A retrospective study of the effect of modified multi-drug therapy in Nepali leprosy patients following the development of adverse effects due to dapsone.

INTRODUCTION: Dapsone Hypersensitivity Syndrome (DHS) occurs in approximately 2% of leprosy patients in Nepal. DHS and other adverse effects of dapsone lead to withdrawal of the drug. METHODS: We reviewed the notes of patients who had dapsone withdrawn from their multi-drug therapy (MDT) following an adverse reaction to the drug between 1990 and 2007. RESULTS: 105 patients were identified from the database and 67 had a documented completion of a modified course of MDT. The majority were treated with rifampicin and clofazimine. All 36 individuals who were slit-skin smear positive had a satisfactory fall in their mean bacterial index. There were no cases of relapse. CONCLUSIONS: Rifampicin and clofazimine appear to be satisfactory treatment for both paucibacillary and multibacillary patients who have to have dapsone stopped because of severe adverse effects

Comparative genomic and phylogeographic analysis of Mycobacterium leprae

Reductive evolution and massive pseudogene formation have shaped the 3.31-Mb genome of Mycobacterium leprae, an unculturable obligate pathogen that causes leprosy in humans. The complete genome sequence of M. leprae strain Br4923 from Brazil was obtained by conventional methods (6 x coverage), and Illumina resequencing technology was used to obtain the sequences of strains Thai53 (38 x coverage) and NHDP63 (46 x coverage) from Thailand and the United States, respectively. Whole-genome comparisons with the previously sequenced TN strain from India revealed that the four strains share 99.995% sequence identity and differ only in 215 polymorphic sites, mainly SNPs, and by 5 pseudogenes. Sixteen interrelated SNP subtypes were defined by genotyping both extant and extinct strains of M. leprae from around the world. The 16 SNP subtypes showed a strong geographical association that reflects the migration patterns of early humans and trade routes, with the Silk Road linking Europe to China having contributed to the spread of leprosy

Safety and efficacy assessment of two new leprosy skin test antigens: randomized double blind clinical study.

New tools are required for the diagnosis of pre-symptomatic leprosy towards further reduction of disease burden and its associated reactions. To address this need, two new skin test antigens were developed to assess safety and efficacy in human trials.A Phase I safety trial was first conducted in a non-endemic region for leprosy (U.S.A.). Healthy non-exposed subjects (n = 10) received three titrated doses (2.5 µg, 1.0 µg and 0.1 µg) of MLSA-LAM (n = 5) or MLCwA (n = 5) and control antigens [Rees MLSA (1.0 µg) and saline]. A randomized double blind Phase II safety and efficacy trial followed in an endemic region for leprosy (Nepal), but involved only the 1.0 µg (high dose) and 0.1 µg (low dose) of each antigen; Tuberculin PPD served as a control antigen. This Phase II safety and efficacy trial consisted of three Stages: Stage A and B studies were an expansion of Phase I involving 10 and 90 subjects respectively, and Stage C was then conducted in two parts (high dose and low dose), each enrolling 80 participants: 20 borderline lepromatous/lepromatous (BL/LL) leprosy patients, 20 borderline tuberculoid/tuberculoid (BT/TT) leprosy patients, 20 household contacts of leprosy patients (HC), and 20 tuberculosis (TB) patients. The primary outcome measure for the skin test was delayed type hypersensitivity induration.In the small Phase I safety trial, reactions were primarily against the 2.5 µg dose of both antigens and Rees control antigen, which were then excluded from subsequent studies. In the Phase II, Stage A/B ramped-up safety study, 26% of subjects (13 of 50) showed induration against the high dose of each antigen, and 4% (2 of 50) reacted to the low dose of MLSA-LAM. Phase II, Stage C safety and initial efficacy trial showed that both antigens at the low dose exhibited low sensitivity at 20% and 25% in BT/TT leprosy patients, but high specificity at 100% and 95% compared to TB patients. The high dose of both antigens showed lower specificity (70% and 60%) and sensitivity (10% and 15%). BL/LL leprosy patients were anergic to the leprosy antigens.MLSA-LAM and MLCwA at both high (1.0 µg) and low (0.1 µg) doses were found to be safe for use in humans without known exposure to leprosy and in target populations. At a sensitivity rate of 20-25% these antigens are not suitable as a skin test for the detection of the early stages of leprosy infection; however, the degree of specificity is impressive given the presence of cross-reactive antigens in these complex native M. leprae preparations.ClinicalTrials.gov NCT01920750 (Phase I), NCT00128193 (Phase II)

