Assessment of Helicobacter pylori positive infected patients according to Clarithromycin resistant 23S rRNA, rpl22 associated mutations and cyp2c19*1, *2, *3 genes pattern in the Early stage of Gastritis.

OBJECTIVE: Clarithromycin resistant Helicobacter pylori (CAM-R) is the main cause of standard triple therapy eradicating failure. Proton pump inhibitors (PPIs) directly pose bacteriocidic activity and prepare the optimum condition for Clarithromycin's best function. In counter with Poor metabolizer subjects, Homozygote Extensive Metabolizers have well characterized by treatment failure. Eventually, determination of CAM-R profile and estimation of PPIs metabolization rate support clinicians in better prescription. So, we explored Helicobacter pylori'mutations in 23S rRNA and rpl22 resistant genes, and cyp2c19 *1, *2, *3 allele variations, and PPIs metabolization patterns in patients, consequently the results reported to the physician. RESULTS: Sixteen out of 96 patients considered to be CAM-R Helicobacter pylori. A2143C (1/16), rpl22 insertion (16/16), and GTG deletion (2/16) recorded in CAM-R strains. P450 2C19 human genotyping demonstrated that the highest proportion of the H. pylori- positive strains infected patients 43/61(70.49%) categorized in Homozygote extensive metabolizer class. The rest (12/61)19.67% classified as Poor metabolizers, and 6/61(9.83%) distinct from Heterozygote extensive metabolizer group. Proportion of poor metabolizers and Heterozygote extensive metabolizer phenotypes between CAM-R strains mentioned to be 10/16(62.5%), and 6/16(37.5%). Cross points between the most frequently distributed allele in CAM-R strains indicated 81.25% for *2, and w2 for 18.75%.

Direct assessment of possible mutations in the 23S rRNA gene encoding macrolide resistance in Chlamydia trachomatis.

Reports of potential treatment failure have raised particular concerns regarding the efficacy of the single dose azithromycin regimen in the treatment of urogenital and anorectal Chlamydia trachomatis (CT) infections. Several factors have been suggested, including heterotypic resistance. Antimicrobial susceptibility testing in CT requires cell culture with serial dilutions of antibiotics, which is laborious and for which there is no standardized testing methodology. One method to partly overcome these difficulties would be to use a genotypic resistance assay, however most current available assays do still require prior CT culture. In order to facilitate the assessment of genotypic resistance directly from clinical samples, without the need for prior culture, the aim of this study was to develop a CT specific PCR assay for the assessment of resistance associated mutations (RAMs) in the 23S rRNA gene, and to evaluate a sample of clinical cases in which CT PCR's remained positive during follow-up despite azithromycin treatment. Neither the in silico analysis nor the analytical specificity testing demonstrated clinically relevant cross-reactivity with other bacterial species. These results in conjunction with the analytical sensitivity demonstrating consistent CT 23S rRNA gene detection in the range of 10e3 IFU/mL, exemplify the assay's apt performance. Although no known macrolide RAMs were detected in the clinical cases, the described assay allows future culture independent macrolide RAM surveillance in CT, and increases accessibility for other laboratories to engage in screening.

Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome.

Microbial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had > 90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The rRNA operon amplicon sequencing of equine fecal samples underestimated compositional percentages of bacterial strains containing unlinked rRNA genes by a fourth to a third, but unlinked rRNA genes had a limited effect on the overall results. The rRNA operon amplicon sequencing with the A519F + U2428R primer set was able to detect some kind of archaeal genomes such as Methanobacteriales and Methanomicrobiales, whereas full-length 16S rRNA with 27F + 1492R could not. Therefore, we conclude that amplicon sequencing targeting the rRNA operon captures more detailed variations of equine microbiota.

Seven-day triple therapy is sufficient to eradicate infection caused by Helicobacter pylori without 23S rRNA point mutation.

