Field assessment of the direct nitrate reductase assay for rapid detection of multidrug-resistant tuberculosis in Honduras.

SETTING: The national TB reference laboratory and four health care units connected to the national laboratory network in Honduras, Central America. OBJECTIVE: To evaluate the performance of the direct nitrate reductase assay (NRA) for rapid, low-cost detection of multidrug-resistant tuberculosis (MDR-TB) in a resource-limited setting. DESIGN: Consecutive smear-positive samples (n = 185) were prospectively analysed with NRA and compared to the proportion method on Löwenstein Jensen medium (PM-LJ) to detect resistance to isoniazid (INH) and rifampicin (RMP). RESULTS: The NRA sensitivity, specificity, positive and negative predictive values for INH and RMP were respectively 100%, 99%, 91%, 100% and 80%, 100%, 100%, 99%. Good agreement was observed between NRA and PM-LJ (κ > 0.8). CONCLUSION: The direct NRA is a reliable alternative for rapid and low-cost identification of MDR-TB cases in resource-limited settings.

Wild-type MIC distributions of four fluoroquinolones active against Mycobacterium tuberculosis in relation to current critical concentrations and available pharmacokinetic and pharmacodynamic data.

OBJECTIVES: To describe wild-type distributions of the MIC of fluoroquinolones for Mycobacterium tuberculosis in relation to current critical concentrations used for drug susceptibility testing and pharmacokinetic/pharmacodynamic (PK/PD) data. METHODS: A 96-stick replicator on Middlebrook 7H10 medium was used to define the MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin for 90 consecutive clinical strains and 24 drug-resistant strains. The MICs were compared with routine BACTEC 460 susceptibility results and with MIC determinations in the BACTEC MGIT 960 system in a subset of strains using ofloxacin as a class representative. PK/PD data for each drug were reviewed in relation to the wild-type MIC distribution. RESULTS: The wild-type MICs of ciprofloxacin, ofloxacin, moxifloxacin and levofloxacin were distributed from 0.125 to 1, 0.25 to 1, 0.032 to 0.5 and 0.125 to 0.5 mg/L, respectively. The MIC data correlated well with the BACTEC 960 MGIT and BACTEC 460 results. PD indices were the most favourable for levofloxacin, followed by moxifloxacin, ofloxacin and ciprofloxacin. CONCLUSIONS: We propose S (susceptible)

Evaluation of the nitrate reductase assay for rapid detection of extensively drug-resistant tuberculosis.

BACKGROUND: New diagnostic tools are needed to support tuberculosis (TB) control strategies, particularly in low- and middle-income countries with a high prevalence of TB. OBJECTIVE: To evaluate the nitrate reductase assay (NRA) for the rapid detection of resistance to isoniazid (INH) and rifampicin (RMP), as well as to second-line drugs such as ofloxacin (OFX) and kanamycin (KM). DESIGN: To determine diagnostic accuracy, 192 selected clinical isolates of Mycobacterium tuberculosis were used to compare NRA with BACTEC 460TB for rapid detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB strains. RESULTS: A good agreement between NRA and the BACTEC 460TB reference method was observed, with good sensitivity and excellent specificity for INH, RMP and OFX. Results for KM were also promising, although the sensitivity for the detection of KM resistance should be improved. CONCLUSION: NRA is a diagnostic tool of promise for the timely detection of M. tuberculosis resistance to first- and second-line drugs. Our study showed a clear potential for the prompt detection of both MDR- and XDR-TB cases. Further studies are needed to optimise the testing of second-line drugs.

Results of the external quality assessment of Mycobacterium tuberculosis drug susceptibility testing in Russia, 2005-2007.

SETTING: External quality assessment (EQA) of Mycobacterium tuberculosis drug susceptibility testing (DST) in bacteriological tuberculosis (TB) laboratories in the Russian Federation. OBJECTIVE: To improve the EQA of DST of first-line anti-tuberculosis drugs using proficiency testing in the Russian Federation. METHOD: Three rounds of DST proficiency testing using Mycobacterium tuberculosis isolates provided by the Swedish Institute for Infectious Disease Control, a World Health Organization Supranational Reference Laboratory (SRL). In total, 42 TB laboratories in the Russian civilian and prison sectors participated in at least one round of proficiency testing, and 17 laboratories participated in all three rounds. RESULTS: Ninety-seven per cent (87/89) of reports were received for the three rounds: 67% of laboratories in the first round and 86% of laboratories in the second round demonstrated >or=95% accuracy for isoniazid, and respectively 72% and 80% of laboratories in the first and second rounds reported >or=95% accuracy for rifampicin. CONCLUSION: Coordination with the SRL network resulted in the introduction of 90 well-characterised strains for EQA in the Russian Federation. Successive rounds of DST proficiency testing have helped to identify highly proficient laboratories that will be used as expert laboratories for proficiency testing in the future.

