Evaluation of a new set of recombinant antigens for the serological diagnosis of human and canine visceral leishmaniasis.

Current strategies for the control of zoonotic visceral leishmaniasis (VL) rely on its efficient diagnosis in both human and canine hosts. The most promising and cost effective approach is based on serologic assays with recombinant proteins. However, no single antigen has been found so far which can be effectively used to detect the disease in both dogs and humans. In previous works, we identified Leishmania infantum antigens with potential for the serodiagnosis of VL. Here, we aimed to expand the panel of the available antigens for VL diagnosis through another screening of a genomic expression library. Seven different protein-coding gene fragments were identified, five of which encoding proteins which have not been previously studied in Leishmania and rich in repetitive motifs. Poly-histidine tagged polypeptides were generated from six genes and evaluated for their potential for diagnosis of VL by ELISA (Enzyme Linked ImmunoSorbent Assay) with sera from infected humans and dogs. None of those was valid for the detection of human VL (26-52% sensitivity) although their performance was increased in the canine sera (48-91% sensitivity), with one polypeptide useful for the diagnosis of canine leishmaniasis. Next, we assayed a mixture of three antigens, found to be best for human or canine VL, among 13 identified through different screenings. This "Mix" resulted in similar levels of sensitivity for both human (84%) and canine (88%) sera. With improvements, this validates the use of multiple proteins, including antigens identified here, as components of a single system for the diagnosis of both forms of leishmaniasis.

Serum cytokines associated with severity and complications of kala-azar.

OBJECTIVES: Recent clinical data suggest that severe kala-azar (or visceral leishmaniasis) is an exaggerated innate immune response mediated by inflammatory cytokines, leading to a systemic inflammatory syndrome similar to what is observed in malaria, sepsis and other diseases. We tested this hypothesis by measuring serum cytokines in individuals with kala-azar. METHODS: We compared patients with severe kala-azar (i.e. hemorrhagic manifestations, n = 38) with patients without evidence of hemorrhage (n = 96). We conducted a detailed clinical and laboratory evaluation, measuring serum IL-1beta, IL-6, IL-8, IL-10, IL-12, interferon-gamma, and TNF-alpha, and markers of disseminated intravascular coagulation (DIC). RESULTS: Infants had higher levels of inflammatory cytokines, while HIV-infected patients had lower concentrations of IL-10 and interferon-gamma. Higher levels of IL-6, interferon-gamma, and IL-8 were found among deceased patients. IL-8 and interferon-gamma were independently associated with bleeding. Several cytokines were associated with different signs of severe clinical and laboratory manifestations, including DIC. IL-6 was highly positively and independently associated with IL-1beta, IL-8, IL-10, and negatively associated with TNF-alpha. IL-1beta and TNF-alpha were also highly independently associated with disease severity. CONCLUSION: In its severe form, kala-azar, a neglected tropical disease, initiates a systemic inflammatory response that leads to DIC and other manifestations. Children may have higher risk of death due to the more intense cytokine release. The data supports the notion that IL-6 is the central cytokine that is associated with lethal disease, but interferon-gamma, IL1beta, IL-8, and TNF-alpha are also involved with disease severity. Inhibition of IL-6 is a potential target of adjuvant therapy for severe or pediatric forms of this disease.