In vitro activity of ciprofloxacin, sparfloxacin, ofloxacin, amikacin and rifampicin against Ghanaian isolates of Mycobacterium ulcerans.

MICs of ciprofloxacin, sparfloxacin, ofloxacin, amikacin and rifampicin were determined for 14 primary clinical isolates and three reference isolates of Mycobacterium ulcerans by modifying a standard agar dilution method for testing Mycobacterium tuberculosis sensitivity. All these antimicrobials were active against every isolate of M. ulcerans. Sparfloxacin exhibited the highest activity and ofloxacin was the least effective. Rifampicin exhibited the broadest range of activity.

Immunoglobulin subclass distribution and diagnostic value of Leishmania donovani antigen-specific immunoglobulin G3 in Indian kala-azar patients.

Visceral leishmaniasis, or kala-azar, a fatal tropical disease, remains problematic, as early diagnosis is difficult and treatment often results in drug resistance and relapse. We have developed a sensitive enzyme-linked immunosorbent assay (ELISA), using leishmanial membrane antigenic extracts (LAg) to detect specific antibody responses in 25 untreated Indian visceral leishmaniasis patients. To investigate the pathogenetic significance of isotype markers in kala-azar, relative levels of specific immunoglobulin G (IgG), IgM, IgA, IgE, and IgG subclasses were analyzed under clinically established diseased conditions. Since LAg showed higher sensitivity for specific IgG than lysate, the immunoglobulin isotype responses were evaluated, with LAg as antigen. Compared to 60 controls, which included patients with malaria, tuberculosis, leprosy, and typhoid and healthy subjects, visceral leishmaniasis patients showed significantly higher IgG (100% sensitivity, 85% specificity), IgM (48% sensitivity, 100% specificity), and IgE (44% sensitivity, 98.3% specificity) responses. Low levels of IgA in visceral leishmaniasis patients contrasted with a 13-fold-higher reactivity in sera from patients with leprosy. Among IgG subclasses, IgG1, -3, and -4 responses were significantly higher in visceral leishmaniasis patients than in the controls. IgG2 response, however, was significantly higher (twofold) in leprosy than even visceral leishmaniasis patients. The rank orders for sensitivity (IgG = IgG1 = IgG3 = IgG4 > IgG2 > IgM > IgE > IgA) and specificity (IgM = IgG3 > IgE > IgG4 > IgG2 > IgG > IgG1 > IgA) for LAg-specific antibody responses suggest the potentiality of IgG3 as a diagnostic marker for visceral leishmaniasis.

Experimental evaluation of possible new short-term drug regimens for treatment of multibacillary leprosy.

Groups of nude mice, with both hind footpads infected with 10(8) Mycobacterium leprae organisms, were treated with 4-week courses of different drug combinations. The effect treatment on each group was evaluated by subinoculating footpad homogenates from the treated mice into groups of normal and nude mice for subsequent regrowth, assessed 1 year later. A combination of rifampin (RMP) with clarithromycin (CLARI), minocycline (MINO), and ofloxacin (OFLO) resulted in the complete killing of M. leprae after 3 weeks of treatment. A combination of sparfloxacin (SPAR) and RMP also resulted in a similar bactericidal effect after 3 weeks of treatment. Other drug combinations showed variable effects. Very little or no effect was observed with any regimen if the treatment was given for less than 2 weeks. World Health Organization (WHO) multidrug therapy (MDT) given for 8 weeks was as effective as the two combinations described above. The results suggest that multidrug combinations consisting of RMP-OFLO (or SPAR)-CLARI (and/or MINO) are as effective as the WHO MDT for the treatment of experimental leprosy. Moreover, they imply that these combinations, which were found to be active in a 4-week experimental treatment protocol, could be administered as treatment to patients for a period of time shorter than the present 2-year regimen without a loss of effectiveness.

Activity of sparfloxacin against Mycobacterium leprae measured by the proportional bactericidal test.

The activity of 25 mg/kg and 50 mg/kg sparfloxacin was measured against Mycobacterium leprae in normal (immunocompetent) mouse foot pads by the proportional bactericidal test. This was compared with the action of 25, 50, and 150 mg/kg ofloxacin by the same method. Sparfloxacin, at both concentrations, was found to be strongly bactericidal by this method, comparable to 150 mg/kg ofloxacin.

An experimental study to evaluate the bactericidal activity of ofloxacin against an established Mycobacterium leprae infection.

The bactericidal effect of a new quinolone, ofloxacin (OFLO), was determined on an established Mycobacterium leprae infection in nude mice. Various drug regimens, including combinations of drugs, were examined for different treatment periods. OFLO and rifampin (RMP) individually failed to produce significant killing after treatment with a single large dose. However, when single large doses of OFLO and RMP were given in combination, a 100-fold reduction in viability was achieved. For a longer period of treatment both of these drugs, at lower doses, produced a moderate reduction in viability. The addition of dapsone to the lower dose of OFLO resulted in a significant reduction in viability, while lower doses of RMP and OFLO together produced a moderate reduction in viability.

Effect of simultaneous administration of interferon-gamma and chemotherapy against Mycobacterium leprae in experimental infection in nude mice.

The possibility of synergy between immunotherapy with recombinant interferon-gamma (IFN-gamma) and chemotherapy with rifampin (RMP) and dapsone (DDS) against Mycobacterium leprae was examined in nude mice. IFN-gamma alone failed to show any effect on the growth of M. leprae in the nude mouse foot pad. No synergy was demonstrable between DDS, either at 0.0001% or at 0.001%, and IFN-gamma. A subinhibitory level of RMP with IFN-gamma was also ineffective, but RMP at 0.006% with IFN-gamma produced a statistically significant enhancement of killing (26-fold) when compared with RMP at 0.006% only. It should be emphasized, however, that results obtained in the immunodeficient nude mouse model may not be comparable to those which might have been given by lepromatous leprosy patients.

