Molecular epidemiology of leprosy based on VNTR typing in Thailand.

Recently about 500 new cases of leprosy have been reported each year in Thailand. In addition to a steady rate of new case detection, Thailand is in Southeast Asia where leprosy is endemic in neighbouring countries; therefore, strain differentiation could be useful in tracing origins and routes of infection, and general leprosy surveillance. To identify suitable markers for differentiation of M. leprae strains in different global geographic regions and to determine the applicability of a systematic genotyping method for tracing leprosy transmission, variable nucleotide tandem repeats (VNTRs) of 14 loci were evaluated using DNA extracts from a total of 97 skin biopsies and slit skin smear samples. The alleles per locus ranged from 2-26 providing adequate strain differentiation. Microsatellite loci (GAA)21, (AT)17 are highly polymorphic followed by (GTA)9, (AC)8a, (AC)8b, and (AC)9. The minisatellites 6-7, 21-3 and 27-5 exhibited a limited number of alleles. The repeat of 23-3 showed no polymorphism. Overall, the strain types can be divided into two distinct Thai groups, according to the alleles at the (GGT)5 and 21-3 loci. However, there are no obvious geographical patterns of distribution of VNTR strain types. Closely matched VNTR profiles found in household members of two multi-case families suggested infection through a common source.

A simplified reverse transcriptase PCR for rapid detection of Mycobacterium leprae in skin specimens.

An RNA-based assay is an additional molecular tool for leprosy diagnosis and determination of the viability of leprosy bacilli. To simplify RNA detection, a one-step reverse transcriptase PCR (RT-PCR) was established and evaluated. RNA and DNA could be isolated simultaneously. With the use of Mycobacterium leprae-specific primers targeting a 171-bp fragment of the M. leprae 16S RNA gene, RT-PCR resulted in detection of M. leprae in both slit skin smears and skin biopsy specimens. To enhance the positive signal, a digoxigenin-labeled DNA was developed, and successfully detected the amplified RT-PCR product. The method is sensitive, as it could detect one leprosy bacillus. When it was used directly on skin specimens collected from leprosy patients, 34 of 36 multibacillary (MB) and 13 of 24 paucibacillary (PB) cases showed positive results. The assay was also effective in monitoring bacterial clearance in leprosy patients during chemotherapy; after treatment with the multidrug therapy for 6 months, resulting in bacterial clearance, 16 of 36 MB patients and three of 24 PB patients tested were still positive for the 16S rRNA gene of M. leprae, suggesting the advisability of a more prolonged treatment course. This form of RT-PCR is of value in terms of simplicity and sensitivity in identifying M. leprae in routine skin specimens, especially when acid-fast bacilli are not discernable.

Gender differences in epidemiological factors associated with treatment completion status of leprosy patients in the most hyperendemic district of Nepal.

The introduction of multidrug therapy (MDT), recommended by WHO, has been a major advance in the treatment of leprosy because of its relatively short treatment course and low rate of relapse. Although leprosy treatment is provided to both sexes equally, in most parts of the world significant differences have been found in treatment status. The main objective of the study was to investigate gender differences in epidemiological factors associated with treatment status of leprosy patients. An analytic cross-sectional study was carried out in the most hyperendemic Dhanusa District, Nepal. Stratified random sampling was applied for selection of the patients. Statistical analysis of the differences in treatment status, between males and females, and among other epidemiological factors of interest was carried out using multiple logistic regression. Chi-square/Fisher's exact test were also used to assess significant differences in values between males and females. There were 580 leprosy patients (385 male and 195 female) aged >15 years registered for MDT between April 1, 2001 to March 31, 2002 in the 16 main health centers of the district. Of the 580 patients, a total of 273 (183 male and 90 female) were included in the study, to collect data on clinical type of leprosy, patterns of physical deformity/disability, site of skin lesions, and socio-demographic information. There were 183 male (68.3% on MB-MDT) and 90 female (61.1% MB-MDT) leprosy patients. We found that 79.2% of male patients completed treatment, while 34.4% female patients did not complete within the given time frame. Significant gender differences among leprosy patients were found in the distribution of disability grades and treatment completion status. However, there was no significant gender difference in the distribution of leprosy types and skin lesion sites. The study also found significant associations between treatment completion status and gender (adjusted OR 2.05, 95% CI: 1.07-3.94), educational status (adjusted OR 2.37, 95% CI: 1.12-4.99), disability grade I (adjusted OR 3.14, 95% CI: 1.23-8.04), and disability grade 0 (adjusted OR 2.92, 95% CI: 1.14-7.47) after adjustment for all other leprosy/demographic factors.

Gender difference in socio-epidemiological factors for leprosy in the most hyper-endemic district of Nepal.