ABSTRACT: Tailored therapy based on dual priming oligonucleotide-based polymerase chain reaction (DPO-PCR) can be considered an alternative to overcome the low eradication rate in high clarithromycin-resistance areas. The triple therapy (TT) duration of the tailored approach in most studies was 7 days for patients without point mutation. However, recent western guidelines have recommended a treatment duration of 14 days. The aim of this study was to compare the success rate of 7 and 14 days of TT for eradicating Helicobacter pylori without point mutation, as determined by DPO-PCR.Between Feb 2016 and Feb 2019, medical records of patients who underwent DPO-PCR were reviewed. Patients without point mutation as determined by DPO-PCR were enrolled in this study. The eradication success rate and adverse events were evaluated.A total of 366 patients without A2142G and A2143G point mutation were enrolled. The success rates of 7-day and 14-day TT were 88.4% (168/190) and 85.9% (151/176) by intention to treat analysis (P = .453) and 90.8% (168/185) and 90.4% (151/167) by per-protocol analysis (P = .900), respectively. The adverse event rates showed no significant difference between the 2 groups.In patients without point mutation based on DPO-PCR results, 7-day TT is as effective as 14-day TT. Therefore, 7 days may be considered as a cost-effective treatment duration in Korea.

Molecular Assessment of Resistance to Clarithromycin in Helicobacter pylori Strains Isolated from Patients with Dyspepsia by Fluorescent In Situ Hybridization in the Center of Iran.

BACKGROUND AND AIMS: Helicobacter pylori is a common infectious bacterium mostly found in gastroduodenal diseases. The increased prevalence of clarithromycin-resistant H. pylori strains is a major challenge in the successful treatment of infections caused by this organism. The present study is aimed at detecting the clarithromycin resistance pattern of H. pylori strains isolated from gastric biopsies and evaluating point mutations of the 23S rRNA gene. Patients and methods. In the present descriptive cross-sectional study, 165 patients with gastrointestinal disorders, who were referred to the Endoscopy Center of Dr. Shariati Hospital of Isfahan, Iran, were enrolled from April to July 2018. H. pylori infection was diagnosed by culture, and susceptibility of the isolates to clarithromycin was assessed by the E-test. Minimum inhibitory concentration (MIC) values were obtained based on EUCAST recommendations. Also, fluorescence in situ hybridization (FISH) was used to determine point mutations associated with clarithromycin resistance. RESULTS: By using culturing, H. pylori was isolated from 50.3% (83/165) gastric biopsy specimens. The overall frequency of resistance to clarithromycin was 25.3% (21/83) by the E-test. In the resistance genotypic analysis, 19 isolates had mutations. The prevalence of A2143G and A2144G mutations was 68.4% (13/19) and 31.5% (6/19), respectively. A2143C mutation was not tracked in any isolate. Two isolates with MIC > 0.5 µg/mL had no mutations that could be related to other mechanisms of resistance. CONCLUSION: As presented in the study, the high prevalence of clarithromycin-resistant H. pylori due to point mutations of the 23S rRNA gene indicates the necessity of revising the standard treatment regimen based on antibiotic susceptibility pattern of each region.

Cost-effectiveness of a tailored Helicobacter pylori eradication strategy based on the presence of a 23S ribosomal RNA point mutation that causes clarithromycin resistance in Korean patients.

