Université Paris Diderot, EA3964, Paris, France.
PLoS Negl Trop Dis. 2012;6(7):e1739. doi: 10.1371/journal.pntd.0001739. Epub 2012 Jul 31.
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.
虽然麻风病可以通过多药疗法有效治疗，但已在全球范围内发现对一线药物（氨苯砜、利福平）和二线药物（氟喹诺酮类药物）的耐药性。由于麻风分枝杆菌不能在体外生长，表型药敏试验需要在小鼠模型中进行为期一年的实验，而这种实验很少进行。抗生素耐药性的遗传学为能够检测麻风病中抗生素耐药性的分子检测提供了基础。
方法/主要发现：开发了一种反向杂交 DNA 条带试验，称为 GenoType LepraeDR 试验。它包括 rpoB、gyrA 和 folP 基因的野生型序列的 DNA 探针，以及分别用于检测利福平、氟喹诺酮类药物和氨苯砜获得性耐药相关突变的探针。通过将 GenoType LepraeDR 试验的结果与先前通过小鼠足垫体内参考药物药敏试验和上述基因区域的 PCR 测序研究的 120 株麻风分枝杆菌菌株的结果进行比较，评估了该试验的性能。耐药菌株的试验结果与 PCR 测序和小鼠足垫试验完全一致：16 株对利福平耐药，22 株对氨苯砜耐药，4 株对氧氟沙星耐药，其 rpoB（10 S456L、1 S456F、1 S456M+L458V、1 H451Y、1 G432S+H451D、1 T433I+D441Y 和 1 Q438V）、folP1（8 P55L、3 P55R、7 T53I、3 T53A、1 T53V）和 gyrA（4 A91V）基因均存在突变。敏感菌株的一致性为 98.3%，有两株菌株在 447 密码子处存在突变，但实际上该突变并未导致耐药，这一点通过体内方法得到了证实。
结论/意义：GenoType LepraeDR 试验是一种商业上可获得的检测方法，可准确检测麻风病病例中的抗生素耐药性。该试验易于操作，可在流行国家实施。
