Methotrexate and prednisolone study in erythema nodosum leprosum (MaPs in ENL) protocol: a double-blind randomised clinical trial.

INTRODUCTION: Erythema nodosum leprosum (ENL) is an immunological complication of leprosy. ENL results in morbidity and disability and if it is not treated can lead to death. The current treatment consists of thalidomide or high doses of oral corticosteroids for prolonged periods. Thalidomide is not available in many leprosy endemic countries. The use of corticosteroids is associated with morbidity and mortality. Identifying treatment regimens that reduce the use of corticosteroids in ENL is essential. Methotrexate (MTX) is used to treat many inflammatory diseases and has been used successfully to treat patients with ENL not controlled by other drugs, including prednisolone and thalidomide. We present the protocol of the 'MTX and prednisolone study in ENL' (MaPs in ENL) a randomised controlled trial (RCT) designed to test the efficacy of MTX in the management of ENL. METHODS AND ANALYSIS: MaPs in ENL is an international multicentre RCT, which will be conducted in leprosy referral centres in Bangladesh, Brazil, Ethiopia, India, Indonesia and Nepal. Patients diagnosed with ENL who consent to participate will be randomly allocated to receive 48 weeks of weekly oral MTX plus 20 weeks of prednisolone or 48 weeks of placebo plus 20 weeks of prednisolone. Participants will be stratified by type of ENL into those with acute ENL and those with chronic and recurrent ENL. The primary objective is to determine whether MTX reduces the requirement for additional prednisolone. Patients' reported outcome measures will be used to assess the efficacy of MTX. Participants will be closely monitored for adverse events. ETHICS AND DISSEMINATION: Results will be submitted for publication in peer-reviewed journals. Ethical approval was obtained from the Observational/Interventions Research Ethics Committee of the London School of Hygiene & Tropical Medicine (15762); The Leprosy Mission International Bangladesh Institutional Research Board (in process); AHRI-ALERT Ethical Review Committee, Ethiopia; Ethics Committee of the Managing Committee of the Bombay Leprosy Project; and The Leprosy Mission Trust India Ethics Committee; the Nepal Health and Research Council and Health Research Ethics Committee Dr. Soetomo, Indonesia. This study is registered at www.clinicaltrials.gov. This is the first RCT of MTX for ENL and will contribute to the evidence for the management of ENL.Trial registration numberNCT 03775460.

Diagnosis and impact of neuropathic pain in leprosy patients in Nepal after completion of multidrug therapy.

OBJECTIVES: Neuropathic pain (NP) can occur as a chronic complication of leprosy neuropathy. NP epidemiology and its impact on patients have not been well documented. This study investigates NP prevalence and impact in the years after patients are declared "released from treatment" (RFT) following multidrug therapy (MDT) completion. METHODS: In this cross-sectional study, 85 RFT patients were recruited within leprosy referral services in Nepal. The Douleur Neuropathique 4 Questionnaire (DN4) was used to screen for NP. Pain severity, impacts on patients' daily activities and mental health were measured by using the Brief Pain Inventory (BPI), Screening of Activity Limitation and Safety Awareness (SALSA), and General Health Questionnaire-12 (GHQ-12) respectively. RESULTS: 96% surveyed had been treated for multibacillary leprosy. 44 (52%) complained of pain of which 30 (68%) were diagnosed with NP. NP was not associated with age, gender, or presence of skin lesions or nerve symptoms at leprosy diagnosis. 70% of patients with NP had either history of or ongoing reactions and 47% had grade 2 disability. Nerve tenderness (p = 0.023) and current reactions (p = 0.018) were significant risk factors for NP. Patients with NP suffered significantly higher intensity pain (p = 0.023) and daily life interference (p = 0.003) and were more likely to have moderate to extreme daily activity limitations (p = 0.005). 13 (43%) exhibited psychological distress, and medications only reduced moderate degree (50-60%) of pain. CONCLUSIONS: In our study, 35% of RFT patients had ongoing NP. Risk factors include nerve tenderness and reaction. They suffer from more daily life interference and psychological distress. Leprosy patient care should include recognition and management of NP.

Effectiveness of 32 versus 20 weeks of prednisolone in leprosy patients with recent nerve function impairment: A randomized controlled trial.

