Molecular identification of streptomycin monoresistant Mycobacterium tuberculosis related to multidrug-resistant W strain.

A distinct branch of the Mycobacterium tuberculosis W phylogenetic lineage (W14 group) has been identified and characterized by various genotyping techniques. The W14 group comprises three strain variants: W14, W23, and W26, which accounted for 26 clinical isolates from the New York City metropolitan area. The W14 group shares a unique IS6110 hybridizing banding motif as well as distinct polymorphic GC-rich repetitive sequence and variable number tandem repeat patterns. All W14 group members have high levels of streptomycin resistance. When the streptomycin resistance rpsL target gene was sequenced, all members of this strain family had an identical mutation in codon 43. Patients infected with the W14 group were primarily of non- Hispanic black origin (77%); all were US-born. Including HIV positivity, 84% of the patients had at least one known risk factor for tuberculosis.

Rapid identification of laboratory contamination with Mycobacterium tuberculosis using variable number tandem repeat analysis.

Compared with solid media, broth-based mycobacterial culture systems have increased sensitivity but also have higher false-positive rates due to cross-contamination. Systematic strain typing is rarely undertaken because the techniques are technically demanding and the data are difficult to organize. Variable number tandem repeat (VNTR) analysis by PCR is rapid and reproducible. The digital profile is easily manipulated in a database. We undertook a retrospective study of Mycobacterium tuberculosis isolates collected over an 18-month period following the introduction of the BACTEC MGIT 960 system. VNTR allele profiles were determined with early positive broth cultures and entered into a database with the specimen processing date and other specimen data. We found 36 distinct VNTR profiles in cultures from 144 patients. Three common VNTR profiles accounted for 45% of true-positive cases. By combining VNTR results with specimen data, we identified nine cross-contamination incidents, six of which were previously unsuspected. These nine incidents resulted in 34 false-positive cultures for 29 patients. False-positive cultures were identified for three patients who had previously been culture positive for tuberculosis and were receiving treatment. Identification of cross-contamination incidents requires careful documentation of specimen data and good communication between clinical and laboratory staff. Automated broth culture systems should be supplemented with molecular analysis to identify cross-contamination events. VNTR analysis is reproducible and provides timely results when applied to early positive broth cultures. This method should ensure that patients are not placed on unnecessary tuberculosis therapy or that cases are not falsely identified as treatment failures. In addition, areas where existing procedures may be improved can be identified.

Rates of torsades de pointes associated with ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin, and moxifloxacin.

STUDY OBJECTIVE: To compare the rates of torsades de pointes associated with ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin, and moxifloxacin administration. DESIGN: Retrospective database analysis. INTERVENTION: Evaluation of reported rates of torsades de pointes in patients who received these quinolones between January 1, 1996, and May 2, 2001. MEASUREMENTS AND MAIN RESULTS: In the United States, 25 cases of torsades de pointes associated with these quinolones (ciprofloxacin 2, ofloxacin 2, levofloxacin 13, gatifloxacin 8, moxifloxacin 0) were identified. Ciprofloxacin was associated with a significantly lower rate of torsades de pointes (0.3 cases/10 million prescriptions, 95% confidence interval [CI] 0.0-1.1) than levofloxacin (5.4/10 million, 95% CI 2.9-9.3, p<0.001) or gatifloxacin (27/10 million, 95% CI 12-53, p<0.001 for comparison with ciprofloxacin or levofloxacin). When the analysis was limited to the first 16 months after initial U.S. approval of the agent, the rates for levofloxacin (16/10 million) and gatifloxacin (27/10 million) were similar (p>0.5). CONCLUSION: Levofloxacin should be administered with caution in patients with risk factors for QT prolongation. Gatifloxacin should be avoided in the same patient population, and the recommended dosage of 400 mg/day should not be exceeded.

Evaluation of the accuracy and reproducibility of a practical PCR panel assay for rapid detection and differentiation of Mycobacterium avium subspecies.

