3. Biomarkers to use in diagnosis

After an incubation period that can range from 3 months to several years, dogs may start presenting clinical signs. However, some dogs remain asymptomatic and never develop any signs [17]. Therefore, dogs can present different forms of the disease: symptomatic, oligosymptomatic and asymptomatic. Dogs that develop few mild symptoms are classified as oligosymptomatic. Even so, it is relevant to mention that it is not consensual that all forms of the disease enable the transmission of the parasite. Infected dogs present non-specific cutaneous alterations such as alopecia, onychogryposis, dermatitis, skin ulceration, anorexia, weight loss and visceral manifestations with splenic, renal and hepatic disorders [18], making an accurate diagnosis impossible. Given that canine disease usually precedes the appearance of human cases and a clear correlation between canine and human infection rates has been demonstrated, CanL must be consid-

The available therapies for leishmaniasis are far from optimal due to their toxicity, high costs, lack of efficacy, lack of access in certain areas, and emerging drug resistance. Treatment efficacy depends on strains and species and there are currently no effective vaccines available for any

Some vaccines exist for veterinary use. Being launched in Portugal in 2011, CaniLeish® was the first vaccine for CanL in the European Union. In Brazil, LeishTec® vaccine was also registered but only offers about 40% of protection against infection. LetiFend® has recently been registered in Europe, but there is limited information available [19]. Despite the efforts, Leishmania vaccinology still has a lot to improve till an effective and universal vaccine is developed [20]. In the absence of human vaccines and due to the zoonotic character of the disease, accurate detection of infection in humans and dogs is crucial for the control of leishmaniasis [21].

Several specific challenges associated to Leishmania infection and leishmaniasis must be overcome. As for any disease, the diagnostic process should be simple, robust, automated, requiring inexpensive reagents and minimal operator intervention without diminishing the fidelity of the results. Notwithstanding, considering that this disease affects mostly poor people in countries with undeveloped and underfinanced health systems, the tests should be cheap and easy to perform in field conditions [22, 23]. Moreover, the detection of asymptomatic infection, often characterized by reduced parasite loads and low specific serology, is essential. In fact, clinical and epidemiological management of Leishmania infections can only be fully successful once a diagnostic test with these characteristics is available [24]. The available tools are adequate for detection of disease, (in conjunction with clinical evaluation) but present limitations for diagnosis of infection in asymptomatic patients and dogs. Therefore, the information of real prevalence of infection and overall burden of disease is believed to be underestimated [22].

Coinfection with HIV is common in VL cases and has a disastrous impact since immunocompromised individuals have more severe manifestations and atypical symptoms that complicate treatment [2]. Pregnant women can be considered a risk population, as HIV-infected patients,

ered as a risk for human health [3].

200 Biomarker - Indicator of Abnormal Physiological Process

2. Importance of diagnosis

form of human leishmaniasis [2].

Every methodology has a detection limit associated but the specificity of the diagnosis platform is mostly influenced by the antigen used, relying on it to produce high confidence results.

Several antigens were proposed overtime but the most common one is crude soluble antigen (CSA) [13, 26]. Although the sensitivity of CSA in Enzyme-Linked Immunosorbent Assay (ELISA) is high, cross-reactivity with other diseases (trypanosomiasis, toxoplasmosis and tuberculosis) occurs frequently, leading to false positive results [27, 28]. Therefore specificity can be low due to cross-reactivity and is not suitable in detecting seropositivity in asymptomatic dogs [29–31]. This is transversal to other serological techniques like Direct Agglutination Test (DAT) or Indirect Fluorescent Antibody Technique (IFAT) which are based on the recognition of the parasite total antigens. The specificity and sensitivity problems associated to conventional assays (such as IFAT, DAT, and CSA ELISA) may be overcome using recombinant polypeptides containing specific epitopes that are able to induce an immune response in most dogs and humans with VL. Hence, there has been a focus on expressing and purifying Leishmania proteins that elicit an immune response in dogs and humans and analyze them and their potential for serological tests [32–34].

The evolution of molecular technology (immunoproteomics) and sequencing of the Leishmania genome in 2005 have been powerful tools for the discovery of recombinant Leishmania proteins to use in the serodiagnosis of human and canine leishmaniasis [35, 36]. Variability in the humoral response concerning different parasite antigens observed in infection suggests that a combination of recombinant proteins can improve the diagnosis efficiency [37]. In fact, each antigen carries often both specific immune-dominant epitopes and other regions that are not important for serological recognition [38]. Therefore, an ideal test would contain a combination of relevant epitopes in a single recombinant antigen, more specific than crude antigen preparation and more sensitive than single epitope-based ELISA [39]. In fact, chimeric multi-epitope proteins are part of the future strategy to look for novel high specific antigens [40]. Several recombinant proteins with high efficacy have reasonable results for the diagnosis of human and canine VL, but development of suitable antigens for diagnosis is still necessary [34].

