4. Diagnosis approaches

Due to its wide range of manifestations, leishmaniasis diagnosis can be difficult [90]. Clinical manifestations can also be confused with other illnesses, often common in Leishmania endemic areas, such as malaria, toxoplasmosis and tuberculosis [91]. Age, medical history and host immune system response are crucial parameters for diagnosis.

Diagnosis of diseased patients and animals can be confirmed with conventional laboratorial techniques, such as visualization of the parasite in tissues by microscopic examination of a stained specimen, or in vitro culture of the parasite from biopsies or aspirates from lesions, lymph nodes, spleen and bone marrow. Other diagnosis methods include molecular detection of parasitic DNA in tissue samples and serological tests that detect anti-Leishmania antibodies [6]. Overall, the available techniques can detect active disease. Still, leishmaniasis has intrinsic

particularities that require not only the detection of symptomatic (diseased) conditions but also asymptomatic (infected, not diseased). This control is important, especially in the context of CanL, as the asymptomatic animals act as reservoirs, and as mentioned above, increase the risk for the human variant of the disease. An already complex scenario rendered more complicated by the need to distinguish treated, exposed and vaccinated. Therefore, leishmaniasis is a very particular disease and one of the many examples where ongoing research for a new and affordable diagnosis approaches is necessary.

#### 4.1. Parasitological diagnosis

The results presented with rKLO8 show increased reactivity with patient sera as compared to rK39 ELISA [85]. The rKLO8 ELISA is more sensitive than the DAT and rK39 strip test, and VL patients from Sudan were tested and have decreased immune responses to rK39, confirming the low sensitivity of rK39 strip test in Sudan. Malaria is common in VL endemic regions of Africa and Asia [86] and is known to be a major cause of cross reactivity to rK39 [84]. Sera of malaria patients were tested and did not give a signal in the rKLO8 ELISA. In conclusion, rKLO8 is a novel recombinant protein of L. donovani with increased reactivity to VL sera from

Traditionally the intrinsic intracellular and surface proteins of the parasites were targeted as primary source of antigens. Recently, secreted proteins are considered an untapped source of possible antigens and are being exploited using combinations of bioinformatic and

Lipid levels are known to vary in acute and chronic infections. In these infections, there is typically a decrease of total cholesterol levels and an increase in the concentration of triglyceride-rich lipoproteins; mainly very low-density lipoproteins. Moreover, apolipoprotein A1, apolipoprotein B and low-density lipoprotein cholesterol levels decrease. In leishmaniasis, this lipid concentration difference may have a prognostic and diagnostic role, as lipids play an important role in the innate and adaptive immune response. Although these have potential as clinical markers, several factors have to be taken into account when interpreting lipids values, such as: genetic and environmental factors, malnutrition, reduced food intake during acute infection, and acute kidney injury and/or acute liver failure (many times associated with this neglected disease), all which influence lipids parameters. More efforts have to be put in the

Due to its wide range of manifestations, leishmaniasis diagnosis can be difficult [90]. Clinical manifestations can also be confused with other illnesses, often common in Leishmania endemic areas, such as malaria, toxoplasmosis and tuberculosis [91]. Age, medical history and host

Diagnosis of diseased patients and animals can be confirmed with conventional laboratorial techniques, such as visualization of the parasite in tissues by microscopic examination of a stained specimen, or in vitro culture of the parasite from biopsies or aspirates from lesions, lymph nodes, spleen and bone marrow. Other diagnosis methods include molecular detection of parasitic DNA in tissue samples and serological tests that detect anti-Leishmania antibodies [6]. Overall, the available techniques can detect active disease. Still, leishmaniasis has intrinsic

Sudan and a valuable candidate to be used in diagnosis in this area [84].

3.10. Secreted proteins

3.11. Lipids

immunoproteomic approaches [87, 88] .

206 Biomarker - Indicator of Abnormal Physiological Process

study of these molecules as disease markers [89].

immune system response are crucial parameters for diagnosis.

