Double Repetitive Element PCR Research Articles

Molecular Mechanism of Multi-Drug Resistance in Mycobacterium tuberculosis

In this study, we demonstrate the clinical applicability of molecular assays for the differential identification of M. tuberculosis isolates by Double Repetitive Element-PCR (DRE-PCR), Duplex PCR (DPCR), Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and IS6110 flanking PCR and for the detection of specific codon mutations in antibiotic-resistant genes, rpoB and katG in 55 MDR-TB and 25 drug-susceptible clinical isolates by Multiplex PCR assays. The MAS-PCR assay was identified as the most prevalent rpoB gene mutations at codon 531 (83.6%), followed by codon 526 (12.7%), and no mutation was found in codon 516. Among the 55 MDR-TB isolates, 49 (89%) isolates had S315T, 5 (9%) had S315N mutations. DRE-PCR and RAPD-PCR generated similar banding of cluster III strains and suggested that MDR-TB strain genotype C may be responsible for the transmission of TB infection among the study population. PCR based differential identification of mtp40 and rpoB DPCR procedures identified two NTM strains among the isolates studied. Genotypic method DRE-PCR was found highly reproducible, followed by RAPD-PCR and mtp40, and rpoB DPCR methods effectively-identified NTM infection in this region. The presence of S315T mutation in katG gene and S531L, H526Y mutations in rpoB gene in MDR-TB isolates proved resistant phenotype. The simplicity of the MAS-PCR assay permits its implementation for the detection of resistance to INH and RIF in clinical laboratories in regions where this mutation is predominant among MDR-TB strains.

High clustering rates of multidrug-resistant Mycobacterium tuberculosisgenotypes in Panama

BackgroundTuberculosis continues to be one of the leading causes of death worldwide and in the American region. Although multidrug-resistant tuberculosis (MDR-TB) remains a threat to TB control in Panama, few studies have focused in typing MDR-TB strains. The aim of our study was to characterize MDR Mycobacterium tuberculosis clinical isolates using PCR-based genetic markers.MethodsFrom 2002 to 2004, a total of 231 Mycobacterium tuberculosis isolates from TB cases country-wide were screened for antibiotic resistance, and MDR-TB isolates were further genotyped by double repetitive element PCR (DRE-PCR), (GTG)5-PCR and spoligotyping.ResultsA total of 37 isolates (0.85%) were resistant to both isoniazid (INH) and rifampicin (RIF). Among these 37 isolates, only two (5.4%) were resistant to all five drugs tested. Dual genotyping using DRE-PCR and (GTG)5-PCR of MDR Mycobacterium tuberculosis isolates revealed eight clusters comprising 82.9% of the MDR-TB strain collection, and six isolates (17.1%) showed unique fingerprints. The spoligotyping of MDR-TB clinical isolates identified 68% as members of the 42 (LAM9) family genotype.ConclusionOur findings suggest that MDR Mycobacterium tuberculosis is highly clustered in Panama’s metropolitan area corresponding to Panama City and Colon City, and our study reveals the genotype distribution across the country.

Clinical and microbiological characteristics of urine culture-confirmed genitourinary tuberculosis at medical centers in Taiwan from 1995 to 2007

All patients with urine culture-confirmed genitourinary tuberculosis (GUTB) diagnosed between 1995 and 2007 at two medical centers in northern Taiwan were included in this retrospective study. Genotypes of 48 preserved Mycobacterium tuberculosis (MTB) isolates from these patients were determined by spoligotyping and double repetitive element PCR (DRE-PCR) analysis. Among the 64 patients, 38 (59.4%) were male with a mean ±SD age of 60.3 ± 16.1years old. The overall mortality rate was 26.2%. Poor prognostic factors included age over 65years (HR = 4.03; 95%; CI: 1.27-12.76), cardiovascular disease (HR = 5.96; 95% CI: 1.98-17.92), receiving steroids (HR = 10.16; 95% CI: 2.27-45.47), not being treated (HR 4.81; 95% CI 1.12-20.67). Spoligotyping and DRE-PCR of the 48 MTB isolates revealed that 20 (41.7%) belonged to the Beijing family and 40 (83.3%) had a clustering pattern. Identification of a Beijing family isolate was not correlated with drug resistance or mortality. Clustering strains were likely to be resistant to isoniazid (OR = 4.71; 95% CI: 1.10 to 23.53). In this study of patients with urine culture-confirmed GUTB, age and coexisting diseases were independently associated with an unfavorable outcome. The Beijing family was the dominant genotype of GUTB isolates, but did not correlate with drug resistance or outcome.

