Robert H. Gelber

Use of Protein Antigens for Early Serological Diagnosis of Leprosy

ABSTRACT Leprosy is a chronic and debilitating human disease caused by infection with the Mycobacterium leprae bacillus. Despite the marked 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. This indicates that M. leprae is still being transmitted and that, without earlier diagnosis, M. leprae infection will continue to pose a health problem. Current diagnostic techniques, based on the appearance of clinical symptoms or of immunoglobulin M (IgM) antibodies that recognize the bacterial phenolic glycolipid I, are unable to reliably identify early-stage leprosy. In this study we examine the ability of IgG within leprosy patient sera to bind several M. leprae protein antigens. As expected, multibacillary leprosy patients provided stronger responses than paucibacillary leprosy patients. We demonstrate that the geographic locations of the patients can influence the antigens they recognize but that ML0405 and ML2331 are recognized by sera from diverse regions (the Philippines, coastal and central Brazil, and Japan). A fusion construct of these two proteins (designated leprosy IDRI diagnostic 1 [LID-1]) retained the diagnostic activity of the component antigens. Upon testing against a panel of prospective sera from individuals who developed leprosy, we determined that LID-1 was capable of diagnosing leprosy 6 to 8 months before the onset of clinical symptoms. A serological diagnostic test capable of identifying and allowing treatment of early-stage leprosy could reduce transmission, prevent functional disabilities and stigmatizing deformities, and facilitate leprosy eradication.

Methods for the Classification of Leprosy for Treatment Purposes

The World Health Organization advocates 2 leprosy treatment regimens on the basis of disease classification (as multibacillary or paucibacillary) by skin lesion count. This method, which, in the Philippines, results in a high prevalence (78%) of patients with multibacillary leprosy, was directly compared with classification using standard histopathological and microbiological criteria in 264 currently untreated patients with leprosy. Of those whose leprosy was classified as paucibacillary, 38%-51% of patients had multibacillary leprosy according to classic criteria and were thus at risk of undertreatment according to World Health Organization recommendations.

The relapse rate in MB leprosy patients treated with 2-years of WHO-MDT is not low.

A group of multibacillary patients is clearly at high risk for relapse following 2-yr WHO-MDT. Relapse is largely confined to BL or LL patients with a high BI initially, and occurs long after the discontinuation of therapy. This important group of patients at risk for treatment failure presents several important issues: the need to identify those at risk and the operational requirements needed for their long term follow-up. Also, this group of patients might well benefit from an alternative antimicrobial regimen from the outset, as well as upon relapse.

Long-term efficacy of 2 year WHO multiple drug therapy (MDT) in multibacillary (MB) leprosy patients.

Relapse rate estimates after 2 year WHO multiple drug therapy (MDT) in multi-bacillary (MB) leprosy vary. Between 1987 and 1994, 500 MB leprosy patients completing 2 year MDT were enrolled in a prospective relapse study. The majority of patients (N = 316) were treated and followed at the physician-staffed Cebu Skin Clinic (CSC), whereas others (N = 184) received therapy from government clinics and were followed by CSC technicians in the field. Relapse definition was an increased bacteriologic index (BI) and new skin lesions, supplemented with mouse footpad inoculations. Through 2002, follow-up was 5368 person-years, with a mean of 10.8 years per patient. The absolute relapse rate was 3% (15/498; 0.28/100 person-years), with a cumulative risk estimate of 3.9% at 15 yrs. For a subset of 217 patients followed for >or=12 yrs or until relapse, relapses occurred in 9% (13/142) attending the CSC, versus 3% (2/75) assessed in the field (p = 0.09). The rate for patients followed at CSC for >or=12 yrs and a pre-treatment BI >or=2.7+ was 13% (13/98). All relapses were BL or LL, with pre-treatment BIs of >or=2.7+. Relapses occurred long after completion of therapy, between 3 and 11 yrs from the midpoint of the examination without relapse to detection, or between 6 to 13 yrs to the actual year of detection, 7 occurring at >or=10 yrs. Lesion material from all relapses contained M. leprae that was rifampin and clofazimine sensitive, whereas 3 showed partial or full dapsone resistance. [Follow-up rigor and time], medical expertise, and pre-treatment bacterial load influence relapse rates after 2 yr MDT.