The Mycobacterium avium subspecies (MAs) include the closely related MAs avium and MAs paratuberculosis. This study was conducted to evaluate the performance of a PCR panel assay as a diagnostic tool to detect and differentiate MAs avium and MAs paratuberculosis infection. Specific oligonucleotides primers derived from the 16 S rRNA (MAs) sequence, insertion elements IS 901 (MAs avium), IS 1245 Mycobacterium avium complex (MAC), IS 900 (MAs paratuberculosis), and the hspX (MAs paratuberculosis) gene sequences were synthesized and used in preassembled PCR reaction mixtures. These five primer sets made up the PCR panel assay. To determine the accuracy of the PCR panel assay for MAs avium and MAs paratuberculosis strain detection and differentiation, lysates of mycobacterial DNA from 120 (n=120) strains were tested with the PCR panel assay by one laboratory (#1). The PCR panel assay specifically detected and differentiated 91/91 (100%) of MAs avium and MAs paratuberculosis strains tested in this study. The PCR panel assay also specifically differentiated all MAs avium and MAs paratuberculosis strains from all but one (M. intracellulare, serovar 23) of the other mycobacterial strains tested. To confirm the accuracy and evaluate the reproducibility of the PCR panel assay, samples were numbered and given to a different laboratory (#2) as 'unknowns' for identification by the PCR panel assay. In this study, the overall accuracy for strain identification using the PCR panel assay was 99.2% (119/120). The reproducibility of the PCR panel assay when comparing data from laboratory #1 with laboratory #2 was found to be 100% (120/120). These results indicate that this 'easy-to-use', rapid PCR method can accurately and reliably detect and differentiate closely related MAs avium and MAs paratuberculosis from each other and from other mycobacterial species. The PCR panel assay can also differentiate mixed cultures of MAs. The simplicity of this PCR method could be beneficial to laboratories that test for members of MA.

Mycobacteria: treatment approaches and mechanisms of resistance.

First-line drugs for tuberculosis treatment include isoniazid, rifampin, ethambutol, streptomycin, and pyrazinamide. Molecular mechanisms for resistance to each of these drugs have been elucidated. In every case, resistance is conferred by mutations in existing genes, not by the acquisition of new genetic material. Drug resistance is a major problem worldwide, but the rates vary widely among countries and within countries. Acquired resistance in previously-treated patients is much more common than primary resistance in patients with no previous treatment. High rates of acquired resistance have been reported from referral centers in Saudi Arabia and Lebanon. Successful TB treatment requires prolonged therapy with at least two active drugs. Directly-observed therapy (DOT) improves success rates and reduces the risk of acquired resistance. TB due to susceptible strains can be cured in over 95% of cases.

Identification of a W variant outbreak of Mycobacterium tuberculosis via population-based molecular epidemiology.

CONTEXT: Typing of Mycobacterium tuberculosis could provide a more sensitive means of identifying outbreaks than use of conventional surveillance techniques alone. Variants of the New York City W strain of M tuberculosis were identified in New Jersey. OBJECTIVE: To describe the spread of the W family of M tuberculosis strains in New Jersey identified by molecular typing and surveillance data. DESIGN: Population-based cross-sectional study. SETTING AND SUBJECTS: All incident culture-positive tuberculosis cases reported in New Jersey from January 1996 to September 1998, for which the W family was defined by insertion sequence (IS) IS6110 DNA fingerprinting, polymorphic GC-rich repetitive sequence (PGRS) typing, spacer oligotyping (spoligotyping), and variable number tandem repeat (VNTR) analysis. MAIN OUTCOME MEASURE: Identification and characterization of W family clones supplemented by surveillance data. RESULTS: Isolates from 1207 cases were analyzed, of which 68 isolates (6%) belonged to the W family based on IS6110 and spoligotype hybridization patterns. The IS6110 hybridization patterns or fingerprints revealed that43 patients (designated group A) shared a unique banding motif not present in other W family isolates. Strains collected from the remaining 25 patients (designated group B), while related to W, displayed a variety of IS6110 patterns and did not share this motif. The PGRS and VNTR typing confirmed the division of the W family into groups A and B and again showed group A strains to be closely related and group B strains to be more diverse. The demographic characteristics of individuals from groups A and B were specific and defined. Group A patients were more likely than group B patients to be US born (91 % vs 24%, P<.001), black (76% vs 16%, P<.001), human immunodeficiency virus positive (40% vs 0%, P = .007), and residents of urban northeast New Jersey counties (P<.001). Patients with group B strains were primarily non-US born, of Asian descent, and more dispersed throughout New Jersey. No outbreak had been detected using conventional surveillance alone. CONCLUSIONS: The implementation of multiple molecular techniques in conjunction with surveillance data enabled us to identify a previously undetected outbreak in a defined geographical setting. The outbreak isolates comprise members of a distinct branch of the W family phylogenetic lineage. The use of molecular strain typing provides a proactive approach that may be used to initiate, and not just augment, traditional surveillance outbreak investigations.

