**4. Immunology**

Asymptomatic cases differ considerably from VL patients, and it is assumed that a mixed profile is crucial not only for the management of parasite replication but also for the preservation of these people' immune state. The increased number of cells expressing different cytokines demonstrates this. However, in VL-endemic areas, the clinical form is frequently asymptomatic, followed by protective immunity with a predominant type 1 T-cell response [50]. Asymptomatic seemed to have mixed profile having an increase of IFN-γ + neutrophils/eosinophils and NK cells, of IL-12+ eosinophils/monocytes, along with increase of IL-4+ neutrophils and NK cells and IL-10+ eosinophils/monocytes [51]. Despite earlier findings of a constant type 1 T-lymphocyte immune response during asymptomatic VL it was recently shown that asymptomatic patients' PBMC generated significant amounts of IL-10 when stimulated with *L. infantum* recombinant antigens [52].

#### *Visceral Leishmaniasis: Asymptomatic Facts DOI: http://dx.doi.org/10.5772/intechopen.101109*

Different findings point to the idea that IL-10 is a key immunomodulator in asymptomatic people, dampening host defense mechanisms and favoring immune response regulation following parasite elimination. Furthermore, CD4+ T cells from asymptomatic patients infected with *L. infantum* have been shown to generate significant amounts of IL-5 [53]. In immunocompetent people, leishmania infection is typically asymptomatic, although the percentage of HIV+ people infected with the parasite who stay asymptomatic is unknown. HIV+ individuals might still have a Th1-type cellular response to Leishmania despite their weakened immune system. These people may be identified using cytokine release tests, which identify IFN-γ in the supernatants of SLA-stimulated PBMC and IFN-γ and IL-2 in SLA-stimulated whole blood. These biomarkers appear to be 100% reliable for detecting asymptomatic immune responders to Leishmania among HIV+ patients [33]. Analyses of cytokine responses in symptomatic and asymptomatic VL patients' peripheral blood mononuclear cells (PBMCs) indicated that the production of Th17 cytokines was highly linked with the asymptomatic status [54, 55]. It was discovered by Carvalho et al. that peripheral blood mononuclear cells (PBMCs) from people with subclinical or asymptomatic infection (positive serology and skin test for Leishmania antigen) react to stimulation with Leishmania antigen by producing IL-2, IFN- and IL-12 [50]. An Indian study found that active disease elicited a mixed IFN-/IL-10 response, but asymptomatic infections (IFN- release assay [IGRA]–positive endemic healthy controls) did not trigger an antigen-driven whole-blood IL-10 response [56]. Surprisingly, the frequency of CD4+ T cells is higher in people with asymptomatic infections who have positive LST [57], Furthermore high levels of IFN- are produced by CD8+ T cells isolated from asymptomatic patients, which implies that CD8+ cells play a role in human resistance to Leishmania infection. Furthermore, researchers discovered that in asymptomatic people, a distinct subpopulation of CD4+ cells that produced both IFN- and IL-5 was important for infection management [22, 53]. A longitudinal study conducted in Sudan recently suggested that Th17 cells may play a protective role in human VL, and it was found that *L. donovani* stimulates the production of IL-17 and IL-22 by exposed PBMCs from healthy and resistant subjects who did not develop VL before or after cytokine response testing. In addition, elevated levels of IFN-γ, C reactive protein, nitric oxide, and IL-12 in the blood have also been reported to offer resistance in asymptomatic patients [58]. Disease resistant endemic individuals have an immune response that shields them against pathogenesis in response to an insect bite which are quite similar to those of VL immunology, and as a result, they cannot be utilized to provide a clear picture of protective immune parameters in asymptomatic patients. Besides the protective immune response, it appears that these people either possess a large number of long-lived memory B cells that continuously secrete antibodies or they are continually exposed to *Leishmania* but do not acquire VL as a result of the protective immunological response [59]. The quantity of antibodies in blood has been connected to the incidence of asymptomatic to symptomatic VL conversion. Although it will be difficult to track these instances, they may aid in the discovery of host immunological mechanisms that influence disease susceptibility and resistance. Strong cell mediated immunity, a broad repertoire of memory T and B cells, and short-lived plasma cells may be linked with the immune biology of resistant asymptomatic infections (they do not show seropositivity for a longer period). Focused research on these people might disclose the characteristics of protective immunity that are needed to build a preventive vaccination candidate. In asymptomatic patients, the levels of ADA, IL-10, and IFN-γ were continuously high, with ADA and IL-10, but not IFN-γ, remaining higher as clinical symptoms progressed into active VL. ADA and IL-10 might be used as a biomarker in the transition from asymptomatic to symptomatic VL [60]. Due to their high innate cellular immunity, many asymptomatic individuals become seronegative without acquiring

VL. IFN- γ became high in asymptomatic infection but dropped after conversion, but TNF-α levels did not alter much at either stage of illness. The cytokine profile might be utilized to better treat VL patients with autoimmune diseases, as well as to identify and protect individuals with asymptomatic infection who are at risk of developing illness. Assays for cytokine release are already being utilized to detect asymptomatic individuals [31, 61]. Cytokines, which operate on macrophages, are receiving a lot of attention these days because of their ability to alter the immune response. Studies on the function of cytokines in asymptomatic infections and/or subclinical VL cases are scarce in the literature, and these investigations generally assess cytokine levels just once, before any clinical symptoms arise. Research into immune responses shows that patients with low levels of IL-10 production have additional flaws. In VL, IL-10 mRNA expression is highly expressed, and this cytokine's involvement in reducing T cell responses in these individuals has been well established. According to the finding, the balance between the production of IFN-γ and IL-10 may be a significant factor in determining whether or not patients develop illness after contracting *L. chagasi* infection even while they are asymptomatic [62].