4. Diagnosis approaches

The gold standard for diagnosis is the microscopic observation of parasites in tissue samples. Parasite rich localizations like bone marrow, skin lesions, liver, lymph nodes and spleen are preferred. Identification of amastigotes by direct examination of aspirates is also possible but must be done by experts since the results are often dependent on the observer [6]. It can also originate false negative results due to the low number of parasites in some samples, particularly in asymptomatic cases. Most often, diagnostic is obtained by observation of the Leishmania amastigote forms in stained microscopic preparations with Giemsa [8]. The best results are obtained with parasite rich regions like spleen aspirates. With bone marrow aspirates, the sensitivity decreases considerably and lymph nodes aspirates have the worst sensitivity ranges [4]. This method requires trained personnel and involves invasive sampling, a risky procedure that can lead to fatal hemorrhage, which can only be performed in a place with access to appropriate medical facilities. Lymph node and bone marrow aspirates are safer; however, material obtained is less concentrated and therefore less sensitive, elevating the risk of false negatives [92]. All these methods include invasive sampling, are time-consuming and impracticable to be performed on a large scale [3].

The culture of infected tissues is another classical diagnostic test, although the major problem with this technique is that different species of Leishmania can have different growth requirements and contaminations are recurrent. The culture is performed through the inoculation of the triturated tissue in adequate media. Both techniques (microscopic exam and culture) have an overall sensitivity of around 85% [3]. The best results are obtained with spleen aspirates (93–98% of sensitivity). When it comes to bone marrow aspirates, the sensitivity comes down to 60 to 85% and worse results are obtained with lymph nodes aspirates (sensitivity ranges between 52 to 58%) [4]. Despite being more sensitive than microscopic examination, it is time consuming and expensive, therefore rarely used for clinical diagnosis [93].

#### 4.1.1. Xenodiagnosis

A positive serological test result is not enough to prove that an infected dog is capable of transmitting the pathogen to the vector. Lack of sensitivity contributes to the lack of diagnostic control efficiency. The only technique that evaluates if an infected mammalian host can transmit the parasite by natural means to the vector is xenodiagnosis [94–96]. Sandflies are placed inside specific containers and placed in contact with dogs for a certain period of time for a blood meal to occur. After the feeding, the sandflies are separated and the feeding rate is calculated. The presence of parasites in the sandfly can be evaluated by direct observation of the promastigote

forms by optical microscopy or Polymerase Chain Reaction (PCR). This test can be highly specific and sensitive, depending on parasite load and procedure implementation [96].

infections [8]. However, this technique is still unreliable (lack of specificity and variable sensitivity). Efforts are being made to improve this tool, as it stands as a promising approach.

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Currently, most clinical and surveillance laboratories use serological techniques to detect pathogen-specific antibodies, since direct methods are often either invasive, potentially fatal or expensive. Leishmania infection is characterized by the presence of a significant humoral response, leading to the production of antibodies against Leishmania species. Serological methods to detect these anti-Leishmania antibodies are useful as alternative diagnosis tests for both human and canine Leishmania infections [107]. The presence of anti-Leishmania antigens/antibodies in both asymptomatic and symptomatic infected dogs has allowed the development of agglutination tests, immunofluorescent serologic tests (such as Western blotting), immunochromatographic tests, and enzyme-linked immunosorbent assays (ELISAs). The sensitivity depends on the methodology but the specificity will depend on the antigen used. Serological tests are the current tests of choice to diagnose CanL. Although, these tests often lack sensitivity and specificity due to dog low specific antibody titers in the early stages of infection. Significant crossreactivity has also been reported [108]. Additionally, after a successful treatment, antibody levels take a while to decrease. This can mask a relapse, making it impossible to diagnose it. The introduction of commercial vaccines against CanL raised concerns since it can lead to antibody production and might prevent accurate results in serological tests for the diagnosis. This may not allow differentiating between Infected and Vaccinated Animals (DIVA) [19]. Most commercially available vaccines against CanL are not DIVA. This difficults the serological identification of

