Clinicobacteriological evaluation of leprosy patients with 1-5 skin lesions.

Background: Mycobacterium leprae is a noncultivable mycobacteria, and diagnosis of the disease is based on its clinical and histopathological characteristics and finding the bacteria in skin scrapings and in biopsies taken from the patients. The aim of this study was to shed light on the clinical classification (based on the number of skin lesions) used extensively in the field where patients classified as paucibacillary (PB) were positive on skin smears and histopathology leading to treatment failure and drug resistance. Methods: In this study, we enrolled untreated 62 leprosy patients with 1-5 skin lesions and did a detailed bacterio-histopathological analysis by slit-skin smears (SSSs) and histopathology. Results: Of 62 patients analyzed, 15 patients came out to be multibacillary (MB) and 47 were PB by SSS and histopathology. Conclusion: The findings of the present study showed that the WHO classification of leprosy based on the number of lesions seems to be inappropriate as it considers a number of MB lesions as PB only, thus misleading the treatment strategies. Hence, it is essential that a comprehensive clinicobacteriological assessment of leprosy cases should be done to ensure the appropriate bacillary status and guiding the appropriate treatment strategy.

Effectiveness of the retreatment of patients with multibacillary leprosy and episodes of erythema nodosum leprosum and/or persistent neuritis: a single-center experience

Abstract: Background: Erythema nodosum leprosum may appear before, during or after treatment of leprosy and is one of the main factors for nerve damage in patients. When it occurs or continues to occur after treatment, it may indicate disease recurrence and a new treatment may be instituted again. Objective: To evaluate the retreatment of patients with multibacillary leprosy who underwent standard treatment with multidrug therapy, but developed or continued to present reactions of erythema nodosum leprosum and/or neuritis 3-5 years after its end. Method: For this objective, a new treatment was performed in 29 patients with multibacillary leprosy who maintained episodes of erythema nodosum and/or neuritis 3-5 years after conventional treatment. Results: In general, we observed that 27 (93.10%) had no more new episodes after a follow up period of eight months to five years. In five of these patients the reason for the retreatment was the occurrence of difficult-to-control neuritis, and that has ceased to occur in all of them. Study limitations: Small number of patients.. Conclusion: In the cases observed, retreatment was an effective measure to prevent the occurrence of erythema nodosum leprosum and/or persistent neuritis.

Mycobacterium leprae Recombinant Antigen Induces High Expression of Multifunction T Lymphocytes and Is Promising as a Specific Vaccine for Leprosy.

Leprosy is a chronic disease caused by M. leprae infection that can cause severe neurological complications and physical disabilities. A leprosy-specific vaccine would be an important component within control programs but is still lacking. Given that multifunctional CD4 T cells [i.e., those capable of simultaneously secreting combinations of interferon (IFN)-γ, interleukin (IL)-2, and tumor necrosis factor (TNF)] have now been implicated in the protective response to several infections, we tested the hypothesis if a recombinant M. leprae antigen-specific multifunctional T cells differed between leprosy patients and their healthy contacts. We used whole blood assays and peripheral blood mononuclear cells to characterize the antigen-specific T cell responses of 39 paucibacillary (PB) and 17 multibacillary (MB) leprosy patients and 31 healthy household contacts (HHC). Cells were incubated with either crude mycobacterial extracts (M. leprae cell sonicate-MLCS) and purified protein derivative (PPD) or recombinant ML2028 protein, the homolog of M. tuberculosis Ag85B. Multiplex assay revealed antigen-specific production of IFN-γ and IL-2 from cells of HHC and PB, confirming a Th1 bias within these individuals. Multiparameter flow cytometry then revealed that the population of multifunctional ML2028-specific T cells observed in HHC was larger than that observed in PB patients. Taken together, our data suggest that these multifunctional antigen-specific T cells provide a more effective response against M. leprae infection that prevents the development of leprosy. These data further our understanding of M. leprae infection/leprosy and are instructive for vaccine development.

Bullous Erythema Nodosum Leprosum as the First Manifestation of Multibacillary Leprosy: A Rare Phenomenon.

Erythema nodosum leprosum (ENL) may uncommonly present before treatment in patients with multibacillary leprosy. Atypical manifestations are known in ENL and may be clinically misleading. Such wide variations in the clinical presentation of leprosy in reaction make histopathology an important tool for supporting clinical diagnosis. We report bullous ENL presenting as the first manifestation of leprosy in a 30-year-old Indian man diagnosed using histopathology.

Leprosy treatment during pregnancy and breastfeeding: A case report and brief review of literature.

Leprosy is a chronic disease which primarily affects the skin, mucous membranes and peripheral nerves due to Mycobacterium leprae. It is now infrequent in Europe and is rarely reported during pregnancy. Leprosy can be exacerbated during pregnancy, and without treatment it can permanently damage the skin, nerves, limbs and eyes. Therefore, it is important to treat leprosy during pregnancy. This article describes a patient with multibacillary lepromatous leprosy who was treated with multidrug therapy during pregnancy and breastfeeding. The patient delivered a healthy baby girl without perinatal complications, and the infant's growth and development were normal during the 1-year follow-up period. Multidrug therapy consisting of dapsone, rifampicine, and clofazimine is highly effective for people with leprosy and considered safe, both for the mother and the child. Antileprosy drugs are excreted into human milk but there is no report of adverse effects except for skin discoloration of the infant due to clofazimine. Multidrug therapy for leprosy patients should be continued unchanged during pregnancy and breastfeeding.

Association of viable Mycobacterium leprae with Type 1 reaction in leprosy.

UNLABELLED: The working hypothesis is that, viable Mycobacterium leprae (M. leprae) play a crucial role in the precipitation of Type 1 reaction (T1R) in leprosy. MATERIAL AND METHODS: A total of 165 new multibacillary patients were studied. To demonstrate presence of viable M. leprae in reactional lesion (T1R+), three tests were used concurrently viz. growth in the mouse foot pad (MFP), immunohistochemical detection of M. leprae secretory protein Ag85, and 16s rRNA--using in situ RT-PCR. Mirror biopsies and non reactional lesions served as controls (T1R-). FINDINGS: A significantly higher proportion of lesion biopsy homogenates obtained at onset, from T1R(+) cases have shown unequivocal growth in MFP, proving the presence of viable bacteria, as compared to T1R(-) (P < 0.005). In contrast, few Mirror biopsies were positive in both T1R(+) and T1R(-). With respect to Ag85, while the overall positivity was higher in T1R(+) (74%), however the intensity of staining (Grade 2+) was disproportionately higher in T1R(+) BT-BB lesions 11/20 (55%). In the rebiopsies obtained during a repeat episode of T1R, Ag 85 as well as 16s rRNA, positivity (62% & 100%) was higher in T1R(+). It is inferred therefore 'viable' bacteria are an essential component in T1R and difference in the quality of bacilli, not the quantity or the ratio of dead to viable play a role in the precipitation of T1R. In conclusion, the findings show that 'metabolically active' M. leprae is a component/prerequisite and the secretory protein Ag 85, might be the trigger for precipitation of T1R.

Association of the solute carrier family 11 member 1 gene polymorphisms with susceptibility to leprosy in a Brazilian sample

Natural resistance-associated macrophage protein 1/solute carrier family 11 member 1 gene (Nramp1/Slc11a1) is a gene that controls the susceptibility of inbred mice to intracellular pathogens. Polymorphisms in the human Slc11a1/Nramp1 gene have been associated with host susceptibility to leprosy. This study has evaluated nine polymorphisms of the Slc11a1/Nramp1 gene [(GT)n, 274C/T, 469+14G/C, 577-18G/A, 823C/T, 1029 C/T, 1465-85G/A, 1703G/A, and 1729+55del4] in 86 leprosy patients (67 and 19 patients had the multibacillary and the paucibacillary clinical forms of the disease, respectively), and 239 healthy controls matched by age, gender, and ethnicity. The frequency of allele 2 of the (GT)n polymorphism was higher in leprosy patients [p = 0.04, odds ratio (OR) = 1.49], whereas the frequency of allele 3 was higher in the control group (p = 0.03; OR = 0.66). Patients carrying the 274T allele (p = 0.04; OR = 1.49) and TT homozygosis (p = 0.02; OR = 2.46), such as the 469+14C allele (p = 0.03; OR = 1.53) of the 274C/T and 469+14G/C polymorphisms, respectively, were more frequent in the leprosy group. The leprosy and control groups had similar frequency of the 577-18G/A, 823C/T, 1029C/T, 1465-85G/A, 1703G/A, and 1729+55del4 polymorphisms. The 274C/T polymorphism in exon 3 and the 469+14G/C polymorphism in intron 4 were associated with susceptibility to leprosy, while the allele 2 and 3 of the (GT)n polymorphism in the promoter region were associated with susceptibility and protection to leprosy, respectively.

Is drug-resistant Mycobacterium leprae a real cause for concern?: First approach to molecular monitoring of multibacillary Colombian patients with and without previous leprosy treatment.

INTRODUCTION: There is no information in Colombia on Mycobacterium leprae primary and secondary drug resistance in regards to the WHO-multidrug therapy regime. On the other hand, public health authorities around the world have issued various recommendations, one of which prompts for the immediate organization of resistance surveillance through simple molecular methods. OBJECTIVE: To determine the prevalence of Mycobacterium leprae drug resistance to rifampicin, ofloxacin and dapsone in untreated and previously treated patients at the Centro Dermatológico Federico Lleras Acosta during the 1985-2004 period. MATERIALS AND METHODS: We conducted a retrospective study which included multibacillary patient biopsies through elective sampling: 381 of them from new patients and 560 from previously treated patients. Using a microtome, we obtained six slides from each skin biopsy preserved in paraffin, and we extracted M. leprae DNA. We amplified three molecular targets through PCR and obtained the patterns of drug resistance to dapsone, rifampicin and ofloxacin by reverse hybridization. Finally, we collected epidemiological, clinical and demographical data for analyses. RESULTS: From 941 samples under study, 4.14% of them were resistant to one or more drugs, and 5.77 and 3.04% had resistant genotypes in new and previously treated patients, respectively. Total resistance for each drug was 0.43% for dapsone, 3.19% for rifampicin and 1.17% for ofloxacin. We found statistically significant differences for rifampicin and for the total population when comparing the results from untreated versus previously treated patients. Two thirds of the resistant samples were resistant to rifampicin alone or combined. CONCLUSIONS: The standard multidrug therapy schemes continue being effective for leprosy cases; however, it is necessary to guarantee adherence and regularity. Surveillance to drug resistance in new and previously treated leprosy cases should be established.

Is drug-resistant Mycobacterium leprae a real cause for concern?: First approach to molecular monitoring of multibacillary Colombian patients with and without previous leprosy treatment / ¿Es la resistencia de Mycobacterium leprae a los medicamentos un verdadero motivo de preocupación?: Primera aproximación a la vigilancia molecular de pacientes colombianos multibacilares con tratamiento previo para lepra y sin él

Introduction: There is no information in Colombia on Mycobacterium leprae primary and secondary drug resistance in regards to the WHO-multidrug therapy regime. On the other hand, public health authorities around the world have issued various recommendations, one of which prompts for the immediate organization of resistance surveillance through simple molecular methods. Objective: To determine the prevalence of Mycobacterium leprae drug resistance to rifampicin, ofloxacin and dapsone in untreated and previously treated patients at the Centro Dermatológico Federico Lleras Acosta during the 1985-2004 period. Materials and methods: We conducted a retrospective study which included multibacillary patient biopsies through elective sampling: 381 of them from new patients and 560 from previously treated patients. Using a microtome, we obtained six slides from each skin biopsy preserved in paraffin, and we extracted M. leprae DNA. We amplified three molecular targets through PCR and obtained the patterns of drug resistance to dapsone, rifampicin and ofloxacin by reverse hybridization. Finally, we collected epidemiological, clinical and demographical data for analyses. Results: From 941 samples under study, 4.14% of them were resistant to one or more drugs, and 5.77 and 3.04% had resistant genotypes in new and previously treated patients, respectively. Total resistance for each drug was 0.43% for dapsone, 3.19% for rifampicin and 1.17% for ofloxacin. We found statistically significant differences for rifampicin and for the total population when comparing the results from untreated versus previously treated patients. Two thirds of the resistant samples were resistant to rifampicin alone or combined. Conclusions: The standard multidrug therapy schemes continue being effective for leprosy cases; however, it is necessary to guarantee adherence and regularity. Surveillance to drug resistance in new and previously treated leprosy cases should be established.

Introducción. Colombia no dispone de información sobre farmacorresistencia primaria y secundaria de Mycobacterium leprae al esquema de terapia múltiple de la Organización Mundial de la Salud (OMS) y las autoridades de salud pública del mundo han emitido varias recomendaciones, entre las cuales está organizar de inmediato la vigilancia a la resistencia empleando métodos moleculares simples. Objetivo. Determinar la prevalencia de la resistencia de M. leprae a rifampicina, ofloxacina y dapsona en pacientes del Centro Dermatológico Federico Lleras Acosta con tratamiento previo y sin él durante el período de 1985 a 2004. Materiales y métodos. Se realizó un estudio retrospectivo. Mediante muestreo electivo se incluyeron biopsias de pacientes multibacilares: 381 de pacientes nuevos y 560 de pacientes previamente tratados. Se obtuvieron con micrótomo seis cortes de cada biopsia de piel incluida en parafina, y se realizó la extracción de ADN de M. leprae. Se llevó a cabo la amplificación de tres blancos moleculares mediante PCR y se obtuvieron los patrones de resistencia a los medicamentos dapsona, rifampicina y ofloxacina por hibridación inversa. Se recolectaron datos epidemiológicos, clínicos y demográficos para llevar a cabo los análisis. Resultados. De las 941 muestras estudiadas, 4,14 % era resistente a uno o más fármacos, y se detectaron 5,77 y 3,04 % con genotipos resistentes en pacientes nuevos y previamente tratados, respectivamente. La resistencia total para cada fármaco fue de 0,43 % a dapsona, 3,19 % a rifampicina y 1,17 % a ofloxacina. Se encontró una diferencia estadísticamente significativa para rifampicina y para la población total al comparar los resultados de los pacientes no tratados con los de los pacientes tratados previamente. Dos tercios de las muestras resistentes lo fueron a rifampicina sola o combinada. Conclusiones. Los esquemas de terapia múltiple estándar siguen siendo efectivos para los casos de lepra; sin embargo, es necesario garantizar el cumplimiento y la regularidad y establecer la vigilancia de la farmacorresistencia en pacientes nuevos y previamente tratados.

Mycobacterium leprae is identified in the oral mucosa from paucibacillary and multibacillary leprosy patients.

In leprosy, the nasal mucosa is considered as the principal route of transmission for the bacillus Mycobacterium leprae. The objective of this study was to identify M. leprae in the oral mucosa of 50 untreated leprosy patients, including 21 paucibacillary (PB) and 29 multibacillary (MB) patients, using immunohistochemistry (IHC), with antibodies against bacillus Calmette-Guérin (BCG) and phenolic glycolipid antigen-1 (PGL-1), and polymerase chain reaction (PCR), with MntH-specific primers for M. leprae, and to compare the results. The material was represented by 163 paraffin blocks containing biopsy samples obtained from clinically normal sites (including the tongue, buccal mucosa and soft palate) and visible lesions anywhere in the oral mucosa. All patients and 158 available samples were included for IHC study. Among the 161 available samples for PCR, 110 had viable DNA. There was viable DNA in at least one area of the oral mucosa for 47 patients. M. leprae was detected in 70% and 78% of patients using IHC and PCR, respectively, and in 94% of the patients by at least one of the two diagnostic methods. There were no differences in detection of M. leprae between MB and PB patients. Similar results were obtained using anti-BCG and anti-PGL-1 antibodies, and immunoreactivity occurred predominantly on free-living bacteria on the epithelial surface, with a predilection for the tongue. Conversely, there was no area of predilection according to the PCR results. M. leprae is present in the oral mucosa at a high frequency, implicating this site as a potential means of leprosy transmission.

Concordance between expected and observed bacilloscopy results of clinical forms of leprosy: a 6-year retrospective study in Recife, State of Pernambuco, Brazil / Concordância entre os resultados observados e esperados da forma clinica da hanseníase de acordo com a baciloscopia: um estudo retrospectivo de seis anos em Recife, Estado de Pernambuco, Brasil

INTRODUCTION: Operational classification of leprosy based on the number of skin lesions was conceived to screen patients presenting severe forms of the disease to enable their reception of a more intense multidrug regimen without having to undergo lymph smear testing. We evaluated the concordance between operational classification and bacilloscopy to define multibacillary and paucibacillary leprosy. METHODS: We selected 1,213 records of individuals with leprosy, who were untreated (new cases) and admitted to a dermatology clinic in Recife, Brazil, from 2000 to 2005, and who underwent bacteriological examination at diagnosis for ratification of the operational classification. RESULTS: Compared to bacilloscopy, operational classification demonstrated 88.6% sensitivity, 76.9% specificity, a positive predictive value of 61.8%, and a negative predictive value of 94.1%, with 80% accuracy and a moderate kappa index. Among the bacilloscopy-negative cases, 23% had more than 5 skin lesions. Additionally, 11% of the bacilloscopy-positive cases had up to 5 lesions, which would have led to multibacillary cases being treated as paucibacillary leprosy if the operational classification had not been confirmed by bacilloscopy. CONCLUSIONS: Operational classification has limitations that are more obvious in borderline cases, suggesting that in these cases, lymph smear testing is advisable to enable the selection of true multibacillary cases for more intense treatment, thereby contributing to minimization of resistant strain selection and possible relapse.

INTRODUÇÃO: A classificação operacional da hanseníase baseada no número de lesões de pele foi concebida para selecionar pacientes que apresentam formas graves da doença para receber regime terapêutico mais intenso com múltiplas drogas sem o exame de baciloscopia da linfa. Nós avaliamos a concordância entre a classificação operacional e a baciloscopia para a definição de hanseníase multibacilar e paucibacilar. MÉTODOS: Nós selecionamos 1.213 registros de indivíduos com hanseníase não tratada (casos novos), atendidos em um Ambulatório de Dermatologia, em Recife, Brasil, no período de 2000 a 2005, que foram submetidos a exame bacteriológico ao diagnóstico para a ratificação da classificação operacional. RESULTADOS: Comparando com a baciloscopia, a classificação operacional baseada no número de lesões cutâneas mostrou sensibilidade de 88,6%, especificidade 76,9%, valor preditivo positivo de 61,8% e valor preditivo negativo de 94,1%, com uma precisão de 80% e um moderado índice kappa. Entre os casos com baciloscopia negativa, 23% tinham mais de cinco lesões de pele, recebendo um tratamento mais intensivo. Além disso, 11% dos casos baciloscopia positiva tinham até cinco lesões, o que induziriam casos multibacilares de serem tratados com hanseníase paucibacilar se a classificação operacional não tivesse sido confirmada pela baciloscopia. CONCLUSÕES: Concluímos que a classificação operacional tem limitações mais visíveis nos casos borderline, sugerindo que, nestes casos, o esfregaço seria aconselhável por permitir que os verdadeiros casos multibacilares fossem selecionados para um tratamento mais intenso, contribuindo para minimizar a seleção de cepas resistentes e uma possível recidiva.

Revalidation of various clinical criteria for the classification of leprosy--a clinic-pathological study.

UNLABELLED: WHO guidelines classify leprosy patients clinically into PB and MB group based on the number of skin lesions (NSL) with > or = 6 skin lesions as a criterion for MB leprosy. Other clinical criteria for classification are based on the number of body areas affected (NBAA) and on size of the largest skin lesions (SLSL). They are also fairly simple and easily practicable in the field. OBJECTIVES: The objective of this study is to explore whether sensitivity and specificity of the WHO classification can be improved by addition of clinical criteria based on NBAA and SLSL to WHO classification. STUDY DESIGN: Among 100 newly diagnosed untreated leprosy patients classified into PB and MB group according to WHO classification, the NSL and NBAA were recorded and the size (longest diameter) of largest skin lesion was measured in centimeters. The Receiver Operator Characteristic (ROC) curves were plotted for each parameter to find the best cut off point (with highest sensitivity and specificity). RESULTS: The sensitivity and specificity of the WHO classification tested, using slit-skin smear (SSS) and skin biopsy results as the gold standard, was found to be 63% and 85% respectively. The ROC curve for NSL found the best cut off of three and more lesions for MB group (sensitivity 90% & specificity 80%). Similarly, ROC curves for NBAA and SLSL found the best cut off points for classification into MB group to be two or more (sensitivity 90% & specificity 75%) and 5 cm or more (sensitivity 87% and specificity 65%) respectively. On combining all these criteria together sensitivity was increased to 98.5% with no significant change in specificity, which was 77.5%. CONCLUSION: The study concluded that the sensitivity of the present clinical classification can be further improved by addition of two other clinical criteria.

Leprosy in transplant recipients: report of a case after liver transplantation and review of the literature.

Leprosy still is an important public health problem in several parts of the world including Brazil. Unlike the diseases caused by other mycobacteria, the incidence and clinical presentation of leprosy seems little affected in immunosuppressed patients. We report the first case, to our knowledge, of a liver transplant patient who developed multi-bacillary leprosy. The patient presented with papules and infiltrated plaques with loss of sensation suggestive of leprosy 3.5 years after living-related liver transplantation for autoimmune hepatitis. A skin biopsy showing non-caseating macrophagic granulomas, neuritis, and intact acid-fast bacilli on Fite-Faraco stain, confirmed the diagnosis of borderline lepromatous leprosy. The donor of the liver did not show any evidence of leprosy. During follow-up, the patient presented 2 episodes of upgrading leprosy type I reactions, 1 mild before leprosy treatment, and 1 moderate 3 months after receiving standard multi-drug treatment (rifampicin, clofazimine, and dapsone). These reactions were accompanied by increase in liver function tests, especially of canalicular enzymes. This reaction occurred despite the patient's triple immunosuppression regimen. The moderate reaction was successfully treated with further immunosuppression (prednisone, 0.5 mg/kg). Currently, the patient is asymptomatic, off leprosy medication, with routine liver transplant follow-up. The dilemmas in diagnosis and management of such a case are discussed and the literature on leprosy in transplant recipients is reviewed.

The effect of corticosteroids usage on bacterial killing, clearance and nerve damage in leprosy; part 3--Study of two comparable groups of 100 multibacillary (MB) patients each, treated with MDT + steroids vs. MDT alone, assessed at 6 months post-release from 12 months MDT.

OBJECTIVE: To investigate effects of therapeutic usage of corticosteroids on M. leprae killing and clearance, on clearance of granuloma and on nerve damage in multibacillary (MB) leprosy patients. DESIGN: From a cohort of 400 untreated MB patients, a comparable group of 100 each receiving MDT + steroids (group A) vs MDT alone (group B) were assessed at 18 months as compared to month zero with respect to clinical and granuloma regression, M. leprae killing and clearance, and nerve functions. Analysis was performed using SPSS version 10.0. The significance of association was tested using Chi square and Fisher's exact tests. RESULTS: Regression of lesions assessed clinically and by histopathology was seen in 52% and 53% patients in group A and 46% and 63% in B respectively (P not significant). Clearance of bacteria assessed by bacteriological index (BI) in slit skin smears (SSS) and extent and intensity of antigen using anti-BCG staining were also comparable in the two groups. Multiplication of M. leprae in the mouse foot pad (MFP) indicating the presence of viable bacilli was seen in 14% and 16% of SSS positive BL-LLs patients in groups A and B respectively (P not significant). The occurrence of viable M. leprae was higher among patients with repeat reaction (19%) than single (11%). Using clinical tests (nerve palpation, monofilament and voluntary muscle testing), the proportion of sensory and motor nerves showing improvement or deterioration were similar in the two groups. However using nerve conduction studies, the overall proportion of nerves showing deterioration (22%) was significantly higher than improvement (9%) (P < 0.001). CONCLUSIONS: Treatment with MDT + corticosteroids does not adversely affect the clearance of granuloma, M. leprae and/or its antigens and M. leprae killing. However the continued presence of viable bacteria in > 14% of BL-LLs patients indicate that 12 months of MDT may be insufficient for complete bacterial killing. In both groups nerve conduction studies indicated that deterioration of nerves was high suggesting, MDT + corticosteroids was not very efficacious in the prevention or reversal of nerve damage. A better immuno-modulatory drug or a modified corticosteroid regime is needed.

VNTR typing studies of Mycobacterium leprae in China: assessment of methods and stability of markers during treatment.

OBJECTIVE: To evaluate the reliability and feasibility of two methods of multilocus variable number of tandem repeat analysis (MLVA) for strain typing of M. leprae, and to study whether short tandem repeat loci are stable and suitable for epidemiological study of leprosy. METHODS: Total DNA was extracted from skin biopsies of 20 new multibacillary (MB) patients from China diagnosed in 2006. To determine the copy numbers of short tandem repeats (STRs) for 13 loci, we amplified each locus individually by PCR, followed by sequence analysis of the amplicons. Separately, the same loci, plus four others were amplified by Multiplex PCRs (MP) using fluorescent primers and the copy number was identified by fragment length analysis (MP-FLA). MLVA was also performed at different times during treatment for a subset of the patients. RESULTS AND CONCLUSIONS: Genetic variability of M. leprae in China can be assessed in microsatellite loci. (GTA)9 and (TTC)21 loci are hypervariable, with array sizes of 25 repeat units or more. The expansion of the (GTA)9 locus is a characteristic of some M. leprae isolates in China. A high level of allele concordance was observed between PCR-sequencing and MP-FLA methods. However, MP-FLA method was cost-effective, rapid, high throughput and suitable for strain typing. Five of the 20 isolates of M. leprae were from patients residing in the same township in Qiubei County, Yunnan, and matched closely by MLVA. Three of these patients are family contacts of previously diagnosed patients, with intra-familial strain types being similar, suggesting infections from common sources and transmission chain(s). The VNTR patterns were highly similar in biopsy and slit skin smears (SSS) before treatment, and in the SSS collected at various time points during treatment. Taken together, VNTR strain typing is a useful tool for study of short range transmission in leprosy.

Long term follow-up results of 1 year MDT in MB leprosy patients treated with standard MDT + once a month Minocycline and Ofloxacin.

BACKGROUND: This study was initiated in consultation with the National Leprosy Eradication Programme (NLEP) in mid nineties to try new treatment regimens for leprosy which were more robust in terms of control of reactions, long term relapses, operationally easier to undertake and feasible in field conditions. It was also envisaged to see if the addition of newer bactericidal drugs would be beneficial. OBJECTIVES: (i) To test the feasibility, safety and response of the patients to the new regimen. (ii) To observe the incidence of reactions during and after stoppage of therapy, for a period of 8-10 years after release from treatment. MATERIALS AND METHODS: A total of one hundred skin smear positive MB patients (15 LL, 35 BL and 50 BB) patients were included in this study. All the patients received the standard MDT + once a month supervised 100 mg of Minocycline and 400 mg of Ofloxacin for 12 months during the treatment phase. Thereafter, the treatment was stopped in all the patients which were followed-up on placebo (B complex tablets). Of these, 70 patients completed the treatment schedule of one year therapy and the post treatment follow-up of 9 to 10 years. RESULTS: All the patients tolerated the drugs well. The clinical response of the patients to the treatment was very good of which 32.85% of cases had history of reactions before starting treatment. During treatment, the incidence of reactions increased marginally to 38.5%, but these were easily controlled with concurrent administration of steroids. After completion of treatment the incidence was much less i.e. 10% and 3% after 1 and 2 years of post treatment follow-up respectively. The overall relapse rate is 5.7% (4/70) with an incidence density of 0.05/100 patient years. Relapses were confirmed by clinical, bacteriological, molecular biological (rRNA probes and 36 kD targeting PCR) as well as ATP bioluminescence. The relapsed patients presented with the appearance of new lesions, slit-skin smears were again found to become positive after becoming negative. Three of the four cases who relapsed had the initial mean BI of 2 to 2.9+ whereas one had the initial mean BI of 1.5+. Also, 2 of the 4 relapsed patients had positive PCR signals at the time of stoppage of treatment. CONCLUSION: The addition of Minocycline and Ofloxacin to the standard FDT has been observed to be a well tolerated. Overall as of now, the incidence of reactions observed with the newer treatment regimen is found to be significantly lower than that of 2 years fixed duration MB-MDT. The efficacy of this regimen regarding bacteriological clearance and relapse rates could not be compared due to non-availability of the results of experience with standard 1 year MDT regimen. However, this regimen appears to be operationally feasible and safe for the users.