It is important to realize that leishmaniasis guidelines cannot always account for individual variation among patients. They are not intended to supplant physician judgment with respect to particular patients or special clinical situations. The IDSA and ASTMH consider adherence to these guidelines to be voluntary, with the ultimate determinations regarding their application to be made by the physician in the light of each patient’s individual circumstances.

Leishmania chagasi, the cause of South American visceral leishmaniasis, must survive antimicrobial responses of host macrophages to establish infection. Macrophage oxidative responses have been shown to diminish in the presence of intracellular leishmania. However, using electron spin resonance we demonstrated that murine and human macrophages produce O2− during phagocytosis of opsonized promastigotes. Addition of the O2− scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl to cultures resulted in increased infection, suggesting that O2− enhances macrophage leishmanicidal activity. The importance of NO· produced by inducible NO synthase (iNOS) in controlling murine leishmaniasis is established, but its role in human macrophages has been debated. We detected NO· in supernatants from murine, but not human, macrophages infected with L. chagasi. Nonetheless, the iNOS inhibitor NG-monomethyl-l-arginine inhibited IFN-γ-mediated intracellular killing by both murine and human macrophages. According to RNase protection assay and immunohistochemistry, iNOS mRNA and protein were expressed at higher levels in bone marrow of patients with visceral leishmaniasis than in controls. The iNOS protein also increased upon infection of human macrophages with L. chagasi promastigotes in vitro in the presence of IFN-γ. These data suggest that O2− and NO· each contribute to intracellular killing of L. chagasi in human and murine macrophages.

We examined the role of immunoglobulin (Ig)G antibodies in mediating host defense to the intracellular parasite, Leishmania. We show that IgG not only fails to provide protection against this intracellular pathogen, but it actually contributes to disease progression. The J_H strain of BALB/c mice, which lack IgG because they have a targeted deletion in the Ig heavy chain (J) locus, were more resistant to infection with Leishmania major than were normal BALB/c mice. However, the passive administration of anti-Leishmania IgG caused J_H mice to develop large lesions containing high numbers of parasites. Antibody administration correlated with an increase in interleukin (IL) 10 production in lesions, and blocking the murine IL-10 receptor prevented antibody-mediated disease exacerbation. In human patients with active visceral leishmaniasis, high IgG levels are predictive of disease. Patients with ongoing disease had high IgG antibody titers and no delayed-type hypersensitivity (DTH) responses to Leishmania antigens. This pattern was reversed upon disease resolution after treatment, resulting in a decrease in total IgG, which was accompanied by a progressive increase in DTH responsiveness. We conclude that IgG can cause a novel form of immune enhancement due to its ability to induce IL-10 production from macrophages.

The Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established. We developed a serial qPCR strategy to identify and rapidly differentiate Leishmania species and quantify parasites in clinical or environmental specimens. SYBR green qPCR is mainly employed, with corresponding TaqMan assays for validation. The screening primers recognize kinetoplast minicircle DNA of all Leishmania species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species-discriminating probes with melt curve analysis. The assay was sufficient to detect Leishmania parasites, make species determinations, and quantify Leishmania spp. in sera, cutaneous biopsy specimens, or cultured isolates from subjects from Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kinetoplast DNA (kDNA) probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between Leishmania species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6-fold between Leishmania species, but the differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi- or minicircles. Thus, serial qPCR is useful for leishmania detection and species determination and for absolute quantification when compared to a standard curve from the same Leishmania species.