Originally created as an alternative to the risky procedure of splenic aspirates, the Direct Agglutination Test (DAT) was the first antibody detection test used in VL diagnosis and has been used for more than 25 years. DAT is a simple semi-quantitative diagnostic tool, with a high sensitivity

DAT detects parasite antibodies in infected blood or serum through direct agglutination. It is a semi-quantitative method based on visual agglutinations obtained by the increased dilution of blood or serum mixed with stained, killed parasites in V-shaped wells [110]. If the result is negative (absence of anti-Leishmania antibodies) DAT antigen accumulates at the bottom of the plate. If there are anti-Leishmania antibodies present, the antigens form a film over the well (positive result) [6, 111]. DAT has some disadvantages; it requires moderate technical expertise (the interpretation of the results depend on the person analyzing the results, creating interobserver discrepancy) [112], serial dilutions must be done (requiring a considerable volume of antigen) and has a relatively long incubation time [112]. DAT results remain positive long after the patient is cured (anti-Leishmania antibodies can persist for years as a result of a VL

Enzyme-linked immunosorbent assay (ELISA) is an important serodiagnosis technique used in almost all infectious diseases, including VL. ELISA assays are based on the detection of

(91–100%), specificity (72–100%), accuracy, reliability and inexpensiveness [4, 109].

infection), making this test inappropriate for detecting relapses [8].

4.3.2. Enzyme-linked immunosorbent assay

animals that become sick or infected in the future [19].

4.3.1. Direct agglutination test

#### 4.1.2. Montenegro skin test

The Montenegro Skin Test evaluates the late cellular hypersensitivity response. A solution with promastigotes is injected intradermally, and a positive result consists in the appearance of a hardened papule, equal or greater than 5 mm after 48 hours of injection. The test is low cost and highly sensitive (can reach over 90%). A positive result is possible within 3 months after infection, and relates to the disease evolution. Specificity is low (around 75%) due to the overall large number of false positive results in cases of unapparent infection and crossreactivity with some pathologies, as well as technical problems [97].

#### 4.2. Molecular methodologies

PCR technology has become an indispensable tool for the diagnosis of many parasitic diseases, including leishmaniasis. It suffers similar limitations to the parasitological tests, where the success is limited by the quality of the sample (parasite rich specific locations), as these techniques are based on the amplification of specific parasitic DNA sequences. Although several DNA targets exist, like rRNA or ITS-1 gene, the most common DNA target is the Leishmania kinetoplast (kDNA) present in the minicircles of the parasite [98]. The diagnostic sensitivity of these approaches is above 95% [99, 100] . In asymptomatic dogs the reported efficacy is lower, less than 70% [100]. Leishmania DNA has been found to be present in the canine oral mucosa, and can possibly be used for diagnostic. Oral swabs have shown positive results in molecular diagnosis of infected dogs. Although, in samples from asymptomatic dogs, the diagnosis test showed lack of sensitivity. Oral swabs combined with conjunctival ones can be used for detection of L. infantum in asymptomatic dogs. The big advantage of this test is that the sample collection is non-invasive, and combining this two swab methods, can significantly contribute for detecting different stages of the infection [101]. The sensitivity of a PCR assay depends on three factors: the physicochemical conditions of the reaction, the concentration of the DNA target and the selected PCR primers [38, 102, 103]. Amplificationbased methods include the conventional PCR and qPCR (quantitative polymerase chain reaction) [104]. Real-time PCR (qPCR) is an innovative approach of target DNA quantification, faster than conventional PCR [102].

Although these techniques have a higher sensitivity for asymptomatic cases and early-stage infections when compared to serological methods, they imply high costs due to sophisticated equipment, reagents and specialized personnel [103, 105, 106].

