Visceral leishmaniasis (VL) is a systemic protozoan disease that is transmitted by phlebotomine sandflies. Poor and neglected populations in East Africa and the Indian sub-continent are particularly affected. Early and accurate diagnosis and treatment remain key components of VL control. In addition to improved diagnostic tests, accurate and simple tests are needed to identify treatment failures. Miltefosine, paromomycin and liposomal amphotericin B are gradually replacing pentavalent antimonials and conventional amphotericin B as the preferred treatments in some regions, but in other areas these drugs are still being evaluated in both mono- and combination therapies. New diagnostic tools and new treatment strategies will only have an impact if they are made widely available to patients.

http://www.bio.umass.edu/micro/klingbeil/590s/Reading/Leishdiag2007.pdf

Visceral leishmaniasis: what are the needs for diagnosis, treatment and control?

The current interest in leishmaniasis stems from the importance of this disease with respect to travel medicine, veterans of Operation Desert Storm, humanitarian concerns, and infection with human immunodeficiency virus. Herein, I review aspects of leishmaniasis that are of practical value to practitioners, including presentation, diagnosis, and chemotherapy; I will emphasize advances in chemotherapy over the last 10 years. Amphotericin B and its new lipid formulations are now competitive with pentavalent antimony as primary therapy for visceral leishmaniasis. Pentamidine, paromomycin, and adjunctive therapy with interferon-gamma are secondary regimens for the treatment of this condition. High-dose long-term regimens of antimony have been shown to be highly effective for the treatment of cutaneous leishmaniasis. Preliminary evidence of efficacy has been observed with short courses of pentamidine for the treatment of Leishmania braziliensis complex disease and topical paromomycin/methylbenzethonium chloride for the treatment of Leishmania major disease.

Human leishmaniasis: clinical, diagnostic, and chemotherapeutic developments in the last 10 years.

The plant hormone abscisic acid (ABA) mediates various responses such as stomatal closure, the maintenance of seed dormancy, and the inhibition of plant growth. All three responses are affected in the ABA-insensitive mutant abi1 of Arabidopsis thaliana, suggesting that an early step in the signaling of ABA is controlled by the ABI1 locus. The ABI1 gene was cloned by chromosome walking, and a missense mutation was identified in the structural gene of the abi1 mutant. The ABI1 gene encodes a protein with high similarity to protein serine or threonine phosphatases of type 2C with the novel feature of a putative Ca2+ binding site. Thus, the control of the phosphorylation state of cell signaling components by the ABI1 product could mediate pleiotropic hormone responses.

https://science.sciencemag.org/content/sci/264/5164/1452.full.pdf

A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana

Over 850 Leishmania-human immunodeficiency virus (HIV) coinfection cases have been recorded, the majority in Europe, where 7 to 17% of HIV-positive individuals with fever have amastigotes, suggesting that Leishmania-infected individuals without symptoms will express symptoms of leishmaniasis if they become immunosuppressed. However, there are indirect reasons and statistical data demonstrating that intravenous drug addiction plays a specific role in Leishmania infantum transmission: an anthroponotic cycle complementary to the zoonotic one has been suggested. Due to anergy in patients with coinfection, L. infantum dermotropic zymodemes are isolated from patient viscera and a higher L. infantum phenotypic variability is seen. Moreover, insect trypanosomatids that are currently considered nonpathogenic have been isolated from coinfected patients. HIV infection and Leishmania infection each induce important analogous immunological changes whose effects are multiplied if they occur concomitantly, such as a Th1-to-Th2 response switch; however, the consequences of the viral infection predominate. In fact, a large proportion of coinfected patients have no detectable anti-Leishmania antibodies. The microorganisms share target cells, and it has been demonstrated in vitro how L. infantum induces the expression of latent HIV-1. Bone marrow culture is the most useful diagnostic technique, but it is invasive. Blood smears and culture are good alternatives. PCR, xenodiagnosis, and circulating-antigen detection are available only in specialized laboratories. The relationship with low levels of CD4+ cells conditions the clinical presentation and evolution of disease. Most patients have visceral leishmaniasis, but asymptomatic, cutaneous, mucocutaneous, diffuse cutaneous, and post-kala-azar dermal leishmaniasis can be produced by L. infantum. The digestive and respiratory tracts are frequently parasitized. The course of coinfection is marked by a high relapse rate. There is a lack of randomized prospective treatment trials; therefore, coinfected patients are treated by conventional regimens. Prophylactic therapy is suggested to be helpful in preventing relapses.

Leishmania and human immunodeficiency virus coinfection: the first 10 years.

Recent epidemiologic studies indicate that leishmaniasis in the Americas is far more abundant and of greater public health importance than was previously recognized. The disease in the New World is caused by a number of different parasite species that are capable of producing a wide variety of clinical manifestations. The outcome of leishmanial infection in humans is largely dependent on the immune responsiveness of the host and the virulence of the infecting parasite strain. This article reviews current concepts of the clinical forms, immunology, pathology, laboratory diagnosis, and treatment of the disease as well as aspects of its epidemiology and control. Recommendations for future research on the disease and its control are made.

Leishmaniases of the New World: current concepts and implications for future research.