BACKGROUND: While prednisolone is commonly used to treat recent nerve function impairment (NFI) in leprosy patients, the optimal treatment duration has not yet been established. In this "Treatment of Early Neuropathy in Leprosy" (TENLEP) trial, we evaluated whether a 32-week prednisolone course is more effective than a 20-week course in restoring and improving nerve function. METHODS: In this multi-centre, triple-blind, randomized controlled trial, leprosy patients who had recently developed clinical NFI (<6 months) were allocated to a prednisolone treatment regimen of either 20 weeks or 32 weeks. Prednisolone was started at either 45 or 60 mg/day, depending on the patient's body weight, and was then tapered. Throughout follow up, NFI was assessed by voluntary muscle testing and monofilament testing. The primary outcome was the proportion of patients with improved or restored nerve function at week 78. As secondary outcomes, we analysed improvements between baseline and week 78 on the Reaction Severity Scale, the SALSA Scale and the Participation Scale. Serious Adverse Events and the need for additional prednisolone treatment were monitored and reported. RESULTS: We included 868 patients in the study, 429 in the 20-week arm and 439 in the 32-week arm. At 78 weeks, the proportion of patients with improved or restored nerve function did not differ significantly between the groups: 78.1% in the 20-week arm and 77.5% in the 32-week arm (p = 0.821). Nor were there any differences in secondary outcomes, except for a significant higher proportion of Serious Adverse Events in the longer treatment arm. CONCLUSION: In our study, a 20-week course of prednisolone was as effective as a 32-week course in improving and restoring recent clinical NFI in leprosy patients. Twenty weeks is therefore the preferred initial treatment duration for leprosy neuropathy, after which likely only a minority of patients require further individualized treatment.

Opening a Can of Worms: Leprosy Reactions and Complicit Soil-Transmitted Helminths.

BACKGROUND: >94% of new annual leprosy cases are diagnosed in populations co-endemic for soil-transmitted helminths (STH). STH can profoundly dysregulate host immune responses towards Th2 bias, which can be restored over time after deworming. We hypothesize that STH co-infection is associated with leprosy reaction (denoted as simply "reaction" herein) occurrence within a co-endemic population. METHODS: A cohort study was performed on a cohort of Nepalese leprosy patients across treatment and diagnostic classifications who were screened by routine fecal smear microscopy and multiplex quantitative PCR (qPCR) for Ascaris lumbricoides (Al), Strongyloides stercoralis (Ss), Ancyclostoma duodenale (Ad) and Necator americanus (Na). RESULTS: Among 145 patients, 55% were positive for ≥1 STH (STH+): 34% Al+, 18% Ss+, 17% Ad+and 5% Na+. Significant inverse STH and reaction relationships were evidenced by the bulk of cases: 63% reaction-negative were STH+ of total cases (p=0.030) while 65% reaction-positive were STH- in new cases (96; p=0.023). Strikingly, the majority of STH+ were reaction-negative, even when considering each species: 59% Al+, 60% Ss+, 62% Ad+and 67% Na+of new leprosy cases. CONCLUSIONS: Absence of STH co-infection is associated with leprosy reaction at diagnosis within a co-endemic population. This is likely due to immune reconstitution effects after deworming or interruption of chronic STH-mediated immune dysregulation.

A leprosy clinical severity scale for erythema nodosum leprosum: An international, multicentre validation study of the ENLIST ENL Severity Scale.

OBJECTIVES: We wished to validate our recently devised 16-item ENLIST ENL Severity Scale, a clinical tool for measuring the severity of the serious leprosy associated complication of erythema nodosum leprosum (ENL). We also wished to assess the responsiveness of the ENLIST ENL Severity Scale in detecting clinical change in patients with ENL. METHODS: Participants, recruited from seven centres in six leprosy endemic countries, were assessed using the ENLIST ENL Severity Scale by two researchers, one of whom categorised the severity of ENL. At a subsequent visit a further assessment using the scale was made and both participant and physician rated the change in ENL using the subjective categories of "Much better", "somewhat better", "somewhat worse" and "much worse" compared with "No change" or "about the same". RESULTS: 447 participants were assessed with the ENLIST ENL Severity Scale. The Cronbach alpha of the scale and each item was calculated to determine the internal consistency of the scale. The ENLIST ENL Severity Scale had good internal consistency and this improved following removal of six items to give a Cronbach's alpha of 0.77. The cut off between mild ENL and more severe disease was 9 determined using ROC curves. The minimal important difference of the scale was determined to be 5 using both participant and physician ratings of change. CONCLUSIONS: The 10-item ENLIST ENL Severity Scale is the first valid, reliable and responsive measure of ENL severity and improves our ability to assess and compare patients and their treatments in this severe and difficult to manage complication of leprosy. The ENLIST ENL Severity Scale will assist physicians in the monitoring and treatment of patients with ENL. The ENLIST ENL Severity Scale is easy to apply and will be useful as an outcome measure in treatment studies and enable the standardisation of other clinical and laboratory ENL research.

