Molecular Epidemiology of Mycobacterium Tuberculosis Strains in the North-West and West of Iran.

BACKGROUND: Identifying Mycobacterium tuberculosis (MTB) transmission type is a key step in the control of this disease. AIM: This study aimed to determine the path and transmission type of MTB and the insertion sequence IS6110 band number and verify their relationship to demographic and clinical risk factors. SUBJECTS AND METHODS: In this cross-sectional study, 64 MTB patients from three border provinces of Iran were selected after full clinical history and physical evaluation design. The drug susceptibility testing was carried out using the standard proportion technique on sputum samples. Isolates tested with restriction fragment length polymorphism technique used IS6110. RESULTS: Recent transmission of disease was 33/50 (66%) based on clustering rate. The IS6110 band number had a significant relationship with drug resistance detected in proportion method tested by univariate linear regression (P < 0.01). Furthermore, the IS6110 band number had association with Bacillus Calmette-Guérin vaccination history (P = 0.02), sex (P < 0.01), and purified protein derivative (PPD) reaction size (P < 0.01) tested by multiple analysis. The risk of recent transmission inferred from the clustering rate was significantly higher in patients from Western provinces compared to those from the North-West province (P = 0.048). However, age (P = 0.39), gender (P = 0.16), vaccination history (P = 0.57), drug susceptibility, and PPD (P < 0.6) were independent of clustering. The largest cluster of up to six subjects was found in the Western provinces. CONCLUSION: Recent MTB transmission was much more common in the West compared to the North-West of Iran. Large MTB clusters with strong epidemiological links may be reflective of a disease outbreak. Correlation noted between the IS6110 band number and vaccination history; PPD size and female gender necessitates further studies.

Revisiting susceptibility testing in MDR-TB by a standardized quantitative phenotypic assessment in a European multicentre study.

OBJECTIVES: Treatment outcome of MDR-TB is critically dependent on the proper use of second-line drugs as per the result of in vitro drug susceptibility testing (DST). We aimed to establish a standardized DST procedure based on quantitative determination of drug resistance and compared the results with those of genotypes associated with drug resistance. METHODS: The protocol, based on MGIT 960 and the TB eXiST software, was evaluated in nine European reference laboratories. Resistance detection at a screening drug concentration was followed by determination of resistance levels and estimation of the resistance proportion. Mutations in 14 gene regions were investigated using established techniques. RESULTS: A total of 139 Mycobacterium tuberculosis isolates from patients with MDR-TB and resistance beyond MDR-TB were tested for 13 antituberculous drugs: isoniazid, rifampicin, rifabutin, ethambutol, pyrazinamide, streptomycin, para-aminosalicylic acid, ethionamide, amikacin, capreomycin, ofloxacin, moxifloxacin and linezolid. Concordance between phenotypic and genotypic resistance was >80%, except for ethambutol. Time to results was short (median 10 days). High-level resistance, which precludes the therapeutic use of an antituberculous drug, was observed in 49% of the isolates. The finding of a low or intermediate resistance level in 16% and 35% of the isolates, respectively, may help in designing an efficient personalized regimen for the treatment of MDR-TB patients. CONCLUSIONS: The automated DST procedure permits accurate and rapid quantitative resistance profiling of first- and second-line antituberculous drugs. Prospective validation is warranted to determine the impact on patient care.

Evaluation of a biphasic media assay for pyrazinamide drug susceptibility testing of Mycobacterium tuberculosis.

OBJECTIVES: Pyrazinamide is a key first-line tuberculosis drug. Reliable drug susceptibility testing (DST) data are of clinical importance, but in vitro testing is challenging since the activity of pyrazinamide is pH sensitive. The BACTEC MGIT 960 is considered the principal reference technique, but Wayne's test is an alternative, although it may be difficult to interpret. A further alternative is the use of a biphasic media assay (BMA). The objective of this work was to evaluate the BMA against the MGIT method and with screening of pncA gene mutations. METHODS: Twenty strains were inoculated in tubes containing 2 mL of Löwenstein-Jensen (LJ) medium and 2 mL of semi-solid Kirchner medium with a critical concentration of 66 mg/L pyrazinamide at a pH of 5.2 or 5.5, incubated for 2 weeks and visually read. The results obtained were compared with MGIT 960 and DNA sequencing. RESULTS: Results were obtained in duplicate for 19 strains. One strain failed to grow on two occasions and only one result was available. Reproducibility was 95%. Eleven of the 19 strains were susceptible to pyrazinamide, whereas 7 were resistant. One strain was susceptible initially and pyrazinamide resistant on repeat testing. At pH 5.5, two strains reported as susceptible at pH 5.2 gave resistant results. CONCLUSIONS: The BMA might serve as a reliable low-cost DST alternative for pyrazinamide, particularly in laboratories using locally made solid media for DST. Its major drawback is the time to result. A reliable and affordable test method for the detection of pyrazinamide resistance is needed, especially in settings where multidrug-resistant tuberculosis is increasing. Proficiency testing should be routinely introduced wherever pyrazinamide DST is performed.

