Clofazimine has delayed antimicrobial activity against Mycobacterium tuberculosis both in vitro and in vivo.

OBJECTIVES: The anti-leprosy drug clofazimine has been shown to have antimicrobial activity against Mycobacterium tuberculosis and has been associated with treatment-shortening activity in both clinical and preclinical studies of TB chemotherapy. However, a reported lack of early bactericidal activity (EBA) in TB patients has raised questions regarding the usefulness of clofazimine as an anti-TB drug. Our objective was to systematically evaluate the EBA of clofazimine in vitro and in vivo to provide insight into how and when this drug exerts its antimicrobial activity against M. tuberculosis. METHODS: We evaluated the 14 day EBA of clofazimine (i) in vitro at concentrations ranging from 4 times below to 4 times above the MIC for M. tuberculosis and (ii) in vivo in infected BALB/c mice at doses ranging from 1.5 to 100 mg/kg/day, and serum clofazimine levels were measured. In both experiments, isoniazid was used as the positive control. RESULTS: In vitro, clofazimine, at any concentration tested, did not exhibit bactericidal activity during the first week of exposure; however, in the second week, it exhibited concentration-dependent antimicrobial activity. In vivo, clofazimine, at any dose administered, did not exhibit bactericidal activity during the first week, and limited antimicrobial activity was observed during the second week of administration. While serum clofazimine levels were clearly dose dependent, the antimicrobial activity was not significantly related to the dose administered. CONCLUSIONS: Our data suggest that clofazimine's delayed antimicrobial activity may be due more to its mechanism of action rather than to host-related factors.

Clofazimine shortens the duration of the first-line treatment regimen for experimental chemotherapy of tuberculosis.

A key drug for the treatment of leprosy, clofazimine has recently been associated with highly effective and significantly shortened regimens for the treatment of multidrug-resistant tuberculosis (TB). Consequently, we hypothesized that clofazimine may also shorten the duration of treatment for drug-susceptible TB. We conducted a controlled trial in the mouse model of TB chemotherapy comparing the activity of the 6-mo standard regimen for TB treatment, i.e., 2 mo of daily rifampin, isoniazid, pyrazinamide, and ethambutol followed by 4 mo of rifampin and isoniazid, with a 4-mo clofazimine-containing regimen: 2 mo of daily rifampin, isoniazid, pyrazinamide, and clofazimine followed by 2 mo of rifampin, isoniazid, and clofazimine. Treatment efficacy was assessed on the basis of Mycobacterium tuberculosis colony counts in the lungs and spleens during treatment and on the proportion of mice with culture-positive relapse 6 mo after treatment cessation. No additive effect of clofazimine was observed after the first week of treatment, but, by the second week of treatment, the colony counts were significantly lower in the clofazimine-treated mice than in the mice receiving the standard regimen. Lung culture conversion was obtained after 3 and 5 mo in mice treated with the clofazimine-containing and standard regimens, respectively, and relapse-free cure was obtained after 3 and 6 mo of treatment with the clofazimine-containing and standard regimens, respectively. Thus, clofazimine is a promising anti-TB drug with the potential to shorten the duration of TB chemotherapy by at least half (3 mo vs. 6 mo) in the mouse model of TB.

Microbiological, histological, immunological, and toxin response to antibiotic treatment in the mouse model of Mycobacterium ulcerans disease.

Mycobacterium ulcerans infection causes a neglected tropical disease known as Buruli ulcer that is now found in poor rural areas of West Africa in numbers that sometimes exceed those reported for another significant mycobacterial disease, leprosy, caused by M. leprae. Unique among mycobacterial diseases, M. ulcerans produces a plasmid-encoded toxin called mycolactone (ML), which is the principal virulence factor and destroys fat cells in subcutaneous tissue. Disease is typically first manifested by the appearance of a nodule that eventually ulcerates and the lesions may continue to spread over limbs or occasionally the trunk. The current standard treatment is 8 weeks of daily rifampin and injections of streptomycin (RS). The treatment kills bacilli and wounds gradually heal. Whether RS treatment actually stops mycolactone production before killing bacilli has been suggested by histopathological analyses of patient lesions. Using a mouse footpad model of M. ulcerans infection where the time of infection and development of lesions can be followed in a controlled manner before and after antibiotic treatment, we have evaluated the progress of infection by assessing bacterial numbers, mycolactone production, the immune response, and lesion histopathology at regular intervals after infection and after antibiotic therapy. We found that RS treatment rapidly reduced gross lesions, bacterial numbers, and ML production as assessed by cytotoxicity assays and mass spectrometric analysis. Histopathological analysis revealed that RS treatment maintained the association of the bacilli with (or within) host cells where they were destroyed whereas lack of treatment resulted in extracellular infection, destruction of host cells, and ultimately lesion ulceration. We propose that RS treatment promotes healing in the host by blocking mycolactone production, which favors the survival of host cells, and by killing M. ulcerans bacilli.

