Identification of asymptomatic Leishmania infections: a scoping review

Background Asymptomatic Leishmania infection may play an important role in the transmission of the parasite in endemic areas. At present there is no consensus on the definition of asymptomatic Leishmania infection, nor is there a safe and accessible gold standard test for its identification. Methods This paper presents a scoping review to summarize definitions of asymptomatic Leishmania infection found in the literature, as well as to detail the approach (molecular, serological, cellular, and/or parasitological tests) used by researchers to identify this asymptomatic population. A scoping review of published and gray literature related to asymptomatic Leishmania infection was conducted; retrieved citations were screened based on predefined eligibility criteria, and relevant data items were extracted from eligible articles. The analysis is descriptive and is presented using tables, figures, and thematic narrative synthesis. Results We conducted a screening of 3008 articles, of which 175 were selected for the full review. Of these articles, we selected 106 that met the inclusion criteria. These articles were published between 1991 and 2021, and in the last 5 years, up to 38 articles were reported. Most of the studies were conducted in Brazil (26%), Spain (14%), India (12%), Bangladesh (10%), and Ethiopia (7%). Of the studies, 84.9% were conducted in the immunocompetent population, while 15.1% were conducted in the immunosuppressed population (HIV, immunosuppressive drugs, and organ transplantation population). We report 14 different techniques and 10 strategies employed by researchers to define asymptomatic Leishmania infection in an endemic area. Conclusions The definition of asymptomatic Leishmania infection is not unified across the literature, but often includes the following criteria: residence (or extended stay) in a Leishmania-endemic area, no reported signs/symptoms compatible with leishmaniasis, and positive on a combination of serological, molecular, cellular, and/or parasitological tests. Caution is recommended when comparing results of different studies on the subject of asymptomatic infections, as the reported prevalence cannot be confidently compared between areas due to the wide variety of tests employed by research groups. More research on the importance of asymptomatic immunosuppressed and immunocompetent Leishmania-positive populations in leishmaniasis epidemiology is required. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-021-05129-y.

Ibarra-Meneses et al. Parasites & Vectors (2022) 15:5 leishmaniasis (CL) and visceral leishmaniasis (VL) are the most severe clinical forms of the disease; CL affects the skin, while VL affects the internal organs of the infected patient [4]. The evolution of the disease occurs progressively over a period of weeks or even months and is influenced by environmental, parasite-, and host-related factors [5]. VL is fatal in 95% of cases if left untreated [2].
Leishmaniasis is endemic in 98 countries; however, official data underestimate the reality of human leishmaniasis due to the low number of mandatory reporter countries (32/98), the large number of cases that are incorrectly diagnosed, official data being obtained exclusively from passive case detection, and the large, unreported asymptomatic population [3,6].
Asymptomatic infection represents approximately 20-60% of Leishmania spp. infection in endemic areas [7,8]. Although the asymptomatic population likely represents the highest proportion of infection, there is no agreed definition of the condition or accurate means by which to detect a subject with asymptomatic Leishmania infection [9]. Some authors define a subject with asymptomatic infection as a healthy individual living in an endemic area who tests positive on a molecular [polymerase chain reaction (PCR), quantitative PCR (qPCR), or loop-mediated isothermal amplification (LAMP)], serological [direct agglutination test (DAT), enzyme-Linked immunosorbent assay (ELISA) test, rK39-immunochromatographic rapid test (rK39-RDT), immunofluorescence antibody test (IFAT), or Western blot (WB)], or cellular test [leishmanin skin test (LST), interferon gamma release assay (IGRA), whole blood assay (WBA), cell proliferation assay (CPA)]; while others consider a combination of the above tests [10][11][12][13][14].
The asymptomatic population is of vital importance for several reasons. Firstly, they may well serve as a reservoir of parasites, presenting a risk to public health through infection of the phlebotomine vector [15]. Immunosuppression is one of the risk factors that can increase progression to clinical manifestation in asymptomatic subjects. Human immunodeficiency virus (HIV) infection, immunosuppressive drugs, and organ transplantation are the most widely studied risk factors for co-infection with Leishmania. HIV infection increases the risk of developing VL by 100-2320 times, while the risk is increased by 20-100 times after treatment with immunosuppressive drugs and after organ transplantation [16,17]. As such, special attention should be paid to this asymptomatic immunosuppressed (IS) population in endemic areas.
Further interest in the asymptomatic population stems from the mystery surrounding leishmaniasis disease progression. It is well known that a large proportion of those infected with Leishmania spp. never demonstrate clinical manifestations of the disease [8]. It has been suggested that progression towards symptomatic VL likely results from a combination of various host, parasite, and sociodemographic factors [18]. A clearer understanding of the manifold factors leading to the development of clinical leishmaniasis could inform the treatment of asymptomatic patients to improve disease outcome, as well as reduce parasite transmission from this potentially significant reservoir [15].
For these myriad reasons, there is an urgent need to establish a specific definition of "asymptomatic Leishmania infection, " such that future studies may contribute to the development of new leishmaniasis control strategies through standardized and methodical means. To this end, our aims in this study are to outline current approaches used to describe asymptomatic Leishmania infection in endemic areas and map out approaches previously used for the study of asymptomatic Leishmania infection in blood banks, epidemiological surveys, and through screening of patients in endemic areas. Furthermore, frequently employed definitions of "asymptomatic Leishmania infection" and their associated diagnostic tests are discussed, such that we may suggest common usage guidelines concerning these topics to inform future studies in the field.