We report the cloning of a Leishmania chagasi antigen gene and an evaluation of leishmaniasis patient antibody responses to the recombinant protein, rK39. rK39 contains a 39-amino acid repeat that is part of a 230-kDa protein predominant in L. chagasi tissue amastigotes. Sequence analyses showed this protein, LcKin, to be related to the kinesin superfamily of motor proteins. Southern blot analyses demonstrated LcKin-related sequences in seven species of Leishmania, with conservation of the repeat between L. chagasi and Leishmania donovani. Serological evaluation revealed that 98% (56 of 57) of Brazilian and 100% (52 of 52) of Sudanese visceral leishmaniasis patients have high antibody levels to the rK39 repeat. Detectable anti-K39 antibody was virtually absent in cutaneous and mucosal leishmaniasis patients and in individuals infected with Trypanosoma cruzi. The data show that rK39 may replace crude parasite antigens as a basis for serological diagnosis of visceral leishmaniasis.

/pdf/molecular-characterization-of-a-kinesin-related-antigen-of-35i3yhrmuo.pdf

Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis.

The diagnosis of visceral leishmaniasis (VL), a serious and often fatal parasitic disease caused by members of the Leishmania donovani complex, remains problematic. Current methods rely on clinical criteria, parasite identification in aspirate material, and serology. The latter methods use crude antigen preparations lacking in specificity. A previously described cloned antigen, rK39, of Leishmania specific for all members of the L. donovani complex (L. chagasi, L. donovani, L. infantum) was very useful in the serodiagnosis by ELISA of both human and canine VL. The present study demonstrated that rK39 seroreactivity correlated with active disease. The sera from early or self-healing infected subjects reacted with leishmanial lysate and were generally nonreactive with rK39. These data demonstrate the utility of rK39 in the serodiagnosis of VL and as an indicator of active disease.

/pdf/rk39-a-cloned-antigen-of-leishmania-chagasi-that-predicts-4ak3ka02bu.pdf

rK39: A Cloned Antigen of Leishmania chagasi that Predicts Active Visceral Leishmaniasis

gp63, an abundant and conserved leishmania cell surface protein, has been implicated in the ability of these parasitic protozoa to infect macrophages in vitro and has shown potential as a protective immunogen in mice. However, little is known regarding human immune responses to this glycoprotein Ag. In this study, human T lymphocyte responses to Leishmania amazonensis native gp63 and to recombinant gp63 (rgp63) produced in Escherichia coli were evaluated in individuals with active or cured cutaneous, mucosal or visceral leishmaniasis. Both native and rgp63 elicited strong proliferative responses in all patients tested. In addition, IFN-gamma was produced in response to stimulation with both forms of the protein. T cell lines generated from PBMC by stimulation with native or rgp63 were phenotypically similar, and proliferated and produced IFN-gamma in response to stimulation with both forms of the molecule. These results suggest that gp63 is a strong T cell immunogen and that the recombinant and native forms can elicit the same type of T cell response from infected patients. In order to compare the immunogenic properties of these two forms of gp63, PBMC from naive (uninfected) donors were sensitized in vitro with native or rgp63. T cell lines generated against rgp63 proliferated in response to rgp63, but failed to proliferate in response to native gp63 or to promastigote lysate. Thus, rgp63 was effective in eliciting T cell responses from patients with active or cured leishmania infection, but did not effectively induce T cell responses under the conditions used.

Human T cell responses to gp63, a surface antigen of Leishmania.

A gene sequence encoding an immunodominant protein with a repetitive epitope from the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, was cloned and expressed. The identified 10-amino acid repeat is present within a high-molecular-weight trypomastigote antigen that appears specific to and conserved among T. cruzi isolates. More importantly, greater than 95% of T. cruzi infection sera, including both chronic and acute Chagas disease, contained elevated levels of antibody to a 15-amino acid synthetic peptide bearing the repetitive B-cell epitope. Considering the wide diversity of T. cruzi parasites, as well as the broad spectrum of clinical manifestations of Chagas disease, such a prevalent immune response among patients is significant and applicable to the control of Chagas disease through the diagnosis of T. cruzi infection.

Identification and synthesis of a major conserved antigenic epitope of Trypanosoma cruzi.

A major surface glycoprotein of Leishmania parasites, gp63, is highly conserved among species and is expressed in both infective and intracellular stages. Although much is known about its role in entry and survival in host macrophages, patient antibody responses to this glycoprotein have not been well characterized. The prevalence of anti-gp63 antibody in sera of leishmaniasis patients was evaluated by ELISA. Sera from most acute visceral leishmaniasis patients (84%) from Brazil and Sudan had notably high levels of antibody to recombinant (r) gp63. Sera from other forms of leishmaniasis and from other diseases did not contain significantly elevated levels of anti-gp63 antibody. These results indicate that rgp63 might be a useful constituent of a defined serologic test for visceral leishmaniasis.

J. M. Burns

Papers

Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis.

rK39: A Cloned Antigen of Leishmania chagasi that Predicts Active Visceral Leishmaniasis

Human T cell responses to gp63, a surface antigen of Leishmania.

Identification and synthesis of a major conserved antigenic epitope of Trypanosoma cruzi.

Antibody Responses of Visceral Leishmaniasis Patients to gp63, a Major Surface Glycoprotein of Leishmania Species