The most common used antigens belong to different protein families, such as the kinesinrelated proteins, heat shock proteins, acidic ribosomal proteins, nuclear proteins, enzymes and other antigens which are associated with parasite function [41].

#### 3.1. Kinesin-related proteins

Kinesins are a family of motor proteins in eukaryotic cells, and constitute part of the microtubule cytoskeleton in Leishmania parasites, important in its growth and differentiation [42–44].

3.2. Heat shock proteins

been characterized.

3.3. Ribosomal proteins

[41, 67].

by sera from the same patients has been detailed [59].

Heat Shock Proteins (HSPs) are a family of proteins that are produced by cells in response to stressful conditions. They are highly conserved molecules that play important roles in protein folding, assembly of protein complexes, and translocation of proteins across cellular compartments. The L. donovani HSP70 was identified after screening of a cDNA library with serum from a patient with VL [58]. It is found in prokaryotic and eukaryotic cells and is a highly conserved protein. HSP70s from other Leishmania species (L. infantum and L. braziliensis [59]) have also

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rHSP70 had high sensitivity and specificity values, being superior to CSA in the diagnosis of CL [60]. The antigenicity of heat shock proteins in general and of HSP70 specifically is not unexpected, since antibodies against the parasite recombinant HSPs have been found in sera of patients with different parasitic diseases [61, 62]. Other authors have described rHSP70 as a preeminent antigen present in Leishmania [63]. Specific antibodies against L. braziliensis rHSP70 or rHSP83 were found in 95% of sera from ML or CL patients and a high recognition of rHSP70

L. infantum-rHSP83 is a possible antigen to be used in the serodiagnosis of leishmaniasis, due to its high specificity and sensitivity and insignificant cross-reactivity with other infectious diseases [48]. Members of the HSP83/90 family have also been described as immunodominant

Another gene, LmSti1, encoding a stress-inducible protein STI1 [65] was strongly recognized in

Ribosomal proteins have been described to induce antibodies in animal and humans infected

Studies have described some Leishmania ribosomal proteins function as immunoregulatory molecules [41, 59]. As a matter of fact, the eukaryotic ribosome is composed of four RNA molecules and more than 70 ribosomal proteins [66]. Furthermore, the acidic ribosomal proteins (P-proteins) have been detailed as prominent antigens during Leishmania infections

The large subunit of ribosomes contains various copies of a protein called P protein. P1, P2 and P0 form a complex notorious for having an essential role in protein synthesis. Three L. infantum antigens (LiP2a, LiP2b and LiP0), homologous to acidic ribosomal proteins, were identified after immunoscreening of a cDNA expression library with CanL serum [68]. They are also

The ribosomal proteins LiP2a, LIP2b, LiP0/LcP0 and LeIF, all conserved antigens of Leishmania,

with the parasite, making them a favorable candidate to assess its diagnosis potential.

antigens during infections caused by L. donovani, L. braziliensis and L. infantum [64].

sera from human patients with CL, VL and post-kala azar CL [48].

recognized by sera from patients with either VL or MCL [41, 69].

have been shown to be recognized by the immune system of the host [70].

Several Leishmania antigens have been characterized, as the recombinant K39 antigen (rK39), a 39-aminoacid-repetitive B-cell epitope of the kinesin-related protein of L. infantum. The rK39 ELISA has shown to be capable of detecting human and canine VL.

The K9 and K26 are two other hydrophilic antigens of L. infantum, and their recombinant antigens (rK9 and rK26) have been used to diagnose VL. The rK28 is a recombinant fusion protein with tandem repeats of K39 kinesin regions and K26, and has been used to detect high levels of antibody responses in infected patients, using ELISA [13, 37].

The rK39, an antigen used for VL diagnosis, is highly conserved among VL species [15]. Several studies carried out in VL endemic areas have demonstrated that the rK39 ELISA is a sensitive and a specific method for the serodiagnosis of human VL [34, 45–47]. An important aspect of the anti-rK39 antibody is that the titer correlates directly with the disease activity, indicating its potential for use in predicting response to therapy [48]. The rk39 has also been tested for ELISA and Flow Cytometry (FC) assays showing high sensitivity and specificity in detecting clinical forms of CanL [49]. rK39 is further addressed in Section 5—Successful diagnosis approaches—from laboratory to field conditions.

