Chemotherapy-associated changes of histopathological features of Mycobacterium ulcerans lesions in a Buruli ulcer mouse model.

Combination chemotherapy with rifampin and streptomycin (RIF-STR) for 8 weeks is currently recommended by the WHO as the first-line treatment for Mycobacterium ulcerans infection (Buruli ulcer). To gain better insight into the mode of action of these antibiotics against established M. ulcerans infection foci and to characterize recovery of local immune responses during chemotherapy, we conducted a detailed histopathological study of M. ulcerans-infected and RIF-STR-treated mice. Mice were inoculated with M. ulcerans in the footpad and 11 weeks later treated with RIF-STR. Development of lesions during the first 11 weeks after infection and subsequent differences in disease progression between RIF-STR-treated and untreated mice were studied. Changes in histopathological features, footpad swelling, and number of CFU were analyzed. After inoculation with M. ulcerans, massive infiltrates dominated by polymorphonuclear leukocytes developed at the inoculation site but did not prevent bacterial multiplication. Huge clusters of extracellular bacteria located in large necrotic areas and surrounded by dead leukocytes developed in the untreated mice. Chemotherapy with RIF-STR led to a rapid drop in CFU associated with loss of solid Ziehl-Neelsen staining of acid-fast bacilli. Development of B-lymphocyte clusters and of macrophage accumulations surrounding the mycobacteria demonstrated the resolution of local immune suppression. Results demonstrate that the experimental M. ulcerans mouse infection model will be a valuable tool to investigate efficacy of new treatment regimens and of candidate vaccines.

Effectiveness of quinine monotherapy for the treatment of Plasmodium falciparum infection in pregnant women in Lambaréné, Gabon.

Pregnant women participating in a longitudinal immuno-epidemiologic survey in Lambaréné, Gabon, and presenting with Plasmodium falciparum parasitemia at monthly blood smear examinations were offered treatment with oral 7-day quinine monotherapy according to national health guidelines. A total of 50 pregnant women were offered 7-day oral quinine sulfate 10 mg/kg thrice daily. Clinical examinations and laboratory tests were performed on Days 28 and 56 to assess the effectiveness of this standard regimen. By Day 28, the effectiveness of the 7-day quinine regimen was 60% (95% confidence interval: 46-72%). We conclude that a 7-day course of quinine has a poor effectiveness and that alternative treatment regimens for malaria in pregnant women should be assessed.

Immunosuppression and treatment-associated inflammatory response in patients with Mycobacterium ulcerans infection (Buruli ulcer).

Buruli ulcer is a necrotizing skin disease caused by Mycobacterium ulcerans. Major necrosis with abundant clusters of extracellularly replicating mycobacteria and only minor leukocyte infiltration are characteristic histopathologic features of the disease. Mycolactone, a cytotoxic macrolide exotoxin of M. ulcerans, plays a key role in the development of this pathology. Antimicrobial therapy, such as rifampicin/streptomycin that was recently introduced, seems to lead to phagocytosis of mycobacteria and massive leukocyte infiltration, which culminates in the development of ectopic lymphoid structures in the lesions. Whereas the curative effect of the antibiotic treatment may be supported by immune defense mechanisms, persisting mycobacterial antigens and immunostimulators occasionally also seem to cause apparent reactivation of the disease. This seems to be related to excessive immunostimulation rather than to incomplete killing of the pathogen.

Development of highly organized lymphoid structures in Buruli ulcer lesions after treatment with rifampicin and streptomycin.

BACKGROUND: Buruli ulcer caused by Mycobacterium ulcerans is an infection of the subcutaneous tissue leading to chronic necrotising skin ulcers. The pathogenesis is associated with the cytocidal and immunosuppressive activities of a macrolide toxin. Histopathological hallmark of progressing disease is a poor inflammatory response despite of clusters of extracellular bacilli. While traditionally wide excision of the infected tissue was the standard treatment, provisional WHO guidelines now recommend an eight week pre-treatment with streptomycin and rifampicin. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a detailed immunohistochemical analysis of tissue samples from Buruli patients who received antibiotic treatment. Cellular immune response along with bacterial load and distribution were monitored. We demonstrate that this treatment leads to the development of highly organized cellular infiltration surrounding areas of coagulative necrosis. Diffuse infiltrates, granulomas and dense lymphocyte aggregation close to vessels were observed. Mycobacterial material was primarily located inside mononuclear phagocytes and microcolonies consisting of extracellular rod-shaped mycobacteria were no longer found. In observational studies some patients showed no clinical response to antibiotic treatment. Corresponding to that, one of five lesions analysed presented with huge clusters of rod-shaped bacilli but no signs of infiltration. CONCLUSIONS/SIGNIFICANCE: Results signify that eight weeks of antibiotic treatment reverses local immunosuppression and leads to an active inflammatory process in different compartments of the skin. Structured leukocyte infiltrates with unique signatures indicative for healing processes developed at the margins of the lesions. It remains to be analysed whether antibiotic resistance of certain strains of M. ulcerans, lacking patient compliance or poor drug quality are responsible for the absent clinical responses in some patients. In future, analysis of local immune responses could serve as a suitable surrogate marker for the efficacy of alternative treatment strategies.

Local activation of the innate immune system in Buruli ulcer lesions.

Buruli ulcer (BU) caused by Mycobacterium ulcerans is a chronic necrotizing disease of the skin and the underlying soft tissue. Fat tissue necrosis accompanied by minimal inflammation is considered the most reliable histopathologic feature of BU. There may be a constant influx of inflammatory cells to the sites of active infection but these are thought to be killed by mycolactone, a polyketide toxin produced by M. ulcerans, through apoptosis and necrosis. Here we describe the spatial correlations between mycobacterial load and the expression of dendritic cell (DC) surface markers (cluster of differentiation (CD)83, CD11c, and CD123), the Toll-like receptor (TLR) 9 and pro- and anti-inflammatory cytokines (IL-8, IL-6, tumor necrosis factor-alpha (TNF-alpha), IFN-alpha, IL-12p40, IL-10, and IFN-gamma) within BU lesions. Although IL-8, IL-6, and TNF-alpha messenger RNA (mRNA) was detectable by real-time PCR in all lesions, the expression of the other cytokines was only found as small foci in some lesions. Correlations of the distribution of mRNA encoding the activation marker CD83 and the DC subset markers CD123 and CD11c indicate that both activated plasmacytoid and myeloid dendritic cells were present in the lesions. Results suggest that M. ulcerans specific immune responses may develop once therapeutic interventions have limited the production of mycolactone.