Proficiency of drug susceptibility testing of Mycobacterium tuberculosis against pyrazinamide: the Swedish experience.

BACKGROUND: Pyrazinamide (PZA) is a key drug in the treatment of tuberculosis (TB), including multidrug-resistant TB. Drug susceptibility testing (DST) of Mycobacterium tuberculosis against PZA is not included in the World Health Organization's yearly proficiency testing. There is an increasing need to establish quality control of PZA DST. OBJECTIVE: To evaluate the performance of PZA DST and to introduce a quality assurance system for the test in Sweden. METHOD: Panels with PZA-susceptible and -resistant isolates were used in three rounds of proficiency testing in all five Swedish clinical TB laboratories and our reference laboratory. All laboratories used the MGIT 960 system. Minimum inhibitory concentrations (MICs) were determined and the pncA gene was sequenced to further characterise the 52 panel strains. RESULTS: Good agreement was seen between the phenotypic PZA DST and pncA sequence data, and MIC determination confirmed high levels of resistance. However, in contrast to other drugs, for which correct proficiency test results were observed, specificity problems occurred for PZA DST in some laboratories. CONCLUSIONS: In Sweden, using panel testing, differences were seen in the proficiency of TB laboratories in correctly identifying PZA susceptibility. Improved results were noted in the third round; PZA has therefore been included in yearly proficiency testing.

Rifampicin-resistant and rifabutin-susceptible Mycobacterium tuberculosis strains: a breakpoint artefact?

OBJECTIVES: It has long been assumed that some rifampicin-resistant Mycobacterium tuberculosis strains are susceptible to, and thus treatable with, rifabutin. However, clinical breakpoints for susceptibility testing of rifabutin as well as the evidence for a clinical effect of rifabutin in rifampicin-resistant strains remains poorly defined. The objective of this study was to re-evaluate the breakpoint for rifabutin in relation to its MIC wild-type distribution and the presence of mutations in rpoB. METHODS: The MIC in 7H10 Middlebrook medium was determined for clinical isolates of M. tuberculosis (n = 95), where a majority were multidrug resistant. Additionally, all strains were screened for rpoB mutations by sequencing and the GenoType MTBDRplus assay. RESULTS: Rifampicin resistance was confirmed by genotypical and/or phenotypical tests in 73 isolates (76.8%). Nineteen isolates, defined as rifampicin resistant and rifabutin susceptible according to the present breakpoint, exhibited significantly higher MICs of rifabutin (0.064-0.5 mg/L) than rifabutin-susceptible isolates without any detectable mutations in rpoB (P < 0.001). These 19 isolates were clearly resistant to rifampicin (MIC 2-256 mg/L) and all but one had mutations in rpoB, with 9 (47.4%) specifically in Asp516Val. CONCLUSIONS: Our results indicate that rifampicin-resistant but rifabutin-susceptible isolates according to the present breakpoints harbour rpoB mutations and have a rifabutin MIC significantly higher than strains without any detectable mutations in rpoB. So far there are no clinical, pharmacological or microbiological data to confirm that such isolates can be treated with rifabutin and we suggest a revision of the current breakpoints.

Reevaluation of the critical concentration for drug susceptibility testing of Mycobacterium tuberculosis against pyrazinamide using wild-type MIC distributions and pncA gene sequencing.