ENLIST 1: An International Multi-centre Cross-sectional Study of the Clinical Features of Erythema Nodosum Leprosum.

Erythema nodosum leprosum (ENL) is a severe multisystem immune mediated complication of borderline lepromatous leprosy and lepromatous leprosy. ENL is associated with skin lesions, neuritis, arthritis, dactylitis, eye inflammation, osteitis, orchitis, lymphadenitis and nephritis. The treatment of ENL requires immunosuppression, which is often required for prolonged periods of time and may lead to serious adverse effects. ENL and its treatment is associated with increased mortality and economic hardship. Improved, evidence-based treatments for ENL are needed; however, defining the severity of ENL and outcome measures for treatment studies is difficult because of the multiple organ systems involved. A cross-sectional study was performed, by the members of the Erythema Nodosum Leprosum International STudy (ENLIST) Group, of patients with ENL attending seven leprosy referral centres in Brazil, Ethiopia, India, Nepal, the Philippines and the United Kingdom. We systematically documented the clinical features and type of ENL, its severity and the drugs used to treat it. Patients with chronic ENL were more likely to be assessed as having severe ENL. Pain, the most frequent symptom, assessed using a semi-quantitative scale was significantly worse in individuals with "severe" ENL. Our findings will determine the items to be included in a severity scale of ENL which we are developing and validating. The study also provides data on the clinical features of ENL, which can be incorporated into a definition of ENL and used for outcome measures in treatment studies.

Differential dermal expression of CCL17 and CCL18 in tuberculoid and lepromatous leprosy.

BACKGROUND: Leprosy is characterized by polar clinical, histologic and immunological presentations. Previous immunologic studies of leprosy polarity were limited by the repertoire of cytokines known at the time. METHODOLOGY: We used a candidate gene approach to measure mRNA levels in skin biopsies from leprosy lesions. mRNA from 24 chemokines and cytokines, and 6 immune cell type markers were measured from 85 Nepalese leprosy subjects. Selected findings were confirmed with immunohistochemistry. PRINCIPAL RESULTS: Expression of three soluble mediators (CCL18, CCL17 and IL-10) and one macrophage cell type marker (CD14) was significantly elevated in lepromatous (CCL18, IL-10 and CD14) or tuberculoid (CCL17) lesions. Higher CCL18 protein expression by immunohistochemistry and a trend in increased serum CCL18 in lepromatous lesions was observed. No cytokines were associated with erythema nodosum leprosum or Type I reversal reaction following multiple comparison correction. Hierarchical clustering suggested that CCL18 was correlated with cell markers CD209 and CD14, while neither CCL17 nor CCL18 were highly correlated with classical TH1 and TH2 cytokines. CONCLUSIONS: Our findings suggest that CCL17 and CCL18 dermal expression is associated with leprosy polarity.

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

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. TRIAL REGISTRATION: ClinicalTrials.gov NCT01920750 (Phase I), NCT00128193 (Phase II).

T-cell regulation in lepromatous leprosy.