From Genome-Based In Silico Predictions to Ex Vivo Verification of Leprosy Diagnosis▿ †

The detection of hundreds of thousands of new cases of leprosy every year suggests that transmission of Mycobacterium leprae infection still continues. Unfortunately, tools for identification of asymptomatic disease and/or early-stage M. leprae infection (likely sources of transmission) are lacking. The recent identification of M. leprae-unique genes has allowed the analysis of human T-cell responses to novel M. leprae antigens. Antigens with the most-promising diagnostic potential were tested for their ability to induce cytokine secretion by using peripheral blood mononuclear cells from leprosy patients and controls in five different areas where leprosy is endemic; 246 individuals from Brazil, Nepal, Bangladesh, Pakistan, and Ethiopia were analyzed for gamma interferon responses to five recombinant proteins (ML1989, ML1990, ML2283, ML2346, and ML2567) and 22 synthetic peptides. Of these, the M. leprae-unique protein ML1989 was the most frequently recognized and ML2283 the most specific for M. leprae infection/exposure, as only a limited number of tuberculosis patients responded to this antigen. However, all proteins were recognized by a significant number of controls in areas of endemicity. T-cell responses correlated with in vitro response to M. leprae, suggesting that healthy controls in areas where leprosy is endemic are exposed to M. leprae. Importantly, 50% of the healthy household contacts and 59% of the controls in areas of endemicity had no detectable immunoglobulin M antibodies to M. leprae-specific PGL-I but responded in T-cell assays to ≥1 M. leprae protein. T-cell responses specific for leprosy patients and healthy household contacts were observed for ML2283- and ML0126-derived peptides, indicating that M. leprae peptides hold potential as diagnostic tools. Future work should concentrate on the development of a sensitive and field-friendly assay and identification of additional peptides and proteins that can induce M. leprae-specific T-cell responses

Number of positive responders and mean induration compared to ECs.

a<p>To allow calculations, the EC percent positive has been changed to 1.0.</p

Diagnostic test statistics – WB-IGRA^a.

a<p>Preliminary unpublished results.</p

Dot plot of induration measurements.

<p>Induration results are provided across five subject groups, including BL/LL leprosy patients (<i>n</i> = 19 in low and <i>n</i> = 20 in high dose group), BT/TT leprosy patients (<i>n</i> = 20), HC (<i>n</i> = 20), TB (<i>n</i> = 20), and ECs (<i>n</i> = 50). Low and high dose groups were combined to show PPD responses: BL/LL leprosy patients (<i>n</i> = 39) and all other groups (<i>n</i> = 40). Mean and standard deviation are shown.</p

Diagnostic test statistics – Skin test.

<p>Diagnostic test statistics were calculated for each test method. Sensitivity (Se) is the likelihood to detect the presence of disease [Total Positive (TP)/TP + False Negative (FN)]. Specificity (Sp) is the likelihood to detect absence of disease [(Total Negative (TN)/TN + False Positive (FP)]. PPD served as an antigen control. Statistics for detecting tuberculosis: Sensitivity is (36/40) 90%, specificity is (41/100) 41%.</p

Phase II, Stage A/B – DTH induration by subject.

<p>Phase II, Stage A/B graph depicting DTH indurations elicited by leprosy skin test antigens at the high dose (1.0 µg) and low dose (0.1 µg), and PPD at 5 TU: A) MLCwA, and B) MLSA-LAM. The first five subjects on both graphs represent subjects from Stage A, and the remaining 45 subjects on both graphs represent subjects from Stage B.</p