Pyrazinamide (PZA) is a potent first-line agent for the treatment of tuberculosis (TB) with activity also against a significant part of drug-resistant Mycobacterium tuberculosis strains. Since PZA is active only at acid pH, testing for susceptibility to PZA is difficult and insufficiently reproducible. The recommended critical concentration for PZA susceptibility (MIC, 100 mg/liter) used in the Bactec systems (460 and MGIT 960) has not been critically evaluated against wild-type MIC distributions in clinical isolates of Mycobacterium tuberculosis. Using the Bactec MGIT 960 system, we determined the PZA MICs for 46 clinical M. tuberculosis isolates and compared the results to pncA sequencing and previously obtained Bactec 460 data. For consecutive clinical isolates (n = 15), the epidemiological wild-type cutoff (ECOFF) for PZA was 64 mg/liter (MIC distribution range, ≤ 8 to 64 mg/liter), and no pncA gene mutations were detected. In strains resistant in both Bactec systems (n = 18), the PZA MICs ranged from 256 to ≥ 1,024 mg/liter. The discordances between pncA sequencing, susceptibility results in Bactec 460, and MIC determinations in Bactec MGIT 960 were mainly observed in strains with MICs close to or at the ECOFF. We conclude that in general, wild-type and resistant strains were clearly separated and correlated to pncA mutations, although some isolates with MICs close to the ECOFF cause reproducibility problems within and between methods. To solve this issue, we suggest that isolates with MICs of ≤ 64 mg/liter be classified susceptible, that an intermediary category be introduced at 128 mg/liter, and that strains with MICs of >128 mg/liter be classified resistant.

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 distributions of seven oral second-line drugs against Mycobacterium tuberculosis.

OBJECTIVES: To determine wild-type minimum inhibitory concentration (MIC) distributions for Mycobacterium tuberculosis, as the background data for defining susceptibility breakpoints are limited. METHODS: We determined wild-type MIC distributions of M. tuberculosis using a 96-stick replicator in Middlebrook 7H10 (7H10) medium for ethionamide (ETH), prothionamide, thiacetazone, cycloserine, rifabutin (RFB), clofazimine and linezolid in consecutive susceptible clinical isolates (n = 78). RESULTS: Tentative epidemiological wild-type cut-offs (ECOFF) were determined for all investigated drugs where World Health Organization recommended critical concentrations for 7H10 are lacking, except for ETH. As the ECOFF was closely related to the non-wild-type strains for ETH and thiacetazone, the use of an intermediary (I) category in drug susceptibility testing could increase reproducibility. The cross-resistance between ETH and isoniazid was 21%. Applying 0.5 mg/l as a breakpoint for RFB classified two non-wild type and rpoB mutated isolates as susceptible for RFB and resistant against rifampicin. CONCLUSIONS: We propose that wild-type MIC distributions should be used as a tool to define clinical breakpoints against second-line drugs. This is increasingly important considering the rapid emergence of drug resistance.

A snapshot of the biodiversity and clustering of Mycobacterium tuberculosis in Oman using spoligotyping.

SETTINGS: National Tuberculosis Reference Laboratory, Central Public Health Laboratory, Ministry of Health, Oman. OBJECTIVE: To use spoligotyping to explore the genetic population structure and clustering of Mycobacterium tuberculosis isolates among nationals and immigrants in Oman. METHODS: Using spoligotyping, we characterised all available isolates from 2007, and randomly selected isolates from 2005 and 2006. A total of 312 clinical isolates from the same number of patients diagnosed with tuberculosis (TB) in 2005-2007 were included in the study. RESULTS: Of 312 isolates, 69% were in clusters ranging from 2 to 38 isolates. The proportion of clustering was 58% among 2005-2006 samples and 67% among 2007 samples, with higher clustering among Omanis than among immigrants. The study showed that M. tuberculosis Indian family lineages, CAS1_Delhi, CAS and EAI5 were the predominant strains. Around 50% of the immigrants shared strains with Omanis. Twelve of the 19 INH-monoresistant strains and the two multidrug-resistant strains were in clusters (P = 0.81). CONCLUSION: This study demonstrates the predominance in Oman of the strain family commonly found on the Indian sub-continent. A high proportion of immigrant strains were in the same clusters as Omani strains. To better ascertain the transmission dynamics of M. tuberculosis, we recommend that stringent molecular and conventional epidemiological methods be applied.

Evaluation of seven tests for the rapid detection of multidrug-resistant tuberculosis in Uganda.