Leprosy which has caused stigma and social ostracism for millennium is nearing elimination worldwide as a public health problem, but the leprosy burden in Nepal is still 4.4 times greater than WHO's target level of less than one case per 10,000 population. Although leprosy affects both the sexes, in most parts of the world males are affected more than females at a ratio of 2:1. The general objective of the study was to investigate the gender difference in socio-epidemiological factors for leprosy. The analytic cross-sectional study was carried out in one of the most hyper endemic district- Dhanusa district of Nepal. Stratified random sampling method was applied for the selection of the patients. Chi-square/Fisher's exact test was applied to assess statistically significant differences in values between males and females. There were 580 leprosy patients (385 male and 195 female) aged above 15 years registered for multi drug therapy between April 1, 2001 to March 31,2002 in the 16 main health centers of the district. Out of 580 patients, 273 patients (183 males and 90 females) were included in the study in order to collect the data on socio-demographics, patient's knowledge on leprosy, treatment seeking behaviour, and social problems faced by the patients. Data were collected using a structured interview schedule. The mean age of the male patients was 45.1 years (range 15-77 years) and female patients were 40.3 years (range 15-75 years). Among male patients 93.4% were married while among female patients 70.0% were married. Among male patients 51.9% were illiterate whereas 71.1% were illiterate among female patients. Most of the patients (69.6%) lived in joint family and the rest in nuclear family. Among male patients, 86.9% had good knowledge about the disease compared to 73.3% among females. This study showed that among the female patients 12.2% were facing high level of social problems, while among male patients only 4.4% were facing the same. About 15% patients had poor treatment seeking behaviors (8.2% among males and 27.8% among females). A significant gender differences among leprosy patients have been found in age distribution, educational status, marital status, caste types, family members, and overall knowledge on the general aspect of leprosy, social problems faced by the patients and treatment seeking behaviour.

T-cell recognition of peptides from the Mycobacterium leprae 35 kDa protein in Thai leprosy patients, healthy contacts, and non-contacts.

The objective of the study was to identify Mycobacterium leprae-specific immunogenic peptides for the development of a skin test reagent. Such a reagent is required for the detection of M. leprae infection and possibly for the diagnosis of patients with active leprosy. For this purpose, we analyzed the in vitro responses of human peripheral blood mononuclear cell (PBMCs) to peptides from the 35 kDa protein of M. leprae. This protein is of interest since it has no homologue within the Mycobacterium tuberculosis complex, although it has a homologue in Mycobacterium avium. The subjects enrolled in the study were paucibacillary (PB) and multibacillary (MB) leprosy patients, healthy contacts, and non-contacts. Seventy-three PB and 124 MB leprosy patients were recruited from four leprosy clinics in Thailand. Fifty-seven healthy contacts were household contacts. Twenty non-leprosy contacts had no family history of or exposure to leprosy. PBMCs from individuals were tested for stimulation with 12 overlapping peptides from the M. leprae 35 kDa protein using the lymphocyte proliferation assay. These peptides were located in four areas containing three to six residues which were distinct for the M. leprae product in comparison to that from M. avium. Four peptides (p60-76, p132-151, p206-224 and p267-286), which were the most permissive from each region and recognized by non-contacts with significantly lower frequencies than other subject groups, were identified. From this preliminary result, we conclude that these four peptides were likely to be M. leprae-specific.

Present status and future vision of the International Medical Co-operation for Leprosy.

In the year 2002, leprosy situation in Thailand has been steadily progress. However, the prevalence rate and percentage of leprosy patients are still quite high in the North-Eastern part of Thailand. Therefore, we have focused our plan of action 2001-2 on "The strengthening of Leprosy Elimination and Prevention of Disability in the North-Eastern Region." The objective of which is to improve and sustain the ability of leprosy related staff to conduct activities such as case finding, complication diagnosis, treatment of disabilities, rehabilitation, supervision and evaluation. The International Medical Co-operation for Leprosy in 2001, we received funds from Netherland Leprosy Relief Association (NLR) for 9 programmes concerning training of leprosy for health officers and assessment of the quality of life for leprosy affected persons living in northeastern colonies. There are 3 training courses of leprosy for new medical doctors, lab technicians from community and provincial hospitals and 2 workshops on Rehabilitation and Development of Leprosy Affected Persons "Quality of Life" under the Germany Leprosy Relief Association (GLRA) support. From Japan we received funding from Sasakawa Memorial Health Foundation (SMHF) for 4 projects in immunological studies since 1997 and 2 projects concerning dental services for Leprosy patients in the north and northeast regions from Umemoto Memorial Dental Service Group (UMDSG). The medical co-operation between Japan and Thailand should increase in many aspects especially, for new chemotherapy, immunotherapy and vaccine study in Leprosy. The future vision of Leprosy, we plan to set up the International Center of Leprosy for medical officers, technicians, etc. for the South-East Asian Countries. You are welcome to join and work together with us.