Abstract
Visceral Leishmaniasis (VL) caused by protozoan parasite Leishmania is a vector borne disease and infection is limited not to human but also to animals worldwide. For infection identification and prevalence in both Leishmania endemic and nonendemic regions, several serological and genetic techniques are used. Although diagnostic techniques and clinical symptoms can establish illness status, it is extremely difficult to diagnose infection in the absence of symptoms. Asymptomatic are healthy people who have an infection but are unaware of it. The epidemiology of asymptomatic Leishmaniasis is critical for its eradication. Only a small percentage of infected people are clinically suspected of having VL, as the majority of them may not show any symptoms and remain asymptomatic. Some asymptomatic infections may go away after a while, or they may linger for years, or they may develop to illness with clinical signs. Asymptomatic infection varies per endemic location, but almost all of them point to this hidden category of parasite infection. It is now critical to understand many factors such as diagnostic markers, genetic markers, and immunological markers along with different risk factors. All of these criteria, as well as some innovative techniques to diagnosing and controlling asymptomatic leishmaniasis, will be covered in this chapter. The main focus will be on asymptomatic condition of Indian Visceral Leishmaniasis, which is caused by Leishmania donovani and spreads via female sand fly P. argentipes biting. The numerous criteria that play a role in asymptomatic to symptomatic conversion in a specific time period will also be discussed in this chapter.
Keywords
- Leishmania
- infection
- asymptomatic
- markers
1. Introduction
Leishmaniasis is a serious public health problem in many countries throughout the world. The illness is caused by numerous intracellular protozoan parasites of the genus
2. Epidemiology
It is essential to understand the global prevalence of asymptomatic leishmaniasis. In addition, it’s critical to look at the variables that contribute to asymptomatic infection. The variation arises because of changes in parasite virulence and host demographic characteristics, as well as from research designs and the tests employed to determine asymptomatic infection. Worldwide epidemiological statistics show that asymptomatic VL can come from both endemic and non-endemic areas. Those who are infected may unintentionally transfer the disease to others. They may go away on their own, or they may develop symptoms at a later time. As per some findings Asymptomatic infections are those who remain seropositive for many (up to 10–12) years without developing into active disease [11, 12], and are more prevalent in VL endemic regions [13]. In the New World, asymptomatic leishmaniasis was considerably more frequent than in the Old World. The higher prevalence might be explained by the greater diversity of leishmaniasis in the New World as a result of the vector’s adoption of new hosts and climate change [14]. Asymptomatic leishmaniasis was less common in children than in the general population. This difference, however, was insignificant on a statistical basis. It was hypothesized that the rise in infection prevalence with age was owing to young children’s reduced exposure to infectious sandfly bites [15]. Some indicators indicate that in VL endemic locations, the ratio of asymptomatic vs. active VL patients varies: 2.4:1 in Sudan, 4:1 in Kenya, 5.6:1 in Ethiopia, between 4:1 and 17:1 in the Indian subcontinent, and 50:1 in Spain [16]. Reports from the other endemic regions also confirm the existence of parasitic DNA in all VL causing species in asymptomatic individuals.
According to a meta-analysis of original articles reporting asymptomatic leishmaniasis, the prevalence of asymptomatic leishmaniasis was 11.3%, 95% confidence interval (CI) 8.6%–14.4%] in general population, 36.7% [95% CI 27.6%–46.8%] in inhabitants living in the same or neighboring household to the symptomatic patients, and 11.8% [95% CI 7.1–19%] in HIV infected patient. Meta-regression analysis also showed no significant change in the prevalence of asymptomatic leishmaniasis during the last 40 years [17]. From 1982 to 2015, the trend of total leishmaniasis’ asymptomatic proportion did not change considerably, according to the meta-regression study [coefficient = 0.0350 (95% CI, −0.0213 to 0.0913), P = 0.2233] [17]. But, while the disease’s geographical range is broad, it is not continuous. The study also suggest, for
3. Diagnosis
Diagnosis of VL is done by serological tests and molecular test along with direct parasite identification technique. Direct techniques are
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, 38, 39].
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 follow-up, 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, 45, 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.
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
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
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
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 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 (
7. Conclusion
Control efforts for leishmaniasis (especially asymptomatic VL), particularly in endemic regions, need a detailed understanding of
When innate immune cells from asymptomatic carriers were stimulated with antigens in vitro, they exhibited a regulated rise in cytokine production that differed from that seen in non-infected subjects. This implies that using more than one diagnostic approach makes it easier to identify a substantial proportion of asymptomatic carriers. One often mentioned flaw in these research is the difficulty in identifying those who are briefly and quietly infected with the parasite. A recent study of asymptomatic people’ innate and adaptive immunity revealed that a mixed cytokine profile is crucial not just for parasite replication control, but also for the preservation of these individuals’ immune state [51]. Only 20% of those infected with
The question of why just a few exposed asymptomatic people acquire full-blown illness symptoms but not all remains unsolved. The major immunological variables that promote the conversion of asymptomatic patients to symptomatic stage of visceral leishmaniasis have been attempted to explicate in several research. It is crucial to note that the use of five diagnostic techniques as a regular strategy would be impractical in endemic situations. Before a particular recommendation can be made, more research is needed to confirm the optimal diagnostic method [61]. The important checkpoints for determining disease resistance or susceptibility are cytokines that control cellular immunity [84, 85]. Despite substantial understanding of host–parasite interactions and immunobiology, reliable protective immunity criteria have yet to be discovered. Asymptomatic instances of human VL can be diagnosed using qPCR using RNA targets. There are also questions about whether or not using RNA as a gene target can help discover asymptomatic instances of VL. By combining this methodology with epidemiological data analysis, it will be possible to improve the detection and treatment of asymptomatic cases. Priority should be given to stepping up efforts to better characterize asymptomatic illness in endemic areas and to develop a uniform case definition for leishmanial infection. Self-clearing infections vs. illness development must have all of their factors examined thoroughly. The problems in parasite and sand fly management methods, as well as changes in the epidemiology of VL in disease-endemic countries, are important threats to its eradication. In addition, the movement of infected but asymptomatic people from endemic areas has resulted in additional infections in non-endemic areas [86]. The xenodiagnosis approach validates whether asymptomatic infected people can be infectious to sand flies and might be a crucial step in determining whether or not to modify the existing VL management strategy. One study used xenodiagnosis to identify VL infection in HIV-positive people and found even asymptomatic patients in the early stages of the infection were able to infect others [87]. Although Xenodiagnosis findings from an Indian investigation indicate that neither asymptomatic nor treated patients were infected by vector sandflies [88]. Asymptomatic infected persons are not now the focus of drug research efforts. This is because the asymptomatic state is not clearly defined, but largely because an intervention is less of a priority as long as the role of asymptomatically infected people in transmission is not clarified. There have been significant gains in reducing infection rates thanks to the eradication programs, but there are still some obstacles to overcome along the way. Considering that humans are the sole reservoir for
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