Detection of antibiotic resistance in leprosy using GenoType LepraeDR, a novel ready-to-use molecular test.
Detection of antibiotic resistance in leprosy using GenoType LepraeDR, a novel ready-to-use molecular test.
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DOI：
10.1371/journal.pntd.0001739
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发表时间：
2012
影响因子：
3.8
通讯作者：
Jarlier V
中科院分区：
文献类型：
--
作者：
Cambau E;Chauffour-Nevejans A;Tejmar-Kolar L;Matsuoka M;Jarlier V
虽然麻风病可通过多种药物治疗有效治疗，但对一线药物（氨苯砜、利福平）和二线药物（氟喹诺酮类）的耐药性在世界范围内均有报道。由于麻风分枝杆菌不在体外生长，表型敏感性测试需要在小鼠模型中进行一年的实验，并且很少进行。抗生素耐药性的遗传学为能够检测麻风抗生素耐药性的分子测试提供了基础。开发了反向杂交DNA试纸条检测作为GenoType LepraeDR检测。它包括rpoB、gyrA和folP基因区域野生型序列的DNA探针，以及分别涉及利福平、氟喹诺酮类和氨苯砜获得性耐药的流行突变的探针。通过比较120 M上的结果，评价了GenoType LepraeDR检测的性能。麻风菌株，先前通过小鼠足垫中的参考药物体内敏感性方法研究了耐药性，并通过PCR测序研究了上述基因区域中的突变。对耐药菌株的检测结果与PCR测序和小鼠足垫试验结果100%一致：16株利福平耐药菌株、22株氨苯砜耐药菌株和4株氧氟沙星耐药菌株发生突变（M. rpoB中S456 L、S456 F、S456 M + L458 V、H451 Y、G432 S + H451 D、T433 I + D441 Y和Q438 V各1例; folP 1中P55 L、P55 R、T53 I、T53 A和T53 V各8例;敏感菌株的一致性为98.3%，两个菌株在密码子447处显示突变，如体内方法所示，该突变实际上不赋予耐药性。GenoType LepraeDR检测是一种市售检测，可准确检测麻风病例的抗生素耐药性。该测试易于执行，可在流行国家实施。虽然麻风病是一种可治愈的疾病，使用抗生素组合一年，但传播仍然活跃，2010年有230 000例新病例。已经描述了耐药性，并且可能会阻止疾病的根除。导致麻风病的传染性病原体麻风分枝杆菌不能在体外生长，抗生素敏感性测试只能在需要一年实验的小鼠足垫模型中进行。因此，这种检测很少进行，麻风病的抗生素耐药性也是未知的。这就是为什么我们决定建立一种新的诊断方法来检测M.麻风病人该检测基于DNA试纸条检测方法，采用多重PCR，然后进行反向杂交。它是作为一种易于使用的测试方法开发的，将在流行国家提供，在这些国家，这种试纸测试已经用于检测结核病的耐药性。新的测试结果，基因型LepraeDR，在120 M上进行。麻风菌株与标准PCR测序和小鼠足垫敏感性测试结果一致。
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. 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. 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. Although leprosy is a curable disease using a combination of antibiotics for one year, the transmission is still active with 230,000 new cases in 2010. Drug resistance has been described and may prevent eradication of the disease. The infectious agent causing leprosy, Mycobacterium leprae, is not growing in vitro and antibiotic susceptibility testing is possible only in the mouse footpad model that requires a one year experiment. Consequently this testing is rarely done and antibiotic resistance rates in leprosy are unknown. This is the reason why we endeavored to set a new diagnosis test that detects for antibiotic resistance in M. leprae. The test is based on the method of a DNA strip test with a multiplex PCR followed by reverse hybridization. It was developed as an easy-to-use test and it will be available in endemic countries, where these kinds of strip tests are already used for detection of drug resistance in tuberculosis. The results of the new test, Genotype LepraeDR, performed on 120 M. leprae strains were concordant with those of the standard PCR sequencing and mouse footpad susceptibility testing.
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影响因子：
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