Population-based resistance of Mycobacterium tuberculosis isolates to pyrazinamide and fluoroquinolones: results from a multicountry surveillance project.

BACKGROUND: Pyrazinamide and fluoroquinolones are essential antituberculosis drugs in new rifampicin-sparing regimens. However, little information about the extent of resistance to these drugs at the population level is available. METHODS: In a molecular epidemiology analysis, we used population-based surveys from Azerbaijan, Bangladesh, Belarus, Pakistan, and South Africa to investigate resistance to pyrazinamide and fluoroquinolones among patients with tuberculosis. Resistance to pyrazinamide was assessed by gene sequencing with the detection of resistance-conferring mutations in the pncA gene, and susceptibility testing to fluoroquinolones was conducted using the MGIT system. FINDINGS: Pyrazinamide resistance was assessed in 4972 patients. Levels of resistance varied substantially in the surveyed settings (3·0-42·1%). In all settings, pyrazinamide resistance was significantly associated with rifampicin resistance. Among 5015 patients who underwent susceptibility testing to fluoroquinolones, proportions of resistance ranged from 1·0-16·6% for ofloxacin, to 0·5-12·4% for levofloxacin, and 0·9-14·6% for moxifloxacin when tested at 0·5 µg/mL. High levels of ofloxacin resistance were detected in Pakistan. Resistance to moxifloxacin and gatifloxacin when tested at 2 µg/mL was low in all countries. INTERPRETATION: Although pyrazinamide resistance was significantly associated with rifampicin resistance, this drug may still be effective in 19-63% of patients with rifampicin-resistant tuberculosis. Even though the high level of resistance to ofloxacin found in Pakistan is worrisome because it might be the expression of extensive and unregulated use of fluoroquinolones in some parts of Asia, the negligible levels of resistance to fourth-generation fluoroquinolones documented in all survey sites is an encouraging finding. Rational use of this class of antibiotics should therefore be ensured to preserve its effectiveness. FUNDING: Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development.

External Quality Assessment for Tuberculosis Diagnosis and Drug Resistance in the European Union: A Five Year Multicentre Implementation Study.

BACKGROUND: External quality assurance (EQA) systems are essential to ensure accurate diagnosis of TB and drug-resistant TB. The implementation of EQA through organising regular EQA rounds and identification of training needs is one of the key activities of the European TB reference laboratory network (ERLTB-Net). The aim of this study was to analyse the results of the EQA rounds in a systematic manner and to identify potential benefits as well as common problems encountered by the participants. METHODS: The ERLTB-Net developed seven EQA modules to test laboratories' proficiency for TB detection and drug susceptibility testing using both conventional and rapid molecular tools. All National TB Reference laboratories in the European Union and European Economic Area (EU/EEA) Member States were invited to participate in the EQA scheme. RESULTS: A total of 32 National TB Reference laboratories participated in six EQA rounds conducted in 2010-2014. The participation rate ranged from 52.9% - 94.1% over different modules and rounds. Overall, laboratories demonstrated very good proficiency proving their ability to diagnose TB and drug-resistant TB with high accuracy in a timely manner. A small number of laboratories encountered problems with identification of specific Non-tuberculous Mycobacteria (NTMs) (N = 5) and drug susceptibility testing to Pyrazinamide, Amikacin, Capreomycin, and Ethambutol (N = 4). CONCLUSIONS: The European TB Reference laboratories showed a steady and high level of performance in the six EQA rounds. A network such as ERLTB-Net can be instrumental in developing and implementing EQA and in establishing collaboration between laboratories to improve the diagnosis of TB in the EU/EEA.

A multisite assessment of the quantitative capabilities of the Xpert MTB/RIF assay.

RATIONALE: The Xpert MTB/RIF is an automated molecular test for Mycobacterium tuberculosis that estimates bacterial burden by measuring the threshold-cycle (Ct) of its M. tuberculosis-specific real-time polymerase chain reaction. Bacterial burden is an important biomarker for disease severity, infection control risk, and response to therapy. OBJECTIVES: Evaluate bacterial load quantitation by Xpert MTB/RIF compared with conventional quantitative methods. METHODS: Xpert MTB/RIF results were compared with smear-microscopy, semiquantiative solid culture, and time-to-detection in liquid culture for 741 patients and 2,008 samples tested in a multisite clinical trial. An internal control real-time polymerase chain reaction was evaluated for its ability to identify inaccurate quantitative Xpert MTB/RIF results. MEASUREMENTS AND MAIN RESULTS: Assays with an internal control Ct greater than 34 were likely to be inaccurately quantitated; this represented 15% of M. tuberculosis-positive tests. Excluding these, decreasing M. tuberculosis Ct was associated with increasing smear microscopy grade for smears of concentrated sputum pellets (r(s) = -0.77) and directly from sputum (r(s) =-0.71). A Ct cutoff of approximately 27.7 best predicted smear-positive status. The association between M. tuberculosis Ct and time-to-detection in liquid culture (r(s) = 0.68) and semiquantitative colony counts (r(s) = -0.56) was weaker than smear. Tests of paired same-patient sputum showed that high viscosity sputum samples contained ×32 more M. tuberculosis than nonviscous samples. Comparisons between the grade of the acid-fast bacilli smear and Xpert MTB/RIF quantitative data across study sites enabled us to identify a site outlier in microscopy. CONCLUSIONS: Xpert MTB/RIF quantitation offers a new, standardized approach to measuring bacterial burden in the sputum of patients with tuberculosis.

Rapid molecular detection of tuberculosis and rifampin resistance.

