Malcolm D. Yates

Evaluation of MGIT 960-Based Antimicrobial Testing and Determination of Critical Concentrations of First- and Second-Line Antimicrobial Drugs with Drug-Resistant Clinical Strains of Mycobacterium tuberculosis

ABSTRACT The objectives of this study were to (i) compare agreement of the MGIT 960 system for first-line drugs with a methodology (the resistance ratio method [RRM]) that had been used in clinical trials, relating drug susceptibility to clinical outcome; (ii) compare the performance of the MGIT 960, RRM, and microtiter plate assay (MPA) methodologies for second-line drug testing; and (iii) define critical concentrations for ciprofloxacin and moxifloxacin for liquid-culture-based testing. The large collection of clinical isolates of Mycobacterium tuberculosis (n = 247) used included 176 (71%) multidrug-resistant isolates. The results for MGIT 960 and the RRM for rifampin and isoniazid (n = 200) were in excellent (99 to 100%) agreement for all strains. For streptomycin, 97% of the results at the critical concentration and 92% at high concentration, and for pyrazinamide 92% of results overall, were concordant, but for ethambutol, fewer than 85% (65% for the critical concentration and 84% for the high concentration) of the MGIT-based results were concordant with those for the RRM. The MGIT 960, RRM, and MPA assays (n = 133) correlated well for most second-line drugs tested. For susceptibility to ofloxacin, the MGIT 960 and MPA results were in full agreement. The amikacin and rifabutin results obtained by MGIT 960 agreed with the RRM results in 131 (99%) cases, and for capreomycin, they agreed for 129 of 133 isolates tested (97%). For prothionamide testing, only a limited number of drug-resistant isolates were available for testing and drawing definitive conclusions. We propose critical concentrations of 1.0 μg/ml and 0.125 μg/ml for ciprofloxacin and moxifloxacin, respectively, for liquid-culture-based testing.

Antibiotic resistant tuberculosis in the United Kingdom: 1993–1999

Background: The re-emergence of tuberculosis as a global health problem over the past two decades, accompanied by an increase in tuberculosis drug resistance, prompted the development of a comprehensive national surveillance system for tuberculosis drug resistance in 1993. Methods: The UK Mycobacterial Resistance Network (Mycobnet), which includes all mycobacterial reference and regional laboratories in the UK, collects a minimum dataset on all individuals from whom an initial isolate of Mycobacterium tuberculosis complex has been isolated and submitted by source hospital laboratories. Data sought include susceptibility to first line antibiotics, demographic, geographical, and risk factor information. Results: There were 25 217 reports of initial isolates of M tuberculosis complex in the UK between 1993 and 1999. All were tested for sensitivity to isoniazid, rifampicin, and ethambutol and 12 692 of the isolates were also tested for sensitivity to pyrazinamide and streptomycin. A total of 1523 (6.1%) isolates were resistant to one or more drugs, 1397 isolates (5.6%) were resistant to isoniazid with or without resistance to other drugs, and 299 (1.2%) were multidrug resistant. Although the numbers of drug resistant isolates increased over the period, the proportions remained little changed. Certain groups of people were at a higher risk of acquiring drug resistant tuberculosis including younger men, residents of London, foreign born subjects, patients with a previous history of tuberculosis and those infected with HIV. Conclusion: Although the proportion of drug resistant tuberculosis cases appears to be stable in the UK at present, more than one in 20 patients has drug resistant disease at diagnosis and more than one in 100 has multidrug resistant disease. Tuberculosis control measures should be strengthened to minimise the emergence of drug resistance through rapid diagnosis, rapid identification of drug resistance, supervised treatment, and maintenance of comprehensive surveillance.

APPLICATION OF A SIMPLE MULTIPLEX PCR TO AID IN ROUTINE WORK OF THE MYCOBACTERIUM REFERENCE LABORATORY

ABSTRACT A PCR specific for spacer regions 33 and 34 of the direct repeat region of the Mycobacterium tuberculosis complex was developed to complement the biochemical differentiation ofM. tuberculosis, Mycobacterium bovis, M. bovis BCG, andMycobacterium africanum subtypes I and II. In addition, this approach was incorporated into a multiplex PCR that included primers specific for IS6110 and the 65-kDa antigen gene in order to differentiate members of the M.tuberculosis complex from atypical mycobacteria.

DNA Fingerprint Changes in Tuberculosis: Reinfection, Evolution, or Laboratory Error?

BACKGROUND DNA fingerprint patterns of Mycobacterium tuberculosis strains vary within individuals and between epidemiologically linked individuals because of pattern evolution, new infections, and laboratory error. We explored the importance of these factors. METHODS Cultures from individuals in northern Malawi who had been diagnosed with tuberculosis (TB) during 1996-2001 were fingerprinted with restriction fragment-length polymorphism (RFLP). Probable laboratory error was inferred by use of dates or isolated positive cultures. Pattern evolution was explored within and between individuals, and the relative importance of relapse and reinfection was estimated in individuals with recurrent TB. RESULTS RFLP results were available for 930 isolates from 806 individuals. The maximum laboratory-error rate was 3.9%. Pattern evolution was more common in linked individuals (17%) than on relapse (11%) or during treatment (3%). Twenty individuals had recurrent TB after completing treatment: in human immunodeficiency virus (HIV)-positive individuals, 7 of 12 recurrences were due to reinfection, compared with 0 of 8 in HIV-negative individuals (P=.01). CONCLUSIONS The rate of DNA fingerprint-pattern evolution is not linear, and rates of change calculated from repeat cultures within individuals may not be applicable to transmission between individuals. The high proportion of recurrence due to reinfection found in HIV-positive individuals suggests that secondary prophylaxis and/or antiretroviral treatments are needed for such individuals.