Time-related decrease of substance P and CGRP in central and peripheral projections of sensory neurones in Mycobacterium leprae infected nude mice: a model for lepromatous leprosy in man.

We have previously shown the depletion of cutaneous calcitonin gene-related peptide (CGRP)- and substance P-containing nerves in human leprosy. The aims of this study were to investigate the temporal effects of leprosy on nerves in skin and spinal cord. Tissues were taken from nude mice, 6 and 12 months after inoculation of Mycobacterium leprae into the hind footpads, and from age-matched controls. Sections were immunostained with antisera to substance P or CGRP. After 6 months of infection, substance P- and CGRP-immunoreactive nerves were reduced in skin from all body areas; by 12 months, the reduction was substantially greater. In the spinal cord, sensory fibres immunoreactive for substance P had decreased compared with controls at 6 and 12 months [by 60 per cent (0.022 mm2) and 80 per cent (0.048 mm2), respectively, P less than 0.001], as with CGRP [30 per cent (0.018 mm2) (P less than 0.02) and 40 per cent (0.028 mm2) (P less than 0.01), respectively]. CGRP immunoreactivity was completely absent in motor neurones after 12 months of infection. Loss of CGRP- and substance P-immunoreactive fibres in skin and spinal cord, and CGRP in motor neurones is in accord with impaired pain sensation and muscle weakness in leprosy.

Hydrogen peroxide and superoxide production by peripheral blood monocytes in leprosy.

Susceptibility to infection with Mycobacterium leprae, the causative organism of leprosy, is the result of a defect in cell-mediated immunity (CMI). The co-operation of macrophages and T lymphocytes is known to be essential for competent CMI response. In this study we have examined peripheral blood monocytes from a range of leprosy patients in an attempt to identify a possible defect in macrophage function. The ability of these cells to produce hydrogen peroxide and superoxide, two bactericidal metabolites of the monocyte/macrophage, has been measured. Monocytes from leprosy patients were found to be capable of producing normal amounts of hydrogen peroxide and superoxide, and no differences in production were found between tuberculoid, lepromatous and control monocytes. These results suggest that macrophages in leprosy are competent, and that probably a T lymphocyte defect contributes to susceptibility to this disease.

Susceptibility of Mycobacterium leprae to the bactericidal activity of mouse peritoneal macrophages and to hydrogen peroxide.

Macrophages from athymic nude mice were infected in vitro with Mycobacterium leprae to study the intracellular fate of this organism. Using the proportional bactericidal test, we have shown that the viability of M. leprae declines rapidly within these macrophages, although results of clearance experiments demonstrate that live and killed organisms are cleared at comparable rates. We have also shown that M. leprae is susceptible to the bactericidal effects of hydrogen peroxide and we suggest that hydrogen peroxide generated by macrophages is responsible for the killing of intracellular M. leprae.

Macrophage activity in Mycobacterium leprae infection.

The outcome of an M. leprae infection is likely to depend upon the balance between the invading organism and the host's immune response. Macrophages are known to play a major role in this response and because M. leprae is an intracellular parasite, being found commonly in the macrophages of infected hosts, we have attempted to examine the macrophage/M. leprae relationship. Our model has been the athymic nude mouse which has been shown to be susceptible to lepromatous infection but whose macrophages when cultured in vitro actually kill phagocytosed M. leprae. We have shown that in vitro this killing effect is probably mediated, at least to some extent, by macrophage-generated hydrogen peroxide. Further, we have examined macrophages from nude and normal mice at various stages of M. leprae infection in time of their ability to produce hydrogen peroxide and superoxide. It would appear from our results that activation of macrophages to produce these two bactericidal metabolites increases with increasing bacterial load. However, it would seem that T-cell mediated mechanisms are also required for effective control of infection as the hyperactive macrophages seen in the nude mouse are unable to control M. leprae growth in contrast to the limited infection seen in normal mice.

Attempts at cultivation of Mycobacterium leprae in macrophages from susceptible animal hosts.

Macrophages from nude mice, nude rats, and armadillos were cultured in vitro and examined for their ability to support Mycobacterium leprae. No significant growth of this organism were observed after over 200 days of culture. No significant benefit was derived from modifying culture conditions or from variations in the source of macrophages or the source of M. leprae.

Effect of rifampin, clofazimine, and B1912 on the viability of Mycobacterium leprae in established mouse footpad infection.

Continuous dietary administration of rifampin to mice with an established Mycobacterium leprae footpad infection reduced the bacillary solid ratio, with an estimated survival half-life of 5-6 days. In rifampin-treated immunosuppressed animals the survival half-life of solid bacilli, in the absence of host immunity, was 12-13 days. Clofazimine and B1912 produced a significant effect on solid ratio only after a lag period of apparently 100 days. The rate of action was considerably slower than that of rifampin. Intermittent (once monthly) administration of both drugs produced effects similar to those of continuous administration.

Effect of rifampin, clofazimine, and B1912 on the viability of Mycobacterium leprae in established mouse footpad infection

Continuous dietary administration of rifampin to mice with an established Mycobacterium leprae footpad infection reduced the bacillary solid ratio, with an estimated survival half-life of 5-6 days. In rifampin-treated immunosuppressed animals the survival half-life of solid bacilli, in the absence of host immunity, was 12-13 days. Clofazimine and B1912 produced a significant effect on solid ratio only after a lag period of apparently 100 days. The rate of action was considerably slower than that of rifampin. Intermittent (once monthly) administration of both drugs produced effects similar to those of continuous administration.