VL patients also have a strong antibody response to K26, which can complement rK39 in a better and more accurately diagnosis of human VL [50]. Specific and independent antibody reactivity to each of these antigens (rK9, rK26 and rK39) have been studied and tested in serodiagnosis of canine VL [31, 51].

Another antigen that has been successfully used in the diagnosis of human VL is rK28. This recombinant fusion antigen was used for ELISA and has shown sensitivity and specificity values similar or higher than those obtained for rK39. Therefore, this antigen has been proposed as a new choice, especially regarding regions where rK39 has shown low levels of sensitivity [52]. In Sudan and in India [53], human serological surveys using rK28 antigen have shown good performance with high sensitivity and specificity [54]. ELISA with rK28 antigen was 100% sensitive and specific in serological diagnosis of CanL [54]. The sensitivity of rK28 was slightly higher than rK39, although no significant differences in the detection of positive dogs were observed, indicating that both antigens may be useful for diagnosing CanL, as previously observed in VL in human [54, 55].

KE16 is another kinesin gene derived from the C-terminus of the kinesin protein from an isolate of L. donovani [56] that has been successfully cloned and expressed. It is used in the rKE16 dipstick test, a one-step rapid immunochromatographic test. Results showed that this antigen can be used as a highly specific and sensitive tool for VL diagnosis, although, in dogs, the average of positive cases among asymptomatic dogs was low [34, 56, 57].

#### 3.2. Heat shock proteins

3.1. Kinesin-related proteins

202 Biomarker - Indicator of Abnormal Physiological Process

tion [42–44].

Kinesins are a family of motor proteins in eukaryotic cells, and constitute part of the microtubule cytoskeleton in Leishmania parasites, important in its growth and differentia-

Several Leishmania antigens have been characterized, as the recombinant K39 antigen (rK39), a 39-aminoacid-repetitive B-cell epitope of the kinesin-related protein of L. infantum. The rK39

The K9 and K26 are two other hydrophilic antigens of L. infantum, and their recombinant antigens (rK9 and rK26) have been used to diagnose VL. The rK28 is a recombinant fusion protein with tandem repeats of K39 kinesin regions and K26, and has been used to detect high

The rK39, an antigen used for VL diagnosis, is highly conserved among VL species [15]. Several studies carried out in VL endemic areas have demonstrated that the rK39 ELISA is a sensitive and a specific method for the serodiagnosis of human VL [34, 45–47]. An important aspect of the anti-rK39 antibody is that the titer correlates directly with the disease activity, indicating its potential for use in predicting response to therapy [48]. The rk39 has also been tested for ELISA and Flow Cytometry (FC) assays showing high sensitivity and specificity in detecting clinical forms of CanL [49]. rK39 is further addressed in Section 5—Successful

VL patients also have a strong antibody response to K26, which can complement rK39 in a better and more accurately diagnosis of human VL [50]. Specific and independent antibody reactivity to each of these antigens (rK9, rK26 and rK39) have been studied and tested in

Another antigen that has been successfully used in the diagnosis of human VL is rK28. This recombinant fusion antigen was used for ELISA and has shown sensitivity and specificity values similar or higher than those obtained for rK39. Therefore, this antigen has been proposed as a new choice, especially regarding regions where rK39 has shown low levels of sensitivity [52]. In Sudan and in India [53], human serological surveys using rK28 antigen have shown good performance with high sensitivity and specificity [54]. ELISA with rK28 antigen was 100% sensitive and specific in serological diagnosis of CanL [54]. The sensitivity of rK28 was slightly higher than rK39, although no significant differences in the detection of positive dogs were observed, indicating that both antigens may be useful for diagnosing CanL, as

KE16 is another kinesin gene derived from the C-terminus of the kinesin protein from an isolate of L. donovani [56] that has been successfully cloned and expressed. It is used in the rKE16 dipstick test, a one-step rapid immunochromatographic test. Results showed that this antigen can be used as a highly specific and sensitive tool for VL diagnosis, although, in dogs,

the average of positive cases among asymptomatic dogs was low [34, 56, 57].

