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Whole-blood nested-PCR amplification of M. leprae-specific DNA for early diagnosis of leprosy.

ABSTRACT: We evaluated the sensitivity and specificity of a nested-polymerase chain reaction (PCR) method for detection of Mycobacterium leprae DNA from whole blood. Whole-blood specimens were subjected to nested-PCR amplification of M. leprae repeat DNA sequences in 49 multibacillary (MB) and 30 paucibacillary (PB) leprosy patients, 96 household contacts (HHCs), 18 tuberculosis (TB) patients, and 35 normal healthy individuals. M. leprae DNA was detected in 95.92% (47/49) of MB, 70% (21/30) of PB, and 6.25% (6/96) of HHC, but it was not detected in 18 TB or 35 normal controls. The sensitivities of the anti-bovine serum albumin (ND-O-BSA) immunoglobulin M (IgM) and antifusion protein of ML0405-ML2331 IgG for MB were 97.96% and 89.8%, and these values for PB were 70% and 53.33%. However, the ND-O-BSA enzyme-linked immunosorbent assay (ELISA) had lower specificity, with relatively high false-positive results for TB patients (16.67%) and normal healthy controls (10%). Based on these promising findings, we propose the use of nested PCR of whole-blood samples along with ELISA test for early detection of leprosy cases.

SUBMITTER: Wen Y

PROVIDER: S-EPMC3752758 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Whole-blood nested-PCR amplification of M. leprae-specific DNA for early diagnosis of leprosy.

Wen Yan Y Xing Yan Y Yuan Lian-Chao LC Liu Jian J Zhang Ying Y Li Huan-Ying HY

The American journal of tropical medicine and hygiene 20130311 5

We evaluated the sensitivity and specificity of a nested-polymerase chain reaction (PCR) method for detection of Mycobacterium leprae DNA from whole blood. Whole-blood specimens were subjected to nested-PCR amplification of M. leprae repeat DNA sequences in 49 multibacillary (MB) and 30 paucibacillary (PB) leprosy patients, 96 household contacts (HHCs), 18 tuberculosis (TB) patients, and 35 normal healthy individuals. M. leprae DNA was detected in 95.92% (47/49) of MB, 70% (21/30) of PB, and 6.2 ...[more]

PMID: 23478578

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Project description:BackgroundLeprosy, caused by Mycobacterium leprae, affects over 200,000 people annually worldwide and remains endemic in the ethnically diverse, mountainous and underdeveloped southwestern provinces of China. Delayed diagnosis of leprosy persists in China, thus, additional knowledge to support early diagnosis, especially early diagnosis of paucibacillary (PB) patients, based on the host immune responses induced by specific M. leprae antigens is needed. The current study aimed to investigate leprosy patients and controls in Southwest China by comparing supernatants after stimulation with specific M. leprae antigens in an overnight whole-blood assay (WBA) to determine whether host markers induced by specific M. leprae antigens improve the diagnosis or discrimination of PB patients with leprosy.Methodology/principal findingsLeprosy patients [13 multibacillary (MB) patients and 7 PB patients] and nonleprosy controls [21 healthy household contacts (HHCs), 20 endemic controls (ECs) and 19 tuberculosis (TB) patients] were enrolled in this study. The supernatant levels of ten host markers stimulated by specific M. leprae antigens were evaluated by overnight WBA and multiplex Luminex assays. The diagnostic value in PB patients and ECs and the discriminatory value between PB patients and HHCs or TB patients were evaluated by receiver operator characteristics (ROC) analysis. ML2044-stimulated CXCL8/IL-8 achieved the highest sensitivity of 100%, with a specificity of 73.68%, for PB diagnosis. Compared to single markers, a 3-marker combination model that included ML2044-induced CXCL8/IL-8, CCL4/MIP-1 beta, and IL-6 improved the diagnostic specificity to 94.7% for PB patients. ML2044-stimulated IL-4 and CXCL8/IL-8 achieved the highest sensitivity (85.71% and 100%) and the highest specificity (95.24% and 84.21%) for discriminating PB patients from HHCs and TB patients, respectively.ConclusionsOur findings suggest that the host markers induced by specific M. leprae antigens in an overnight WBA increase diagnostic and discriminatory value in PB patients with leprosy, with a particularly strong association with interleukin 8.

