The ability of Leishmania and Plasmodium parasites to manipulate host immunity and co-inhabit part of the same lymphoid tissues suggests the possibility that the two diseases may interact with each other, when co-occurring in the same host. This is demonstrated for the first time in naturally co-infected patients by the pilot study presented here. Comparative analysis of cytokine profiles from co- and mono-infected patients highlighted substantial variations in the immune response mounted upon co-infection, confirming the ability of L. donovani and P. falciparum to mutually interact at the immunological level. Patients harboring both leishmanial and malarial parasites responded with an overall increase in type-1 and pro-inflammatory cytokine release, which partly reflected the effect elicited by VL (TNF, IFN-γ) and malaria (IL-2), and partly resulted from a synergistic interaction of the two diseases upon each other (IL-17A). Secretion of IL-13 in co-infected patients significantly exceeded the amounts found in VL patients and displayed positive correlations with most of the examined cytokines. Although this trend can be seen as an attempt of the immune system to contain the effects elicited by type-1 and pro-inflammatory cytokines (these patients exhibited some of the highest concentrations of IFN-γ, TNF and IL-17A), the finding remains poorly representative, reflecting the response of 4 patients only, with the remaining co-infected patients (n = 11) displaying negligible levels of IL-13 just as most of VL and malaria patients.
Measurement of IL-17A levels allowed to distinguish the co-infected patients from both VL and malaria mono-infected counterparts, indicating that both diseases synergistically concurred to its up-regulation. Better known for its pro-inflammatory effects in allergic and autoimmune conditions, IL-17 has been recently implicated in the protective immunity towards bacterial, fungal and protozoan infections , where it is thought to mediate recruitment of neutrophils to the epithelial and mucosal surfaces and induce production of antimicrobial peptides [55, 56]. Its release by CD4+ Th17 cells has been associated with resistance to human VL  and positive resolution of murine L. donovani infections , suggesting that Th17 and Th1 cytokines may play complementary roles in parasite clearance. Hence, the increased concentrations of IL-17A found in the co-infected vs. the VL mono-infected cohort, besides the already elevated IFN-γ and IL-12p70, may be indicative of a favorable, possibly improved, prognosis for VL, though the present data do not allow to draw conclusions in this respect. In support of this speculation is the finding of a recent study conducted in Barbar el Fugarra, a Sudanese village situated only a few tens of kilometers away from Tabarak Allah Hospital (where patients in this study were recruited), in which peripheral blood mononuclear cells (PBMCs) isolated from VL-seropositive individuals who did not develop disease at any time during the 6-year survey, secreted significantly higher IL-17 levels when challenged with L. donovani in comparison with VL-seropositive individuals who became symptomatic within 6 months from the evaluation . Interestingly, malaria appeared as the major trigger of this IL-17A up-regulation in co-infected patients, given that no relationship between patients’ demographic and clinical variables and the corresponding IL-17A serum level could be identified in this group (nor in any other group). Expansion of IL-17-producing cells (either CD4+ T cells, CD8+ T cells or macrophages) and related cytokines (IL-17, IL-22 and IL-23) has been observed in P. vivax natural infections  as well as P. berghei, P. chabaudi and P. fragile animal models [60–62], where these interleukins have been shown to reduce parasite intensity and protect against fatal outcomes [59–61]. Conversely, a clear role of IL-17 immunity in P. falciparum infections is yet to be demonstrated. Transcriptional profiling of PBMCs isolated from P. falciparum-infected patients has recently highlighted a Th17/Thαβ driven bias in the immune response mounted against malaria, with up-regulation of several Th17- and neutrophil-related genes [63, 64] and induction of a NK cell-mediated humoral response via interferon α and β . Triggering of this Thαβ immunity, in particular, was shown to inhibit the IL-12 driven Th1 response , necessary for boosting clearance of malaria parasites [29, 65–67], particularly during the pre-erythocytic stage, when cell-mediated immunity is essential for control of infection. If induction of a Th17 response may, therefore, indirectly impair host ability to contain malaria through suppression of macrophage activities, the IFN-γ dominant response elicited by VL may partially compensate for this deficiency and act as a pre-priming stimulus upon Plasmodium infection, for the development of malaria adaptive immunity (via NKT cells, e.g.) and the nitric oxygen-dependent suppression of intra-hepatocytic forms. The above-shown data confirm the leading presence of IFN-γ (P <0.0001), followed by TNF (P <0.05) and IL-4 (P <0.05), in the sera of VL patients as compared with the malaria ones, and clearly identify a shift towards type-1/pro-inflammatory polarization when malaria co-occurred with VL. In addition, a significantly reduced P. falciparum infection intensity was observed among co-infected patients, suggesting improved tolerance of these individuals to the malaria disease. Whether this reduced susceptibility resulted from the VL-driven pre-immune response remains to be demonstrated. The pioneer work of Adler et al. on co-infected hamsters  highlighted a reduced proliferation of P. berghei for effect of the Leishmania infection, supporting the idea of a VL-triggered cross-immunity against malaria, whereas the more recent mouse model data [46–48] seem to suggest the opposite conclusion. It is worthy to note that animals were challenged with blood-stage parasites rather than with sporozoites, bypassing the naturally occurring liver phase against which cellular immunity is most effective and most likely to be developed in response to VL (Leishmania parasites visceralize in the liver, too). Moreover, mice and hamsters are not equally representative models of the VL disease, whose clinico-pathological features in humans are better reproduced by the golden hamster model .
The exploratory nature of this survey implies its design and findings are limited by the small sample sizes and the lack of subject matching between groups, although no significant difference in the distribution of patients’ demographic and clinical features was observed. Diagnosis of VL in clinical suspects was confirmed by serology, according to the national policy, precluding any analysis on parasite loads and their link with cytokine profiles. Assessment of malaria parasitaemia, on the contrary, was performed on peripheral blood films, but the low sensitivity of microscopy observation inevitably limits its reliability as a quantitative assay. Moreover, in the absence of a molecular screening of the recruited individuals, the risk of sub-microscopy malaria infections being carried by the VL patients and/or apparently healthy controls cannot be excluded. Malaria mono- and co-infected patients exhibited different P. falciparum blood parasitaemias. Whether these differences are linked to their particular diagnosis, however, is unknown, as patients were recruited sequentially and discernment between clinical and sub-clinical co-infection cases is not possible if one of the two diseases manifests with symptoms. Therefore, recruitment of asymptomatic, but parasitaemic individuals for each of the two infections may be useful to control for non-homogeneous group-wise comparisons. Absence of pre-existent disorders was based on patient reporting only, with no diagnostic procedure performed, other than those ones aimed to confirm VL or malaria. Finally, longitudinal rather than cross-sectional assessments should be endorsed, as they could help identifying those fundamental associations amongst parasite load, cytokine response and clinical picture which are keys to the interpretation of present data. Similar studies may not only clarify the exact role of the VL-malaria co-infection on P. falciparum proliferation, but they would be pivotal for understanding the clinical implications that arise from the different cytokine profiles.