BACKGROUND AND AIM: The Helicobacter pylori eradication rate using conventional triple therapy has decreased due to clarithromycin (CAM) resistance in H. pylori. Recently, dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) can be used to detect H. pylori and point mutations in the 23S ribosomal RNA gene causing CAM resistance. This study aimed to evaluate the success rate and cost-effectiveness of tailored H. pylori eradication using DPO-PCR. METHODS: The H. pylori-positive patients diagnosed by a rapid urease test or DPO-PCR were enrolled from a single academic hospital. The patients with positive rapid urease test results received a CAM-based triple regimen. In the tailored therapy group that underwent DPO-PCR testing, patients with A2142G and/or A2143G point mutations were treated with a bismuth-containing quadruple regimen. The cost-effectiveness of H. pylori eradication success was evaluated according to the average cost per patient and the incremental cost-effectiveness ratio. RESULTS: A total of 243 patients were allocated to the triple therapy group and 124 patients to the tailored therapy group. The first-line eradication rate of H. pylori was significantly higher in the tailored therapy group than in the conventional triple therapy group (92.7% vs 76.5%, P < 0.001). The average costs per patient for tailored therapy were $307.37 and $299.59 for first-line and second-line treatments, respectively. Compared with triple therapy, the incremental cost-effectiveness ratios of tailored therapy were $3.96 and -$3.81 per patient for first-line and second-line treatments, respectively. CONCLUSION: In Korea, tailored H. pylori eradication using DPO-PCR may be more cost-effective than conventional triple therapy.

Isolation, characterization, and antibiotic sensitivity assessment of Gallibacterium anatis biovar haemolytica, from diseased Egyptian chicken flocks during the years 2013 and 2015.

Gallibacterium anatis biovar haemolytica constitutes a part of the normal microflora in the upper respiratory and genital tracts of healthy chickens, but it is also associated with different pathological conditions. In the current study, 102 commercial chicken flocks suffering from respiratory disease and/or drop in egg production were investigated for the presence of G. anatis during 2013 and 2015. These flocks comprised 8 breeder, 32 layer, and 62 broiler flocks. By culture method, 20 flocks were found positive: one isolate derived from broiler breeders, 6 isolates from layers, and 13 isolates from broilers. G. anatis biovar haemolytica was identified by phenotyping and PCR. Additionally, partial genome sequencing of 11 isolates (5 layer isolates of 2013 and 6 broiler isolates of 2015) based on 16S rRNA and 23S rRNA gene sequences was performed and revealed 96.5% to 100% genetic relatedness. Antibiotic sensitivity of these isolates revealed that the 2013 isolates were highly susceptible to florfenicol while the isolates of 2015 were highly susceptible to cefotaxime. Gallibacterium anatis biovar haemolytica is a newly introduced bacteria in Egypt causing salpingitis, peritonitis, drop in egg production, and/or respiratory signs.

Sybr Green- and TaqMan-Based Quantitative PCR Approaches Allow Assessment of the Abundance and Relative Distribution of Frankia Clusters in Soils.

The nodule-forming actinobacterial genus Frankia can generally be divided into 4 taxonomic clusters, with clusters 1, 2, and 3 representing nitrogen-fixing strains of different host infection groups and cluster 4 representing atypical, generally non-nitrogen-fixing strains. Recently, quantitative PCR (qPCR)-based quantification methods have been developed for frankiae of clusters 1 and 3; however, similar approaches for clusters 2 and 4 were missing. We amended a database of partial 23S rRNA gene sequences of Frankia strains belonging to clusters 1 and 3 with sequences of frankiae representing clusters 2 and 4. The alignment allowed us to design primers and probes for the specific detection and quantification of these Frankia clusters by either Sybr Green- or TaqMan-based qPCR. Analyses of frankiae in different soils, all obtained from the same region in Illinois, USA, provided similar results, independent of the qPCR method applied, with abundance estimates of 10 × 105 to 15 × 105 cells (g soil)-1 depending on the soil. Diversity was higher in prairie soils (native, restored, and cultivated), with frankiae of all 4 clusters detected and those of cluster 4 dominating, while diversity in soils under Alnus glutinosa, a host plant for cluster 1 frankiae, or Betula nigra, a related nonhost plant, was restricted to cluster 1 and 3 frankiae and generally members of subgroup 1b were dominating. These results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees.IMPORTANCE Root nodule formation by the actinobacterium Frankia is host plant specific and largely, but not exclusively, correlates with assignments of strains to specific clusters within the genus. Due to the lack of adequate detection and quantification tools, studies on Frankia have been limited to clusters 1 and 3 and generally excluded clusters 2 and 4. We have developed tools for the detection and quantification of clusters 2 and 4, which can now be used in combination with those developed for clusters 1 and 3 to retrieve information on the ecology of all clusters delineated within the genus Frankia Our initial results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees.

