Jack T. Crawford

Nosocomial transmission of multidrug-resistant Mycobacterium tuberculosis : a risk to patients and health care workers

OBJECTIVE To determine the factors associated with the development of multidrug-resistant tuberculosis among patients at a New York City Hospital and to investigate possible nosocomial transmission. DESIGN A retrospective case-control study and tuberculin skin test survey. PATIENTS Twenty-three patients with tuberculosis whose isolates were resistant to at least isoniazid and rifampin (case patients) were compared with patients with tuberculosis whose isolates were susceptible to all agents tested (controls). Tuberculin skin test conversion rates were compared among health care workers assigned to wards where patients with tuberculosis were frequently or rarely admitted. SETTING A large, teaching hospital in New York City. MEASUREMENTS Mycobacterium tuberculosis isolates from case patients and controls were typed by restriction fragment length polymorphism analysis. RESULTS Case patients were younger (median age, 34 compared with 42 years; P = 0.006), more likely to be seropositive for HIV (21 of 23 compared with 11 of 23 patients; odds ratio, 11.5; 95% CI, 1.9 to 117), and more likely to have had a previous hospital admission within 7 months before the onset of tuberculosis (19 of 23 compared with 5 of 23 patients; odds ratio, 17.1; CI, 3.3 to 97), particularly on one ward (12 of 23 compared with 0 of 23 patients; odds ratio, undefined; P = 0.002). Health care workers assigned to wards housing case patients were more likely to have tuberculin skin test conversions than were health care workers assigned to other wards (11 of 32 compared with 1 of 47 health care workers; P less than 0.001). Few (6 of 23) case patients were placed in acid-fast bacilli isolation, and no rooms tested had negative pressure. Of 16 available multidrug-resistant isolates obtained from case patients, 14 had identical banding patterns by restriction fragment length polymorphism analysis. In contrast, M. tuberculosis isolates from controls with drug-susceptible tuberculosis had patterns distinct from each other and from those of case patients. CONCLUSIONS These data suggest nosocomial transmission of multidrug-resistant tuberculosis occurred from patient to patient and from patient to health care worker and underscore the need for effective acid-fast bacilli isolation facilities and adherence to published infection control guidelines in health care institutions.

ethA, inhA, and katG Loci of Ethionamide-Resistant Clinical Mycobacterium tuberculosis Isolates

ABSTRACT Ethionamide (ETH) is a structural analog of the antituberculosis drug isoniazid (INH). Both of these drugs target InhA, an enzyme involved in mycolic acid biosynthesis. INH requires catalase-peroxidase (KatG) activation, and mutations in katG are a major INH resistance mechanism. Recently an enzyme (EthA) capable of activating ETH has been identified. We sequenced the entire ethA structural gene of 41 ETH-resistant Mycobacterium tuberculosis isolates. We also sequenced two regions of inhA and all or part of katG. The MICs of ETH and INH were determined in order to associate the mutations identified with a resistance phenotype. Fifteen isolates were found to possess ethA mutations, for all of which the ETH MICs were ≥50 μg/ml. The ethA mutations were all different, previously unreported, and distributed throughout the gene. In eight of the isolates, a missense mutation in the inhA structural gene occurred. The ETH MICs for seven of the InhA mutants were ≥100 μg/ml, and these isolates were also resistant to ≥8 μg of INH per ml. Only a single point mutation in the inhA promoter was identified in 14 isolates. A katG mutation occurred in 15 isolates, for which the INH MICs for all but 1 were ≥32 μg/ml. As expected, we found no association between katG mutation and the level of ETH resistance. Mutations within the ethA and inhA structural genes were associated with relatively high levels of ETH resistance. Approximately 76% of isolates resistant to ≥50 μg of ETH per ml had such mutations.

Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis Isolates with Low Copy Numbers of IS6110 by Using Mycobacterial Interspersed Repetitive Units

ABSTRACT A study set of 180 Mycobacterium tuberculosis and Mycobacterium bovis isolates having low copy numbers of IS6110 were genotyped using the recently introduced method based on the variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR). The results were compared with results of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The isolates were collected in Michigan from 1996 to 1999 as part of a project to genotype all isolates from new cases of tuberculosis in the state. Twelve MIRU loci were amplified, and the amplicons were analyzed by agarose gel electrophoresis to determine the copy number at each MIRU locus. MIRU-VNTR produced more distinct patterns (80 patterns) than did IS6110 RFLP (58 patterns), as would be expected in this study set. Spoligotyping identified 59 patterns. No single method defined all unique isolates, and the combination of all three typing methods generated 112 distinct patterns identifying 90 unique isolates and 90 isolates in 22 clusters. The results confirm the potential utility of MIRU-VNTR typing and show that typing with multiple methods is required to attain maximum specificity.

