Transcriptomic Analysis of Mycobacterium leprae-Stimulated Response in Peripheral Blood Mononuclear Cells Reveal Potential Biomarkers for Early Diagnosis of Leprosy.

We aimed to identify an unique host transcriptional signature in peripheral blood mononuclear cells (PBMCs) in response to Mycobacterium leprae antigens to distinguish between patients with leprosy and non-leprosy controls for early diagnosis of the disease. Sixteen individuals were enrolled in the discovery cohort [eight patients with leprosy, comprising four multibacillary (MB) and four paucibacillary (PB); and eight non-leprosy controls, comprising four healthy house contacts (HHCs) and four endemic controls (ECs)]. The differences in the transcriptome response of PBMCs to M. leprae sonicate antigen were evaluated between leprosy patients and non-leprosy controls, and 12 differentially expressed genes (CCL2/MCP-1, IL-8, JAKM, ATP, ND1, SERP, FLJ10489, LINC00659, LOC34487, LOC101928143, MIR22, and NCF1C) were identified. The accuracy of the 12 differentially expressed genes was further validated for the diagnosis of leprosy using real-time quantitative PCR in 82 individuals (13 MB, 10 PB, 37 HHCs, and 22 ECs) in the validation cohort. We found that a 5 gene signature set IL-8, CCL2/MCP-1, SERP, LINC00659 and FLJ10489 had a suitable performance in discriminating leprosy from ECs. In addition, elevated expression of IL-8, CCL2/MCP-1, SERP and LINC00659 was associated with MB diagnosis compared with ECs, whereas increased expression of IL-8, CCL2/MCP-1, SERP and FLJ10489 was found to be useful biomarkers for PB diagnosis from ECs. Moreover, we found decreased expression of NCF1C among leprosy patients could distinguish leprosy from HHCs, whereas higher expression of CCL2 among MB than PB could distinguish different leprosy patients. In conclusion, among the 12 candidate host genes identified, a three gene signature IL-8, CCL2/MCP-1, and SERP showed the best performance in distinguishing leprosy patients from healthy controls. These findings may have implications for developing a rapid blood-based test for early diagnosis of leprosy.

Host immune responses induced by specific Mycobacterium leprae antigens in an overnight whole-blood assay correlate with the diagnosis of paucibacillary leprosy patients in China.

BACKGROUND: Leprosy, 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 FINDINGS: Leprosy 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. CONCLUSIONS: Our 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.

Evaluation of antigen-specific immune responses for leprosy diagnosis in a hyperendemic area in China.

OBJECTIVE: To evaluate antigen-specific immune responses for leprosy diagnosis in a hyperendemic area in China. METHODS: Eighty-three leprosy patients and 161 non-leprosy controls were enrolled from Hani-yi Autonomous Prefecture of Honghe, Yunnan Province, China. Leprosy patients were divided into multibacillary (MB, n = 38), paucibacillary (PB, n = 23), and post-multi-drug therapy (MDT, n = 22) groups. Controls were divided into the following groups: healthy household contacts (HHC, n = 119), tuberculosis (TB, n = 11), and endemic controls (EC, n = 31). The NDO-LID Rapid Test, M. leprae antigen-specific ELISA and antigen-specific IFN-γ secretion in a whole blood assay (WBA) were used to evaluate these subjects. RESULTS: The NDO-LID Rapid Test achieved higher positive response rates in MB than in PB patients[94.7%(36/38) vs 65.2%(15/23)], and these rates were higher than those observed by ELISA using anti-LID-1[92.1%(35/38) vs 52.2%(12/23)], anti-NDO-LID[92.1%(35/38) vs 47.8% (11/23)], and anti-ND-O-BSA[89.5%(34/38) vs 60.9%(14/23)]. However, the NDO-LID Rapid Test also showed a higher positive response rate in the EC group (33.3%,10/31), which was higher than the rates observed for anti-NDO-LID (12.9%,4/31) and anti-ND-O-BSA (16.1%,5/31). M. leprae antigen-specific ELISA demonstrated relatively high specificity (86.84-97.37%) but low sensitivity (15.97-72.73%) in discriminating between leprosy patients and non-leprosy controls by ROC curve analysis. In contrast, M. leprae antigen-specific IFN-γ secretion detection achieved higher positive response rates in PB than in MB patients (positive ratio of MB vs PB: 40% vs 56% for LID-1, 28.6% vs 47.8% for ML89, 31.4% vs 60.7% for ML2044, and 31.4 vs 47.8% for ML2028) and could distinguish MB from EC when stimulated with ML89(AUC = 0.6664) and PB fromTB when stimulated with ML2044 and ML2028(AUC = 0.7549 and 0.7372, respectively). CONCLUSION: The NDO-LID Rapid Test and M. leprae antigen-specific ELISA are useful tools to assist in the diagnosis of leprosy patients, especially MB patients, although the former had higher sensitivity but lower specificity than the latter. M. leprae antigen-specific IFN-γ release assessed by WBA has diagnostic value for distinguishing PB from TB but not for distinguishing PB from HHC or EC. Screening novel M. leprae-specific antigens, combining different M. leprae antigens and a multi-cytokine analyte model may be needed for more effective diagnosis of leprosy.