Prevalence and genetic diversity of endosymbiotic bacteria infecting cassava whiteflies in Africa.

BACKGROUND: Cassava provides over half of the dietary requirement for more than 200 million poor in Africa. In recent years, cassava has been affected by an epidemic of a virus disease called cassava brown streak disease (CBSD) that is spreading in much of eastern and central Africa, affecting food security and the economic development of the poor. The viruses that cause CBSD are transmitted by the insect vector whitefly (Bemisia tabaci), which have increased to very high numbers in some African countries. Strains of endosymbiotic bacteria infecting whiteflies have been reported to interact specifically with different whitefly populations with varied effects on its host biology and efficiency of virus transmission. The main aim of this study was therefore to investigate the prevalence and diversity of the secondary endosymbiotic bacteria infecting cassava whiteflies with a view to better understand their role on insect population dynamics and virus disease epidemics. RESULTS: The genetic diversity of field-collected whitefly from Tanzania, Malawi, Uganda and Nigeria was determined by mitochondrial DNA based phylogeny and restriction fragment length polymorphism. Cassava in these countries was infected with five whitefly populations, and each one was infected with different endosymbiotic bacteria. Incidences of Arsenophonus, Rickettsia, Wolbachia and Cardinium varied amongst the populations. Wolbachia was the most predominant symbiont with infection levels varying from 21 to 97%. Infection levels of Arsenophonus varied from 17 to 64% and that of Rickettsia was 0 to 53%. Hamiltonella and Fritschea were absent in all the samples. Multiple locus sequence typing identified four different strains of Wolbachia infecting cassava whiteflies. A common strain of Wolbachia infected the whitefly population Sub-Saharan Africa 1-subgroup 1 (SSA1-SG1) and SSA1-SG2, while others were infected with different strains. Phylogeny based on 16S rDNA of Rickettsia and 23S rDNA of Arsenophonus also identified distinct strains. CONCLUSIONS: Genetically diverse bacteria infect cassava whiteflies in Africa with varied prevalence across different host populations, which may affect their whitefly biology. Further studies are required to investigate the role of endosymbionts to better understand the whitefly population dynamics.

Low fitness cost of the multidrug resistance gene cfr.

The recently described rRNA methyltransferase Cfr that methylates the conserved 23S rRNA residue A2503, located in a functionally critical region of the ribosome, confers resistance to an array of ribosomal antibiotics, including linezolid. A number of reports of linezolid-resistant cfr-positive clinical strains indicate the possible rapid spread of this resistance mechanism. Since the rate of dissemination and the efficiency of maintenance of a resistance gene depend on the fitness cost associated with its acquisition, we investigated the fitness cost of cfr expression in a laboratory Staphylococcus aureus strain. We found that acquisition of the cfr gene does not produce any appreciable reduction in the cell growth rate. Only in a cogrowth competition experiment was some loss of fitness observed because Cfr-expressing cells slowly lose to the cfr-negative control strain. Interestingly, cells expressing wild-type and catalytically inactive Cfr had very similar growth characteristics, indicating that the slight fitness cost associated with cfr acquisition stems from expression of the Cfr polypeptide rather than from the modification of the conserved rRNA residue. In some clinical isolates, cfr is coexpressed with the erm gene, which encodes a methyltransferase targeting another 23S rRNA residue, A2058. Dimethylation of A2058 by Erm notably increases the fitness cost associated with the Cfr-mediated methylation of A2503. The generally low fitness cost of cfr acquisition observed in our experiments with the laboratory S. aureus strain offers a microbiological explanation for the apparent spread of the cfr gene among pathogens.

Assessment of clarithromycin susceptibility in strains belonging to the Mycobacterium abscessus group by erm(41) and rrl sequencing.

