactobacillus rhamnosus due to yogurt ingestion?

A young man who ate large quantities of probiotic yogurt developed endocarditis and septic arthritis caused by Lactobacillus rhamnosus. The pathogenic isolate could not be distinguished from the yogurt microflora using methods routinely used in the clinical microbiology laboratory. Only by using more appropriate methodology, including PCR, the pathogen could be distinguished from the yogurt isolate.

Application of blood-based polymerase chain reaction for detection of Chlamydia pneumoniae in acute respiratory tract infections.

The aim of this study was to investigate whether blood-based polymerase chain reaction could serve as a diagnostic tool to identify individuals with acute respiratory Chlamydia pneumoniae infection. Respiratory specimens and peripheral blood mononuclear cells of 58 patients were analyzed using nested polymerase chain reaction and cell culture. Fifteen patients were polymerase chain reaction-positive for Chlamydia pneumoniae. Nine patients were positive in only the respiratory specimen; two in both the respiratory and blood sample (time intervals between onset of symptoms and sample collection, 3-10 days and 3-4 weeks, respectively); and four in only the blood sample. Detection of Chlamydia pneumoniae DNA in peripheral blood mononuclear cells does not seem to be a suitable marker for acute respiratory Chlamydia pneumoniae infection.

Microbiological irrigation water quality of the Marchfeld canal system.

The Marchfeld basin with a size of approximately 1000 km2 represents the Austrian "granary". To prevent shortage of water as a result of increased ground water removal for irrigation, industrial purposes and drinking water supply, a canal being 18 km in length was constructed from the Danube to the center of the Marchfeld. From there, water is further distributed via two creeks (Russbach and Stempfelbach). This study was intended to evaluate whether the surface water of the Marchfeld canal system can be classified into hygienic-microbiological categories as proposed by DIN (Deutsche Industrienorm) standards for irrigation water. For this purpose, water sampled monthly from three different sampling sites from 1996 to 1999 was examined for E. coli and enterococci. In addition, water samples were examined for salmonella twice a year from 1996 to 1998 and for cryptosporidia six times during the year 1999. Though the water showed varying degrees of fecal load, the results of the examinations revealed that only one of the three sampling sites showed constant quality levels according to the DIN classification system over prolonged periods of time. However, exceeding of the limit values was occasionally observed indicating the need for regular bacteriological examinations. The high variation of the results from the other sampling sites hardly permits a definite classification in one of the quality classes.

Urease prevents adherence of Helicobacter pylori to Kato III gastric epithelial cells.

The role of urease in Helicobacter pylori adherence to and internalization by Kato III cells was investigated. Kato III cells were incubated with wild-type strains (N6 or P1), with isogenic mutants lacking urease (N6ureB::TnKm or P1ureA::TnMax5) or producing the inactive apoprotein (N6ureG::TnKm), and with urease-positive clones recovered after complementation of N6ureB::TnKm with ureAB. Bacteria were stained with the green fluorescent dye PKH2, and the bacteria load of cells was analyzed by flow cytometry. With mutants lacking urease, the bacteria load was considerably increased, in comparison with the corresponding parental strains (P<.001). With clone K2(3), producing larger amounts of urease than N6, a significant reduction of bacteria load was observed, in comparison with the wild type (P<.001). N6ureG::TnKm showed adherence characteristics similar to those of N6. The role of urease in internalization was not clear. Thus, urease significantly inhibits H. pylori adherence to Kato III cells by a mechanism largely independent of enzymatic activity.

Genetic complementation of the urease-negative Helicobacter pylori mutant N6ureB::TnKm.

Helicobacter pylori produces urease composed of the structural subunits UreA and UreB. Isogenic mutants produced by shuttle mutagenesis from the wild-type strain N6 are widely used in the literature. We describe the genetic complementation of the mutant N6ureB::TnKm by stable transformation with the vector pHel2 containing the cloned genes ureA and ureB and their specific promoter sequence. The orientation of the cloned insert was found to be crucial for urease expression. The majority of complemented clones functionally expressed urease at higher levels than did N6. Homologous recombination between chromosomal and cloned genes occurred at a frequency of 5%.

Multicenter comparison trial of DNA extraction methods and PCR assays for detection of Chlamydia pneumoniae in endarterectomy specimens.

The reported rate of detection of Chlamydia pneumoniae DNA within atherosclerotic lesions by PCR varies between 0 and 100%. In this study, identical sets of coded experimental atheroma samples (n = 15) and spiked controls (n = 5) were analyzed by 16 test methods in nine centers by means of PCR. The positive controls were correctly identified to levels of 1, 0.1, and 0.01 inclusion bodies of C. pneumoniae/ml of tissue homogenate by 16 (100%), 11 (69%), and 3 (19%) of the test methods, respectively. Three out of 16 negative controls (19%) were rated positive. Positivity rates for atheroma samples varied between 0 and 60% for the different test methods, with the maximum concordant result for positivity being only 25% for one carotid artery sample. There was no consistent pattern of positive results among the various laboratories, and there was no correlation between the detection rates and the sensitivity of the assay used.

Implementation of a molecular typing system to support epidemiological investigations in the tuberculosis health care system in Vienna.