Should the 'bleach microscopy method' be recommended for improved case detection of tuberculosis? Literature review and key person analysis.

SETTING: It has been proposed that the sensitivity of direct sputum smear microscopy can be improved if sputum is liquefied with sodium hypochlorite (NaOCl or household bleach), and concentrated by centrifugation before acid-fast staining. OBJECTIVE: To summarise the results of the studies of the bleach method for improved sensitivity of sputum microscopy and to describe the opinions and knowledge of key persons in National Tuberculosis Control Programmes (NTPs) about this method. DESIGN: We searched Medline, EMBASE and Web of Science for studies comparing the bleach method to direct sputum smear microscopy in low- or middle-income countries. Each study was assessed regarding methodology and field applicability. We also sent out questionnaires concerning the bleach method to key persons in NTPs in 85 countries. RESULTS: In 15 of the 19 studies identified there was a statistically significant improvement in the proportion of positive tests or sensitivity ranging from 7-253%. The majority (73%) of the key persons had heard of the bleach method. Forty-four per cent thought it could improve case detection in their countries, while 49% did not know; 93% of them would promote the bleach method; the most common reasons for doing so would be recommendations from the WHO or the IUATLD, or favourable studies performed in their own country. The bleach method was used routinely in only three countries. CONCLUSION: There is enough evidence to recommend the evaluation and introduction of the bleach method in most settings where mycobacterial culture is not performed routinely.

Evaluation of the BacT/ALERT 3D system for recovery and drug susceptibility testing of Mycobacterium tuberculosis.

We evaluated the BacT/ALERT 3D system for recovery and drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB). Of 2659 clinical specimens, MTB was detected in 92 using BacT/ALERT, compared to 94 using Löwenstein-Jensen culture. Detection time was 25% shorter with BacT/ALERT. Sensitivities were 92%, 96%, 78% and 100% for resistance to rifampicin, isoniazid, streptomycin and ethambutol, respectively, while specificity was 100% for all antibiotics, when BacT/ALERT was compared with the BACTEC 460 method on 50 MTB isolates. The BacT/ALERT system is fully automated and creates no radioactive waste. It seems to be a valid alternative for primary isolation, but further evaluation is needed regarding DST.

In vitro activity of thiacetazone on mycobacterial species belonging to the Mycobacterium tuberculosis complex.

Thiacetazone, despite frequent side-effects, may still be considered for the treatment of new tuberculosis cases when there is a shortage of drugs and for the management of multidrug-resistant tuberculosis. Fifty-four strains of M. tuberculosis complex were characterised based on the minimum inhibitory concentration (MIC) of thiacetazone and the growth pattern in the presence of different concentrations of the drug. The results showed that the MIC of thiacetazone to type II M. africanum strains was significantly higher than for other strains in the study (P < 0.01). Thiacetazone showed a paradoxical effect on 63% of strains where lower concentrations exhibited a better inhibiting activity than higher concentrations.

Antimicrobial susceptibility of Mycobacterium marinum determined by E-test and agar dilution.

Mycobacterium marinum is recognized as a cutaneous pathogen requiring antibiotic treatment. We compared the E-test with a reference agar dilution method for susceptibility testing of M. marinum to amikacin, ciprofloxacin, clarithromycin, doxycycline, rifampicin, trimethoprim-sulfamethoxazole and ethambutol. MICs obtained after 6 d showed agreement between the E-test and agar dilution within +/- 2 dilutions in 95% of all cases for amikacin, ciprofloxacin, doxycycline and rifampicin. Inhibitory concentrations of trimethoprim-sulfamethoxazole were difficult to define using the E-test because of gradually decreased growth in the presence of increasing concentrations. For clarithromycin, results were generally 1-3 dilution steps lower with the E-test and for ethambutol they were often > 3 dilution steps lower. These differences always appeared in the low MIC range and did not affect the categorization of the strains as susceptible to these 2 antimicrobial agents. All strains were interpreted as susceptible to all tested antibiotics, except for doxycycline, according to recommended breakpoints. Overall, our results suggest that the E-test can be considered an alternative for susceptibility testing of certain antibacterial agents against M. marinum.