BCG-mediated protection against Mycobacterium ulcerans infection in the mouse.

BACKGROUND: Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) is widely used to reduce the risk of childhood tuberculosis and has been reported to have efficacy against two other mycobacterial diseases, leprosy and Buruli ulcer caused by M. ulcerans (Mu). Studies in experimental models have also shown some efficacy against infection caused by Mu. In mice, most studies use the C57BL/6 strain that is known to develop good cell-mediated protective immunity. We hypothesized that there may be differences in vaccination efficacy between C57BL/6 and the less resistant BALB/c strain. METHODS: We evaluated BCG vaccine efficacy against challenge with ∼3×10(5)M. ulcerans in the right hind footpad using three strains: initially, the Australian type strain, designated Mu1617, then, a Malaysian strain, Mu1615, and a recent Ghanaian isolate, Mu1059. The latter two strains both produce mycolactone while the Australian strain has lost that capacity. CFU of both BCG and Mu and splenocyte cytokine production were determined at intervals after infection. Time to footpad swelling was assessed weekly. PRINCIPAL FINDINGS: BCG injection induced visible scars in 95.5% of BALB/c mice but only 43.4% of C57BL/6 mice. BCG persisted at higher levels in spleens of BALB/c than C57BL/6 mice. Vaccination delayed swelling and reduced Mu CFU in BALB/c mice, regardless of challenge strain. However, vaccination was only protective against Mu1615 and Mu1617 in C57BL/6 mice. Possible correlates of the better protection of BALB/c mice included 1) the near universal development of BCG scars in these mice compared to less frequent and smaller scars observed in C57BL/6 mice and 2) the induction of sustained cytokine, e.g., IL17, production as detected in the spleens of BALB/c mice whereas cytokine production was significantly reduced, e.g., IL17, or transient, e.g., Ifnγ, in the spleens of C57BL/6 mice. CONCLUSIONS: The efficacy of BCG against M. ulcerans, in particular, and possibly mycobacteria in general, may vary due to differences in both host and pathogen.

Rifapentine, moxifloxacin, or DNA vaccine improves treatment of latent tuberculosis in a mouse model.

RATIONALE: Priorities for developing improved regimens for treatment of latent tuberculosis (TB) infection include (1) developing shorter and/or more intermittently administered regimens that are easier to supervise and (2) developing and evaluating regimens that are active against multidrug-resistant organisms. OBJECTIVES AND METHODS: By using a previously validated murine model that involves immunizing mice with Mycobacterium bovis bacillus Calmette-Guérin to augment host immunity before infection with virulent Mycobacterium tuberculosis, we evaluated new treatment regimens including rifapentine and moxifloxacin, and assessed the potential of the Mycobacterium leprae heat shock protein-65 DNA vaccine to augment the activity of moxifloxacin. MEASUREMENTS: Quantitative spleen colony-forming unit counts, and the proportion of mice with culture-positive relapse after treatment, were determined. MAIN RESULTS: Three-month, once-weekly regimens of rifapentine combined with either isoniazid or moxifloxacin were as active as daily isoniazid for 6-9 mo. Six-month daily combinations of moxifloxacin with pyrazinamide, ethionamide, or ethambutol were more active than pyrazinamide plus ethambutol, a regimen recommended for latent TB infection after exposure to multidrug-resistant TB. The combination of moxifloxacin with the experimental nitroimidazopyran PA-824 was especially active. Finally, the heat shock protein-65 DNA vaccine had no effect on colony-forming unit counts when given alone, but augmented the bactericidal activity of moxifloxacin. CONCLUSIONS: Together, these findings suggest that rifapentine, moxifloxacin, and, perhaps, therapeutic DNA vaccination have the potential to improve on the current treatment of latent TB infection.