Regulatory T (Treg) cells are known for their role in maintaining self-tolerance and balancing immune reactions in autoimmune diseases and chronic infections. However, regulatory mechanisms can also lead to prolonged survival of pathogens in chronic infections like leprosy and tuberculosis (TB). Despite high humoral responses against Mycobacterium leprae (M. leprae), lepromatous leprosy (LL) patients have the characteristic inability to generate T helper 1 (Th1) responses against the bacterium. In this study, we investigated the unresponsiveness to M. leprae in peripheral blood mononuclear cells (PBMC) of LL patients by analysis of IFN-γ responses to M. leprae before and after depletion of CD25+ cells, by cell subsets analysis of PBMC and by immunohistochemistry of patients' skin lesions. Depletion of CD25+ cells from total PBMC identified two groups of LL patients: 7/18 (38.8%) gained in vitro responsiveness towards M. leprae after depletion of CD25+ cells, which was reversed to M. leprae-specific T-cell unresponsiveness by addition of autologous CD25+ cells. In contrast, 11/18 (61.1%) remained anergic in the absence of CD25+ T-cells. For both groups mitogen-induced IFN-γ was, however, not affected by depletion of CD25+ cells. In M. leprae responding healthy controls, treated lepromatous leprosy (LL) and borderline tuberculoid leprosy (BT) patients, depletion of CD25+ cells only slightly increased the IFN-γ response. Furthermore, cell subset analysis showed significantly higher (p = 0.02) numbers of FoxP3+ CD8+CD25+ T-cells in LL compared to BT patients, whereas confocal microscopy of skin biopsies revealed increased numbers of CD68+CD163+ as well as FoxP3+ cells in lesions of LL compared to tuberculoid and borderline tuberculoid leprosy (TT/BT) lesions. Thus, these data show that CD25+ Treg cells play a role in M. leprae-Th1 unresponsiveness in LL.

Comparison of two rapid tests for anti-phenolic glycolipid-I serology in Brazil and Nepal

The diagnosis of leprosy continues to be based on clinical symptoms and early diagnosis and treatment are critical to preventing disability and transmission. Sensitive and specific laboratory tests are not available for diagnosing leprosy. Despite the limited applicability of anti-phenolic glycolipid-I (PGL-I) serology for diagnosis, it has been suggested as an additional tool to classify leprosy patients (LPs) for treatment purposes. Two formats of rapid tests to detect anti-PGL-I antibodies [ML immunochromatography assay (ICA) and ML Flow] were compared in different groups, multibacillary patients, paucibacillary patients, household contacts and healthy controls in Brazil and Nepal. High ML Flow intra-test concordance was observed and low to moderate agreement between the results of ML ICA and ML Flow tests on the serum of LPs was observed. LPs were "seroclassified" according to the results of these tests and the seroclassification was compared to other currently used classification systems: the World Health Organization operational classification, the bacilloscopic index and the Ridley-Jopling classification. When analysing the usefulness of these tests in the operational classification of PB and MB leprosy for treatment and follow-up purposes, the ML Flow test was the best point-of-care test for subjects in Nepal and despite the need for sample dilution, the ML ICA test yielded better performance among Brazilian subjects. Our results identified possible ways to improve the performance of both tests.

Human beta-defensin 3 is up-regulated in cutaneous leprosy type 1 reactions.

BACKGROUND: Leprosy, a chronic granulomatous disease affecting the skin and nerves, is caused by Mycobacterium leprae (M. leprae). The type of leprosy developed depends upon the host immune response. Type 1 reactions (T1Rs), that complicate borderline and lepromatous leprosy, are due to an increase in cell-mediated immunity and manifest as nerve damage and skin inflammation. Owing to the increase in inflammation in the skin of patients with T1Rs, we sought to investigate the activation of the innate immune system during reactionary events. Specifically, we investigated the expression levels of human beta-defensins (hBDs) 2 and 3 in the skin of patients with T1Rs, in keratinocytes, and in macrophages stimulated with M. leprae and corticosteroids. RESULTS: Skin biopsies from twenty-three patients with Type 1 reactions were found to have higher transcript levels of hBD3 as compared to fifteen leprosy patients without Type 1 reactions, as measured by qPCR. Moreover, we observed that keratinocytes but not macrophages up-regulated hBD2 and hBD3 in response to M. leprae stimulation in vitro. Corticosteroid treatment of patients with T1Rs caused a suppression of hBD2 and hBD3 in skin biopsies, as measured by qPCR. In vitro, corticosteroids suppressed M. leprae-dependent induction of hBD2 and hBD3 in keratinocytes. CONCLUSIONS: This study demonstrates that hBD3 is induced in leprosy Type 1 Reactions and suppressed by corticosteroids. Furthermore, our findings demonstrate that keratinocytes are responsive to M. leprae and lend support for additional studies on keratinocyte innate immunity in leprosy and T1Rs. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN31894035.