Identification of novel genetic loci GAL3ST4 and CHGB involved in susceptibility to leprosy.

Leprosy has long been thought to have a strong genetic component, and so far, only positional cloning and genomewide association studies have been used to study the genetic susceptibility to leprosy,while whole exome sequencing (WES) approach has not yet been applied. In this study, we used WES approach on four leprosy patients and four healthy control relatives from two leprosy families. We found three new susceptible loci of leprosy, one in GAL3ST4 and two in CHGB. We went on to validate the findings of WES using 151 leprosy cases and 226 healthy controls by Sanger sequencing. Stratified by gender, GAL3ST4 was found to be the susceptible gene only for the female population, and CHGB48 and CHGB23 were susceptibile to leprosy for the male population, respectively). Moreover, the gene expression levels of the three susceptible loci were measured by real-time PCR after the stimulation by M. leprae antigens in the PBMC (peripheral blood mononuclear cells) of 69 healthy people. The results showed that the female subjects with high frequent genotype in GAL3ST4 had a fivefold elevated expression. We suggest the polymorphisms in GAL3ST4 in different population are associated with increased risk of leprosy.

Characterization of Mycobacterium leprae Genotypes in China--Identification of a New Polymorphism C251T in the 16S rRNA Gene.

Leprosy continues to be prevalent in some mountainous regions of China, and genotypes of leprosy strains endemic to the country are not known. Mycobacterium lepromatosis is a new species that was discovered in Mexico in 2008, and it remains unclear whether this species exists in China. Here, we conducted PCR- restriction fragment length polymorphism (RFLP) analysis to classify genotypes of 85 DNA samples collected from patients from 18 different provinces. All 171 DNA samples from skin biopsies of leprosy patients were tested for the presence of Mycobacterium leprae and Mycobacterium lepromatosis by amplifying the 16S rRNA gene using nested PCR, followed by DNA sequencing. The new species M. lepromatosis was not found among the 171 specimens from leprosy patients in 22 provinces in China. However, we found three SNP genotypes among 85 leprosy patients. A mutation at C251T in the 16S rRNA gene was found in 76% of the strains. We also found that the strains that showed the 16S rRNA C251T mutation belonged to SNP type 3, whereas strains without the point mutation belonged to SNP type 1. The SNP type 3 leprosy strains were observed in patients from both the inner and coastal regions of China, but the SNP type 1 strains were focused only in the coastal region. This indicated that the SNP type 3 leprosy strains were more prevalent than the SNP type 1 strains in China. In addition, the 16S rRNA gene sequence mutation at C251T also indicated a difference in the geographical distribution of the strains. To our knowledge, this is the first report of a new polymorphism in 16S rRNA gene in M. leprae in China. Our findings shed light on the prevalent genotypes and provide insight about leprosy transmission that are important for leprosy control in China.