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.

Alterações hematológicas, hemostáticas e bioquímicas induzidas pela clofazimina e claritromicina, em doses única e múltiplas, em ratos / Hematological, hemostatic and biochemical alterations induced by clofazimine and clarithromycin, in single and multiple doses, in rats

Claritromicina e clofazimina têm sido utilizadas no tratamento da hanseníase, tuberculose e infecções causadas pelo complexo Mycobacterium avium. Como os dados sobre a toxicidade de esquemas terapêuticos que incluem estes fármacos são escassos, este estudo teve como objetivo determinar os efeitos adversos destas terapias, por meio da avaliação dos parâmetros hematológicos, hemostáticos e bioquímicos. Os fármacos foram administrados em ratos machos Wistar, em monoterapia, em regime de doses única e múltipla. Claritromicina provocou aumento de leucócitos mono e polimorfonucleares. Ambos os fármacos inverteram a proporção entre células mono e polimorfonucleares e provocaram aumento do número de células polimorfonucleares e células em degeneração. Clofazimina e claritromicina prolongaram o tempo de protrombina e claritromicina também prolongou o tempo de tromboplastina parcial ativa. Claritromicina causou aumento de bilirrubinas total e direta e, ambos os fármacos, elevaram os níveis plasmáticos de gama-glutamiltransferase. Portanto, clofazimina e claritromicina induzem alterações hematológicas, hemostáticas e hepáticas.

Clarithromycin and clofazimine have been used to treat leprosy, tuberculosis and infections caused by the Mycobacterium avium complex. Since there is a scarcity of data on the toxicity of therapeutic regimens that include these drugs, this study had the aim of determining the adverse effects of these therapies, through evaluation of hematological, hemostatic and biochemical parameters. The drugs were administered to male Wistar rats, as monotherapy, in regimens of single and multiple doses. Clarithromycin caused increases in the numbers of mononuclear and polymorphonuclear leukocytes. Both of the drugs inverted the proportions between mononuclear and polymorphonuclear cells and increased the numbers of polymorphonuclear cells and degenerating cells. Clofazimine and clarithromycin prolonged the prothrombin time and clarithromycin also prolonged the activated partial thromboplastin time. Clarithromycin caused increases in total and direct bilirubin. Both of the drugs increased the plasma levels of gamma-glutamyltransferase. Therefore, clofazimine and clarithromycin induce hematological, hemostatic and hepatic changes.

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.

Reverse line probe assay for the rapid detection of rifampicin resistance in Mycobacterium leprae.

Mutations in the rpoB gene of 40 biopsy isolates of Mycobacterium leprae were analyzed by reverse hybridization-based line probe assay after PCR, and nine distinct single-nucleotide substitutions were found. Among them, a 3-nucleotide substitution was found in two, and 2-nucleotide substitutions were found in seven isolates. This is a new finding of multiple mutations in a single point of the rpoB gene for rifampicin resistance. This investigation demonstrates that the pattern of mutations in the rpoB gene for rifampicin resistance in Nepal involves more variety.

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.

Assessment of viability by normal mouse foot-pad and bacillary ATP bioluminescence assay in multibacillary cases treated with an MDT regimen using conventional as well as newer drugs like minocycline and ofloxacin.

The therapeutic effect of a drug regimen of conventional drugs as well as newer drugs like ofloxacin and minocycline in smear-positive multibacillary (MB) leprosy cases was assessed by mouse foot-pad and ATP bioluminiscence methods. Biopsies were taken before starting treatment and after one year of treatment. They were processed for viability assessment by normal mouse foot-pad inoculation and bacillary ATP assay techniques. The test regimen was quite effective in its anti-bacterial effect as it was found to result in loss of bacillary viability in all the cases, as assessed by both methods.

Search for newer antileprosy drugs.

In 1991 World Health Organization proclaimed the goal of global elimination of leprosy as a public health problem by year 2000 by implementing multidrug therapy (MDT). Since then the prevalence rate has declined by 85%. However, during the same period the incidence rate of leprosy has remained constant or even has been increasing. This suggests that it will take a long time for the eradication of leprosy and that without in-vitro cultivation of M. leprae, eradication of leprosy is not likely to be achieved. While in-vitro cultivation is a long-term goal, as an immediate measure, there is an urgent need for the development of newer drugs and newer multidrug therapy regimens. Using the in-vitro system for screening potential antileprosy drugs and also using the mouse foot-pad system we have evaluated several compounds in four classes of drugs--dihydrofolate reductase inhibitors, fluoroquinolones, rifampicin analogues and phenazines--and identified at least two compounds that appear to be more potent than dapsone, rifampicin and clofazimine. Newer combinations of rifampicin analogues and fluoroquinolones have also been identified that seem to be better than the combination of rifampicin and ofloxacin.

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.

Ofloxacin containing combined drug regimens in the treatment of multibacillary leprosy.

The results of ofloxacin containing combined drug regimens in the treatment of 60 multibacillary leprosy cases from January 1989 to June 1995 are reported. The objective of the trial is to compare the antileprotic property of ofloxacin and rifampicin in multibacillary leprosy patients and to study the killing rate of M. leprae by ofloxacin and rifampicin before mass treatment can be recommended. The complications and side-effects of ofloxacin and rifampicin were of a mild nature and both drugs were well tolerated. Moderate to marked clinical improvement was noticed in a short period with ofloxacin containing regimens in multibacillary leprosy patients. No persisters were detected in any of the 33 specimens (of mouse footpads) that had been obtained after treatment for 6 months. Ofloxacin if added to the currently used WHO recommended MB-MDT regimen may shorten the duration of treatment. Ofloxacin, therefore, may be considered as a suitable alternative in suspected/proven rifampicin resistant cases and where rifampicin is contraindicated. The results were evaluated on the basis of the clinical conditions, mycobactericidal effectiveness, signs of drug toxicity and side effects.

Comparative in vitro activities of rifamycin analogues against Mycobacterium leprae.

Comparative activities of various rifamycin analogues against leprosy were studied by evaluating their effects on in vitro growth of Mycobacterium leprae in DH medium as described earlier. Among the seven analogues studied, KRM-1648 was found to be the most potent in inhibiting the growth of rifampicin-sensitive strains of M. leprae, MIC being 0.05 microgram/ml. This was followed by KRM-2312 and T9 (MIC of each being 0.1 microgram/ml) and rifabutin (MIC, 0.2 microgram/ml). Rifampicin, along with KRM-1657 and KRM-1668, were least effective, with MIC for each being 0.4 microgram/ml. The effects of each at their respective MICs were bactericidal. The results were similar for rifampicin-resistant strains of M. leprae, but the MICs were higher than those obtained with rifampicin-sensitive strains of M. leprae. Thus, even though rifampicin has been the first-line drug in the treatment of leprosy, the results in present studies suggest that other rifamycin analogues are available that are more potent than rifampicin against both rifampicin-sensitive as well as rifampicin-restraint strains of M. leprae.

In vivo activities of novel benzoxazinorifamycins against Mycobacterium leprae.

Among the four newly synthesized benzoxazinorifamycin derivatives, KRM-1648 and KRM-2312 completely inhibited the multiplication of rifampicin-sensitive as well as rifampicin-resistant strains of M. leprae in the foot-pads of mice. Both were found to be more potent than rifampicin and were bactericidal. In combination with ofloxacin, another potent bactericidal drug against M. leprae, both KRM-1648 and KRM-2312 exhibited synergism. Thus, combination of one of these benzoxazinorifamycin derivatives and ofloxacin in multidrug regimens is worth evaluating in clinical trials.

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.