Evaluation of a Two-Step Approach for Large-Scale, Prospective Genotyping of Mycobacterium tuberculosis Isolates in the United States

ABSTRACT Genotyping of Mycobacterium tuberculosis isolates is useful in tuberculosis control for confirming suspected transmission links, identifying unsuspected transmission, and detecting or confirming possible false-positive cultures. The value is greatly increased by reducing the turnaround time from positive culture to genotyping result and by increasing the proportion of cases for which results are available. Although IS6110 fingerprinting provides the highest discrimination, amplification-based methods allow rapid, high-throughput processing and yield digital results that can be readily analyzed and thus are better suited for large-scale genotyping. M. tuberculosis isolates (n = 259) representing 99% of culture-positive cases of tuberculosis diagnosed in Wisconsin in the years 2000 to 2003 were genotyped by using spoligotyping, mycobacterial interspersed repetitive unit (MIRU) typing, and IS6110 fingerprinting. Spoligotyping clustered 64.1% of the isolates, MIRU typing clustered 46.7% of the isolates, and IS6110 fingerprinting clustered 29.7% of the isolates. The combination of spoligotyping and MIRU typing yielded 184 unique isolates and 26 clusters containing 75 isolates (29.0%). The addition of IS6110 fingerprinting reduced the number of clustered isolates to 30 (11.6%) if an exact pattern match was required or to 44 (17.0%) if the definition of a matching IS6110 fingerprint was expanded to include patterns that differed by the addition of a single band. Regardless of the genotyping method chosen, the addition of a second or third method decreased clustering. Our results indicate that using spoligotyping and MIRU typing together provides adequate discrimination in most cases. IS6110 fingerprinting can then be used as a secondary typing method to type the clustered isolates when additional discrimination is needed.

Transfer of a Mycobacterium tuberculosis Genotyping Method, Spoligotyping, from a Reverse Line-Blot Hybridization, Membrane-Based Assay to the Luminex Multianalyte Profiling System

ABSTRACT Spoligotyping using Luminex technology was shown to be a highly reproducible method suitable for high-throughput analysis. Spoligotyping of 48 isolates using the traditional membrane-based assay and the Luminex assay yielded concordant results for all isolates. The Luminex platform provides greater flexibility and cost effectiveness than the membrane-based assay.

A rapid method for screening antimicrobial agents for activities against a strain of Mycobacterium tuberculosis expressing firefly luciferase.

We developed a rapid method to screen the efficacy of antimicrobial agents against Mycobacterium tuberculosis. A restriction fragment carrying a promoterless firefly luciferase gene was cloned into a 4,488-bp shuttle vector, pMV261, and luciferase was expressed under the control of a mycobacterial heat shock promoter. The resulting plasmid, pLUC10, was introduced by electroporation into the avirulent strain M. tuberculosis H37Ra. Luciferase assays of sonic lysates of Triton X-100-treated cells of M. tuberculosis H37Ra(pLUC10) yielded bioluminescence in excess of 1,000 relative light units/approximately 10(9) tubercle bacilli, compared with 0.0025 for the same number of parental cells. A 48-h microdilution antimicrobial agent-screening assay using this strain was developed. Images

Human Tuberculosis due to Mycobacterium bovis in the United States, 1995-2005

BACKGROUND Understanding the epidemiology of human Mycobacterium bovis tuberculosis (TB) in the United States is imperative; this disease can be foodborne or airborne, and current US control strategies are focused on TB due to Mycobacterium tuberculosis and airborne transmission. The National TB Genotyping Services work has allowed systematic identification of M. tuberculosis-complex isolates and enabled the first US-wide study of M. bovis TB. METHODS Results of spacer oligonucleotide and mycobacterial interspersed repetitive units typing were linked to corresponding national surveillance data for TB cases reported for the period 2004-2005 and select cases for the period 1995-2003. We also used National TB Genotyping Service data to evaluate the traditional antituberculous drug resistance-based case definition of M. bovis TB. RESULTS Isolates from 165 (1.4%) of 11,860 linked cases were identified as M. bovis. Patients who were not born in the United States, Hispanic patients, patients <15 years of age, patients reported to be HIV infected, and patients with extrapulmonary disease each had increased adjusted odds of having M. bovis versus M. tuberculosis TB. Most US-born, Hispanic patients with TB due to M. bovis (29 [90.6%] of 32) had extrapulmonary disease, and their overall median age was 9.5 years. The National TB Genotyping Services data indicated that the pyrazinamide-based case definitions sensitivity was 82.5% (95% confidence interval; 75.3%-87.9%) and that data identified 14 errors in pyrazinamide-susceptibility testing or reporting. CONCLUSIONS The prevalence of extrapulmonary disease in the young, US-born Hispanic population suggests recent transmission of M. bovis, possibly related to foodborne exposure. Because of its significantly different epidemiologic profile, compared with that of M. tuberculosis TB, we recommend routine surveillance of M. bovis TB. Routine surveillance and an improved understanding of M. bovis TB transmission dynamics would help direct the development of additional control measures.