Protocol and registration
We developed the protocol for the scoping review in line with the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P). The protocol was developed before beginning the search and was reviewed and approved by all members of the review team.

Eligibility criteria
For this scoping review, we sought to identify primary studies reporting on asymptomatic Leishmania infection within human populations in an endemic area (see Additional file 1: Table S1 for the list of endemic countries). Eligible studies could include populations of any age, sex, and health status. Only studies in which a diagnostic technique was employed to identify Leishmania in asymptomatic people were considered eligible. We included articles in English, French, Spanish, and Portuguese, and from any period. The eligible study designs included surveillance studies, cross-sectional studies, cohort and case-control studies, and interventional studies. Articles were excluded if they reported on studies of symptomatic Leishmania infection, involved only animal populations, or did not include a diagnostic technique. Studies not based on primary data, such as reviews and modelling studies, were also ineligible for this scoping review.
For our study, we considered subjects as asymptomatically infected with Leishmania if they met the following criteria: no signs/symptoms of leishmaniasis (based on clinical examination by a medical professional and/or medical history as declared by the patient), positive on at least one diagnostic test (serological, molecular, cellular, and/or parasitological), and residence (or history of extended stay) in an area of leishmaniasis endemicity.

Information sources
To identify potentially relevant articles, we conducted a detailed search of the PubMed, Web of Science, and LILACS databases. All three bibliographic sources were searched on 11 August 2021. We also conducted a manual search of eligible articles for potentially relevant articles that may have been missed in the bibliographic search.

Search
Our search strategy combined the broad terms "Leishmania" and "asymptomatic. " We included alternate terms within each concept to improve the sensitivity of the search. No restrictions on language or publication period were included in the search. The initial search strategy was developed by AVIM, CO, and AC, and was reviewed by CFP. The final search strategy was modified via an iterative and consultative process involving all members of the review team. For this scoping review, we did not include gray literature.
The complete search syntax for the PubMed search is presented below, combining Medical Subject Headings (MeSH) and free-text search (Additional file 2: Table S2).

Selection of sources of evidence
The citations returned from the electronic database searches were imported into EndNote, where duplicate records were identified and deleted. The de-duplicated citations were subsequently imported into COVIDENCE, where further de-duplication was carried out and articles were assessed for eligibility.
AVIM and AC independently screened the titles and abstracts of records to identify potentially relevant studies. Subsequently, AVIM, AC and VW independently read the full text of the potentially relevant articles and selected those that met the eligibility criteria. At each stage of the selection process, two reviewers had to independently agree on the assignment of each article. The agreement was high between voting members, with 95% agreement at the title and abstract stage, and 81-88% at the full-text stage. Disagreement in voting at each stage was resolved by CFP following consultation with the voting pair.

Data charting process
Predefined data items were identified by the review team through a consultative process during the planning of the scoping review and subsequently integrated into an extraction form. The data extraction form was designed and piloted in Excel using some eligible papers and modified as required. One member of the team (AVIM, AC, and VW) conducted the initial data extraction, and a second member crosschecked the extracted data to ensure completeness and accuracy (AVIM, AC, and VW). Discordance in extracted data was resolved by consensus between the reviewers.

Data items
In Table 1 below, we present details of the data items collected as part of the scoping review.

Outcomes and prioritization
The outcome of interest in our review is a description of the common definitions of "asymptomatic Leishmania infection" found in the literature, as well as the techniques used for the detection of this population in various endemic areas. No other outcomes are considered for prioritization.

Critical appraisal of individual sources of evidence
We did not conduct a formal critical appraisal of included articles as part of this scoping review, as the aim of this review was to describe the scope of research and not to present pooled study results.