Spread of drug-resistant pulmonary tuberculosis in Estonia.

Restriction fragment length polymorphism (RFLP) analysis of 209 Mycobacterium tuberculosis clinical isolates obtained from newly detected pulmonary tuberculosis patients (151 male and 58 female; mean age, 41 years) in Estonia during 1994 showed that 61 isolates (29%) belonged to a genetically closely related group of isolates, family A, with a predominant IS6110 banding pattern. These strains shared the majority of their IS6110 DNA-containing restriction fragments, representing a predominant banding pattern (similarity, >65%). This family A comprised 12 clusters of identical isolates, and the largest cluster comprised 10 strains. The majority (87.5%) of all multidrug-resistant (MDR) isolates, 67.2% of all isolates with any drug resistance, but only 12% of the fully susceptible isolates of M. tuberculosis belonged to family A. These strains were confirmed by spoligotyping as members of the Beijing genotype family. The spread of Beijing genotype MDR M. tuberculosis strains was also frequently seen in 1997 to 1999. The members of this homogenous group of drug-resistant M. tuberculosis strains have contributed substantially to the continual emergence of drug-resistant tuberculosis all over Estonia.

Characterization of isoniazid-resistant strains of Mycobacterium tuberculosis on the basis of phenotypic properties and mutations in katG.

Forty isoniazid-resistant Mycobacterium tuberculosis isolates were characterized on the basis of phenotypic properties (i.e., catalase activity, MIC of isoniazid, and growth pattern in the presence of 7 different concentrations of isoniazid) and alterations in the katG gene (codons 315 and 463). Three different growth patterns could be distinguished: concentration-dependent inhibition of growth was observed in 29 strains, similar growth at all concentrations was seen in 7 strains, and enhanced growth at low concentrations of isoniazid was evident in 4 strains. The MIC of isoniazid was < or = microg/ml for 29 of 40 strains. Mutation at codon 315 of the katG was detected in 28 of 40 strains. However, only one of the seven strains for which the MIC of isoniazid was > or = 16 microg/ml had mutation at this codon. Five of these seven strains for which the MIC was > or = 16 microg/ml had no catalase activity. The results indicate that the MIC of isoniazid for a majority of strains is below the level achievable in serum. Therefore, isoniazid may be beneficial for the treatment of some cases of multidrug-resistant tuberculosis. Determination of catalase activity aids in the detection of isolates for which MICs are high and could, in conjunction with molecular methods, provide rapid detection of most isoniazid-resistant strains.

In vitro activity of PR-39, a proline-arginine-rich peptide, against susceptible and multi-drug-resistant Mycobacterium tuberculosis.

We have investigated the in vitro activity of antimicrobial peptides against Mycobacterium tuberculosis using a radiometric method and cfu determinations. PR-39, a proline-arginine-rich antibacterial peptide from porcine leucocytes, was found to be active against drug-susceptible as well as multi-drug-resistant (MDR) clinical isolates of M. tuberculosis. The activity of PR-39 was concentration dependent, with 80% growth inhibition of M. tuberculosis H37Rv at 50 mg/L. The MDR M. tuberculosis strains E1380/94 and P34/95 were less susceptible to PR-39, with 39 and 49% growth inhibition at 50 mg/L peptide, respectively, suggesting a lower susceptibility than strain H37Rv and drug-susceptible clinical isolates. Reduction of counts of M. tuberculosis H37Rv and the MDR M. tuberculosis strain E1380/94 by PR-39 indicated that the growth inhibition seen in the radiometric assay is due to a mycobactericidal effect of the peptide. These observations suggest that antimicrobial peptides may play an important role in host defence against MDR M. tuberculosis.

Tuberculosis as an occupational hazard for health care workers in Estonia.