Clarithromycin was the drug of choice for Mycobacterium abscessus infections until inducible resistance due to erm(41) was described. Because M. abscessus was split into M. abscessus sensu stricto, Mycobacterium massiliense, and Mycobacterium bolletii, we looked for erm(41) in the three species and determined their clarithromycin susceptibility levels. Ninety strains were included: 87 clinical strains from cystic fibrosis patients (61%) and others (39%), representing 43 M. abscessus, 30 M. massiliense, and 14 M. bolletii strains identified on a molecular basis, and 3 reference strains. Clarithromycin and azithromycin MICs were determined by broth microdilution and Etest with a 14-day incubation period. Mutations in rrl (23S rRNA gene) known to confer acquired clarithromycin resistance were also sought. erm(41) was detected in all strains but with two deletions in all M. massiliense strains. These strains were indeed susceptible to clarithromycin (MIC(90) of 1 µg/ml) except for four strains with rrl mutations. M. abscessus strains harbored an intact erm(41) but had a T/C polymorphism at the 28th nucleotide: T28 strains (Trp10 codon) demonstrated inducible clarithromycin resistance (MIC(90) of >16 µg/ml), while C28 strains (Arg10) were susceptible (MIC(90) of 2 µg/ml) except for two strains with rrl mutations. M. bolletii strains had erm(41) sequences similar to the sequence of the T28 M. abscessus group, associated with inducible clarithromycin resistance (MIC(90) of >16 µg/ml). erm(41) sequences appeared species specific within the M. abscessus group and were fully concordant with clarithromycin susceptibility when erm(41) sequencing was associated with detection of rrl mutations. Clarithromycin-resistant strains, including the six rrl mutants, were more often isolated in cystic fibrosis patients, but this was not significantly associated with a previous treatment.

Assessment of linezolid resistance mechanisms among Staphylococcus epidermidis causing bacteraemia in Rome, Italy.

OBJECTIVES: To characterize linezolid resistance among blood cultured Staphylococcus epidermidis from patients at the Polyclinic Agostino Gemelli (2006-08). Isolates also showed elevated MICs of macrolide, lincosamide and streptogramin (MLS) compounds, which were investigated. METHODS: Ten S. epidermidis exhibiting linezolid MICs ≥ 4 mg/L were included. Isolates were screened for cfr mutations in 23S rRNA, L3, L4 and L22, and MLS genes by PCR/sequencing. Ribosomal proteins were compared with those from a linezolid-susceptible (MIC, 1 mg/L) clinical strain and ATCC 12228. cfr location was determined by Southern blot/hybridization. The cfr strain was submitted to plasmid curing. Epidemiology was assessed by PFGE and multilocus sequence typing (MLST). RESULTS: S. epidermidis displayed linezolid MICs of 4 or 8 mg/L, except for strain 4303A (MIC, 64 mg/L). These organisms and a linezolid-susceptible strain exhibited L3 Leu101Val compared with ATCC 12228. Isolates also showed L3 Phe147Leu and Ala157Arg, and L4 Asn158Ser. Strain 12375A possessed L4 Lys68Arg. Isolates were wild-type for 23S rRNA and L22. cfr was plasmid located in strain 4303A and the plasmid-cured strain exhibited a linezolid MIC (4 mg/L) similar to that for cfr-negative strains (4-8 mg/L). All organisms harboured erm(A) and msr(A), while vga(A) was detected in several isolates. All isolates were clonally related and ST-23. CONCLUSIONS: L3 Phe147Leu and/or Ala157Arg appeared responsible for the elevated linezolid MIC, since adjacent alterations have been associated with resistance. L4 Asn158Ser has been reported in a linezolid-susceptible isolate and Lys68Arg detected here did not seem to provide an additive effect. Acquisition of cfr markedly increased (8- to 16-fold) the linezolid MICs. vga(A) was associated with higher MICs of quinupristin/dalfopristin and retapamulin.