Comparison of two rapid tests for anti-phenolic glycolipid-I serology in Brazil and Nepal.

The diagnosis of leprosy continues to be based on clinical symptoms and early diagnosis and treatment are critical to preventing disability and transmission. Sensitive and specific laboratory tests are not available for diagnosing leprosy. Despite the limited applicability of anti-phenolic glycolipid-I (PGL-I) serology for diagnosis, it has been suggested as an additional tool to classify leprosy patients (LPs) for treatment purposes. Two formats of rapid tests to detect anti-PGL-I antibodies [ML immunochromatography assay (ICA) and ML Flow] were compared in different groups, multibacillary patients, paucibacillary patients, household contacts and healthy controls in Brazil and Nepal. High ML Flow intra-test concordance was observed and low to moderate agreement between the results of ML ICA and ML Flow tests on the serum of LPs was observed. LPs were "seroclassified" according to the results of these tests and the seroclassification was compared to other currently used classification systems: the World Health Organization operational classification, the bacilloscopic index and the Ridley-Jopling classification. When analysing the usefulness of these tests in the operational classification of PB and MB leprosy for treatment and follow-up purposes, the ML Flow test was the best point-of-care test for subjects in Nepal and despite the need for sample dilution, the ML ICA test yielded better performance among Brazilian subjects. Our results identified possible ways to improve the performance of both tests.

A phase two randomised controlled double blind trial of high dose intravenous methylprednisolone and oral prednisolone versus intravenous normal saline and oral prednisolone in individuals with leprosy type 1 reactions and/or nerve function impairment.

BACKGROUND: Leprosy Type 1 reactions are a major cause of nerve damage and the preventable disability that results. Type 1 reactions are treated with oral corticosteroids and there are few data to support the optimal dose and duration of treatment. Type 1 reactions have a Th1 immune profile: cells in cutaneous and neural lesions expressing interferon-γ and interleukin-12. Methylprednisolone has been used in other Th1 mediated diseases such as rheumatoid arthritis in an attempt to switch off the immune response and so we investigated the efficacy of three days of high dose (1 g) intravenous methylprednisolone at the start of prednisolone therapy in leprosy Type 1 reactions and nerve function impairment. RESULTS: Forty-two individuals were randomised to receive methylprednisolone followed by oral prednisolone (n = 20) or oral prednisolone alone (n = 22). There were no significant differences in the rate of adverse events or clinical improvement at the completion of the study. However individuals treated with methylprednisolone were less likely than those treated with prednisolone alone to experience deterioration in sensory function between day 29 and day 113 of the study. The study also demonstrated that 50% of individuals with Type 1 reactions and/or nerve function impairment required additional prednisolone despite treatment with 16 weeks of corticosteroids. CONCLUSIONS: The study lends further support to the use of more prolonged courses of corticosteroid to treat Type 1 reactions and the investigation of risk factors for the recurrence of Type 1 reaction and nerve function impairment during and after a corticosteroid treatment. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN31894035.

Single lesion multibacillary leprosy, a treatment enigma: a case report.

INTRODUCTION: Leprosy exhibits a wide spectrum of presentation, varying from the tuberculoid to the lepromatous pole, with immunologically unstable borderline forms in-between, depending upon the immune status of the individual. The clinical system of classification for the purpose of treatment includes the number of skin lesions and nerves involved as the basis for classifying the patients into multibacillary and paucibacillary. CASE PRESENTATION: A 20-year-old man belonging to a moderately endemic leprosy area in the Terai region of Nepal reported a large single, hypopigmented, well defined anaesthetic lesion on his left thigh extending to his knee which had been present for 2 years. There was no other nerve involvement. Clinical diagnosis was tuberculoid leprosy and immunological lateral flow test for anti-Phenolic glycolipid-I antibody was positive. Six months of paucibacillary multidrug treatment was advised immediately. However, the patient was reclassified as multibacillary on the basis of a positive skin smear and appropriate treatment of 24 months multibacillary multidrug regimen was commenced after only 1 week. Slit skin smear examination for Mycobacterium leprae from the lesion revealed a bacterial index of 4+ while it was negative from the routine sites. Histopathological examination from skin biopsy of the lesion further supported the bacterial index of the lesion granuloma which was 2+ and the patient was diagnosed as borderline tuberculoid. Bacteriological, histological, and immunological findings of this patient were borderline tuberculoid leprosy and he should have been treated with multibacillary regimen from the beginning. Five months after commencement of treatment, the patient developed a leprae reaction of Type 1 or reversal reaction with some nerve function impairment and enlargement of the lateral popliteal nerve of the left leg. This reversal reaction was managed by standard oral prednisolone whilst continuing the multibacillary multidrug regimen. CONCLUSION: This case illustrates and emphasizes the importance of slit-skin smear and biopsy as routine in all new cases to help differentiate multibacillary from paucibacillary for correct treatment. It further suggests that there are factors yet undetermined which play a significant role in determining the host response to M. leprae which is a remaining challenge in this disease.