Mycobacterium tuberculosis Beijing Genotype, Northern Malawi

In a 7-year population-based study in Malawi, we showed that Beijing genotype tuberculosis (TB) increased as a proportion of TB cases. All the Beijing genotype strains were fully drug sensitive. Contact histories, TB type, and case-fatality rates were similar for Beijing and non-Beijing genotype TB.

Evaluation of a Rapid PCR-Based Epidemiological Typing Method for Routine Studies of Mycobacterium tuberculosis

ABSTRACT Restriction fragment length polymorphism (RFLP) based on the insertion sequence IS6110 is used to investigate episodes of suspected transmission of infection of tuberculosis but usually takes a number of weeks from receipt of request to obtain a result. Often investigations would benefit from a more rapid method, possibly one containing an amplification step. The method employed uses a simple DNA extraction followed by a PCR step involving a single primer. Restriction enzyme analysis was performed when the patterns obtained from the PCR products were indistinguishable, especially when only single similar-size bands were obtained. The isolates used were strains of Mycobacterium tuberculosis submitted for epidemiological investigations as part of (i) possible contact-outbreak (22 episodes involving between 2 and 20 patients), (ii) possible incidents of laboratory cross-contamination (21 episodes), and (iii) possible change in drug resistance pattern or a case of reinfection (1 patient). The PCR products giving similar patterns were then subjected to restriction enzyme analysis. In conclusion it has been shown that this method is rapid, with results within 1 to 2 days of the request being received; is reproducible; and gives the same results as does RFLP. The restriction enzyme analysis stage has improved the efficiency of the technique.

Progression Toward an Improved DNA Amplification-Based Typing Technique in the Study of Mycobacterium tuberculosis Epidemiology

ABSTRACT While high-copy-number IS6110-based restriction fragment length polymorphism (HCN-RFLP) is the gold standard for typing most Mycobacterium tuberculosis strains, the time taken for culturing and low throughput make it impractical for large-scale prospective typing of large numbers of isolates. The development of a new method, mycobacterial interspersed repetitive units (MIRU), a variation of the original variable-number tandem repeat (VNTR) technique, may provide a viable alternative. Panels based on the original 12-loci MIRU (12MIRU), a combination of 12MIRU and remaining ETR loci (15MIRU-VNTR), and an extended panel with an additional 10 novel regions (25VNTR) were used to study three populations with varying degrees of epidemiological data. MIRU discrimination increased with panel size and the addition of spoligotyping. Combining these two techniques enabled a reduction in the panel size from 25 to 14 loci without a significant loss in discrimination. However, 25VNTR alone or in combination with spoligotyping still possessed weaker discrimination than RFLP for high-copy-number isolates.

Evaluation and Utilization as a Public Health Tool of a National Molecular Epidemiological Tuberculosis Outbreak Database within the United Kingdom from 1997 to 2001

ABSTRACT The aim of this study was to develop a national model and analyze the value of a molecular epidemiological Mycobacterium tuberculosis DNA fingerprint-outbreak database. Incidents were investigated by the United Kingdom PHLS Mycobacterium Reference Unit (MRU) from June 1997 to December 2001, inclusive. A total of 124 incidents involving 972 tuberculosis cases, including 520 patient cultures from referred incidents and 452 patient cultures related to two population studies, were examined by using restriction fragment length polymorphism IS6110 fingerprinting and rapid epidemiological typing. Investigations were divided into the following three categories, reflecting different operational strategies: retrospective passive analysis, retrospective active analysis, and retrospective prospective analysis. The majority of incidents were in the retrospective passive analysis category, i.e., the individual submitting isolates has a suspicion they may be linked. Outbreaks were examined in schools, hospitals, farms, prisons, and public houses, and laboratory cross-contamination events and unusual clinical presentations were investigated. Retrospective active analysis involved a major outbreak centered on a high school. Contact tracing of a teenager with smear-positive pulmonary tuberculosis matched 14 individuals, including members of his class, and another 60 cases were identified in schools clinically and radiologically and by skin testing. Retrospective prospective analysis involved an outbreak of 94 isoniazid-resistant tuberculosis cases in London, United Kingdom, that began after cases were identified at one hospital in January 2000. Contact tracing and comparison with MRU databases indicated that the earliest matched case had occurred in 1995. Subsequently, the MRU changed to an active prospective analysis targeting linked isoniazid-monoresistant isolates for follow up. The patients were multiethnic, born mainly in the United Kingdom, and included professionals, individuals from the music industry, intravenous drug abusers, and prisoners.

HIV and the risk of tuberculosis due to recent transmission over 12 years in Karonga District Malawi.

Summary Tuberculosis (TB) patients with strains common to other recent cases (‘clustering’) suggest recent transmission. HIV status and age may affect proportions clustered. We investigated TB clustering by HIV and age in a population-based study in Malawi. Among 746 patients, HIV infection increased the proportion clustered. Sex-period-adjusted odds ratios for the association of HIV and clustering were 1.26 (95% CI 0.4–4.1) for ages 15–25 years, 1.40 (0.9–2.3) for 25–50 years and 10.44 (2.3–47.9) for >50 years and remained stable over two periods examined. These results suggest that HIV increases the proportion of TB due to recent transmission in the elderly.