Identification of Francisella tularensis by whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry: fast, reliable, robust, and cost-effective differentiation on species and subspecies levels.

Francisella tularensis, the causative agent of tularemia, is a potential agent of bioterrorism. The phenotypic discrimination of closely related, but differently virulent, Francisella tularensis subspecies with phenotyping methods is difficult and time-consuming, often producing ambiguous results. As a fast and simple alternative, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was applied to 50 different strains of the genus Francisella to assess its ability to identify and discriminate between strains according to their designated species and subspecies. Reference spectra from five representative strains of Francisella philomiragia, Francisella tularensis subsp. tularensis, Francisella tularensis subsp. holarctica, Francisella tularensis subsp. mediasiatica, and Francisella tularensis subsp. novicida were established and evaluated for their capability to correctly identify Francisella species and subspecies by matching a collection of spectra from 45 blind-coded Francisella strains against a database containing the five reference spectra and 3,287 spectra from other microorganisms. As a reference method for identification of strains from the genus Francisella, 23S rRNA gene sequencing was used. All strains were correctly identified, with both methods showing perfect agreement at the species level as well as at the subspecies level. The identification of Francisella strains by MALDI-TOF MS and subsequent database matching was reproducible using biological replicates, different culture media, different cultivation times, different serial in vitro passages of the same strain, different preparation protocols, and different mass spectrometers.

23S rRNA mutation A2074C conferring high-level macrolide resistance and fitness cost in Campylobacter jejuni.

To examine the development of macrolide resistance in Campylobacter jejuni and assess the fitness of the macrolide-resistant mutants, two macrolide-susceptible C. jejuni strains, American Type Culture Collection (ATCC) 33291 and H1, from different geographic areas were exposed to tylosin in vitro. Multiple mutant strains were obtained from the selection. Most of the high-level macrolide-resistant strains derived from the selection exhibited the A2074C transversion in all three copies of 23S rRNA and displayed strong stability in the absence of antibiotic selection pressure. The competition experiments demonstrated that the strains containing the A2074C transversion imposed a fitness cost in competition mixtures. In addition, the fitness cost of the mutation was not ameliorated after approximately 500 generations of evolution under laboratory conditions. These findings indicate that the A2074C transversion in C. jejuni is not only correlated with stable and high-level macrolide resistance but also associated with a fitness cost.

Linezolid resistance in Staphylococcus aureus: gene dosage effect, stability, fitness costs, and cross-resistances.

Linezolid resistance in Staphylococcus aureus is typically associated with mutations in the 23S rRNA gene. Here we show that the accumulation of a single point mutation, G2576T, in the different copies of this gene causes stepwise increases in resistance, impairment of the biological fitness, and cross-resistance to quinupristin-dalfopristin and chloramphenicol.

Frequency of development and associated physiological cost of azithromycin resistance in Chlamydia psittaci 6BC and C. trachomatis L2.

Azithromycin is a major drug used in the treatment and prophylaxis of chlamydial infections. Spontaneous azithromycin-resistant mutants of Chlamydia psittaci 6BC were isolated in vitro in the plaque assay at a frequency of about 10(-8). Isogenic clonal variants with A(2058)C, A(2059)G, or A(2059)C mutations in the unique 23S rRNA gene (Escherichia coli numbering system) displayed MICs for multiple macrolides (i.e., azithromycin, erythromycin, josamycin, and spiramycin) at least 100 times higher than those of the parent strain and were also more resistant to the lincosamide clindamycin. Chlamydia trachomatis L2 variants with a Gln-to-Lys substitution in ribosomal protein L4 at position 66 (E. coli numbering system), conferring an eightfold decrease in azithromycin and erythromycin sensitivities and a fourfold decrease in josamycin and spiramycin sensitivities, were isolated following serial passage in subinhibitory concentrations of azithromycin. Each mutation was stably maintained in the absence of selection but severely affected chlamydial infectivity, as determined by monitoring the development of each isolate over 46 h in the absence of selection, in pure culture or in 1:1 competition with the isogenic parent. Data in this study support the hypothesis that the mechanisms which confer high-level macrolide resistance in chlamydiae carry a prohibitive physiological cost and may thus limit the emergence of highly resistant clones of these important pathogens in vivo.