Selection of antigens and development of prototype tests for point-of-care leprosy diagnosis.

Leprosy can be a devastating chronic infection that causes nerve function impairment and associated disfigurement. Despite the recent reduction in the number of registered worldwide leprosy cases as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains relatively stable. The diagnosis of leprosy is currently based on the appearance of clinical signs and requires expert clinical, as well as labor-intensive and time-consuming laboratory or histological, evaluation. For the purpose of developing an effective, simple, rapid, and low-cost diagnostic alternative, we have analyzed the serologic antibody response to identify Mycobacterium leprae proteins that are recognized by leprosy patients. More than 100 recombinant antigens were analyzed in a protein array format to select those with discriminatory properties for leprosy diagnosis. As expected, multibacillary leprosy patients recognized more antigens with stronger antibody responses than paucibacillary leprosy patients. Our data indicate, however, that multibacillary patients can be distinguished from paucibacillary patients, and both of these groups can be segregated from endemic control groups. We went on to confirm the diagnostic properties of antigens ML0405 and ML2331 and the LID-1 fusion construct of these two proteins by enzyme-linked immunosorbent assay. We then demonstrated the performance of these antigens in rapid test formats with a goal of developing a point-of-care diagnostic test. A serological diagnostic test capable of identifying and allowing treatment of leprosy could reduce transmission, prevent functional disabilities and stigmatizing deformities, and facilitate leprosy eradication.

Development and evaluation of a novel multiple-primer PCR amplification refractory mutation system for the rapid detection of mutations conferring rifampicin resistance in codon 425 of the rpoB gene of Mycobacterium leprae.

Rifampicin-resistant Mycobacterium leprae is regularly reported and drug resistance is a major threat for the elimination of leprosy. There is an urgent need for a simple method that can detect rifampicin resistance in clinical isolates. This study developed a multiple-primer PCR amplification refractory mutation system, a simple, reliable and economical method for clinical specimens that allowed the rapid detection of mutations in the nucleotides of the codon for Ser425 of the M. leprae rpoB gene, mutation of which to Leu, Met or Phe is associated with rifampicin resistance. The approach involved a multiple-primer PCR in which both mutant-specific and normal sets of primers were included in the reaction. The mutant-specific primer was complementary to the corresponding sequence of the wild-type gene except for one additional deliberate mismatch at the fourth nucleotide from the 3'-OH terminus. A single mismatch has little influence on the yield of PCR products, but if there are two mismatches as a result of mutation at the position being tested, the mutant-specific primer will not function in PCR under appropriate conditions, leading to no yield of PCR product from the mutant allele. The assay was evaluated successfully using a panel of plasmids and M. leprae reference strains carrying the wild-type or known rpoB mutations. The assay was subsequently applied to M. leprae DNA extracts from skin biopsies taken from patients. In all biopsy samples, the wild-type allele was detected for Ser425. The PCR results correlated with rifampicin susceptibility, as also measured by the traditional in vivo mouse footpad technique.

Vaccination with DNA of the Mycobacterium tuberculosis 85B antigen protects mouse foot pad against infection with M. leprae

A DNA vaccine composed of the gene for the common mycobacterial secreted protein antigen 85B was demonstrated to protect the mouse foot pad against infection with Mycobacterium leprae. The protective effect was demonstrated by a 61%-88% reduction in the bacterial number, a protective effect less than that of BCG. The same DNA vaccine has been shown to protect mice against M. tuberculosis infection, and the importance of testing other candidate tuberculosis vaccines for their potential to protect against leprosy is discussed.