Synthesis of results
We used a descriptive approach in the synthesis and presentation of the scoping review findings. We present an overview of the study selection process through narrative and PRISMA flowchart. A summary of the included articles by year of publication, study country, study setting, study aim(s), and population characteristics is presented in a descriptive table. We subsequently describe how researchers had operationalized and defined asymptomatic Leishmania infection and summarize these strategies using a thematic approach. We used a combination of deductive and inductive processes to obtain the definitions based on explicit or implicit case definitions obtained from included articles. We further identified test-test comparison pairs based on a reduced number of eligible articles where two or more tests were used in parallel. Leishmania microscopy and culture were collectively classified as "parasitology tests, " while WBA, LST and CPA tests were categorized as "cellular immunology tests. " The other tests included in this study-serological and molecular-were considered individually. Using the suite of network commands in Stata [16], we constructed a network showing comparisons made between tests among eligible studies. We inspected linkages (or interconnectivity) between test types in the network to identify potential gaps in test comparison. Using a combination of narrative synthesis, graphs, and network diagrams, we demonstrate how specific tests were used independently or in combination in the included studies.

Searching for an asymptomatic definition
The diagram used in this scoping review for the selection of articles is shown in Fig. 1, following the Tricco et al. guideline [19]. Initially, the first search in the three chosen databases yielded 4290 articles. After eliminating duplicates, 3008 articles were selected for screening by title and abstract. After the first screening, 175 articles were retrieved. Of these 175 articles, 69 were discarded because they did not meet the inclusion criteria described above. Thus, in total, we selected 106 articles for this scoping review.
The number of studies on the identification of the asymptomatic Leishmania populations in endemic areas has increased over the years. Figure 2 shows an exponential increase in the number of studies carried out in 5-year periods. Between 1991 and 1996, only two studies were published describing asymptomatic Leishmania infection, while in the current period (2017-2021), as of August 2021, 38 articles had been published.

Description of the asymptomatic studies included
Information from the 106 studies describing asymptomatic Leishmania infection in endemic areas is described in Table 2. All the studies included were primary studies, where subjects did not manifest any symptoms or signs of the disease. The age of subjects ranged from 2 years to > 60 years. Among studies, 94.3% were associated with asymptomatic infection in endemic areas of L. infantum The country or countries where the study was conducted Objective of study As outlined by the authors. This was extracted verbatim and subsequently thematically characterized into analyzable data items, e.g., prevalence survey, test validation, etc.
Population description Description of the study population, including the methods of selection, e.g., household contacts, blood donors and volunteers Population size The size of the sampled population The clinical status of patients is associated with their immunological status. In this scoping review, the majority of studies (84.9%) were conducted in immunocompetent (IC) subjects. That said, 11.3% of the studies were conducted in HIV patients and 2.8% in solid organ transplant (SOT) recipients, and only 1.9% were identified as having been conducted in IS individuals. The studies in HIV patients were carried out in Spain (n = 4), Brazil (n = 3), Ethiopia (n = 2), France (n = 1), Italy (n = 1), Iran (n = 1), and Morocco (n = 1). Meanwhile, the three studies in drug-IS populations were carried out in Spain (n = 2) and France (n = 1). Finally, the three studies in SOT recipients were carried out in Brazil (n = 1), Italy (n = 1), and Spain (n = 1). Within the 84.9% of works investigating the IC population, 7.8% describe CL, and 2.2% postkala-azar dermal leishmaniasis (PKDL). We found seven studies in IC populations with various cutaneous manifestations in seven countries: Brazil (n = 2), Tunisia (n = 2), Colombia (n = 1), Peru (n = 1), and Venezuela (n = 1). Meanwhile, only two studies described PKDL (India and Bangladesh). The vast majority of studies included in this review investigated VL.
A total of 22.6% of included studies were performed in blood banks to determine the prevalence of asymptomatic infection, and 39.6% were performed in volunteer subjects where screening of the population was carried  To include patients in blood bank studies, surveys, or clinical trials, knowledge of their previous clinical history is vital. In this scoping review, we found that 9.4% of the studies included subjects with a previous history of leishmaniasis in their asymptomatic group; 59.4% of the studies excluded those subjects who had suffered from leishmaniasis in the past, while 33% did not describe the history of leishmaniasis in the study population.