Project description:BACKGROUND:Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and second-line (fluoroquinolones) drugs has been described worldwide. However, the characteristics of drug resistance in Southwest China remain unknown. Furthermore, the sensitivity of polymerase chain reaction (PCR)/sequencing for resistance detection is limited, especially for paucibacillary (PB) leprosy patients. The current study aimed to develop a nested PCR/sequencing and TaqMan SNP Genotyping Assay to increase the sensitivity of the method used to detect drug resistance in Mycobacterium leprae and to reveal the nature of M. leprae drug resistance in Southwest China. METHODOLOGY/PRINCIPAL FINDINGS:Seventy-six specimens, including skin biopsy (n = 64), formalin-fixed paraffin-embedded (FFPE) (n = 11) and skin-slit smear (SSS) (n = 1) samples from multibacillary (MB, n = 70) and PB (n = 6) leprosy patients from Southwest China, were included in this study. The presence of mutations in drug resistance-determining regions (DRDRs) of the rpoB, folP1, and gyrA genes, which are associated with rifampicin, dapsone, and quinolone resistance, respectively, was detected by PCR/sequencing, as recommended by the WHO, and the nested PCR and TaqMan SNP Genotyping Assay developed in this study. Mutations in the folP gene were detected in 19 (25.00%) samples, indicating dapsone-resistant M. leprae, with one (1.31%) sample showing mutations in two genes, folP and gyrA, reflecting multidrug-resistant strains to dapsone and ofloxacin. However, no rpoB mutation was detected. Compared with PCR/sequencing, nested PCR increased the sensitivity of detecting rpoB (from 51.39% to 78.94% for leprosy patients and from 0.00% to 50.00% for PB), gyrA (from 75.00% to 80.26% for leprosy patients and from 50.00% to 66.67% for PB), and folP1 (from 5.26% to 84.21% for leprosy patients and from 0.00% to 66.67% for PB). Moreover, the TaqMan SNP Genotyping Assay showed greater sensitivity for folP1 detection (from 5.26% to 78.94-86.84% for leprosy patients and from 0.00% to 33.33%-83.33% for PB patients) than the PCR/sequencing method. In addition, the latter method was able to more easily distinguish heterozygous genotypes and mutant homozygous genotypes from homozygous genotypes. CONCLUSIONS/SIGNIFICANCE:Nested PCR/sequencing and the TaqMan SNP Genotyping Assay are rapid and highly sensitive methods for detecting drug resistance in leprosy cases. The current study revealed that diamino-diphenylsulfone (DDS; also known as dapsone) resistance in M. leprae, as indicated by folP1 gene detection, is still the most concerning form of drug resistance in leprosy patients from Southwest China.

Project description:BackgroundPreimplantation genetic diagnosis (PGD) remains nowadays a valid alternative for couples at high-risk of having a child with a genetic disease and for women older than 37-40 years with the high risk of chromosomal aneuploidies in the embryos. However the use of PGD for high penetrance recessive, dominant and X-liked disorders occurring in early life is documented, debate exists regarding its appropriateness in lower penetrance and late-onset cancer susceptibility syndromes. The data regarding the efficacy of different molecular techniques used in PGD are still lacking. We therefore sought to assess the different molecular techniques used in PGD for detecting three most frequent BRCA1 gene mutations: 5382insC, 185delAG and C61G.MethodsAnonymous donors of the oocytes and control healthy blood samples were extracted and analyzed in the Fertility and Reproductive Center Invicta in Gdansk. Preimplantation genetic diagnosis for the most frequent mutations: 185delAG, 5382insC, C61G in BRCA 1 gene was carried out on single, unfertilized oocytes, in metaphase of second meiotic division, not qualified to IVF. Positive mutation controls were represented by cell lines from the Coriell Institute for Medical Research: GM14090 (185delAG), GM14097 (C61G), GM13715 (5382insC).ResultsRepeatability of the results acquired from the WGA analysis for the mutation 5382insC was 38%. The repeatability of the nested-PCR analysis in the second round of the amplification was labile for the mutation 5382insC and 185delAG and was ranged from 47% to 57%. However, the repeatability for the mutation C61G was 100%.ConclusionsOur results suggest that the nested-PCR technique remains more sensitive and specific method as compared to WGA. WGA performed on the single cells did not reflect expected results. The repeatability of the WGA methodology remains questionable, and any analysis attempt does not guarantee reliable results. Further evaluation is strongly needed to propose the most accurate molecular technique used in PGD for detecting three most frequent BRCA1 gene mutations: 5382insC, 185delAG and C61G.

Dataset Information

Whole-blood nested-PCR amplification of M. leprae-specific DNA for early diagnosis of leprosy.

Publications

Whole-blood nested-PCR amplification of M. leprae-specific DNA for early diagnosis of leprosy.

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