Molecular characterization of Mycobacterium tuberculosis isolates from Łódź, Poland: Analysis by IS 6110 restriction fragment length polymorphism and double-repetitive-element PCR

The aim of the present study was to characterize Mycobacterium tuberculosis strains isolated in the area of Łódź, Poland, from 1996 to 2000. Two hundred sixty three isolates from 250 patients with tuberculosis were analysed by IS6110 restriction fragment length polymorphism (RFLP) and the double-repetitive-element PCR (DRE-PCR) method when indicated. The isolates were found to show a great heterogeneity and only 52 strains (20.8%) occurred in 20 clusters of 2-5 identical clones. Despite this diversity of IS6110 RFLP patterns, a computer analysis of similarities revealed a high level of relatedness (at least 90%) among 38.4% of different patterns. Most of the patients with clustered strains showed no apparent epidemiologic links with other patients whose strains had the same pattern. Utilisation of the DRE-PCR analysis as an additional typing test allowed to differentiate M. tuberculosis strains with a discriminating capacity similar to that of the IS6110 RFLP. Also, DRE-PCR differentiated nine strains that were indistinguishable by the RFLP analysis. Both methods used for the molecular characterization of M. tuberculosis clinical isolates showed similar discriminating ability. DRE-PCR analysis proved a simple, rapid and cost-effective adjunct to the IS6110 RFLP reference method. It could be applied as a screening test, thus, reducing the number of isolates that need further subtyping with the IS6110 RFLP to those initially clustered.

Genotypic Diversity among Isoniazid-Resistant Isolates of Mycobacterium tuberculosis from Rashid Hospital in Dubai, United Arab Emirates

Objective: To perform molecular fingerprinting for strain relatedness among isoniazid-resistant Mycobacterium tuberculosis isolates recovered from tuberculosis (TB) patients in the United Arab Emirates (UAE). Materials and Methods: Thirty-two consecutive isoniazid-resistant M. tuberculosis strains isolated from TB patients (4 natives and 28 expatriates) at Rashid Hospital, Dubai, UAE were analyzed. The typing was carried out by touchdown double-repetitive-element PCR (DRE-PCR). The status of R463 or L463 polymorphism and the presence of S315T mutation in the katG gene were also determined for isolates exhibiting cluster pattern in DRE-PCR. Results: All the 32 isolates (28 from expatriate patients and 4 from UAE nationals) exhibited 21 distinct patterns in DRE-PCR with 20 of 32 isolates exhibiting unique patterns and the remaining 12 exhibiting cluster ‘A’ pattern. All the isolates (19 of 19) yielding two or more DNA fragments in DRE-PCR were unique strains. The genotypic heterogeneity among 10 of the 12 cluster isolates was suggested by the varying susceptibility of these isolates to anti-TB drugs, presence of R463 or L463 polymorphism in the katG gene and the presence or absence of S315T mutation in the katG gene. Conclusion: The genotypic diversity among isoniazid-resistant M. tuberculosis isolates mostly recovered from expatriate patients indicates that most expatriates were infected with a unique strain imported, most likely, from their country of origin and that their latent infection was reactivated in UAE.

Épidémiologie moléculaire de la tuberculose dans l’agglomération angevine étudiée par une stratégie d’association de 3 méthodes de génotypage par PCR

The new genotyping methods efficiently complement classical epidemiological investigation in order to attempt a global approach to TB control. In the present work, we have studied the genomic diversity of Mycobacterium tuberculosis isolated during the year 1998 within the district of Angers, France (260,000 inhabitants distributed in 29 districts), in order to identify recent transmission events and any related risk factors. The methods used included “spacer oligonucleotide typing” or spoligotyping, “variable number of DNA tandem repeats” or VNTR, and “double repetitive element PCR” or DRE-PCR. The resulting spoligotyping and VNTR results were also feeded to international databases and compared with >10,000 isolates for spoligotyping and 500 isolates for VNTR, representative of about 60 countries. The results obtained underlined that most of the TB cases in our setting probably reflected reactivation cases, as clustered cases indicative of potential events of recent transmission were rare. Furthermore, interrogation of international databases showed that most of the isolates from the Angers region belonged to major conserved families of TB isolates representative of Europe, with only rare cases of Asian origin, or those previously reported in specific epidemies reported from elsewhere.

Drug resistance and genotypes of strains of Mycobacterium tuberculosis isolated from human immunodeficiency virus-infected and non-infected tuberculosis patients in Bauru, São Paulo, Brazil.