SETTING: Tuberculosis incidence has been increasing in the Baltic states since the 1990s, accompanied by the emergence of drug resistance, including multidrug resistance (MDR). In this changing situation, the potential threat of nosocomial spread of tuberculosis to other patients and health care workers (HCW) has remained unrecognised. OBJECTIVE: To investigate the risk of tuberculosis in health care workers in Estonia. DESIGN: Cases of tuberculosis registered among HCWs from 1994 to 1998 were evaluated. The case records were analysed retrospectively and combined with bacteriological data including data on drug resistance. RESULTS: Sixty-seven HCWs (23 physicians, 23 nurses and seven laboratory technicians, 12 assistant nurses and two cleaners), all of whom tested negative for human immunodeficiency virus, were diagnosed as having active tuberculosis. The incidence of tuberculosis among HCWs (mean 91/100,000/year) was 1.5 to three times higher than in the general population. In a chest hospital in charge of regional tuberculosis care, the incidence was 30 to 90 times higher, and was highest among physicians. In 49 HCWs tuberculosis was confirmed by culture. Among these, drug resistance was detected in 23 (49%), 18 (38%) of whom had MDR tuberculosis. CONCLUSIONS: Health care workers, especially those working in a chest hospital where tuberculosis patients were treated, were found to be at an elevated risk of tuberculosis. MDR tuberculosis poses a particular threat which is difficult to combat.

Comparison of Mycobacterium avium complex (MAC) strains from pigs and humans in Sweden by random amplified polymorphic DNA (RAPD) using standardized reagents.

Infections with atypical mycobacteria belonging to the Mycobacterium avium/intracellulare complex (MAC) can cause infection in both animals and humans. Using a standardized reagents commercial kit for random amplified polymorphic DNA (RAPD) analysis, 49 MAC strains isolated from 32 slaughter pigs and 17 humans in Sweden were identified and sorted out, yielding 6 RAPD types. By combining the results of RAPD primers 4 and 5 and the primer IS1245A, we found that pigs and humans may be infected with the same types of MAC strains, since 14 strains from humans and 8 strains from pigs were essentially identical and together, comprised RAPD type 2, the largest group of strains (44.8% of strains). With respect to grouping of strains, serotype and RAPD type were uncorrelated, except for serotype 20 and RAPD type 6. Using standardized beads, RAPD analysis is a reproducible technique for typing MAC strains, as the indistinguishable banding patterns obtained with repeated analyses of two isolates from each strain in this study demonstrate. However, primer selection and DNA purity were crucial for differentiating closely related strains.

Mycobacterium interjectum: a new pathogen in humans?

Molecular genetic techniques have increased the number of species recognized within the genus Mycobacterium. The clinical significance of these is uncertain and their pathogenic potential has still to be proven. We describe here a case of mycobacterial lymphadenitis in a Swedish boy, which supports the role of M. interjectum as a human pathogen.

Standardization of antituberculosis drug resistance surveillance in Europe. Recommendations of a World Health Organization (WHO) and International Union Against Tuberculosis and Lung Disease (IUATLD) Working Group.

Surveillance of antituberculosis drug resistance is an essential tool for evaluating the quality of tuberculosis control programmes. Consensus-based recommendations on uniform reporting of antituberculosis drug resistance surveillance data in Europe have been developed by a Working Group of the World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease (IUATLD). Laboratories should use standardized methods for testing drug susceptibility with a quality assurance programme including national and international proficiency testing. The proportion of drug resistance, particularly resistance to isoniazid, rifampicin or both (multidrug resistance) among all definite, i.e. culture-positive, tuberculosis cases at the start of treatment is the major indicator of interest. It should be calculated separately among patients treated previously and among those who have never been treated with > or = 1 month of combined antituberculosis drugs. The Working Group recommends that, in countries in which resources allow, laboratories report drug susceptibility test results on all isolates of the Mycobacterium tuberculosis complex. Test results of the specimen at the start of treatment and clinical data from the notification should be linked using a suitable identifier. Results should be presented by calendar year and analysed by age, sex, place of birth, site of disease and sputum smear results. In countries in which a routine system cannot be organized, representative surveys or sentinel systems are possible alternatives. In some countries, the annual prevalence of multidrug-resistant tuberculosis may be estimated through a national laboratory reporting system.

Improved sputum microscopy for a more sensitive diagnosis of pulmonary tuberculosis.

