Detection of antibiotic resistance in leprosy using GenoType LepraeDR, a novel ready-to-use molecular test

Abstract

Background: Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and to second-line drugs (fluoroquinolones) was described worldwide. Since Mycobacterium leprae is not growing in vitro, phenotypic susceptibility testing requires a one year experiment in the mouse model and this is rarely performed. Genetics on antibiotic resistance provide the basis for molecular tests able to detect for antibiotic resistance in leprosy.

Methodology/principal findings: A reverse hybridization DNA strip test was developed as the GenoType LepraeDR test. It includes DNA probes for the wild-type sequence of regions of rpoB, gyrA and folP genes and probes for the prevalent mutations involved in acquired resistance to rifampin, fluoroquinolones and dapsone, respectively. The performances of the GenoType LepraeDR test were evaluated by comparing its results on 120 M. leprae strains, previously studied for resistance by the reference drug in vivo susceptibility method in the mouse footpad and for mutations in the gene regions described above by PCR-sequencing. The results of the test were 100% concordant with those of PCR sequencing and the mouse footpad test for the resistant strains: 16 strains resistant to rifampin, 22 to dapsone and 4 to ofloxacin with mutations (numbering system of the M. leprae genome) in rpoB (10 S456L, 1 S456F, 1 S456M + L458V, 1 H451Y, 1 G432S + H451D, 1 T433I + D441Y and 1 Q438V), in folP1 (8 P55L, 3 P55R, 7 T53I, 3 T53A, 1 T53V) and gyrA (4 A91V), respectively. Concordance was 98.3% for the susceptible strains, two strains showing a mutation at the codon 447 that in fact was not conferring resistance as shown by the in vivo method.

Conclusions/significance: The GenoType LepraeDR test is a commercially available test that accurately detects for antibiotic resistance in leprosy cases. The test is easy to perform and could be implemented in endemic countries.

Figure 1. Mutations conferring resistance in Mycobacterium leprae are detected by the GenoType LepraeDR DNA strip test.

Lane 1 is a negative control (only the CC band). Lanes 2 to 11 showed various profiles for resistant strains: lane 2, rpoB mutation S456L with wild type gyrA and folP1 alleles; lane 3, wild type rpoB and gyrA alleles with a folP1 mutation to be defined; lane 4, rpoB mutation S456L with a wild type gyrA allele but a mutation in folP1; lane 5, rpoB mutation (Q438V) with wild type gyrA and folP1 alleles; lane 6, wild type rpoB and gyrA alleles with a P55L mutation in folP1; lane 7, rpoB mutation S456L with a A91V gyrA mutation and a P55L mutation in folP; lane 8 and lane 9, wild type rpoB and gyrA alleles with a P55L mutation in folP1 ; lane 10 and lane 11, rpoB mutation S456L with wild type gyrA and folP1 alleles. The numbering system used is that of the Mycobacterium leprae genome strain NT (n°NC 002677).

Figure 2. Mycobacterium leprae susceptible strains showed a wild type profile in the GenoType LepraeDR test.

Lane 1 to 16 (except lane 8) showed wild type profiles for susceptible M. leprae strains. Lane 8 showed a multiresistant profile with mutations in rpoB, gyrA and folP1 genes. Lanes 17 and 18 showed result of negative controls.