**3. Diagnosis**

Diagnosis of VL is done by serological tests and molecular test along with direct parasite identification technique. Direct techniques are *Leishmania* parasite isolation from spleen, bone marrow, or blood for microscopy or culture. Different serological tests are ELISA, direct agglutination test (DAT), immunofluorescence antibody test (IFAT), indirect immunofluorescence (IIF), western blot (WB), rK39 immuno-chromatographic strip test while molecular tests include polymerase chain reaction (PCR), qRT-PCR and k-DNA southern blot whereas immunological test include Montenegro Skin Test (MST)/Leishmania skin test (LST) or Interferon Gamma Release Assay (IGRA). The lack of a good biomarker makes defining *Leishmania* asymptomatic infections extremely difficult. It's also unclear how to distinguish parasite persistence in an asymptomatically infected person from new infections that develop after the first episode, i.e. old parasites eliminated by the immune response followed by new infectious parasite populations that would follow the same destiny. Asymptomatic infections cannot be diagnosed with a single, widely approved test. It cannot be diagnosed using any standard or commercially available methods. Patients who are infected with *Leishmania* do not show any symptoms, but tests such as the polymerase chain reaction (PCR) or leishmania skin test (LST)/Montenegro skin test (MST) are positive regardless of whether they show any symptoms [22]. Population-based demographic and immunological surveys showed high but variable prevalence of leishmanial antibodies in the population of Bihar [26]. Serological test DAT/ELISA can perform in epidemiological studies as is noninvasive. But is indirect methodology and tells antibody response due to *Leishmania* infection. Because of the varying durations between infection and seroconversion (ranging from 3 months to 7 years), serology may not be a useful predictor of infection when employed in cross-sectional research [19]. The most often used techniques include an intradermal skin test that indicates the cellular immune response associated with prior exposure to *Leishmania* and the identification of anti-Leishmania antibodies, a less specific indication of infection or continuing illness [27]. Conventional PCR is direct tool as show presence of *Leishmania* specific DNA in interest of samples (blood, buccal swab, urine). Sensitivity and specificity vary for selection of primers. These above mentioned techniques are in use worldwide in epidemiological study to know prevalence of *Leishmania* infection in healthy individuals. But when data compare is compare with active *Leishmania* cases tough to distinguish healthy infection, i.e. asymptomatic. Using DAT seroconversion as a measure of infection, studies found that asymptomatic infection was nine times more common than acute VL illness in high-endemic foci in Bihar [19]. However, in any longitudinal epidemiological investigation, seroconversion should be the primary criteria for detecting asymptomatic infection. The variation in the ratio of VL cases versus asymptomatic cases in different *L. donovani* and *L. infantum* endemic areas from 2.4:1 in Sudan to 50:1 in Spain [28] reflects variations in parasite virulence and host features, However, this may also be due to variations in research design and the methodologies used to diagnose asymptomatic infection. Cell immunity generally lasts for several years, and in some cases, for the rest of a person's life [29, 30]. Serological indicators, on the other hand, can go from positive

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

to negative in as little as four months after the initial sample is examined [22]. In endemic locations where mean parasitemia levels are low or intermittent, serology is generally unreliable for identifying silent Leishmania infection [6]. Cytokine release assays are useful for detecting asymptomatic individuals among immunocompetent subjects in VL-endemic areas; they can also detect the same among immunosuppressed subjects following solid organ transplantation [31]. When compared with the reference test SLA-lymphoproliferative assay, IL-2 appears as a new, 100% sensitive and specific marker for asymptomatic individuals with a positive cellular response (compared with 100% and 84.78%, respectively, for IFN-γ) [32]. Some laboratory tests, including SLA-stimulated PBMC assay, may be difficult to perform under certain conditions. In contrast, the WBA holds much promise as a test at the point-of-care level [33]. The WHO recently recommended screening healthy populations for leishmaniasis infection using SLA-stimulated blood. There are ways to diagnose those who have asymptomatic *Leishmania* infection by whole blood stimulation with the soluble *Leishmania* antigen (SLA), followed by plasma cytokine and chemokine measurements. Combining these diagnostic tests with molecular studies might assist in estimating the real scope of the *Leishmania* outbreak in the endemic region. CXCL10 and CXCL9 DPS were shown to be reliable indicators for identifying asymptomatic individuals in *L. infantum* and *L. donovani* endemic regions. In distant areas, it makes samples more accessible and reduces the cost of epidemiological and epidemic investigations [34].