ELISA has shown to be capable of detecting human and canine VL.

levels of antibody responses in infected patients, using ELISA [13, 37].

diagnosis approaches—from laboratory to field conditions.

serodiagnosis of canine VL [31, 51].

previously observed in VL in human [54, 55].

Heat Shock Proteins (HSPs) are a family of proteins that are produced by cells in response to stressful conditions. They are highly conserved molecules that play important roles in protein folding, assembly of protein complexes, and translocation of proteins across cellular compartments.

The L. donovani HSP70 was identified after screening of a cDNA library with serum from a patient with VL [58]. It is found in prokaryotic and eukaryotic cells and is a highly conserved protein. HSP70s from other Leishmania species (L. infantum and L. braziliensis [59]) have also been characterized.

rHSP70 had high sensitivity and specificity values, being superior to CSA in the diagnosis of CL [60]. The antigenicity of heat shock proteins in general and of HSP70 specifically is not unexpected, since antibodies against the parasite recombinant HSPs have been found in sera of patients with different parasitic diseases [61, 62]. Other authors have described rHSP70 as a preeminent antigen present in Leishmania [63]. Specific antibodies against L. braziliensis rHSP70 or rHSP83 were found in 95% of sera from ML or CL patients and a high recognition of rHSP70 by sera from the same patients has been detailed [59].

L. infantum-rHSP83 is a possible antigen to be used in the serodiagnosis of leishmaniasis, due to its high specificity and sensitivity and insignificant cross-reactivity with other infectious diseases [48]. Members of the HSP83/90 family have also been described as immunodominant antigens during infections caused by L. donovani, L. braziliensis and L. infantum [64].

Another gene, LmSti1, encoding a stress-inducible protein STI1 [65] was strongly recognized in sera from human patients with CL, VL and post-kala azar CL [48].

#### 3.3. Ribosomal proteins

Ribosomal proteins have been described to induce antibodies in animal and humans infected with the parasite, making them a favorable candidate to assess its diagnosis potential.

Studies have described some Leishmania ribosomal proteins function as immunoregulatory molecules [41, 59]. As a matter of fact, the eukaryotic ribosome is composed of four RNA molecules and more than 70 ribosomal proteins [66]. Furthermore, the acidic ribosomal proteins (P-proteins) have been detailed as prominent antigens during Leishmania infections [41, 67].

The large subunit of ribosomes contains various copies of a protein called P protein. P1, P2 and P0 form a complex notorious for having an essential role in protein synthesis. Three L. infantum antigens (LiP2a, LiP2b and LiP0), homologous to acidic ribosomal proteins, were identified after immunoscreening of a cDNA expression library with CanL serum [68]. They are also recognized by sera from patients with either VL or MCL [41, 69].

The ribosomal proteins LiP2a, LIP2b, LiP0/LcP0 and LeIF, all conserved antigens of Leishmania, have been shown to be recognized by the immune system of the host [70].

The L. infantum acidic ribosomal proteins, LiP2a and LiP2b, have disease-specific antigenic determinants identified by more than 80% of canine VL [70]. Engineered LiP2a and LiP2b recombinant proteins are shown to be useful as tools to discriminate between VL and Chagas disease [71]. L. infantum P0 ribosomal protein is also recognized by a high percentage of the sera from dogs with VL [70].

recovered cases of VL in both humans and dogs. Leishmania cysteine proteinases had already

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A recombinant cysteine proteinase from L. chagasi, rLdccys1, has shown to be a good biomarker for the different stages of both human and CanL. ELISA assays showed high sensitivity and specificity. Moreover, the fact that it is possible to detect clinical and subclinical forms of the disease in canines indicates that this biomarker is important in the control of CanL in

Histones are structural proteins involved in the organization and function of DNA within the eukaryotic nucleus. There are four main classes of histones: H2A, H2B, H3 and H4 which form the nucleosomal core unit of chromatin. These proteins are among the most conserved proteins in eukaryotic organisms, maintaining sequence and function in trypanosomatids. Histones are prominent antigens found in animals during Leishmania infection that trigger a specific immune response and antibody production [39]. Also, sera from children infected with VL specifically recognize the L. infantum H2A and H2B histones, with high specificity and sensi-

A2 proteins of L. donovani are only present in the amastigote stage, and help visceralization of parasites in the mammalian host. They are overexpressed in the amastigote stage in L. donovani as well as L. mexicana species complex, including L. amazonensis, but not in L. tropica or L. braziliensis species complexes. Anti-A2 antibodies were detected in human and dog sera suffering from active VL [79, 80]. Also, it was found that rA2 is more sensitive when compared to the rK39 and rK26 antigens, for serological detection of asymptomatic infection in dogs [34].