SETTINGS: National Tuberculosis (TB) Reference Laboratory and Department of Medical Microbiology, College of Health Sciences, Makerere University, Kampala, Uganda. OBJECTIVE: To evaluate head-to-head rapid tests for drug susceptibility testing (DST) of Mycobacterium tuberculosis against rifampicin (RMP) and isoniazid (INH) in a resource-limited setting. METHODS: Thirty-one well-characterised strains of M. tuberculosis were tested with the nitrate reductase assay (NRA), microscopic observation drug susceptibility (MODS), MGIT 960 (Mycobacterium Growth Indicator Tube 960), Genotype MTBDRplus, Alamar blue, MTT and resazurin assays. The proportion method on Löwenstein-Jensen medium was used as the reference test. RESULTS: NRA correctly identified the resistant strains, with 100% sensitivity and specificity. MGIT 960 detected all multidrug-resistant strains but missed one RMP-monoresistant strain. Genotype MTBDRplus detected all RMP-resistant strains, but the sensitivity for detection of INH resistance was lower (88%). Sensitivity and specificity ranged from 86% to 100% for MODS and from 57% to 100% for the Alamar blue, MTT and resazurin assays. Test results were obtained within 2-14 days. CONCLUSION: In the study setting, NRA, MGIT 960 and Genotype MTBDRplus gave excellent detection of multidrug-resistant tuberculosis, with significantly shorter time to results compared to conventional testing.

Wild-type MIC distributions for aminoglycoside and cyclic polypeptide antibiotics used for treatment of Mycobacterium tuberculosis infections.

The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed +/-1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.

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)

Genetic characterisation of drug-resistant Mycobacterium tuberculosis in rural China: a population-based study.

OBJECTIVES: A population-based study was performed to characterise the genotype and phenotype of drug-resistant tuberculosis (TB) in the year 2004-2005 in two Chinese rural counties with different durations of DOTS implementation, Deqing and Guanyun. METHODS: Mycobacterium tuberculosis strains were isolated from respectively 164 and 187 patients registered at local TB dispensaries of Deqing and Guanyun. Drug susceptibility profiling and DNA sequencing were performed on the isolates. RESULTS: A total of 223 isolates from 223 patients were identified as resistant to first-line drugs, of which 53 were multidrug-resistant TB (MDR-TB, i.e., resistant to isoniazid [INH] and rifampicin [RMP]). Mutations in katG were identified in 81 of 131 INH-resistant isolates (61.8%), all of which harboured the mutation in codon 315. Mutations related to RMP resistance occurred mostly in codon 531, 526 and 516 of the rpoB gene. Seventy-eight of the 115 streptomycin-resistant isolates carried a mutation in the rpsL gene at codon 43 or 88. A mutation in codon 306 of embB occurred in 21 ethambutol (EMB) resistant and 19 EMB-susceptible isolates. CONCLUSIONS: Our data indicated that DNA sequencing of specific codons of the rpoB gene should be effective for predicting RMP resistance and MDR-TB in rural China.

Rates and mechanisms of resistance development in Mycobacterium tuberculosis to a novel diarylquinoline ATP synthase inhibitor.

R207910 (also known as TMC207) is an investigational drug currently in clinical studies for the treatment of multidrug-resistant (MDR) tuberculosis. It has a high degree of antimycobacterial activity and is equally effective against drug-susceptible and MDR Mycobacterium tuberculosis isolates. In the present study, we characterized the development of resistance to R207910 in vitro. Ninety-seven independent R207910-resistant mutants were selected from seven different clinical isolates of M. tuberculosis (three drug-susceptible and four MDR isolates) at 10x, 30x, and 100x the MIC. At a concentration of 0.3 mg/liter (10x the MIC), the mutation rates ranged from 4.7 x 10(-7) to 8.9 x 10(-9) mutations per cell per division, and at 1.0 mg/liter (30x the MIC) the mutation rate ranged from 3.9 x 10(-8) to 2.4 x 10(-9). No resistant mutants were obtained at 3 mg/liter (100x the MIC). The level of resistance ranged from 0.12 to 3.84 mg/liter for the mutants identified; these concentrations represent 4- to 128-fold increases in the MICs. For 53 of the resistant mutants, the atpE gene, which encodes a transmembrane and oligomeric C subunit of the ATP synthase and which was previously shown to be involved in resistance, was sequenced. For 15/53 mutants, five different point mutations resulting in five different amino acid substitutions were identified in the atpE gene. For 38/53 mutants, no atpE mutations were found and sequencing of the complete F0 ATP synthase operon (atpB, atpE, and atpF genes) and the F1 ATP synthase operon (atpH, atpA, atpG, atpD, and atpC genes) from three mutants revealed no mutations, indicating other, alternative resistance mechanisms. Competition assays showed no measurable reduction in the fitness of the mutants compared to that of the isogenic wild types.