The immunological determinants such as Adenosine deaminase (ADA), Interferon gamma (IFN-γ), Tumor Necrosis Factor alpha (TNF-α) and Interleukin 10 (IL-10) were examined to predict probable biomarkers for conversion to symptomatic VL. Asymptomatic cases were also earlier reported to harbor the parasite in their blood [35, 36]. Many immunological methods such as direct agglutination test (DAT) and lateral flow immune-chromatographic tests, such as rK39 and rkE-16 have been introduced to screen large number of individuals in endemic areas [37–39]. *Leishmania infantum/chagasi* infection is endemic in Sicily. Approximately 47% of residents live in areas at risk of infection. The prevalence of asymptomatic carriers is unknown. In asymptomatic subjects, IFAT showed sensitivity (30.1%) higher than rK39-ELISA (26.3%) for the detection of cryptic infection, even though a lower specificity was reported (63.4 vs. 76.3%).

Molecular methods are the most suited due to the lack of a gold standard and the limitations of conventional diagnostic procedures, where parasitology is ethically impractical for persons without symptoms and serological tests do not discriminate between past and present illness. Recent molecular methods, such as conventional polymerase chain reaction (PCR) and quantitative real time PCR assay (qPCR), have made considerable advances in screening, diagnosis, and post-therapy followup, allowing for better sensitivity than prior serological assays. Quantitative PCR (qPCR) is now a days promising tool for detection and quantification of Leishmania and able to describe threshold as well as reference value for asymptomatic infection.

There are several types of molecular methodologies, and the choice of use should be based on what results are expected to be achieved. While in the conventional Polymerase Chain Reaction (cPCR) the results are only qualitative, quantitative products can be obtained in the Real-Time technique (qPCR), such as the levels of parasitic DNA circulating in the blood [40]. The sensitivity of the assays may vary according to the types of targets and samples used. The most used amplification targets are: kinetoplast DNA (kDNA) [41, 42], internal non-coding spacer region (ITS-1) [43] and the smaller ribosomal subunits (SSU - rRNA) [44–46]. Sudarshan et al. [35] when performing a qPCR, analyzed the level of circulating parasites to differentiate a possible disease progression. They obtained a minimum level of detection of 0.001 parasitic genomes/mL of blood and 34.79% of positive samples by the technique,

using the kDNA and hydrolysis probes of the TaqMan type, as a target and method of visualizing the products. Likewise, Kaushal et al. [6, 47] (S. Das et al., 2014; Kaushalet al., 2017, [6, 47] (S. Das et al., 2014; Kaushal et al., 2017) (Das et al. 2014, Kaushal et al. 2017, ([6, 47], when carrying out a study to detect asymptomatic individuals, obtained an amount < 5 parasites/mL of blood and a positive sample rate of 21.54%, using kDNA as a target and SYBR Green I as a result detection system. Sudarshan et al. [48], affirm that Leishmania DNA may be used as a marker of infection since it is detected before the seroconversion of antibodies. Individuals can be diagnosed as seronegative when they are tested before the development of immunity or when it is in a very low quantity, not being identified by serological methods. Similar data were also suggested by Costa et al. [27] and Bhattarai et al. [49], wherein asymptomatic infections detected by molecular methods have been observed in seronegative people. This demonstrates that possibly due to the limitations of serological methods, molecular tests are more suitable for the identification of asymptomatic cases. Although parasitic DNA is considered the first infection marker before immunological conversion [35], there are controversies regarding its use. The limitation of the use of DNA as a target is found in a possible detection of the genetic material of the parasite when it is already dead, although this is discussed, the half-life of the nucleic acid in the body is around 24 hours, which can cause flaws in distinguishing viable parasites from detecting fragments of lifeless parasites. Lack of standardization of a methodology still becomes a gap that can lead mainly to problems and delays in detection of the cases. Moreover, the use of nanoparticle techniques represents a trend for diagnosis, immunotherapy, and programs to eliminate VL. These methodologies bring a new approach with new forms of diagnosis and drugs, where improvements in efficacy and less toxicity can be observed. There will be continuous and significant improvements to all their current roles in diagnostics and will also provide multiple roles in terms of recognizing other DNA or materials, using fluorophores or other active molecules. it is reasonable to have a lower value of serum hemoglobin, hematocrit, and albumin among symptomatic patients. So, they would be considered as a marker of symptomatic diseases rather than a risk or protective factor. Studies are going on to define asymptomatic as yet there is no or very less agreement between different markers. Although Gold standard for Leishmaniasis detection is parasitological confirmation by microscopy which need splenic aspirate. But for asymptomatics it is not possible as ethical issues are very high because of invasive nature of samples. As the use of spleen or bone marrow aspirate is not ethical in asymptomatic subjects, the negative predictive value (NPV) cannot be exactly evaluated.