Newer drugs in leprosy

During the last 15 years, new drugs active against Mycobacterium leprae have been identified. All of them belong to the fluoroquinolone, cycline and macrolide drug families. In the mouse model and in humans, minocycline, ofloxacin, and clarithromycin have demonstrated, individually or in combination, antileprosy activities much superior to those of the standard drugs dapsone and clofazimine. In humans, a single dose of the combination ofloxacin 400 mg + minocycline 100 mg was able to kill 68% to 98% of viable M. leprae and a single dose of ROM, a three-drug combination of rifampin 600 mg + ofloxacin 400 mg + minocycline 100 mg, was killing more than 99% of viable M. leprae. As a result of a double-blind, control, clinical trial, the Seventh Report of the WHO Expert Committee on Leprosy recommended in 1997 the use of single-dose ROM for the treatment of patients with single-lesion paucibacillary leprosy. Recently moxifloxacin, a new fluoroquinolone, and rifapentine, a long-lasting rifamycin derivative, have demonstrated in the mouse model highly promising antileprosy activities, justifying their assessment in humans.

Study of 39 documented relapses of multibacillary leprosy after treatment with rifampin

Among 39 strains of Mycobacterium leprae isolated from patients with multibacillary leprosy who relapsed after treatment with rifampin (RMP), 22 strains were resistant to RMP and 17 were susceptible. All of the RMP-resistant strains were recovered from patients who had been treated with more than 50 doses of RMP, usually given as monotherapy. RMP-susceptible strains were recovered from only six patients who had received more than 50 doses of RMP, and from 11 patients who had received no more than seven doses. The median time to relapse after the beginning of RMP therapy was 9 years (range 1-12 years) among the patients harboring RMP-resistant strains of M. leprae, and the median time to relapse after discontinuation of RMP treatment was 7 years (range 1-11 years) among the patients harboring RMP-susceptible strains. These data suggest that monotherapy with more than a few doses of RMP can be responsible for the emergence of RMP-resistant strains of M. leprae, thus emphasizing the need to employ RMP only in combination with other effective drugs in the chemotherapy of multibacillary leprosy

Epidemiología de la lepra en Guadalupe de 1970 a 1983 / The epidemiology of leprosy in Guadeloupe from 1970 to 1983

Se estudiaron la prevalencia y la evolución de la incidencia anual de lepra en Guadalupe entre 1970 y 1983. El análisis de los datos acopiados en el servicio de control de lepra indicó que en 1981 la prevalencia era de 380 por cada 100 000 habitantes, calculada según el número de enfermos incluidos en el archivo activo del registro departamental durante 12 años en el caso de enfermos paucibacilares y durante toda la vida en el caso de enfermos multibacilares. De 1970 a 1983 la incidencia anual disminuyó de 24,0 a 13,0 por 100 000 habitantes. La disminución fue más importante en las formas de lepra paucibacilar que en las de lepra multibacilar, y mucho más importante en menores que en mayores de 15 años. El análisis de la forma de detección reveló que 80% de los enfermos se encontraron mediante la búsqueda pasiva (enfermos sintomáticos que acudieron a consulta), 10% mediante la búsqueda entre la población escolar y 10% mediante la búsqueda entre los contactos domiciliarios de enfermos conocidos. A partir de 1980 se inoculó a ratones con Mycobacterium leprae provenientes de biopsias de enfermos multibacilares para efectuar cultivos y estudiar la sensibilidad del microorganismo a la dapsona y a la rifampicina. Las 16 cepas de M. leprae provenientes de pacientes que padecían recidivas de lepra multibacilar demonstraron ser resistentes a la dapsona, y 15 de estas pusieron de manifiesto una elevada resistencia. De las 19 cepas de M. leprae provenientes de casos nuevos que nunca habían recebido tratamiento, solo ocho eran sensibles a la dapsona. Todas las cepas de M. leprae, tanto de pacientes con recidivas como de casos nuevos, resultaron ser sensibles a la rifampicina. En términos generales, los diferentes parámetros considerados indican que desde el punto devista epidemiológico la lepra ha tenido una evolución favorable en Guadalupe. Por ende, se deben mantener la infraestructura y la organización actual del control de lepra, aunque la frecuencia de la resistencia a la dapsona impone una utilización estricta de la poliquimioterapia