Leishmania homolog of receptors for activated C Kinase (LACK) was identified after a search for parasite antigens recognized by a protective Th1 clone derived from the spleen of BALB/c mice that had been vaccinated with a soluble extract of L. major promastigotes. It has been proposed that the LACK protein contains an immunodominant epitope that acts as a target of

Sudan has the highest number of reported cases in East Africa [82], in particular in eastern and central regions. Field tests based on rK39 commonly have a high reliability in various countries [83, 84]; however, the low sensitivity in Sudan limits its use in this region. A novel L. donovaniderived recombinant immunodominant protein (rKLO8) was tested for detection of VL in

been used as vaccines targets and in chemotherapy [76].

3.8. Leishmania homolog of receptors for activated C kinase

endemic areas [77, 78].

3.6. Nuclear proteins

tivity in ELISA assays.

early immune responses [41, 81].

3.9. rKLO8

Sudan.

3.7. A2 proteins

The acidic ribosomal proteins (P1, P2 and P0) are recognized by antibodies frequently found in sera from systemic lupus erythematosus (SLE) patients. These autoantibodies recognize an amino acid sequence located at the C-terminal ends of the three P proteins [72]. Furthermore, patients with chronic Chagas heart disease produce a strong humoral response against the Cterminal of Trypanosoma cruzi ribosomal P proteins. Leishmania ribosomal P2a and P2b Cterminal end of the proteins is also well conserved and these proteins are recognized by sera from both SLE and Chagas disease patients [69]. The anti-P antibodies produced during the Leishmania infection do not recognize the conserved C-terminal domain of the P proteins. Therefore, to avoid cross-reactivity, engineered versions of the recombinant proteins LiP2a and LiP2b, without the C-terminal ends, have been found to be useful for the diagnosis of MCL and VL [69].

#### 3.4. Peroxiredoxins

Tryparedoxins belong to a particular class of oxidoreductases related to thioredoxins and found in trypanosomatids. They bear oxidoreductase activity toward disulfide bridges and are crucial to the parasite, as they are expressed in all stages of development [73]. These proteins are believed to be involved in Leishmania detoxification of peroxides.

LiTXN1 protein is present in the cytosol and upregulated in the infectious forms of the parasite, indicating that it plays an important role during infection. LiTXN1 preferentially reduces the cytosolic L. infantum peroxiredoxins, LicTXNPx1 and LicTXNPx2 [73].

LicTXNPx antigen is highly immunogenic during both human and canine infections [74]. High antibody titers are found during the Leishmania infection and these decrease after its resolution [75].

Anti-LicTXNPx antibodies are present in both symptomatic and asymptomatic experimental canine infections, making this antigen a good candidate marker and a prognostic indicator for monitoring the response to CanL treatment [75]. An ELISA with both LicTXNPx and rK39 antigens (LAM-ELISA) was performed to improve specificity and sensitivity of this methodology and presented promising results. This test associated with DAT may be a valuable tool for screening CanL [22, 30].

#### 3.5. Cysteine proteinases

This family of proteins is associated to disease progression. Activity of cysteine proteases can be found in parasite surface or inside the macrophage endoplasmatic reticulum. Domains of cysteine proteinases (CP), type I (CPB) and type II (CPA), were used to diagnose active and recovered cases of VL in both humans and dogs. Leishmania cysteine proteinases had already been used as vaccines targets and in chemotherapy [76].

A recombinant cysteine proteinase from L. chagasi, rLdccys1, has shown to be a good biomarker for the different stages of both human and CanL. ELISA assays showed high sensitivity and specificity. Moreover, the fact that it is possible to detect clinical and subclinical forms of the disease in canines indicates that this biomarker is important in the control of CanL in endemic areas [77, 78].

#### 3.6. Nuclear proteins

The L. infantum acidic ribosomal proteins, LiP2a and LiP2b, have disease-specific antigenic determinants identified by more than 80% of canine VL [70]. Engineered LiP2a and LiP2b recombinant proteins are shown to be useful as tools to discriminate between VL and Chagas disease [71]. L. infantum P0 ribosomal protein is also recognized by a high percentage of the

The acidic ribosomal proteins (P1, P2 and P0) are recognized by antibodies frequently found in sera from systemic lupus erythematosus (SLE) patients. These autoantibodies recognize an amino acid sequence located at the C-terminal ends of the three P proteins [72]. Furthermore, patients with chronic Chagas heart disease produce a strong humoral response against the Cterminal of Trypanosoma cruzi ribosomal P proteins. Leishmania ribosomal P2a and P2b Cterminal end of the proteins is also well conserved and these proteins are recognized by sera from both SLE and Chagas disease patients [69]. The anti-P antibodies produced during the Leishmania infection do not recognize the conserved C-terminal domain of the P proteins. Therefore, to avoid cross-reactivity, engineered versions of the recombinant proteins LiP2a and LiP2b, without the C-terminal ends, have been found to be useful for the diagnosis of

Tryparedoxins belong to a particular class of oxidoreductases related to thioredoxins and found in trypanosomatids. They bear oxidoreductase activity toward disulfide bridges and are crucial to the parasite, as they are expressed in all stages of development [73]. These

LiTXN1 protein is present in the cytosol and upregulated in the infectious forms of the parasite, indicating that it plays an important role during infection. LiTXN1 preferentially

LicTXNPx antigen is highly immunogenic during both human and canine infections [74]. High antibody titers are found during the Leishmania infection and these decrease after its

Anti-LicTXNPx antibodies are present in both symptomatic and asymptomatic experimental canine infections, making this antigen a good candidate marker and a prognostic indicator for monitoring the response to CanL treatment [75]. An ELISA with both LicTXNPx and rK39 antigens (LAM-ELISA) was performed to improve specificity and sensitivity of this methodology and presented promising results. This test associated with DAT may be a valuable tool for

This family of proteins is associated to disease progression. Activity of cysteine proteases can be found in parasite surface or inside the macrophage endoplasmatic reticulum. Domains of cysteine proteinases (CP), type I (CPB) and type II (CPA), were used to diagnose active and

proteins are believed to be involved in Leishmania detoxification of peroxides.

reduces the cytosolic L. infantum peroxiredoxins, LicTXNPx1 and LicTXNPx2 [73].

sera from dogs with VL [70].

204 Biomarker - Indicator of Abnormal Physiological Process

MCL and VL [69].

3.4. Peroxiredoxins

resolution [75].

screening CanL [22, 30].

3.5. Cysteine proteinases

Histones are structural proteins involved in the organization and function of DNA within the eukaryotic nucleus. There are four main classes of histones: H2A, H2B, H3 and H4 which form the nucleosomal core unit of chromatin. These proteins are among the most conserved proteins in eukaryotic organisms, maintaining sequence and function in trypanosomatids. Histones are prominent antigens found in animals during Leishmania infection that trigger a specific immune response and antibody production [39]. Also, sera from children infected with VL specifically recognize the L. infantum H2A and H2B histones, with high specificity and sensitivity in ELISA assays.

#### 3.7. A2 proteins

A2 proteins of L. donovani are only present in the amastigote stage, and help visceralization of parasites in the mammalian host. They are overexpressed in the amastigote stage in L. donovani as well as L. mexicana species complex, including L. amazonensis, but not in L. tropica or L. braziliensis species complexes. Anti-A2 antibodies were detected in human and dog sera suffering from active VL [79, 80]. Also, it was found that rA2 is more sensitive when compared to the rK39 and rK26 antigens, for serological detection of asymptomatic infection in dogs [34].

#### 3.8. Leishmania homolog of receptors for activated C kinase

Leishmania homolog of receptors for activated C Kinase (LACK) was identified after a search for parasite antigens recognized by a protective Th1 clone derived from the spleen of BALB/c mice that had been vaccinated with a soluble extract of L. major promastigotes. It has been proposed that the LACK protein contains an immunodominant epitope that acts as a target of early immune responses [41, 81].

#### 3.9. rKLO8

Sudan has the highest number of reported cases in East Africa [82], in particular in eastern and central regions. Field tests based on rK39 commonly have a high reliability in various countries [83, 84]; however, the low sensitivity in Sudan limits its use in this region. A novel L. donovaniderived recombinant immunodominant protein (rKLO8) was tested for detection of VL in Sudan.

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

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

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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 imprac-

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

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

consuming and expensive, therefore rarely used for clinical diagnosis [93].

affordable diagnosis approaches is necessary.

ticable to be performed on a large scale [3].

4.1.1. Xenodiagnosis

4.1. Parasitological diagnosis

#### 3.10. Secreted proteins

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 immunoproteomic approaches [87, 88] .

#### 3.11. Lipids

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 study of these molecules as disease markers [89].
