5. Successful diagnosis approaches-from laboratory to field conditions

Currently, the scientific community is investing in developing VL tests based on antigen detection, such as rapid diagnostic tests.

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 molecules that allow detection by the naked eye.

#### 5.1. Immunochromatographic Tests

These Immunochromatographic Tests (ICTs) usually based on immunochromatography with a dipstick, are used for VL diagnosis using protein from Leishmania as the antigen [110]. The RDTs for VL, above all but not exclusively the rK39-based ICTs, seem to be the present solution for field diagnosis in field settings due to their ease of use, convenience and cost, making them potentially favorable to increase patients' access to VL diagnosis and treatment [110]. There is a limited number of commercially available RDTs for VL [110] (Table 2).

sensitive (67–100%) and specific (70–100%) [34]. In the Indian subcontinent (India and Nepal), the test proved to have a high sensitivity and specificity but in East Africa (Sudan, Kenya and Ethiopia) the results were not so remarkable [34, 123]. These results in East Africa are not entirely understood; however, lower antibody levels for rK39 in this region might be the reason for these decreased values of sensitivity and specificity [31]. Several studies have been carried out using the dipstick form of rK39 in dogs. The results revealed an overall good sensitivity in symptomatic dogs, although the number of studies for a proper analysis is too small for a detailed comparison [34, 124]. This recombinant antigen can also be used in a latex agglutination test, where latex is used for antigen absorption. The attachment of molecules to latex particles can be accomplished through either physical adsorption or covalent coupling. However, the sensitivity of this latex test is relatively low [34]. In comparison to DAT and ELISA, dipstick rK39 is a better choice for field conditions, sufficiently sensitive and highly

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specific method for the diagnosis of active VL in humans as well as in dogs [34].

detect asymptomatic cases [31].

detects the same antigen present in urine [125].

6. Conclusion and remarks

tion for field use.

5.2. Latex agglutination kit

The diagnosis of CanL remains a problem due to lack of sensitivity or specificity in the current diagnostic tests. Nevertheless, rapid tests like the immunochromatographic-dipstick test using rK39 and rK26 proteins of L. infantum [32] as antigens seem to be most appropriate for diagnosis of symptomatic cases of CanL but lack sensitivity for asymptomatic dogs. Research should continue in order to develop a more sensitive and specific recombinant assay able to

Latex Agglutination Kit (KATex) is a simple, noninvasive, rapid, reliable and easily executable extensively used in diagnosis of L. donovani in endemic areas as Sudan, India and Nepal. It is not commonly used in L. infantum endemic areas [125]. Latex beads coated with anti-Leishmania antibodies detect the presence of a low molecular weight, heat-stable glycoconjugate antigen in urine [126]. This antigen is present in both promastigote and amastigote forms of VL patients with active infection. It has a high specificity (from 82 to 100%) and a sensitivity that varies from 47 to 95%. Although, for immunocompromised patients, the sensitivity and specificity have been reported at 85–100% and at 96–100%, respectively. The antigen may be detectable from one to 6 months after treatment. The method is appropriate for the diagnosis of primary VL, for monitoring the efficacy of treatment and for the detection of sub-clinical infection. KAtex test was also tested with oral fluids, which demonstrated the usefulness of oral-fluid collection in the detection of both Leishmania antibodies and DNA [125]. Sensitivity was higher in saliva that in urine, but specificity was lower. The immunoassay probably

Currently, several biomarkers for diagnosis are available in research laboratories, with expensive and sophisticated equipment requiring trained operators, precluding their direct applica-

The scientific technological advances in recombinant antigens as reagents for the serological diagnosis of VL have resulted in high sensitivity and specificity of the serological tests. Several recombinant proteins have been shown to be useful for the diagnosis of Leishmania infection both in humans and dogs [34]. Recombinant antigens such as the above mentioned rK9, rK16, rK26, rK28 and rk39 have been evaluated for its potential use in rapid diagnostic tests in field conditions.

Of all commercial RDTs, the rK39 is the most widely used. The rK39 rapid immunechromatographic dipstick test was developed to meet the need for diagnosis in field conditions. Among the recombinant antigens, this one showed promising diagnosis and has been extensively tested in the last 5 years with IC tests in several leishmaniasis endemic areas [60, 122], being associated with several commercial applications (Table 2). It is an easy and qualitative test able to detect anti-Leishmania circulating antibodies, not requiring scientifically trained personnel. The test procedure involves adding the patient's blood or serum with diluent buffer on the strip [110]. These rK39 ICTs give an immediate result (typically between 10 and 20 minutes) and give a binary reading (positive or negative) [110]. This test has been approved by the Food and Drug Administration and tested by the World Health Organization in many endemic countries. The rapid rK39 immunochromatographic dipstick test is both


Data representative of available information in 2016.

Table 2. Immunochromatographic tests available for Leishmania diagnosis.

sensitive (67–100%) and specific (70–100%) [34]. In the Indian subcontinent (India and Nepal), the test proved to have a high sensitivity and specificity but in East Africa (Sudan, Kenya and Ethiopia) the results were not so remarkable [34, 123]. These results in East Africa are not entirely understood; however, lower antibody levels for rK39 in this region might be the reason for these decreased values of sensitivity and specificity [31]. Several studies have been carried out using the dipstick form of rK39 in dogs. The results revealed an overall good sensitivity in symptomatic dogs, although the number of studies for a proper analysis is too small for a detailed comparison [34, 124]. This recombinant antigen can also be used in a latex agglutination test, where latex is used for antigen absorption. The attachment of molecules to latex particles can be accomplished through either physical adsorption or covalent coupling. However, the sensitivity of this latex test is relatively low [34]. In comparison to DAT and ELISA, dipstick rK39 is a better choice for field conditions, sufficiently sensitive and highly specific method for the diagnosis of active VL in humans as well as in dogs [34].

The diagnosis of CanL remains a problem due to lack of sensitivity or specificity in the current diagnostic tests. Nevertheless, rapid tests like the immunochromatographic-dipstick test using rK39 and rK26 proteins of L. infantum [32] as antigens seem to be most appropriate for diagnosis of symptomatic cases of CanL but lack sensitivity for asymptomatic dogs. Research should continue in order to develop a more sensitive and specific recombinant assay able to detect asymptomatic cases [31].

#### 5.2. Latex agglutination kit

5.1. Immunochromatographic Tests

212 Biomarker - Indicator of Abnormal Physiological Process

Data representative of available information in 2016.

Table 2. Immunochromatographic tests available for Leishmania diagnosis.

These Immunochromatographic Tests (ICTs) usually based on immunochromatography with a dipstick, are used for VL diagnosis using protein from Leishmania as the antigen [110]. The RDTs for VL, above all but not exclusively the rK39-based ICTs, seem to be the present solution for field diagnosis in field settings due to their ease of use, convenience and cost, making them potentially favorable to increase patients' access to VL diagnosis and treatment [110]. There is a

The scientific technological advances in recombinant antigens as reagents for the serological diagnosis of VL have resulted in high sensitivity and specificity of the serological tests. Several recombinant proteins have been shown to be useful for the diagnosis of Leishmania infection both in humans and dogs [34]. