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.

Acometimento hepático na síndrome sulfona dapsona-induzida durante tratamento de hanseníase / Hepatic involvement in sulfone dapsone-induced syndrome during treatment of leprosy

A hanseníase é uma doença endêmica no Brasil e constitui grave problema de saúde pública. Em números absolutos, o Brasil é o segundo país que mais registra novos casos da doença por ano no mundo. O tratamento da hanseníase compreende: quimioterapia específica, supressão dos surtos reacionais, prevenção de incapacidades físicas, reabilitação física e psicossocial. A síndrome sulfona é uma condição multissistêmica potencialmente grave que pode ocorrer durante o tratamento de algumas dermatoses, entre elas a hanseníase. Relatamos um caso de síndrome de hipersensibilidade à dapsona (SHD) em um paciente masculino, de 32 anos, ocorrida durante o tratamento de hanseníase multibacilar...

Suppression of ROS generation by 4,4'-diaminodiphenylsulfone in non-phagocytic human diploid fibroblasts

The action mode of 4,4'-diaminodiphenylsulfone (DDS) is still under debate, although it has long been used in treatment of several dermatologic diseases including Hansen's disease. In this study, we tested the effect of DDS as an antioxidant on paraquat-induced oxidative stress in non-phagocytic human diploid fibroblasts (HDFs). Overall, preincubation of HDFs with DDS prevented the oxidative stress and the resulting cytotoxic damages caused by paraquat in these cells. The specific effects of DDS in paraquat-treated HDFs are summarized as follows: a) reducing the expression of NADPH oxidase 4 (NOX4) by inhibiting paraquat-induced activation of PKC; b) inhibiting paraquat-induced decreases in mitochondrial complex protein levels as well as in membrane potentials; c) consequently, inhibiting the generation of cytosolic and mitochondrial superoxide anions. Taken together, these findings suggest that DDS would suppress the radical generation in non-phagocytic HDFs during oxidative stress, and that DDS might have the extended potential to be used further in prevention of other oxidative stress-related pathologies.

Lepromatous leprosy of histoid type.

Histoid leprosy is a variant of lepromatous leprosy, which develops as a result of resistance to dapsone monotherapy. Here we report two cases of lepromatous leprosy of histoid type, one with typical and another with atypical presentations.

Malaria, leprosy and dapsone.

Although the preventive action of dapsone against P. falciparum malaria was known for many years, there was no report about the incidence of P. falciparum malaria in leprosy patients treated with dapsone, especially from areas of Southeast Asia where both leprosy and malaria are endemic. Therefore, two clinic-based malaria surveys were undertaken at a gap of 12 years, comprising 506 lepromatous leprosy patients and 499 febrile nonleprosy control subjects. Both the surveys showed that the lepromatous patients treated with MDT had only P. vivax malaria (incidence comparable to the febrile nonleprosy controls) with complete freedom from P. falciparum. On the contrary, control sujects not taking any-leprosy drugs and staying with the leprosy patients at the same beggars' home, had both P. vivax and P. falciparum malaria. It is postulated that dapsone provided protection against P. falciparum among leprosy patients.

Drug resistant-Mycobacterium leprae--results of mouse footpad studies from a laboratory in south India.

Out of 265 biopsies of leprosy patients received at the Experimental Pathology Laboratory of Schieffelin Leprosy Research and Training Centre from 1987 to 1997 for evaluating resistant strains of M. leprae, using the mouse footpad technique, 49 showed resistant strains of M leprae to varying concentrations of dapsone, rifampicin and clofazimine. 23 (47%) of these were from a control area. With 369 skin-smear positive multibacillary (MB) patients as the risk group (denominator), 23 (6.23%) were resistant to one or more drugs. 18 (4.88%) had dapsone resistance, 5 (1.36%) were resistant to rifampicin and 9 (2.44%) had resistance to low concentrations of clofazimine (0.0001%). Out of the 23 biopsies with drug resistance from the control area, primary dapsone resistance was seen in 7 (30%) biopsies and secondary dapsone resistance in 11 (48%). Primary rifampicin resistance was seen in 4 (17.4%) patients, secondary rifampicin resistance in 1 (4.35%) and primary clofazimine resistance in 7 (30%). 3 (13%) of the strains showed secondary clofazimine resistance. One biopsy had resistant strains to all the three drugs. In a control area where properly supervised effective multidrug therapy (MDT) was regularly administered over the years, the emergence of drug resistance is negligible. It may not be the case if the content, duration and regularity of the drug regimen were not satisfactory. Aware of the possible shortcomings in mass administration of MDT, it is emphasized that mouse footpad studies on drug resistance should be made available at least in endemic areas where the incidence of the disease has not changed despite good MDT coverage in order to monitor the emergence of drug resistance. Research into molecular biological identification of drug resistant-M.leprae should be intensified. These steps would help to institute timely measures to check the spread of any drug-resistant organisms in the community.

Drug resistance in leprosy: lessons from past and future perspective.

Some recent studies indicate that the problem of drug resistance in leprosy is very much there but the exact picture is not clear. In the emerging scenario with increasing number of new cases with low bacterial load, the conventional in-vivo and most of current in-vitro methods for determination of drug resistance may not help. It is pointed out that newer molecular approaches may be more useful and that it will be important to undertake studies to develop such tools.

Dapsone resistance does not appear to be associated with a mutation in the dihydropteroate synthase-2 gene of Mycobacterium leprae.

Evidence suggests that resistance to dapsone (DDS) in Mycobacterium leprae is related to the enzyme dihydropteroate synthase (DHPS). Two M. leprae genes (folP-1 and folP-2) encoding DHPS-1 and DHPS-2, respectively, have been identified through the M. leprae genome project. We have studied DDS-susceptible and resistant strains of M. leprae to determine whether the DDS-resistant phenotype is associated with a mutation(s) in folP-2 and to establish the number of genomic copies of the gene encoding DHPS-2 (folP-2). RFLP analysis of genomic DNA from DDS-susceptible and resistant strains of M. leprae exhibited a unique 4.2 kb restriction fragment consistent with a single genomic copy of folP-2 in both phenotypes. DNA encoding folP-2 was amplified by PCR and sequenced from two susceptible and two resistant strains of M. leprae. The folP-2 sequences from these strains were identical indicating that resistance to DDS was not associated with mutation(s) in the gene encoding DHPS-2.

Inhibition of multiplication of leprosy bacilli in nude mice by simultaneous adminstration of new rifamycin derivative krm-1648 and sparfloxacin combined with dapsone

The inhibition of multiplication of mycobacterium leprae inculated in to foot pads of nude mice by oral administration of new rifamycin derivative KRM-1648 and a new quinoline, sparfloxacin [SPFX] combined with dapsone [DDS]: was examined. When these drugs administered simultaneously once weekly, the use of 0.6 mg/Kg of KRM, 10 mg/kg of sparfloxacin and 0.001/ Dapsone, entirely prevented the multiplication of M. leprae even the number of non solid bacilli detected was less than that inoculated into each foot pad. This study revealed the possible future use of this multi-drug regimen in the chemotherapy of leprosy patient

The anti-inflammatory activity of some sulphonamides in albino rats.

Clinically equivalent doses of dapsone, sulphasalasine and sulphamethizole in albino rats showed significant (P < 0.05) anti-inflammatory activity in carrageenan and cotton pellet induced inflammation. Their activity was comparable to that of aspirin (200 mg/kg) and was confirmed by granuloma histology. Further, these compounds also showed significant (P < 0.01) analgesic activity which was comparable to that of aspirin. The ulcer index for sulphamethizole was comparable to that of control animals, whereas dapsone and sulphasalazine showed significant ulcerogenicity (P < 0.01). Other sulphonamides like sulphadiazine, sulphanilamide, sulphamoxole and cotrimoxazole did not show significant anti-inflammatory and analgesic activities.

Improving patient compliance--a multicentre evaluation of the 'DDS tile test'.

The feasibility and utility of the "DDS tile test" under field conditions was assessed in 112 leprosy centres in Maharashtra. About 10% of the 2952 urine samples tested negative for dapsone. Feed back information from 54 centres one year later showed that the test could be performed easily under field conditions and also that counselling of patients showing poor compliance helped to improve drug compliance in over 80% of cases.

Effect of drug treatment on electroneurological measures of peripheral nerve function in leprosy patients.