#### 4.3. Serological diagnosis

Serological tests are based on the screening of antigen or antibodies. Antigen detection should be more specific for diagnosis that antibody-based immunodiagnostic tests. Thus, antigen levels are expected to correlate with parasite burden, being useful when antibody prediction is deficient. This approach should avoid cross-reactivity and distinguish active from past infections [8]. However, this technique is still unreliable (lack of specificity and variable sensitivity). Efforts are being made to improve this tool, as it stands as a promising approach.

Currently, most clinical and surveillance laboratories use serological techniques to detect pathogen-specific antibodies, since direct methods are often either invasive, potentially fatal or expensive. Leishmania infection is characterized by the presence of a significant humoral response, leading to the production of antibodies against Leishmania species. Serological methods to detect these anti-Leishmania antibodies are useful as alternative diagnosis tests for both human and canine Leishmania infections [107]. The presence of anti-Leishmania antigens/antibodies in both asymptomatic and symptomatic infected dogs has allowed the development of agglutination tests, immunofluorescent serologic tests (such as Western blotting), immunochromatographic tests, and enzyme-linked immunosorbent assays (ELISAs). The sensitivity depends on the methodology but the specificity will depend on the antigen used. Serological tests are the current tests of choice to diagnose CanL. Although, these tests often lack sensitivity and specificity due to dog low specific antibody titers in the early stages of infection. Significant crossreactivity has also been reported [108]. Additionally, after a successful treatment, antibody levels take a while to decrease. This can mask a relapse, making it impossible to diagnose it. The introduction of commercial vaccines against CanL raised concerns since it can lead to antibody production and might prevent accurate results in serological tests for the diagnosis. This may not allow differentiating between Infected and Vaccinated Animals (DIVA) [19]. Most commercially available vaccines against CanL are not DIVA. This difficults the serological identification of animals that become sick or infected in the future [19].

#### 4.3.1. Direct agglutination test

forms by optical microscopy or Polymerase Chain Reaction (PCR). This test can be highly

The Montenegro Skin Test evaluates the late cellular hypersensitivity response. A solution with promastigotes is injected intradermally, and a positive result consists in the appearance of a hardened papule, equal or greater than 5 mm after 48 hours of injection. The test is low cost and highly sensitive (can reach over 90%). A positive result is possible within 3 months after infection, and relates to the disease evolution. Specificity is low (around 75%) due to the overall large number of false positive results in cases of unapparent infection and cross-

PCR technology has become an indispensable tool for the diagnosis of many parasitic diseases, including leishmaniasis. It suffers similar limitations to the parasitological tests, where the success is limited by the quality of the sample (parasite rich specific locations), as these techniques are based on the amplification of specific parasitic DNA sequences. Although several DNA targets exist, like rRNA or ITS-1 gene, the most common DNA target is the Leishmania kinetoplast (kDNA) present in the minicircles of the parasite [98]. The diagnostic sensitivity of these approaches is above 95% [99, 100] . In asymptomatic dogs the reported efficacy is lower, less than 70% [100]. Leishmania DNA has been found to be present in the canine oral mucosa, and can possibly be used for diagnostic. Oral swabs have shown positive results in molecular diagnosis of infected dogs. Although, in samples from asymptomatic dogs, the diagnosis test showed lack of sensitivity. Oral swabs combined with conjunctival ones can be used for detection of L. infantum in asymptomatic dogs. The big advantage of this test is that the sample collection is non-invasive, and combining this two swab methods, can significantly contribute for detecting different stages of the infection [101]. The sensitivity of a PCR assay depends on three factors: the physicochemical conditions of the reaction, the concentration of the DNA target and the selected PCR primers [38, 102, 103]. Amplificationbased methods include the conventional PCR and qPCR (quantitative polymerase chain reaction) [104]. Real-time PCR (qPCR) is an innovative approach of target DNA quantification,

Although these techniques have a higher sensitivity for asymptomatic cases and early-stage infections when compared to serological methods, they imply high costs due to sophisticated

Serological tests are based on the screening of antigen or antibodies. Antigen detection should be more specific for diagnosis that antibody-based immunodiagnostic tests. Thus, antigen levels are expected to correlate with parasite burden, being useful when antibody prediction is deficient. This approach should avoid cross-reactivity and distinguish active from past

specific and sensitive, depending on parasite load and procedure implementation [96].

reactivity with some pathologies, as well as technical problems [97].

4.1.2. Montenegro skin test

208 Biomarker - Indicator of Abnormal Physiological Process

4.2. Molecular methodologies

faster than conventional PCR [102].

4.3. Serological diagnosis

equipment, reagents and specialized personnel [103, 105, 106].

Originally created as an alternative to the risky procedure of splenic aspirates, the Direct Agglutination Test (DAT) was the first antibody detection test used in VL diagnosis and has been used for more than 25 years. DAT is a simple semi-quantitative diagnostic tool, with a high sensitivity (91–100%), specificity (72–100%), accuracy, reliability and inexpensiveness [4, 109].

DAT detects parasite antibodies in infected blood or serum through direct agglutination. It is a semi-quantitative method based on visual agglutinations obtained by the increased dilution of blood or serum mixed with stained, killed parasites in V-shaped wells [110]. If the result is negative (absence of anti-Leishmania antibodies) DAT antigen accumulates at the bottom of the plate. If there are anti-Leishmania antibodies present, the antigens form a film over the well (positive result) [6, 111]. DAT has some disadvantages; it requires moderate technical expertise (the interpretation of the results depend on the person analyzing the results, creating interobserver discrepancy) [112], serial dilutions must be done (requiring a considerable volume of antigen) and has a relatively long incubation time [112]. DAT results remain positive long after the patient is cured (anti-Leishmania antibodies can persist for years as a result of a VL infection), making this test inappropriate for detecting relapses [8].

#### 4.3.2. Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) is an important serodiagnosis technique used in almost all infectious diseases, including VL. ELISA assays are based on the detection of antibodies present in blood or serum. An antigen/recombinant protein is used to coat the plate and samples are added and incubated. A secondary antibody conjugated with an enzyme recognizes and binds to the primary antibody. After adding a substrate, product susceptible to colorimetric determination is developed and the results can be measured by optical density techniques.

The flow cytometry technique can quantify the antibodies against Leishmania surface antigens, restraining potential cross-reactivity against more conserved intracellular structures [117] and showing potential to monitor post chemotherapy VL, in order to evaluate the success of

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As described before, every diagnosis technique has its downsides. Parasite demonstration in tissue smears and culture provide definitive diagnosis of VL, but generally have a lower sensitivity than serologic methods with a high risk associated. Microscopy techniques lacks sensitivity, whereas culture requires a longer time to obtain a result and is vulnerable to

Molecular diagnostic tools like PCR and real-time PCR are quite sensitive and specific although difficult to perform in field conditions and costly [8]. Its use remains largely restricted to some

Serological tests are often not sensitive enough to detect asymptomatic individuals, having to be combined with classical methods of diagnosis to confirm. ELISA, IFAT, DAT and rK39 immunochromatographic strip test (ICT) are highly sensitive and specific when analyzing active VL in immunocompetent individuals. This does not happen when titers decline and parasite charge is lower. Due to this, false negative results frequently happen in immunocom-

In that way, the big challenge in the field is to isolate, recombine, produce and identify new proteins capable of detecting asymptomatic leishmaniasis cases and early stages of infection in VL endemic regions, using safe techniques that are easy, fast, sensitive and with a fair

The importance of serological methods has been rising in vector-borne diseases, due to the low sensitivity associated to the microscopic methods [52]. Molecular biology developments over the years have led to advances in techniques that aided in VL control and had significant

The main problem with leishmaniasis diagnosis is the identification of markers that can detect the presence of low titres of antibodies. Thus, new markers are needed to contribute for a more accurate diagnosis capable of detecting asymptomatic cases and early stages of disease in large

