Pseudo-Outbreak of Pre-Extensively Drug-Resistant (Pre-XDR) Tuberculosis in Kinshasa: Collateral Damage Caused by False Detection of Fluoroquinolone Resistance by GenoType MTBDR sl

Abstract

f Fluoroquinolones are the core drugs for the management of multidrug-resistant tuberculosis (MDR-TB). Molecular drug susceptibility testing methods provide considerable advantages for scaling up programmatic management and surveillance of drugresistant TB. We describe here the misidentification of fluoroquinolone resistance by the GenoType MTBDRsl (MTBDRsl) (Hain Lifescience GmbH, Nehren, Germany) line probe assay (LPA) encountered during a feasibility and validation study for the introduction of this rapid drug susceptibility test in Kinshasa, Democratic Republic of Congo. The double gyrA mutation 80Ala and 90Gly represented 57% of all fluoroquinolone mutations identified from MDR-TB patient sputum samples, as confirmed by DNA sequencing. This double mutation was previously found to be associated with susceptibility to fluoroquinolones, yet it leads to absent hybridization of a wild-type band in the MTBDRsl and is thus falsely scored as resistance. Our findings suggest that MTBDRsl results must be interpreted with caution when the interpretation is based solely on the absence of a wild-type band without confirmation by visualization of a mutant band. Performance of the MTBDRsl LPA might be improved by replacing the gyrA wild-type probes by additional probes specific for well-documented gyrA mutations that confer clinically relevant resistance.

MTBDR plus and MTBDR sl Assays: Absence of Wild-Type Probe Hybridization and Implications for Detection of Drug-Resistant Tuberculosis

“…Six percent of FQ resistance, as classified by MTBDRsl, was due to the sole absence of WT probe hybridization. Our results contrast significantly with the results of two studies conducted in the Congo and the Democratic Republic of Congo, where over 60% of FQ resistance was attributable to the sole absence of WT probe hybridization (17,18). Our results more closely resemble those reported by Brossier et al (33) and Huang et al (34), in which less than 20% of FQ resistance was attributable to the sole absence of WT probe hybridization.…”

“…Although MTBDRsl detects resistance to FQ, SLID, and ethambutol, phenotypic resistance to ethambutol was not performed as part of this study, and consequently only resistance to FQ and SLID were assessed. MTBDRsl probes detect mutations in codons 85 to 97 of the gyrA gene positions 1401 to 1402 and position 1484 of the rrs gene (17,23). Mutations associated with FQ resistance are detected by mutation probes gyrAMUT1(A90V), gyrAMUT2(S91P), gyrAMUT3A(D94A), gyrAMUT3B(D94N/Y), gyrAMUT3C(D94G), and gyrAMUT3D(D94H); mutations associated with resistance to SLID are detected by mutation probes rrsMUT1(A1401G) and rrsMUT2(G1484T).…”

“…(ii) If no MUT probes hybridize on the test strip and one or more WT probes also do not hybridize, the presence of an unknown mutation in that region is inferred, and the specimen is classified as resistant. Two recent studies assessing the performance of the MTBDRsl assay detected an unusually high proportion of FQresistant specimens that were eventually determined to be falsely positive after comparison to sequencing and/or conventional culture results (17,18). False-positive (FP) FQ resistance results from the two studies were determined to be the result of absent WT probe hybridization (interpreted to mean the presence of a nonwild-type or unknown mutation sequence) rather than direct detection of a known mutation (17,18).…”

“…Two recent studies assessing the performance of the MTBDRsl assay detected an unusually high proportion of FQresistant specimens that were eventually determined to be falsely positive after comparison to sequencing and/or conventional culture results (17,18). False-positive (FP) FQ resistance results from the two studies were determined to be the result of absent WT probe hybridization (interpreted to mean the presence of a nonwild-type or unknown mutation sequence) rather than direct detection of a known mutation (17,18). Given the significant impact of FQ resistance on MDR-TB treatment and the potential for the sole absence of WT probe hybridization to increase the number of false-positive test results, we analyzed individual probe performance to further understand the underlying subtleties of the MTBDRplus v2 and MTBDRsl assay results (19).…”

Some Synonymous and Nonsynonymous gyrA Mutations in Mycobacterium tuberculosis Lead to Systematic False-Positive Fluoroquinolone Resistance Results with the Hain GenoType MTBDR sl Assays

“…tuberculosis lineage 4), we speculated that the gyrA double mutant strains might constitute a subgroup of the Uganda genotype (16, 17). This hypothesis appeared to be consistent with the results of two studies from the Republic of the Congo and the Democratic Republic of the Congo, which reported the highest frequency of these double mutants (in 60% [9/15] versus 7.2% [15/209] of MDR TB cases from Brazzaville and Pointe-Noire versus Kinshasa, respectively) (7, 8). This was further supported by mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) results (7, 15).…”