OBJECTIVE: To investigate whether drug treatment improves the electroneurological measures of affected peripheral nerve function in leprosy patients. DESIGN: Clinical status of patients determined on the first visit by an investigator administered, pre-designed questionnaire, followed by measurement of motor conduction velocity (MCV) and distal latency (DL) of ulnar, median, common peroneal and posterior tibial nerves bilaterally in patients referred consecutively from the dermatology unit and leprosy clinic, Teaching Hospital, Galle. MCV and DL measurements were repeated after 6 to 12 months of treatment. SETTING: Department of Physiology, Faculty of Medicine, University of Ruhuna, Galle. SUBJECTS: 24 diagnosed leprosy patients; tuberculoid, lepromatous and borderline in clinical type. INTERVENTIONS: Based on clinical typing. Tuberculoid (paucibacillary) type rifampicin 600 mg monthly and dapsone 100 mg daily for six months. Lepromatous and borderline (multibacillary) type rifampicin 600 mg and clofazimine 300 mg monthly and dapsone 100 mg and clofazimine 50 mg daily for 24 months. RESULTS: DL in all 4 nerves and MCV in 3 nerves tested were significantly different (p > 0.001) to those for the normal population and remained so after 6 to 12 months of treatment. The DL in the ulnar nerve showed significant improvement (p < 0.05) after treatment. When analysed in each patient individually, before and after treatment, the MCV showed an improvement in 48 to 72% of patients and the DL in 41 to 59%, but differences were not significant. CONCLUSIONS: Electroneurological recovery (return to normal state) of the affected peripheral nerves of leprosy patients does not occur after 6 to 12 months of drug treatment. The significant (p < 0.05) improvement (becoming better) of ulnar nerve DL indicates that, if at all, electroneurologically detectable improvement of nerve function occurs in the early stages of nerve damage, and that it may take longer than one year after starting treatment.

Influence of the rapid acetylator phenotype on the emergence of DDS resistant Mycobacterium leprae

Determinou-se o fenótipo acetilador da insoniazida de 21 hansenianos suspeitos de terem desenolvido resistência à DDS. A resitência do Mycobacterium leprae desses pacientes foi investigada por intermédio de testes no coxim plantar de camundongos da estirpe BALB/c. Nossos resultados indicam que o surgimento de resistência à DDS no M. leprae dos acetiladores rápidos é mais provável do que nos bacilos dos acetiladores lentos. Parece plausível sugerir que os acetiladores rápidos recebem maiores doses de DDS do que os lentos, visto que a concentraçäo bacilar nas lesöes de pacientes com M. leprae sensível à DDS nos acetiladores rápidos foi mais de 17 vezes maior do que nos lentos

In vitro and in vivo interactions of drugs used in multidrug therapy in leprosy.

Interactions of different drugs commonly used in multiple drug therapy were evaluated using both in vitro culture (cell-free as well as macrophage) system and mouse footpad. No additive effects were obtained in the in vitro system when dapsone was combined with either rifampicin or clofazimine, while a strong antagonism was observed when clofazimine was combined with rifampicin but not with rifabutin. In the mouse footpad system, a strong synergism was obtained when clofazimine was combined with either rifampicin or rifabutin, but significant antagonism was observed with the combination of clofazimine and dapsone.

Effect of brodimoprim on Mycobacterium leprae in vitro and in mouse foot-pads.

The new in vitro screening system reported earlier was adopted to determine anti-M. leprae activity of a dihydrofolate reductase inhibitor, brodimoprim, and the results were compared with those obtained using mouse foot-pad technique. Even though the MIC of brodimoprim against M. leprae was very high compared to other commonly used anti-leprosy drugs, in combination with dapsone it showed a remarkable synergistic activity in inhibiting the growth of M. leprae at concentrations much lower than the MICs of each of the drugs used singly. Similar effects were also demonstrated in mouse foot-pad experiments.

An in vitro culture method for screening new drugs against Mycobacterium leprae.

An in vitro culture system has been devised for the maintenance and growth of M. leprae in a cell-free medium. Cells from four-week old cultures could be transferred to fresh medium and normal growth was observed in subcultures. Using this system, the M.I.Cs of dapsone and rifampicin were determined. Dapsone at 25 ng/ml and rifampicin at 300 ng/ml completely inhibited the growth of host-grown as well as in vitro-adapted M. leprae. It was further shown that the effects of both the drugs were bactericidal; this observation was subsequently confirmed using mouse foot-pad technique.

Primary dapsone resistance as assessed by uptake of labelled thymidine by the macrophage resident Mycobacterium leprae.

12 untreated lepromatous leprosy patients were screened for primary dapsone resistance by the uptake of labelled thymidine by macrophage resident M. leprae. There were found to harbour primary dapsone resistant strains of M. leprae and another three partially resistant strains to the drug. This rapid, simple and reliable method should be used routinely to screen leprosy patients, for drug resistance.