5. Successful diagnosis approaches-from laboratory to field conditions

Currently, the scientific community is investing in developing VL tests based on antigen detec-

Rapid diagnostic tests (RDTs) are equipment-free diagnostic devices that are adequate for field conditions. The results of this test can be read easily and in a short amount of time. Most RDTs work by capturing either an antigen or an antibody on a solid surface and then attaching

promised patients and asymptomatic Leishmania infections [120].

progress in surveillance and diagnosis of this disease [52].

treatment [118].

contamination [119].

cost.

hospitals and research centers [90].

screening studies [38, 121].

tion, such as rapid diagnostic tests.

molecules that allow detection by the naked eye.

This tool is frequently used to detect anti-Leishmania antibodies due to its high sensitive and specificity as well as good reproducibility and high throughput screening of large number of samples, at an affordable expense. However, the specificity of this technique depends on the antigen used. ELISA's major disadvantage is its inadaptability in poor-resource field conditions and requiring specialized operators.

#### 4.3.3. Indirect fluorescent antibody technique

Indirect Fluorescent Antibody Technique (IFAT) is an extremely valuable tool that allows assessment of anti-Leishmania antibody titers produced by infected individuals. It is a quite useful method in epidemiological studies and in clinical practice [113]. Promastigote or amastigote forms have been used as a source for antigens used by IFAT [25]. IFAT is based on the use of fluorophores to detect antibodies that recognize the parasites. It involves the use of a primary antibody that binds to the antigen and allows the formation of an antigen-antibody complex. Posteriorly, the binding of a fluorophore-conjugated secondary antibody results in an amplified signal that can be examined by fluorescence microscopy. IFAT is the serological gold standard for diagnosis of CanL in most countries. This analytical methodology differs from ELISA as it resorts to the whole body parasite as antigen [113]. Although having a high specificity and sensitivity (100% and 90%, respectively), IFAT sensitivity is lower for asymptomatic infections when compared to ELISA [29, 114].

#### 4.3.4. Immunochromatographic tests

Immunochromatographic (IC) tests are recurrently used in large-scale screenings. The results obtained from IC tests consider the epidemiological context and do not require antibody titration [115]. IC test is based on chromatographic and relies on capillary action. An antigenantibody complex is formed and entrapped on the membrane. The sample migrates across this membrane, the antibodies present in the blood recognize the antigen and the labeled antibody present in the membrane resulting in the formation of a colored product. IC tests are inexpensive, practical, rapid and suitable for field use. However, these tests frequently lead to false positives with patients in endemic regions that have already been infected in the past. Resultantly, this technique does not work in the diagnosis of relapsed patients [115]. Much like ELISA, the specificity and sensitivity of IC test is dependent on the antigen used.

#### 4.3.5. Flow cytometry

Flow Cytometry (FC) is an emerging technique for the diagnostic of several infectious diseases [116]. It has a high throughput capacity, possibility of quantification, high reproducibility and sensitivity and potential for multiplexing [49]. This technique analyses and sorts cells or particle suspensions in a controlled fluid stream, through the measurement of fluorescence and scatter induced illumination, allowing the acquisition of structural and functional data.

The flow cytometry technique can quantify the antibodies against Leishmania surface antigens, restraining potential cross-reactivity against more conserved intracellular structures [117] and showing potential to monitor post chemotherapy VL, in order to evaluate the success of treatment [118].

antibodies present in blood or serum. An antigen/recombinant protein is used to coat the plate and samples are added and incubated. A secondary antibody conjugated with an enzyme recognizes and binds to the primary antibody. After adding a substrate, product susceptible to colorimetric determination is developed and the results can be measured by optical density

This tool is frequently used to detect anti-Leishmania antibodies due to its high sensitive and specificity as well as good reproducibility and high throughput screening of large number of samples, at an affordable expense. However, the specificity of this technique depends on the antigen used. ELISA's major disadvantage is its inadaptability in poor-resource field condi-