Tests used for identification of asymptomatic Leishmania infection
The inconsistent definition of asymptomatic Leishmania infection further demonstrates the lack of consensus in the techniques used. Figure 4 shows an example of the different tissues (blood, serum, urine) used in combination with the tests researchers employed to define the asymptomatic population (Table 2). Parasitological (culture and microscopy), molecular (PCR, qPCR, LAMP), serological (ELISA, RDT, DAT, IFAT, WB, KAtex), and cellular (LST, CPA, WBA) techniques were employed. Likewise, it was described that the cellular immunity of asymptomatic patients was associated with a Th1-type cellular response, where elevated levels of interferongamma (IFN-γ) were produced both in serum and in stimulated plasma and supernatant. It was also found that this group of asymptomatic subjects produced high levels of tumor necrosis factor (TNF), interleukin-2 (IL-2), interferon gamma-induced protein 10 (IP-10, IP-10/CXCL10), monokine induced by gamma interferon (MIG/CXCL9), monocyte chemoattractant protein-1 (MCP-1/CCL2), neopterin, and soluble CD40 ligand (sCD40L), while low levels of IL-10, IL-4, and IL-17 were detected.

Definition of "asymptomatic infection"
As shown in Fig. 4, we report more than 10 different techniques used for the detection of the asymptomatic population in question. "Asymptomatic Leishmania infection" frequently describes a subject in a Leishmania-endemic area testing positive by a molecular or serological or cellular test, with no signs or symptoms of the disease. However, within the different groups of techniques used, there exist multiple approaches for identification and numerous test combinations for detection of asymptomatic infection. Table 3 shows the different approaches used by researchers to define asymptomatic Leishmania infection; 10 different ways to describe these subjects are reported. The first approach involves a combination of four techniques (serological, molecular, cellular, and parasitological). The second and third strategies involve a combination of three techniques (type 2; serological, molecular, and cellular; and type 3; serological, molecular, and parasitological). Meanwhile, types 4 (serological and molecular), 5 (serological and cellular), and 6 (molecular and cellular) used a combination of two techniques. Finally, 33 studies employed a single technique [serological (type 7), molecular (type 8), cellular (type 9), parasitological (type 10)] for the detection of the asymptomatic population in an endemic area.

Markers for asymptomatic infection
Fifty percent of the studies included in this scoping review used an ELISA for identification of asymptomatic Leishmania infection, followed by PCR (40%), RDT (35%), IFAT (30%), DAT (29%), and LST (22%). To determine how multiple tests are applied in parallel, we identified 78 eligible studies where at least two tests were used on either the entire population or a subset thereof. Inspection of the network (Fig. 5) showed a high degree of interconnectivity between several of the tests. The highest density of interconnectivity was observed for DAT, RDT, IFAT, PCR, and ELISA, where links were found to all other tests. LAMP was the test method with the least interconnectivity within the network, linking only with ELISA, RDT, IFAT, and PCR once each.
A combination of serological and molecular techniques was the most common combination of detection approaches among the studies included in this scoping review. However, it is important to note that the results mentioned above do not consider the antigen or target used. Figure 6a summarizes the diversity of commercial brands that exist for the rK39 rapid diagnostic test (rK39-RDT). Sixty-three percent of the studies that employed RDT used the rK39 Kalazar Detect ™ rapid test (InBios), while 11% used IT Leish (BioRad), 6% used the SD BIOLINE Leishmania Ab Test (Bioline/Abbott), 6% used OnSite Leishmania IgM/IgG Combo test (CTK Biotech, Inc.), 3% used the Leti Laboratories test, and 11% of articles did not describe the commercial brand in question. Figure 6b shows the diversity of antigens employed for the detection of antibodies by ELISA. Thirty-four percent of the studies that used ELISA for identification of the asymptomatic population used soluble Leishmania antigen (SLA), another 34% used the rK39 antigen, while 16% used crude Leishmania antigen (CSA), 4% used recombinant kinesin 26 (rK26) antigen, 3% used Leishmania promastigotes, and 1% used recombinant kinesin 28 (rK28), rTR18, and rKR95 antigen. For the molecular techniques described (Fig. 6c), nine different targets were identified. Of these, 57% of studies used kinetoplast DNA as their target (kDNA), 21% the small subunit 18S rRNA gene (ssu 18S rRNA), and 11% and 3% used internal transcribed spacer (ITS) 1 and 2, respectively. Meanwhile, 1% of studies employed the glucose-6-phosphate dehydrogenase gene (g6pd gene), mini-exon repeat, REPL repeat, DNA polymerase alpha (DNApα), and the telomeric region.