BACKGROUND: Global control of tuberculosis is hampered by slow, insensitive diagnostic methods, particularly for the detection of drug-resistant forms and in patients with human immunodeficiency virus infection. Early detection is essential to reduce the death rate and interrupt transmission, but the complexity and infrastructure needs of sensitive methods limit their accessibility and effect. METHODS: We assessed the performance of Xpert MTB/RIF, an automated molecular test for Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF), with fully integrated sample processing in 1730 patients with suspected drug-sensitive or multidrug-resistant pulmonary tuberculosis. Eligible patients in Peru, Azerbaijan, South Africa, and India provided three sputum specimens each. Two specimens were processed with N-acetyl-L-cysteine and sodium hydroxide before microscopy, solid and liquid culture, and the MTB/RIF test, and one specimen was used for direct testing with microscopy and the MTB/RIF test. RESULTS: Among culture-positive patients, a single, direct MTB/RIF test identified 551 of 561 patients with smear-positive tuberculosis (98.2%) and 124 of 171 with smear-negative tuberculosis (72.5%). The test was specific in 604 of 609 patients without tuberculosis (99.2%). Among patients with smear-negative, culture-positive tuberculosis, the addition of a second MTB/RIF test increased sensitivity by 12.6 percentage points and a third by 5.1 percentage points, to a total of 90.2%. As compared with phenotypic drug-susceptibility testing, MTB/RIF testing correctly identified 200 of 205 patients (97.6%) with rifampin-resistant bacteria and 504 of 514 (98.1%) with rifampin-sensitive bacteria. Sequencing resolved all but two cases in favor of the MTB/RIF assay. CONCLUSIONS: The MTB/RIF test provided sensitive detection of tuberculosis and rifampin resistance directly from untreated sputum in less than 2 hours with minimal hands-on time. (Funded by the Foundation for Innovative New Diagnostics.)

Unequal distribution of resistance-conferring mutations among Mycobacterium tuberculosis and Mycobacterium africanum strains from Ghana.

Isoniazid (INH) and rifampicin (RMP) resistance in Mycobacterium tuberculosis complex (MTC) isolates are mainly based on mutations in a limited number of genes. However, mutation frequencies vary in different mycobacterial populations. In this work, we analyzed the distribution of resistance-associated mutations in M. tuberculosis and M. africanum strains from Ghana, West Africa. The distribution of mutations in katG, fabG1-inhA, ahpC, and rpoB was determined by DNA sequencing in 217 INH-resistant (INH(r)) and 45 multidrug-resistant (MDR) MTC strains isolated in Ghana from 2001 to 2004. A total of 247 out of 262 strains investigated (94.3%) carried a mutation in katG (72.5%), fabG1-inhA (25.1%), or ahpC (6.5%), respectively. M. tuberculosis strains mainly had katG 315 mutations (80.1%), whereas this proportion was significantly lower in M. africanum West-African 1 (WA1) strains (43.1%; p<0.05). In contrast, WA1 strains showed more mutations in the fabG1-inhA region (39.2%, p<0.05) compared to M. tuberculosis strains (20.9%). In 44 of 45 MDR strains (97.8%) mutations in the 81-bp core region of the rpoB gene could be verified. Additionally, DNA sequencing revealed that 5 RMP-susceptible strains also showed mutations in the rpoB hotspot region. In conclusion, although principally the same genes were affected in INH(r)M. tuberculosis and M. africanum strains, disequilibrium in the distribution of mutations conferring resistance was verified that might influence the efficiency of molecular tests for determination of resistance.

Multidrug- and extensively drug-resistant tuberculosis, Germany.

We evaluated risk factors and treatment outcomes associated with multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) in Germany in 2004-2006. In 177 (4%) of 4,557 culture-positive TB cases, Mycobacterium tuberculosis isolates were identified as MDR TB; an additional 7 (0.15%) met criteria for XDR TB. Of these 184 patients, 148 (80%) were born in countries of the former Soviet Union. In patients with XDR TB, hospitalization was longer (mean +/- SD 202 +/- 130 vs. 123 +/- 81 days; p = 0.015) and resistance to all first-line drugs was more frequent (36% vs. 86%; p = 0.013) than in patients with MDR TB. Seventy-four (40%) of these 184 patients received treatment with linezolid. Treatment success rates ranged from 59% for the entire cohort (59% for MDR TB and 57% for XDR TB) to 87% for those with a definitive outcome (n = 125; 89% for MDR TB and 80% for XDR TB). Extensive drug susceptibility testing and availability of second- and third-line drugs under inpatient management conditions permit relatively high treatment success rates in MDR- and XDR TB.

Multidrug-resistant tuberculosis in central Asia.

Multidrug-resistant tuberculosis (MDR-TB) has emerged as a major threat to TB control, particularly in the former Soviet Union. To determine levels of drug resistance within a directly observed treatment strategy (DOTS) program supported by Médecins Sans Frontières in two regions in Uzbekistan and Turkmenistan, Central Asia, we conducted a cross-sectional survey of smear-positive TB patients in selected districts of Karakalpakstan (Uzbekistan) and Dashoguz (Turkmenistan). High levels of MDR-TB were found in both regions. In Karakalpakstan, 14 (13%) of 106 new patients were infected with MDR-TB; 43 (40%) of 107 previously treated patients were similarly infected. The proportions for Dashoguz were 4% (4/105 patients) and 18% (18/98 patients), respectively. Overall, 27% of patients with positive smear results whose infections were treated through the DOTS program in Karakalpakstan and 11% of similar patients in Dashoguz were infected with multidrug-resistant strains of TB on admission. These results show the need for concerted action by the international community to contain transmission and reduce the effects of MDR-TB.