Multiplex approach for screening genetic markers of microbial indicators.

Genetic markers are expected to provide better specificity in epidemiological studies and potentially serve as better indicators of waterborne pathogens. Methods used to assess genetic markers of emerging microbial indicators include pulsed field gel electrophoresis, polymerase chain reaction (PCR), and microarrays. This paper outlines a high-throughput approach to screen for such genetic markers using a set of theoretical and experimental screening tools. The theoretical screening involves evaluating genes related to the ribosomal RNA and specific functions from emerging indicator groups, followed by experimental validation with appropriate sampling schemes and high-throughput and economical screening methods, such as microarrays, real time PCR, and on-chip PCR. Analysis of a wide range of samples covering temporal variability in location, host, and waterborne disease outbreaks is essential. The proposed approach is expected to shorten the time and cost associated with searching for new genetic markers of emerging indicators by at least 10-fold.

Assessment of Southern blot ribotyping for differentiation of Leptospira strains isolated from field rats.

A Southern blot ribotyping based on EcoRV and HindIII digestion with two 16S and 23S rDNA probes for differentiating 27 Leptospira serovars was developed. The results between ribotyping and serotyping among 40 leptospiral strains isolated from field rats trapped in the northeastern region of Thailand during 1999-2000, were compared. A combination of Southern blot ribotyping, using EcoRV or HindIII digestion with both 16S and 23S rDNA as the probes, successfully typed 27 Leptospira serovars into 24 ribotypes with the discriminatory index (D) values of 0.99. The 16S- and 23S-EcoRV ribopatterns produced 17 and 9 profiles, respectively, with D values of 0.95 and 0.63, respectively. Ribopatterns of HindIII from both specific probes yielded 17 patterns. The D values of 16S- and 23S-HindIII ribopatterns were 0.94 and 0.93, respectively. With EcoRV digestion, the 16S rDNA probe was more discriminative than the 23S rDNA probe for differentiating Leptospira serovars. Moreover, the 16S-EcoRV (11 profiles), 16S-HindIII (11 profiles), and 23S-HindIII (10 profiles) ribopatterns produced higher numbers of distinct and unique profiles than the 23S-EcoRV (5 profiles). The results showed 100% concordance between ribotyping and serotyping, leading to all 40 isolates being successfully typed. The current study revealed that ribotyping as a quick and powerful tool for differentiating Leptospira serovars, has potential value in epidemiological studies.

Routine use of real-time PCR for detection of Helicobacter pylori and of clarithromycin resistance mutations.

OBJECTIVES: We previously showed that real-time PCR was a reliable technique for coupled detection of Helicobacter pylori and clarithromycin resistance mutations directly from biopsies. After one year of use, we compared its performances to those of histology, which remains the most employed method for H. pylori detection from gastric biopsies. MATERIALS AND METHODS: 518 subjects underwent endoscopy during the year 2003 with biopsies taken for H. pylori detection by histology, PCR, and in case of discrepancy between the two techniques, by culture. RESULTS: The prevalence of infection, defined as positive PCR and histology, and in case of discrepancy as a positive culture, was 30% (163/518). The percentage of concordance between the two tests was 87.8% (455/518). The sensitivity, specificity, positive and negative predictive values of PCR were 98.2%, 97.5%, 94.7%, and 99.1%, respectively. The corresponding performances of histology were 87.7%, 91.3%, 82.2%, and 94.2%, respectively (p<0.001). The prevalence of clarithromycin resistance was 30%. CONCLUSIONS: PCR is more accurate in routine than histology and permits easy determination of clarithromycin resistance, which is useful in countries like France where the prevalence of resistance is high.