Discussion
Our scoping review included a total of 106 articles from 19 countries in five of six different WHO regions. There has been a marked increase in studies conducted on the subject of asymptomatic Leishmania infections in recent years (2017-2021) versus past decades, possibly due to increased awareness of their potential significance in the epidemiology of leishmaniasis. Most of the studies included in our review were conducted in Brazil (26%) and India (12%); this is likely because leishmaniasis is very widespread in these countries, which constitute two of the six countries responsible for more than 90% of VL cases around the world [3]. The vast majority (84.9%) of studies included in this review explore asymptomatic Leishmania infection in IC populations. Different mathematical modeling studies have shown that this asymptomatic IC population is less infective than the population with active disease, and their role in disease transmission is still under investigation [15,63]. The issue remains of major concern and could have a substantial impact on the spread of this parasite [63,91].
With respect to populations under treatment with immunosuppressive drugs, despite the low number of studies reported (n = 3), seven different tests and two different strategies were employed for detection of the asymptomatic population [85][86][87]. This same pattern was found with SOT recipients: five different tests and three different strategies were used in the three studies reported in this scoping review [17,88,119]. These data confirm the lack of consensus on defining and detecting asymptomatic infection in these populations.
In total, this scoping review identified 14 different tests used for detection of parasites, parasite load, antibodies/antigens, and cellular immune response, resulting in nine different overall approaches for defining asymptomatic infections in IC populations. There is a complex relationship between the various tests used for the detection of asymptomatic populations, as demonstrated by the interconnectivity in our network. The nine strategies employed can be separated into two main categories: strategies 1-6 involved independent combinations of serological, molecular, cellular, and/or parasitological tests to identify the asymptomatic cohort; on the other hand, strategies 7, 8, and 9 involved the use of a single test type to detect asymptomatic individuals. These two very different, wide categories broaden the detection range, leading to a lack of consensus concerning the identification of the asymptomatic population in Leishmania-endemic areas. Furthermore, the use of different strategies makes it difficult to compare the same population in the same (or different) endemic areas. Importantly, this complication lies not only in the large number of tests employed, but also in the numerous targets (molecular tests) or antigens (serological and cellular tests) used by each group. We report the use of more than six types of rapid test (rK39-RDT) from different manufacturers, and the effectiveness of these commercial tests varies between regions [124]. Moreover, nine different antigens were utilized to detect antibodies using ELISA alone [125], and nine different targets were employed to identify the parasite with molecular tools [126]. This substantial variation in detection methods coupled with the plethora of definitions of "asymptomatic Leishmania infection" makes an accurate determination of prevalence in any region near impossible.

Common usage guidelines
Standardization of the term "asymptomatic Leishmania infection" is key to improving the outcome of future studies and allowing accurate comparison between the results Fig. 6 Network demonstrating comparisons of various tests employed for the detection of asymptomatic Leishmania infection within articles included in the scoping review. In the network diagram, the nodes represent the test type and the edges (lines) indicate that two test types have been compared in at least one study of different works. According to the literature, subjects are often considered as asymptomatically infected with Leishmania under the following conditions: residence (or extended stay) in a Leishmania-endemic area, no reported signs/symptoms compatible with leishmaniasis, and positive on a combination of serological, molecular, cellular, and/or parasitological tests. We consider this an appropriate definition. While a medical history of leishmaniasis may complicate interpretation of certain tests, we feel it does not preclude the development of an asymptomatic infection if the subject was previously considered "cured. " More research regarding how many years following a reported VL episode a patient can be considered "asymptomatic" is necessary; as such, it is recommended that the conditions of a study population with VL history be explicitly stated. Furthermore, we recommend caution when comparing results of different studies on the subject of asymptomatic infections, as the reported prevalence cannot confidently be compared between areas due to the wide variety of tests employed by research groups. With respect to future studies, we suggest that researchers use the most sensitive and specific test available to them, and interpret their results within the framework of other studies employing the same technique and target. We recommend a minimum of two different detection methods employed in parallel for identification of asymptomatic Leishmania infections among a study population.
Of note, for this work, we did not analyze the results/ methodology of each piece of literature included in the review; this was beyond the scope of our study. There may therefore be limitations with respect to the quality of the methodology employed in each individual article. In the future, we will perform a meta-analysis in order to compare the sensitivity and specificity of different diagnostic tests employed by research groups for the detection of asymptomatic Leishmania-infected populations in endemic areas (Table 4).

Conclusions
Asymptomatic Leishmania infection remains poorly understood; the lack of baseline tests for its detection means that its prevalence is likely underestimated, and its epidemiological role remains unknown. This scoping review was performed in order to inform researchers of the different approaches that exist for identification of asymptomatic Leishmania infection. It also highlights the need to standardize the definition of this population in order to reach a consensus for future work strategies in endemic areas, especially in IS populations.