## **5. Genetics**

For many years, there has been speculation that, in VL, the Leishmania genotypic differences involve in asymptomatic or symptomatic forms of the disease. There was findings that the Leishmania Internal Transcribed Spacer 1 (ITS1) from symptomatic VL and asymptomatic cases has significant genetic differences in southern Iran [63]. Several investigations have shown that the genotypic characteristics of symptomatic and asymptomatic VL patients might differ [64, 65]. Researchers identified significant genetic diversity between *Leishmania* species isolated from asymptomatic and symptomatic patients, particularly those with HIV/ VL coinfection, in a study done in southern France. The study also discovered that asymptomatic isolates had a modest polymorphism in their parasite genome [64]. In Southern France, MLMT showed parasite genotype appear to differs in *Leishmania* patients compared with asymptomatic related carriers [66].

Aside from the parasite genotype, the host's genetic background may have a role in determining whether VL is asymptomatic or symptomatic [67]. Study also linked symptomatic VL to a gene that codes for a receptor for transforming growth factor beta (TGF-β) whereas the asymptomatic is connected to gene encoding II-a receptor for the Fc fragment of IgG [67]. However, the association between SNP/ HLA genotyping and progression from asymptomatic or seroconversion to VL overt disease has been insignificant [68]. Polymorphism at SLC11A1 has been shown to be linked [69, 70] and associated in regulating susceptibility with human VL in Sudan. However, no evidence of such an association was found in an Indian population [71]. Few studies indicate that host genetic association and development of clinical symptoms is linked to NRAMP1, TNF-α, IL-4 and IFN-γ receptor (IFNGR1)], TGF β, IL-8, C-X-C chemokine receptor 1 (CXCR1) and C-X-C chemokine receptor 2 (CXCR2), IL-2R, Delta-like1 (DLL1), and mannan binding lectin genes [48, 69, 72, 73]. In one of the recent most studies on asymptomatic VL, were able to link several HLA-DRβ allele groups with the progression of VL [68].

#### **6. Other risk factors**

Besides, serological methodologies performed on individuals without symptoms may have low sensitivity due to a weak humoral response [74]. Risk factors have

#### *Visceral Leishmaniasis: Asymptomatic Facts DOI: http://dx.doi.org/10.5772/intechopen.101109*

been analyzed by some studies, taking into account that contact with the parasite is necessary, but it is not sufficient for the development of the active disease. These characteristics can play an important role in the cycle of asymptomatic individuals [6]. The male gender is one of the individual factors that demonstrate a positive association with asymptomatic infection [75]. Although other hosts and parasite variables may be additional causes, the conversion of asymptomatic infections to symptomatic VL also indicates the survival of parasites in these people [76]. The extrinsic variables such as age and nutritional state, as well as a weakened host immunological system, are thought to be significant in the progression from asymptomatic to symptomatic infection. Poor dietary status has been linked to an increased chance of developing clinical VL in addition to hereditary risk. The relationship between malnutrition and the course of VL at the cellular level is poorly understood. A better understanding of these mechanisms might open new opportunities for prevention or therapeutic dietary intervention [16].

There were evidences that suspected individuals living in households with family history, were at particularly high risk of infection. Although the cohort studied did not contain population-specific genetic markers, the addition of such factors might help predict outcomes when molecular diagnostics and serodiagnostic testing are combined. Even if they have a competent immune response, persons who have come into touch with the parasite do not inevitably acquire the symptomatic version of the condition [16]. Age, genetic, immunological, and dietary features, the existence of other diseases, and vector density are all potential risk factors for the disease's development [75], and type of "asymptomatic" definition applied to the study [28]. Despite being practical and easy, methodologies handling have some limitations: (i) do not differentiate past disease from recent [75, 77] (ii) there is the possibility of cross-reactivity with other related parasites [78]. Asymptomatic infection is usually observed in family members or in direct contact to clinical VL cases, suggesting that family members are at risk of infection. In a research from India, it is discovered that family members of VL patients had 1.8 times the risk of becoming infected as compared to those who did not have VL in the house. Kala-azar patients were younger (*P* < 0.001) and reported lower red meat consumption (*P* < 0.01) than asymptomatic seropositive individuals. Retinol and zinc levels were lower in current kala-azar patients and those who later developed kala-azar compared with uninfected and asymptomatically infected subjects. The characteristics that help determine whether an infection leads to overt disease appear to include age and dietary factors such as intake of iron- and zinc-rich red meat [79, 80]. Kala-azar patients were younger (P < 0.001) and reported lower red meat consumption (P < 0.01) than asymptomatic seropositive individuals. In comparison to uninfected and asymptomatically infected people, active kala-azar patients and those who later acquired kala-azar had decreased retinol and zinc levels. In contrast with different groups, kala-azar patients had greater CRP values. A population at increased risk of symptomatic illness may have a low red meat intake and low zinc and retinol levels.
