Development and evaluation of the phenotypic 2G test to detect drug-resistant TB

Abstract

Background: Early diagnosis of TB with drug susceptibility testing (DST) is critical to achieve successful treatment outcomes. We aimed to develop and test a novel colorimetric, 12-well, thin-layer agar-based test to assess its accuracy for TB diagnosis and DST in a clinical setting in Southern Mozambique.

Methods: Development of the first prototype of the second generation (2G) test in the laboratory setting followed by a cross-sectional diagnostic accuracy study with consecutive recruitment of subjects with microbiologically confirmed TB using GeneXpert MTB/RIF Ultra.

Results: In the laboratory setting, the 2G test showed 100% accuracy in detecting resistance of genotypically characterised drug-resistant Mycobacterium tuberculosis strains. In the clinical setting, the sensitivity of the 2G test to detect M.tb complex versus Xpert and Mycobacteria Growth Indicator Tube (MGIT) culture using fresh sputa was 45.9% and 45.2%, respectively. The 2G test sensitivity versus MGIT decreased to 23.1% when using frozen decontaminated sputum samples.

Conclusion: In the clinical setting, the 2G test showed a low sensitivity versus Xpert and MGIT. The 2G test sensitivity was lower when frozen instead of fresh sputa was used. Despite these results, important information was collected to further improve this 2G test prototype and its implementation in resource-constrained settings.

Keywords: DR-TB; Mozambique; Mycobacterium tuberculosis; drug susceptibility testing; thin-layer culture; tuberculosis.

Figure 1.

Evolution of the 1G test into the first prototype of the 2G test. Critical concentrations for each of the drugs are as follows: isoniazid (INH), 0.2 μg/mL; rifampicin (RIF), 1 μg/mL; pyrazinamide (PZA), 1,100 μg/mL; ethambutol (EMB), 7.5 μg/mL; bedaquiline (BDQ), 0.25 μg/mL; linezolid (LZD), 1 μg/mL; amikacin (AMK), 2 μg/mL; protionamide (PTO), 10 μg/mL; cycloserine (CYL), 32 μg/mL; moxifloxacin (MFX), 0.5 μg/mL; and clofazimine (CLO), 1 μg/mL. Drug-susceptible (DS) well without drug (control well).

Figure 2.

A: Results of the 2G test using different DR-M.tb strains in the laboratory setting. (Black arrows show Mycobacterium tuberculosis growth). Left plate: Growth of a DR-M.tb strain with resistance to INH, RIF, AMK, and MXF. Middle plate: Growth of a DR-M.tb strain with resistance to INH, RIF, and MXF. Right plate: Growth of a DR-M.tb strain with resistance to AMK. B: Days to a positive 2G test result using 21 DR-M.tb strains previously stored under optimal and non-optimal conditions.

Figure 3.

Study participants flow and tests performed using fresh and frozen sputa. ^AThose with positive Xpert results at TB diagnosis. MGIT = Mycobacteria Growth Indicator Tube; P = positive; N = negative; C = contaminated; U = uninterpretable; NP = not performed; n = sample size.

Figure 4.

Interpretation of 2G test results obtained in the clinical setting using sputum samples from Xpert RIF susceptible and RIF resistant TB cases. A: The 2G test interpretation is growth of an MDR-M.tb complex strain with additional resistance to LNZ, AMK, PRO, and CYL. The sputum sample comes from a patient that is HIV negative with negative AFB; after microscopic evaluation, acid-fast bacilli growth on wells A1, A2, B4, and C2 lacks cord factor suggesting non-M.tb complex bacilli. The Xpert confirmatory result is ‘Trace’ (RIF resistance was confirmed with the Xpert diagnostic result of ‘Low’). No MGIT DST and LPA results were available. This is probably a mixed M.tb complex + NTM infection case. B: The 2G test interpretation is growth of an M.tb complex strain with monoresistance to CYL; the sputum sample comes from a patient that is HIV negative, with AFB 2+, an Xpert confirmatory result of ‘High’ with RIF susceptibility (no rpoB mutation). MGIT DST and LPA results were not available. C: The 2G test interpretation is growth of an MDR-M.tb complex strain with additional CYL resistance; the sputum sample comes from a patient that is HIV positive with AFB 2+. Growth on A1–A3 and C2 wells is compatible with acid-fast bacilli from M.tb complex showing cord factor in the microscopic evaluation. The confirmatory Xpert result is ‘High’ with RIF susceptibility (no rpoB mutation). MGIT result is Mycobacterium tuberculosis complex (MTBC), and MGIT DST shows additional resistance to INH and streptomycin. LPA result showed susceptibility to INH and RIF. This is an INH monoresistance detection by MGIT and the 2G test (missed by Xpert and LPA), with a potential RIF resistance detected by 2G but not confirmed by MGIT DST. D: The 2G test interpretation is growth of an MDR-M.tb complex strain with additional resistance to LNZ, AMK, PRO, and CYL. The sputum sample comes from a patient that is HIV negative with AFB 2+. Growth on A1, A2, B4, and C2 shows AFB lacking cord factor on the microscopy suggesting non-M.tb complex bacilli. Xpert confirmatory result is ‘Low’ with RIF susceptibility (no rpoB mutation). MGIT result is MTBC, but MGIT DST result was NTM, and LPA results showed susceptibility to INH and RIF. This is probably a mixed infection case, showing M.tb complex and NTM growth. MGIT result was initially MTBC, and when re-culturing for DST gave an NTM result.