Diagnosis of tuberculosis in low-income countries is hindered by the low sensitivity of direct sputum smear microscopy. We compared an improved method based on liquefaction of sputum with NaOCl followed by centrifugation with standard direct smear in a central hospital and at peripheral health centres in Honduras. Specificity was high and sensitivity significantly better with the NaOCl method. Moreover, this technique is safe, inexpensive and easy to perform. We recommend its implementation to enable rapid, sensitive laboratory diagnosis of pulmonary tuberculosis, especially in resource-poor settings where culture is not possible.

European recommendations on surveillance of antituberculosis drug resistance.

Antituberculosis drug resistance, whose extent in Europe is not well documented, is a serious threat to tuberculosis control. The aim of the recent European recommendations on antituberculosis drug resistance surveillance, issued by a working group compos

Evolution and clonal traits of Mycobacterium tuberculosis complex in Guinea-Bissau.

Two hundred twenty-nine consecutive isolates of Mycobacterium tuberculosis complex from patients with pulmonary tuberculosis in Guinea-Bissau, which is located in West Africa, were analyzed for clonal origin by biochemical typing and DNA fingerprinting. By using four biochemical tests (resistance to thiophene-2-carboxylic acid hydrazide, niacin production, nitrate reductase test, and pyrazinamidase test), the isolates could be assigned to five different biovars. The characteristics of four strains conformed fully with the biochemical criteria for M. bovis, while those of 85 isolates agreed with the biochemical criteria for M. tuberculosis. The remaining 140 isolates could be allocated into one of three biovars (biovars 2 to 4) representing a spectrum between the classical bovine (biovar 1) and human (biovar 5) tubercle bacilli. By using two genotyping methods, restriction fragment length polymorphism analysis with IS6110 (IS6110 RFLP analysis) and spoligotyping, the isolates could be separated into three groups (groups A to C) of the M. tuberculosis complex. Group A (n = 95), which contained the majority of classical human M. tuberculosis isolates, had large numbers of copies of IS6110 elements (mean number of copies, 9) and a distinctive spoligotyping pattern that lacked spacers 33 to 36. Isolates of the major group, group B (n = 119), had fewer IS6110 copies (mean copy number, 5) and a spoligotyping pattern that lacked spacers 7 to 9 and 39 and mainly comprised isolates of biovars 1 to 4. Group C isolates (n = 15) had one to three IS6110 copies, had a spoligotyping pattern that lacked spacers 29 to 34, and represented biovar 3 to 5 isolates. Four isolates whose biochemical characteristics conformed with those of M. bovis clustered with the group B isolates and had spoligotype patterns that differed from those previously reported for M. bovis, in that they possessed spacers 40 to 43. Interestingly, isolates of group B and, to a certain extent, also isolates of group C showed a high degree of variability in biochemical traits, despite genotypic identity in terms of IS6110 RFLP and spoligotype patterns. We hypothesize that isolates of groups B and C have their evolutionary origin in West Africa, while group A isolates are of European descent.

Drug resistance in Mycobacterium tuberculosis strains isolated from re-treatment cases of pulmonary tuberculosis in Ethiopia: susceptibility to first-line and alternative drugs.

SETTING: Addis Ababa Tuberculosis Demonstration and Training Center, Ethiopia. OBJECTIVES: To determine the pattern of drug resistance among re-treatment cases of pulmonary tuberculosis (TB), to determine the risk factors associated with multi-drug resistant (MDR) TB, and to propose re-treatment regimens based on the patterns of susceptibility to first-line and alternative drugs. DESIGN: One hundred and seven Mycobacterium tuberculosis strains isolated from an equal number of re-treatment cases of pulmonary TB were included in the study. Drug susceptibility was determined by the Bactec method. RESULTS: About 50% of the strains were resistant to one or more of the first-line drugs and 12% of the strains were multi-drug resistant, i.e., resistant to both isoniazid and rifampicin. Previous treatment with rifampicin was the most important predictor of MDR-TB. All MDR strains were susceptible to amikacin, ciprofloxacin, ethambutol, ethionamide and clofazimine. CONCLUSION: The WHO re-treatment regimen would theoretically be effective for the treatment of all non-MDR-TB patients in this study. A proposed 12-month re-treatment regimen for MDR-TB patients would include a fluoroquinolone in combination with streptomycin, pyrazinamide, isoniazid, ethambutol and clofazimine. There is an urgent need for more research to define safe and inexpensive treatment regimens for MDR-TB patients in low-income countries.