Second-line drug susceptibility breakpoints for Mycobacterium tuberculosis using the MODS assay.

OBJECTIVE: To establish breakpoint concentrations for the fluoroquinolones (moxifloxacin [MFX] and ofloxacin [OFX]) and injectable second-line drugs (amikacin [AMK], kanamycin [KM] and capreomycin [CPM]) using the microscopic observation drug susceptibility (MODS) assay. SETTING: A multinational study conducted between February 2011 and August 2012 in Peru, India, Moldova and South Africa. DESIGN: In the first phase, breakpoints for the fluoroquinolones and injectable second-line drugs (n = 58) were determined. In the second phase, MODS second-line drug susceptibility testing (DST) as an indirect test was compared to MGIT™ DST (n = 89). In the third (n = 30) and fourth (n = 156) phases, we determined the reproducibility and concordance of MODS second-line DST directly from sputum. RESULTS: Breakpoints for MFX (0.5 µg/ml), OFX (1 µg/ml), AMK (2 µg/ml), KM (5 µg/ml) and CPM (2.5 µg/ml) were determined. In all phases, MODS results were highly concordant with MGIT DST. The few discrepancies suggest that the MODS breakpoint concentrations for some drugs may be too low. CONCLUSION: MODS second-line DST yielded comparable results to MGIT second-line DST, and is thus a promising alternative. Further studies are needed to confirm the accuracy of the drug breakpoints and the reliability of MODS second-line DST as a direct test.

Evaluation of a rapid screening test for rifampicin resistance in re-treatment tuberculosis patients in the Eastern Cape.

BACKGROUND AND OBJECTIVES: Patients with multidrug-resistant (MDR) tuberculosis (TB) are at high risk of treatment failure. It is anticipated that early identification of MDR-TB and appropriate treatment will improve patient outcome and disease control. We evaluated the rapid detection of rifampicin resistance in previously treated TB patients, directly from acidfast bacilli (AFB)-positive sputum using a phage-based test, FASTPlaque-Response (Biotec Laboratories Ltd, Ipswich, UK). The ability of rifampicin resistance to predict MDR-TB was also determined. DESIGN: A prospective study was done comparing performance of the rapid phage test with conventional culture and drug susceptibility testing (DST) in AFB-positive TB patients. SETTING: Five primary health clinics and one TB referral centre in the Port Elizabeth Metropolitan area, Eastern Cape. OUTCOME MEASURES: Sensitivity, specificity and overall accuracy of the phage test were determined compared with gold standard culture and DST. Discrepant results were resolved by molecular detection of mutations conferring rifampicin resistance. The proportion of rifampicin-resistant strains that were MDR was also determined. RESULTS: Previously treated patients were at a high risk of MDRTB (35.7%). Sensitivity, specificity and overall accuracy of FASTPlaque-Response for rifampicin resistance determination were 95.4% (95% confidence interval (CI): 91.0 - 99.8%), 97.2% (95% CI: 94.5 - 99.9%) and 96.5% (95% CI: 94.1 - 98.9%) respectively compared with conventional DST (unresolved), calculated for specimens that had both FASTPlaque-Response and conventional DST results available. FASTPlaque-Response results were available in 2 days instead of 28 - 85 days with conventional DST. However, only 70.6% of FASTPlaque-Response results were interpretable compared with 86.3% of conventional DST results. The majority (95.5%) of rifampicinresistant strains were MDR-TB. CONCLUSIONS: Rapid detection of rifampicin resistance using FASTPlaque-Response could contribute to improved management of patients at risk of MDR-TB, such as previously treated patients. However, improvement in control of specimen-related contamination is needed to ensure that a higher proportion of FASTPlaque-Response results are interpretable. Where indicated, early modification of therapy could improve patient prognosis and reduce disease transmission.

Development of an antimicrobial formulation for control of specimen-related contamination in phage-based diagnostic testing for tuberculosis.