Phenotypic Characterization of pncA Mutants of Mycobacterium tuberculosis

ABSTRACT We examined the correlation of mutations in the pyrazinamidase (PZase) gene (pncA) with the pyrazinamide (PZA) resistance phenotype with 60 Mycobacterium tuberculosis isolates. PZase activity was determined by the method of Wayne (L. G. Wayne, Am. Rev. Respir. Dis. 109:147–151, 1974), and the entirepncA nucleotide sequence, including the 74 bp upstream of the start codon, was determined. PZA susceptibility testing was performed by the method of proportions on modified Middlebrook and Cohn 7H10 medium. The PZA MICs were ≥100 μg/ml for 37 isolates, 34 of which had alterations in the pncA gene. These mutations included missense substitutions for 24 isolates, nonsense substitutions for 3 isolates, frameshifts by deletion for 4 isolates, a three-codon insertion for 1 isolate, and putative regulatory mutations for 2 isolates. Among 21 isolates for which PZA MICs were <100 μg/ml, 3 had the same mutation (Thr47→Ala) and 18 had the wild-type sequence. For the three Thr47→Ala mutants PZA MICs were 12.5 μg/ml by the method of proportions on 7H10 agar; two of these were resistant to 100 μg of PZA per ml and the third was resistant to 800 μg of PZA per ml by the BACTEC method. In all, 30 different pncA mutations were found among the 37 pncA mutants. No PZase activity was detected in 35 of 37 strains that were resistant to ≥100 μg of PZA per ml or in 34 of 37 pncA mutants. Reduced PZase activity was found in the three mutants with the Thr47→Ala mutation. This study demonstrates that mutations in the pncA gene may serve as a reliable indicator of resistance to ≥100 μg of PZA per ml.

Simultaneous Infection with Multiple Strains of Mycobacterium tuberculosis

Drug-susceptible and drug-resistant isolates of Mycobacterium tuberculosis were recovered from 2 patients, 1 with isoniazid-resistant tuberculosis (patient 1) and another with multidrug-resistant tuberculosis (patient 2). An investigation included patient interviews, record reviews, and genotyping of isolates. Both patients worked in a medical-waste processing plant. Transmission from waste was responsible for at least the multidrug-resistant infection. We found no evidence that specimens were switched or that cross-contamination of cultures occurred. For patient 1, susceptible and isoniazid-resistant isolates, collected 15 days apart, had 21 and 19 restriction fragments containing IS6110, 18 of which were common to both. For patient 2, a single isolate contained both drug-susceptible and multidrug-resistant colonies, demonstrating 10 and 11 different restriction fragments, respectively. These observations indicate that simultaneous infections with multiple strains of M. tuberculosis occur in immunocompetent hosts and may be responsible for conflicting drug-susceptibility results, though the circumstances of infections in these cases may have been unusual.

The molecular epidemiology of tuberculosis in New York City: the importance of nosocomial transmission and laboratory error

SETTING During the 1980s, New York City experienced a rapid increase of tuberculosis cases, more than 40% of which were human immunodeficiency virus (HIV)-associated. OBJECTIVE To better define the molecular epidemiology of tuberculosis in New York City. DESIGN We collected an isolate from every patient in New York City with a positive culture for Mycobacterium tuberculosis, including both incident and prevalent cases, in April 1991. Restriction fragment length polymorphism (RFLP) analysis using IS6110 was performed and the clinical, demographic, epidemiologic, and drug susceptibility patterns of patients were correlated with RFLP results. RESULTS Of 441 patients, 12 (3%) had laboratory, clinical, and RFLP evidence of falsely positive cultures. The remaining 429 patients had 252 distinct RFLP patterns. Patients with clustered 1-3 band isolates did not share demographic or drug susceptibility patterns. Eliminating these patients from the analysis, 344 patients remained, of whom 126 (37%) belonged to one of 31 clusters ranging in size from 2-17 patients (median cluster size = 3). Clustering was more common among patients with multidrug-resistant isolates (53%), African Americans (44%), and the homeless (49%), but was not associated with HIV infection or acquired immune deficiency syndrome (AIDS), Multidrug-resistance, being African American, and homelessness remained independently associated with clustering in multivariate analysis. Of 79 patients in clusters of > or = 4 patients, 25 (32%) had identifiable epidemiologic linkages; 17 (74%) of these patients, and 6% of all cases, were documented to have been nosocomially associated. CONCLUSION A small but non-negligible proportion (3%) of New York City patients had falsely positive cultures for M. tuberculosis as a result of laboratory error. More than one third of all patients and most patients with multidrug-resistance in April 1991 had clustered RFLP patterns, suggesting recent transmission of M. tuberculosis. Homelessness, multidrug-resistance, and being African American independently increased the risk of clustering. Most of the identified epidemiologic linkages and 6% of all cases resulted from transmission in hospitals.