Tuberculosis continues to be one of the predominant infectious diseases. Effective control of its spread requires that sources of infection and routes of transmission be disclosed as quickly as possible. At present such investigations are still performed by conventional epidemiological methods. In the recent past, however, molecular typing systems were added to the spectrum of epidemiological tools. Unfortunately, they were applied to retrospective investigations rather than used as an aid in the health care system. In this study, 515 Mycobacterium tuberculosis strains isolated during 1997 and 1998 in Vienna were analysed by spoligotyping, a molecular technique requiring no further cultivation of mycobacteria. The study was aimed to assess the suitability of the method as a quick means of disclosing new cases. Thus, clusters obtained by spoligotyping were analysed along with demographic and epidemiological data and compared with clusters obtained by conventional epidemiological techniques alone. In addition, spoligotype-forming clusters were matched with an international database containing spoligotypes from four different studies. Of 515 isolates, 107 showed an unique pattern. The remaining 408 isolates were distributed into two large clusters of 82 and 73 isolates and into 49 smaller ones consisting of 2 to 33 isolates each. The two spoligotypes forming the large clusters were identical with the most prevalent spoligotypes in the world. Therefore, for the tuberculosis authorities, information was only gained by excluding rather than tracing possible ways of transmission. Twenty-two of the 49 spoligotypes forming smaller clusters were identical with strains found in other parts of the world. Seventeen of 22 infection chains assumed by conventional investigations were confirmed by spoligotyping. In small clusters, an additional 24 infections were assumed due to similarities such as living conditions or socioeconomic status. In 27 clusters, all patients sharing the same strain belong to the same country or geographical area. In conclusion, spoligotyping proved suitable as an early guide in conventional investigations to trace routes of M. tuberculosis transmission in a community. However, when a strain isolated from a patient belongs to a spoligotype shared by many isolates, a second molecular typing method is required.

Susceptibility testing of Candida species: comparison of NCCLS microdilution method with Fungitest.

Fungitest is a new commercially available and easy-to-perform breakpoint test system using six antifungal agents. We compared this test with a modified standard method described by the National Committee for Clinical Laboratory Standards (NCCLS). One hundred isolates of Candida species were tested with both methods. Based on the same breakpoints, the correlation of qualitative results between the reference method and Fungitest was high. Best results were obtained after incubation of Fungitest for 48 h. Overall agreement was high, an excellent correlation was given with amphotericin B and flucytosine (100% and 99%, respectively), whereas itraconazole showed only 86% concordance. When Fungitest was read after 24 h the agreement was lower ranging from 100% to 75%. Some of the breakpoints used with Fungitest differ from the breakpoints recommended by NCCLS, whereas others have not been elaborated by the NCCLS. The adaptation of Fungitest breakpoints to NCCLS and determination of further breakpoints have to be discussed before Fungitest can be recommended for routine use.

Two enzyme immunoassays and PCR for detection of Helicobacter pylori in stool specimens from pediatric patients before and after eradication therapy.

This study of pediatric patients was intended to determine the suitability of stool PCR and two antigen enzyme immunoassays (EIAs; Premier Platinum HpSA and the novel FemtoLab H. pylori), which detect Helicobacter pylori antigens in feces, as pretreatment diagnostic tools and especially as posttreatment control. Forty-nine H. pylori-infected children with dyspepsia received eradication therapy. Successful treatment was determined by a negative [(13)C]urea breath test 4 and 12 weeks after discontinuation of therapy. Fecal specimens were collected prior to eradication therapy as well as 4 weeks after the end of treatment. Successfully treated children delivered stool samples at 6, 8, and 12 weeks posttreatment also. Specimens were examined by seminested PCR and Premier Platinum HpSA and were reexamined by both EIAs as soon as FemtoLab H. pylori was available. In the first test series, the overall sensitivities of PCR and Premier Platinum HpSA were 93.0 and 91.1%, respectively. With specimens collected at 4 weeks after treatment, the respective specificities were 68.8 and 79.3%. After longer follow-up periods, however, they gradually increased to 100 and 96.9%, respectively. In the new test series, Premier Platinum HpSA delivered a considerably lower number of false-positive results (4 versus 18), indicating intertest variations. The overall test sensitivity was 94.6%, and the overall specificity was 97.5%. FemtoLab H. pylori showed an excellent performance with an overall sensitivity and specificity of 98.2 and 98.1%, respectively. Thus, in contrast to PCR, both EIAs were shown to be suitable for early posttreatment control.

Drug resistance rates of Mycobacterium tuberculosis strains in Austria between 1995 and 1998 and molecular typing of multidrug-resistant isolates. The Austrian Drug Resistant Tuberculosis Study Group.

In this study the drug resistance pattern of 3559 Mycobacterium tuberculosis strains isolated in Austria between 1995 and 98 was evaluated. Of these strains, 165 (4.6%) were resistant to one or more drugs, 113 (3.2%) to one of the tested drugs and 53 (1.5%) to two or more drugs. Monodrug resistance was observed most often to isoniazid (56 strains), followed by streptomycin (44 strains). Resistance to rifampicin or ethambutol alone was rarely seen (12 strains and 1 strain, respectively). Of the 53 strains resistant to 2 or more drugs, 25 were resistant to isoniazid and streptomycin, while 17 were multidrug resistant. Molecular typing revealed a large diversity among the multidrug-resistant strains.