AIMS: To develop and evaluate an antimicrobial supplement for use with phage-based tests for rapid detection of drug resistance of tuberculosis (TB). METHODS AND RESULTS: An antimicrobial formulation containing nystatin, oxacillin and aztreonam (NOA) (final concentrations of 50,000 IU l(-1), 2 mg l(-1), and 30 mg l(-1) respectively) was developed. This formulation was tested for its influence on detection of a number of Mycobacterium tuberculosis (MTB) strains using the phage amplification (FASTPlaque) assay. Addition of the supplement did not lead to significant reduction in assay sensitivity. Antimicrobial efficacy was assessed with a range of Gram-positive and -negative organisms. The NOA supplement had a broad antimicrobial effect. The supplement was tested for its effect on growth of MTB culture, and on determination of rifampicin resistance using the phage-based methodology (FASTPlaque-Response). NOA did not significantly affect the growth of a range of rifampicin susceptible and resistant MTB strains, nor did it have an adverse effect on the number of interpretable results, nor the ability to discriminate between rifampicin susceptibility and resistance. CONCLUSION, SIGNIFICANCE AND IMPACT OF STUDY: Use of NOA antimicrobial supplement with rapid phage-based tests for TB will increase the proportion of interpretable results obtained, and enable their wider implementation in disease-endemic countries by improved control of specimen-related contamination.

Rapid indication of multidrug-resistant tuberculosis from liquid cultures using FASTPlaqueTB-RIF, a manual phage-based test.

SETTING: A Mycobacteriology Reference Laboratory in Johannesburg, South Africa. OBJECTIVE: To determine the ability of FASTPlaqueTB-RIF, a rapid bacteriophage-based test, to correctly identify rifampicin susceptibility in clinical strains of Mycobacterium tuberculosis after growth in the Bactec 460 semi-automated liquid culture system. DESIGN: A comparative study of FASTPlaqueTB-RIF and conventional drug susceptibility methods, with selection bias to include sufficient rifampicin-resistant strains. RESULTS: Rifampicin susceptibility results were available for 133 strains of M. tuberculosis. Using the Bactec 460 method, 42 of these strains were rifampicin-resistant and 91 strains were rifampicin-susceptible. A further one strain was found to have a mutation in the rpoB gene which was strongly indicative of rifampicin resistance. Sensitivity, specificity and overall accuracy for the FASTPlaqueTB-RIF were respectively 100%, 98.8% and 99.2% for detection of rifampicin resistance; 95.3% (41/43) of the rifampicin-resistant strains were also resistant to isoniazid (multidrug-resistant). CONCLUSION: FASTPlaqueTB-RIF offers performance comparable to the Bactec 460 method, with results available within 2 days and without the need for specialised equipment. This makes FASTPlaqueTB-RIF a rapid test for rifampicin resistance suitable for widespread application. A combination of the FAST-PlaqueTB-RIF test with semi-automated liquid culture reduces the time required to report susceptibility results, enabling rapid and appropriate management of patients with MDR-TB. Rifampicin resistance was a good predictor of multidrug resistance in this population.

Characterisation of the pncA gene in Mycobacterium tuberculosis isolates from Gauteng, South Africa.

SETTING: The use of pyrazinamide (PZA) is important for the treatment of Mycobacterium tuberculosis as it is bactericidal to semi-dormant mycobacteria that are not affected by other drugs. The incidence of resistance to PZA and other drugs used in the treatment of M. tuberculosis is increasing in South Africa. OBJECTIVE: To characterise the pncA gene of M. tuberculosis isolates from Gauteng, South Africa, and to develop a rapid diagnostic method. DESIGN: The pncA gene and the putative regulatory gene were characterised by sequence analysis in a total of six PZA susceptible and 15 resistant isolates. The association with classical PZA susceptibility testing and PZase activity was determined. RESULTS: All PZA-resistant isolates were PZase negative as well as resistant to at least one other anti-tuberculosis drugs. Mutations were identified throughout the length of the pncA gene in 10/15 PZA-resistant isolates. Five lacked PZase activity, but the wild type pncA sequence was present. In all six PZase-positive strains, a PZA-susceptible pattern was obtained on BACTEC and the wild type pncA sequence was present. CONCLUSION: Sequencing is an effective means to identify mutations in the pncA gene in M. tuberculosis and therefore resistance to PZA. The fact that some PZA-resistant M. tuberculosis isolates lack mutations in the pncA gene suggests that alternative mechanisms for drug resistance exist. In PZase negative strains with no genetic changes which are resistant to 100 microg/ml and susceptible to 300 microg/ml, 300 microg/ml may be a more reliable breakpoint.