ML0405 and ML2331 Are Antigens of Mycobacterium leprae with Potential for Diagnosis of Leprosy

ABSTRACT Despite the success of multidrug therapy in reducing the number of registered leprosy cases worldwide, evidence suggests that Mycobacterium leprae continues to be transmitted. A serological diagnostic test capable of identifying and allowing treatment of early-stage disease could reduce transmission and prevent the onset of the disability, a common complication of the disease in later stages. Serological diagnosis based on antibody recognition of phenolic glycolipid I (PGL-I) cannot reliably identify individuals with lower bacterial indices (BI). One strategy that might improve this situation is the provision of highly specific serological antigens that may be combined with PGL-I to improve the sensitivity of diagnosis. Using serological expression cloning with a serum pool of untreated lepromatous leprosy (LL) patients, we identified 14 strongly reactive M. leprae proteins, 5 of which were previously unstudied. We present results suggesting that two of these proteins, ML0405 and ML2331, demonstrate the ability to specifically identify LL/borderline lepromatous (BL) patients on the basis of immunoglobulin G (IgG) reactivity. In a household contact study, LL index cases were identified on the basis of this reactivity, while household contacts of these patients demonstrated undetectable reactivity. At a serum dilution of 1:800, suitable to reduce background PGL-I IgM reactivity, two BL patients with a BI of <4 showed anti-human polyvalent immunoglobulin G, A, and M reactivity measured with a combination of ML0405, ML2331, and natural disaccharide O-linked human serum albumin (NDOHSA) (synthetic PGL-I) that was markedly higher than IgM reactivity to NDOHSA alone. We suggest that ML0405 and ML2331 may have utility in serological leprosy diagnosis.

Analysis of Antibody Responses to Mycobacterium leprae Phenolic Glycolipid-I, Lipoarabinomannan and Recombinant Proteins to Define Disease-Subtype Specific Antigenic Profiles in Leprosy.

ABSTRACT A simple serodiagnostic test based on the Mycobacterium leprae-specific phenolic glycolipid I(PGL-I), for individuals with leprosy is nearly universally positive in leprosy patients with high bacillary loads but cannot be used as a stand-alone diagnostic test for the entire spectrum of the disease process. For patients with early infection with no detectable acid-fast bacilli in lesions or with low or no antibody titer to PGL-I, as in those at the tuberculoid end of the disease spectrum, this diagnostic approach has limited usefulness. To identify additional M. leprae antigens that might enhance the serological detection of these individuals, we have examined the reactivity patterns of patient sera to PGL-I, lipoarabinomannan (LAM), and six recombinant M. leprae proteins (ML1877, ML0841, ML2028, ML2038, ML0380, and ML0050) by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Overall, the responses to ML2028 (Ag85B) and ML2038 (bacterioferritin) were consistently high in both multibacillary and paucibacillary groups and weak or absent in endemic controls, while responses to other antigens showed considerable variability, from strongly positive to completely negative. This analysis has given a clearer understanding of some of the differences in the antibody responses between individuals at opposite ends of the disease spectrum, as well as illustrating the heterogeneity of antibody responses toward protein, carbohydrate, and glycolipid antigens within a clinical group. Correlating these response patterns with a particular disease state could allow for a more critical assessment of the form of disease within the leprosy spectrum and could lead to better patient management.

Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines

ABSTRACT To address the persisting problem of leprosy in Cebu, Philippines, we compiled a database of more than 200 patients who attend an established referral skin clinic. We described the patient characteristics in conventional demographic parameters and also applied multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) and single nucleotide polymorphism (SNP) typing for Mycobacterium leprae in biopsied skin lesion samples. These combined approaches revealed that transmission is ongoing, with the affected including the young Cebuano population under 40 years of age in both crowded cities and rural areas of the island. The emergence of multicase families (MCF) is indicative of infection unconstrained by standard care measures. For the SNPs, we designed a low-cost PCR-restriction fragment length polymorphism typing method. MLVA in M. leprae was highly discriminatory in this population yet could retain broad groups, as defined by the more stable SNPs, implying temporal marker stability suitable for interpreting population structures and evolution. The majority of isolates belong to an Asian lineage (SNP type 1), and the rest belong to a putative postcolonial lineage (SNP type 3). Specific alleles at two VNTR loci, (GGT)5 and 21-3, were highly associated with SNP type 3 in this population. MLVA identified M. leprae genotype associations for patients with known epidemiological links such as in MCFs and in some villages. These methods provide a molecular database and a rational framework for targeted approaches to search and confirm leprosy transmission in various scenarios.

A clinical trial of minocycline in lepromatous leprosy.

population injected drugs in the six months before arrest.45 If we assume from our data that 8% of them were HIV positive that gives an estimated total of 203 HIV positive male drug injectors in the sentenced population. The corresponding figures for women (about 168 recent injectors, and 15% HIV positive) suggest a total of 25 HIV positive women in prison. At any time, therefore, the total number of HIV positive prisoners may be about 228 in a sentenced population of about 36 000. If drug injecting is found as commonly among remand prisoners as amongst those sentenced the total number of HIV positive drug injectors in the prison system (both sentenced and on remand) is probably about 285 out of a prison population ofabout 45 000, or 1 in 158. It is more difficult to estimate the prevalence of HIV infection among non-injecting prisoners. We found that 17% of the non-injecting subjects were HIV positive but it is difficult to make a projection when prevalence is low. On the basis of the evidence for drug injectors, we conclude that the prevalence of HIV infection in the prison population is not less than about 1 in 158 (0 6%). This estimate must be compared with the prison departments figure of roughly 50 prisoners known to be positive for HIV antibody per year.

Detection of Mycobacterium leprae and the potential for monitoring antileprosy drug therapy directly from skin biopsies by PCR

An improved protocol for PCR analysis of Mycobacterium leprae-infected tissues, based on enzymatic lysis, has been developed and used to demonstrate the feasibility of using PCR for detecting M. leprae in routine skin biopsies taken from leprosy patients throughout the clinical spectrum. Of 92 multibacillary patients tested, 99% were PCR-positive using gel electrophoresis or DNA hybridization to detect the amplified product. Similar analysis of paucibacillary patients, in which only one of 27 biopsies had demonstrable AFB microscopically, gave a positivity rate of 74%. No PCR signals were demonstrated from skin biopsies from seven patients with non-leprosy dermatoses and one AIDS patient with a disseminated atypical mycobacteriosis. Evaluation of leprosy patients with antileprosy drug therapy prior to biopsy demonstrated that PCR signals were either greatly diminished or absent after 2 months of continuous antibiotic therapy. PCR was also able to detect the presence of M. leprae in tissues of patients receiving antibacterial therapy when patients were suspected of harbouring drug-resistant M. leprae.

Reactions following completion of 1 and 2 year multidrug therapy (MDT).

We evaluated the incidence, severity, and duration of reactional states in 139 multibacillary (MB) leprosy patients in the first 2 years after the completion of the 1 year regimen of multidrug therapy (MDT) currently recommended by the World Health Organization (WHO) and compared those findings with 295 MB leprosy patients treated with the same regimen previously recommended for 2 years. During the first year after the completion of 1 year MDT, patients experienced 1 or more reactional states 27% of the time, the vast majority being lepra type 1 reactions (reversal reactions, RR), whereas patients who received 2 year MDT experienced a reactional state during that time period only 8% of the time (P < 0.001). Furthermore, during the first year after the completion of therapy, and during the first 2 years, both the number of reactional states and reversal reactions were significantly (P < or = 0.004) more frequent, severe, of longer duration, and more commonly associated with neuritis.