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.

The added value of a European Union tuberculosis reference laboratory network--analysis of the national reference laboratory activities.

National reference laboratories (NRL) and other laboratories are the cornerstones of well-functioning tuberculosis programmes and surveillance activities. However, the scope and activity of NRL services for mycobacterial identification and drug susceptibility testing (DST) has not been examined in detail across the European Union (EU), nor has the added value of cooperation and networking at the European level been explored with regard to strengthening laboratory services. Therefore, the European Centre for Disease Prevention and Control (ECDC) has commissioned a survey to explore these issues and to identify areas of work that could bring added value by supporting networking activities of tuberculosis (TB) reference laboratories in the EU. Structured questionnaires were sent to TB reference laboratory experts in the EU and European Economic Area (EEA) countries, and in three additional countries selected on the basis of their networking activities with EU projects and other initiatives (Switzerland, Croatia and Israel). The compiled results describe the activities and structure of 32 NRLs (29 countries replied, a response rate of 91%). The analysis of the survey led to the following recommendations for strengthening TB laboratory services: (1) implementing of the published European standards for TB laboratory services with respect to infrastructure, national reference functions, biosafety, human resources, quality assurance, operational research (including evaluation of new medical diagnostics), accuracy and speed, appropriately trained staff; (2) ensuring that laboratories only perform activities for which they have demonstrated proficiency; (3) implement validated and standardised second-line drug susceptibility testing (DST), including drugs used to define extensively drug-resistant tuberculosis (XDR TB); (4) aiming to identify Mycobacterium tuberculosis complex (MTBC) and rifampicin (RIF) resistance in over 90% of cultures and cases from smear-positive sputum directly within one to two working days. To realise some of the above recommendations and to strengthen links of TB surveillance and microbiology activities in the EU, a list of suggested generic areas of activities for an EU network of reference laboratories is presented. Such a network would build on and link to existing networks and initiatives at the European and global level.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis (EUCAST document E.DEF 8.1)--report of the Subcommittee on Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis of the European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID).

This review describes the methods available for drug susceptibility testing of Mycobacterium tuberculosis. The methods have been developed over several decades and are restricted to specialised centres in most European countries, as they are technically demanding, require appropriate isolation facilities and can be difficult to interpret. The absolute concentration, resistance ratio and proportion methods can all give accurate results, provided that they are carefully quality-controlled and standardised. Automated rapid culture and molecular methods have been evaluated at large reference centres and in multicentre collaborations, and perform well for testing susceptibility to most first- and second-line anti-tuberculosis drugs. Accuracy is more important than rapid testing, and this is most reliably achieved if drug susceptibility tests are done in a small number of well-equipped, experienced laboratories that participate and perform well in an international drug susceptibility testing quality assessment scheme. The WHO Supranational Laboratory Quality Control Network offers a global scheme that assesses the ability of participating laboratories to identify isoniazid, rifampicin, ethambutol and streptomycin resistance. Second-line drug resistance testing is currently being standardised, and such testing should only be performed at the national reference laboratories in western and central European countries because of the relatively small number of cases and the concomitant difficulty of maintaining testing proficiency in multiple centres performing small numbers of tests. There is a need to expand international external quality assessment to include second-line drug susceptibility testing.

Recommended standards for modern tuberculosis laboratory services in Europe.

The principles underpinning these standards are that any tuberculosis laboratory-based diagnostic procedure should be performed by appropriately trained staff, working to standardised operating procedures in appropriately equipped and safe laboratories, against clear national and international proficiency and quality standards. Quality should be the pre-eminent criteria, not cost. The standards are technologically feasible, but initially may not be within the financial capacity of all laboratories. There is a requirement for government and international donors to adequately fund an appropriate safe infrastructure to enable staff to deliver accurate and timely results at whatever level of activity they are performing. There is a need for national reference laboratories to train a new cadre of mycobacterial laboratory experts. This will require the funding of appropriate individuals at these centres to train and assist in the implementation of good laboratory practice and evaluation to build sustainable capacity. Further operational research is needed to establish the optimal configuration of new technologies to determine isoniazid, rifampicin and second-line drug susceptibility in mycobacterial cultures and also, increasingly, directly on specimens. Improved integration of laboratory medicine as a core part of all tuberculosis programmes is needed to achieve and maximise the potential of new developments.