Little is known about transmission and drug resistance of tuberculosis (TB) in Bauru, State of S o Paulo. The objective of this study was to evaluate risk factors for transmission of Mycobacterium tuberculosis strains in this area. Strains were collected from patients attended at ambulatory services in the region and susceptibility towards the main first line antibiotics was determined and fingerprinting performed. A total of 57 strains were submitted to susceptibility testing: 23 (42.6%) were resistant to at least one drug while 3 (13%) were resistant against both rifampicin and isoniazide. Resistant strains had been isolated from patients that had not (n = 13) or had (n = 9) previously been submitted to anti-TB treatment, demonstrating a preoccupying high level of primary resistance in the context of the study. All strains were submitted to IS6110 restriction fragment length polymorphism (IS6110-RFLP) and double repetitive element PCR (DRE-PCR). Using IS6110-RFLP, 26.3% of the strains were clustered and one cluster of 3 patients included 2 HIV-infected individuals that had been hospitalized together during 16 days; clustering of strains of patients from the hospital was however not higher than that of patients attended at health posts. According to DRE-PCR, 55.3% belonged to a cluster, confirming the larger discriminatory power of IS6110-RFLP when compared to DRE-PCR, that should therefore be used as a screening procedure only. No clinical, epidemiological or microbiological characteristics were associated with clustering so risk factors for transmission of TB could not be defined in the present study.

Molecular fingerprinting of isoniazid-resistant Mycobacterium tuberculosis isolates from chest diseases hospital in Kuwait.

Touchdown double-repetitive-element-PCR (DRE-PCR) was carried out for typing 38 consecutive isoniazid-resistant Mycobacterium tuberculosis strains isolated at Chest Diseases Hospital, Kuwait, during 1998-2000. The polymorphism at codon 463 in the katG gene was also determined and correlated with genotypic relationships among the isolates. The isolates exhibited 21 distinct patterns in DRE-PCR. Nearly half of the isolates (18 of 38) exhibited unique patterns. Majority of isolates (16 of 20) yielding multiple DNA fragments in DRE-PCR were unique strains while most of the isolates (16 of 18) yielding a single DNA fragment in DRE-PCR clustered together. The prevalence of L463 in the katG gene was much higher in isolates from Middle Eastern (mostly Kuwaiti) patients than is reported for this ethnic group. The data indicate the possibility of some strains of South Asian/Southeast Asian origin spreading among local populations.

Molecular typing of Mycobacterium tuberculosis based on variable number of tandem DNA repeats used alone and in association with spoligotyping.

Fingerprinting based on variable numbers of tandem DNA repeats (VNTR), a recently described methodology, was evaluated for molecular typing of Mycobacterium tuberculosis in an insular setting. In this study, VNTR fingerprinting was used alone or as a second-line test in association with spoligotyping, double-repetitive-element PCR (DRE-PCR), and IS6110 restriction fragment length polymorphism (RFLP) analysis, and the discriminatory power for each method or the combination of methods was compared by calculating the Hunter-Gaston discriminative index (HGI). The results obtained showed that in 6 out of 12 (50%) cases, VNTR-defined clusters were further subdivided by spoligotyping, compared to 7 out of 18 (39%) cases where spoligotyping-defined clusters were further subdivided by VNTR. When used alone, VNTR was the least discriminatory method (HGI = 0.863). Although VNTR was significantly more discriminatory when used in association with spoligotyping (HGI = 0.982), the combination of spoligotyping and DRE-PCR (HGI = 0.992) was still the most efficient among rapid, PCR-based methodologies, giving results comparable to IS6110 RFLP analysis. Nonetheless, VNTR typing may provide additional phylogenetical information that may be helpful to trace the molecular evolution of tubercle bacilli.

Molecular fingerprinting of mycobacterium tuberculosis isolates obtained in havana, cuba, by IS6110 restriction fragment length polymorphism analysis and by the double-repetitive-element PCR method.

Mycobacterium tuberculosis sputum isolates from 38 patients, obtained in the first 6 months of 1997 in Havana, Cuba, were characterized by IS6110 restriction fragment length polymorphism (RFLP) analysis and the double-repetitive-element PCR (DRE-PCR) method. Among 41 strains from 38 patients, 24 and 25 unique patterns, and 5 and 4 cluster patterns, were found by the RFLP and DRE-PCR methods, respectively. Patients within two of these clusters were found to be epidemiologically related, while no relation was observed in patients in the other clusters. The DRE-PCR method is rapid, and it was as discriminating as IS6110 RFLP analysis in identifying an epidemiological association. Its simplicity makes the technique accessible for subtyping of M. tuberculosis strains in laboratories not equipped to perform RFLP analysis.