Indirect Fluorescent Antibody Technique (IFAT) is an extremely valuable tool that allows assessment of anti-Leishmania antibody titers produced by infected individuals. It is a quite useful method in epidemiological studies and in clinical practice [113]. Promastigote or amastigote forms have been used as a source for antigens used by IFAT [25]. IFAT is based on the use of fluorophores to detect antibodies that recognize the parasites. It involves the use of a primary antibody that binds to the antigen and allows the formation of an antigen-antibody complex. Posteriorly, the binding of a fluorophore-conjugated secondary antibody results in an amplified signal that can be examined by fluorescence microscopy. IFAT is the serological gold standard for diagnosis of CanL in most countries. This analytical methodology differs from ELISA as it resorts to the whole body parasite as antigen [113]. Although having a high specificity and sensitivity (100% and 90%, respectively), IFAT sensitivity is lower for asymp-

Immunochromatographic (IC) tests are recurrently used in large-scale screenings. The results obtained from IC tests consider the epidemiological context and do not require antibody titration [115]. IC test is based on chromatographic and relies on capillary action. An antigenantibody complex is formed and entrapped on the membrane. The sample migrates across this membrane, the antibodies present in the blood recognize the antigen and the labeled antibody present in the membrane resulting in the formation of a colored product. IC tests are inexpensive, practical, rapid and suitable for field use. However, these tests frequently lead to false positives with patients in endemic regions that have already been infected in the past. Resultantly, this technique does not work in the diagnosis of relapsed patients [115]. Much like

Flow Cytometry (FC) is an emerging technique for the diagnostic of several infectious diseases [116]. It has a high throughput capacity, possibility of quantification, high reproducibility and sensitivity and potential for multiplexing [49]. This technique analyses and sorts cells or particle suspensions in a controlled fluid stream, through the measurement of fluorescence and scatter induced illumination, allowing the acquisition of structural and functional data.

ELISA, the specificity and sensitivity of IC test is dependent on the antigen used.

techniques.

tions and requiring specialized operators.

210 Biomarker - Indicator of Abnormal Physiological Process

4.3.3. Indirect fluorescent antibody technique

tomatic infections when compared to ELISA [29, 114].

4.3.4. Immunochromatographic tests

4.3.5. Flow cytometry

As described before, every diagnosis technique has its downsides. Parasite demonstration in tissue smears and culture provide definitive diagnosis of VL, but generally have a lower sensitivity than serologic methods with a high risk associated. Microscopy techniques lacks sensitivity, whereas culture requires a longer time to obtain a result and is vulnerable to contamination [119].

Molecular diagnostic tools like PCR and real-time PCR are quite sensitive and specific although difficult to perform in field conditions and costly [8]. Its use remains largely restricted to some hospitals and research centers [90].

Serological tests are often not sensitive enough to detect asymptomatic individuals, having to be combined with classical methods of diagnosis to confirm. ELISA, IFAT, DAT and rK39 immunochromatographic strip test (ICT) are highly sensitive and specific when analyzing active VL in immunocompetent individuals. This does not happen when titers decline and parasite charge is lower. Due to this, false negative results frequently happen in immunocompromised patients and asymptomatic Leishmania infections [120].

In that way, the big challenge in the field is to isolate, recombine, produce and identify new proteins capable of detecting asymptomatic leishmaniasis cases and early stages of infection in VL endemic regions, using safe techniques that are easy, fast, sensitive and with a fair cost.

The importance of serological methods has been rising in vector-borne diseases, due to the low sensitivity associated to the microscopic methods [52]. Molecular biology developments over the years have led to advances in techniques that aided in VL control and had significant progress in surveillance and diagnosis of this disease [52].

The main problem with leishmaniasis diagnosis is the identification of markers that can detect the presence of low titres of antibodies. Thus, new markers are needed to contribute for a more accurate diagnosis capable of detecting asymptomatic cases and early stages of disease in large screening studies [38, 121].