Bone marrow cultures for the diagnosis of mycobacterial and fungal infections in patients infected with the human immunodeficiency virus.

SETTING: University medical center. OBJECTIVE: To determine the value of bone marrow cultures for mycobacteria and fungi in patients infected with the human immunodeficiency virus (HIV). DESIGN: Retrospective review of charts and laboratory records. RESULTS: From 1992-1996, 1225 bone marrow specimens were submitted for mycobacterial and fungal cultures. The number of specimens submitted,declined sharply from 435 in 1992 to 94 in 1996 (P = 0.002 for trend). The yield remained stable. Thirty-one of 1225 specimens grew mycobacteria or fungi; 26 isolates were from 24 HIV-infected patients. These 24 patients were infected with Mycobacterium avium complex (19), M. tuberculosis (one), M. chelonae (one), Histoplasma capsulatum (two), and Cryptococcus neoformans (one). All 24 HIV-infected patients had a culture submitted from at least one other site within 4 weeks of the positive bone marrow culture. The identical organism was grown from another site (usually blood) in 18 of these 24 patients. The bone marrow culture provided the only positive result in six patients and the first positive result in eight patients. CONCLUSIONS: Utilization of bone marrow cultures for mycobacteria and fungi declined at our institution. Bone marrow and blood cultures were highly concordant. However, the majority of positive bone marrow cultures provided useful information.

Complex transmission dynamics of clonally related virulent Mycobacterium tuberculosis associated with barhopping by predominantly human immunodeficiency virus-positive gay men.

Limited data suggest that measures to reduce tuberculosis transmission should be based on locations rather than on personal contacts. Molecular epidemiologic methods (analysis of IS6110 patterns, spoligotypes, variable numbers of tandem DNA repeats, and automated DNA sequence data) identified a cohort of 48 persons who were infected with progeny of the same Mycobacterium tuberculosis strain. Epidemiologic investigation documented that a large proportion of the patients were gay white human immunodeficiency virus-positive men. Most practiced barhopping, an activity that involved patronizing many bars in the same neighborhood each night. Few subjects were directly linked to more than 1 or 2 other persons by conventional investigation methods, which shows that the transmission dynamics were unusually complex compared with most previously described episodes of strain spread. The data support the concept that identification of locations where pathogen dissemination likely occurs may provide additional strategies for targeted tuberculosis control.

The GroES antigens of Mycobacterium avium and Mycobacterium paratuberculosis.

The GroES antigen provokes a strong immune response in human beings with tuberculosis or leprosy. We cloned and sequenced the Mycobacterium avium and Mycobacterium paratuberculosis GroES genes. M. avium and M. paratuberculosis have identical GroES sequences which differ from other mycobacterial species. This supports the current formal designation of M. paratuberculosis as M. avium subsp. paratuberculosis. Immunodominant epitopes from Mycobacterium tuberculosis GroES are conserved in M. avium, but some Mycobacterium leprae epitopes are distinct. GroES is unlikely to be specific as a serologic or skin test reagent, but may be an appropriate component of a broad mycobacterial vaccine.

Protection against Mycobacterium avium by DNA vaccines expressing mycobacterial antigens as fusion proteins with green fluorescent protein.

Mycobacterium avium causes disseminated disease in humans with AIDS, paratuberculosis in ruminants, lymphadenopathy in swine, and tuberculosis in birds. We constructed DNA vaccines expressing mycobacterial antigens as fusion proteins with enhanced green fluorescent protein (EGFP). Plasmids p65K-EGFP, p85A-EGFP, and p85B-EGFP expressed the M. avium 65-kDa antigen, the Mycobacterium bovis BCG 85A antigen, and the M. avium 85B antigen, respectively, as EGFP fusion proteins. We visualized protein expression directly in cultured murine fibroblasts and intact muscle. p65K-EGFP expressed fusion protein in a diffuse cytoplasmic pattern, and p85A-EGFP and p85B-EGFP produced a speckled pattern. We vaccinated C57BL/6 mice with three doses of plasmid DNA and then challenged them intraperitoneally with M. avium. Negative controls received saline, and positive controls received one dose of BCG vaccine. Mice in all groups developed disseminated infection with a high burden of organisms. Compared to negative controls, mice vaccinated with p85A-EGFP had an eightfold reduction in spleen M. avium CFU at 4 weeks after infection and a fourfold reduction at 8 weeks, reductions similar to those generated by BCG vaccine. Mice vaccinated with p65K-EGFP had a fourfold CFU reduction at 4 weeks and no effect at 8 weeks. This is the first report of DNA vaccines expressing foreign antigens as fusion proteins with EGFP and the first report of successful DNA vaccination against M. avium.

Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility.

In this study, the currently known typing methods for Mycobacterium tuberculosis isolates were evaluated with regard to reproducibility, discrimination, and specificity. Therefore, 90 M. tuberculosis complex strains, originating from 38 countries, were tested in five restriction fragment length polymorphism (RFLP) typing methods and in seven PCR-based assays. In all methods, one or more repetitive DNA elements were targeted. The strain typing and the DNA fingerprint analysis were performed in the laboratory most experienced in the respective method. To examine intralaboratory reproducibility, blinded duplicate samples were included. The specificities of the various methods were tested by inclusion of 10 non-M. tuberculosis complex strains. All five RFLP typing methods were highly reproducible. The reliability of the PCR-based methods was highest for the mixed-linker PCR, followed by variable numbers of tandem repeat (VNTR) typing and spoligotyping. In contrast, the double repetitive element PCR (DRE-PCR), IS6110 inverse PCR, IS6110 ampliprinting, and arbitrarily primed PCR (APPCR) typing were found to be poorly reproducible. The 90 strains were best discriminated by IS6110 RFLP typing, yielding 84 different banding patterns, followed by mixed-linker PCR (81 patterns), APPCR (71 patterns), RFLP using the polymorphic GC-rich sequence as a probe (70 patterns), DRE-PCR (63 patterns), spoligotyping (61 patterns), and VNTR typing (56 patterns). We conclude that for epidemiological investigations, strain differentiation by IS6110 RFLP or mixed-linker PCR are the methods of choice. A strong association was found between the results of different genetic markers, indicating a clonal population structure of M. tuberculosis strains. Several separate genotype families within the M. tuberculosis complex could be recognized on the basis of the genetic markers used.

Phenotypic and genotypic characterization of Mycobacterium africanum isolates from West Africa.

The Mycobacterium tuberculosis complex includes M. tuberculosis, M. bovis, M. africanum, and M. microti. Most clinical isolates are M. tuberculosis or M. bovis. These species can be distinguished by phenotypes and genotypes. However, there is no simple definition of M. africanum, and some authors question the validity of this species. We analyzed 17 human isolates from Sierra Leone, identified as M. africanum by biochemical and growth characteristics. We sequenced polymorphic genes and intergenic regions. We amplified DNA from six loci with variable numbers of tandem repeats (VNTRs) and determined the exact number of repeats at each locus in each strain. All M. africanum isolates had the ancestral CTG Leu at katG codon 463. Drug-resistant M. africanum isolates had katG and rpoB mutations similar to those found in drug-resistant M. bovis and M. tuberculosis. Fourteen Sierra Leone M. africanum isolates (designated group A) had katG codon 203 ACC Thr, also found in M. africanumT (the T indicates type strain) from Senegal. Group A isolates clustered with M. africanumT by VNTR analysis. Three M. africanum isolates (group B) had katG codon 203 ACT Thr, found in M. tuberculosisT, and clustered with M. tuberculosisT by VNTR analysis. Phenotypic identification of M. africanum yielded a heterogeneous collection of strains. Genotypic analyses identified a cluster (M. africanum group A) which included M. africanumT and was distinct from the rest of the M. tuberculosis complex. Future studies of M. africanum should include both phenotypic and genotypic analyses.

Evolutionary bottlenecks in the agents of tuberculosis, leprosy, and paratuberculosis.

Parasitic mycobacteria cause important human and animal diseases including tuberculosis, leprosy, and paratuberculosis. Several methods demonstrate a high degree of sequence conservation in three parasitic mycobacterial species (Mycobacterium tuberculosis, M. leprae, and M. avium subspecies paratuberculosis). Each of these species has completely conserved deoxyribonucleic acid (DNA) sequence in an internal transcribed spacer. In contrast, several species of environmental mycobacteria (M. intracellulare, M. kansasii, M. gordonae, and M. scrofulaceum) have substantial strain-to-strain variation in this region. These data suggest that each of the parasitic species has gone through a recent evolutionary bottleneck. Comparisons of tandem-repeat DNA from ancient and modern mycobacterial strains may allow this hypothesis to be tested directly.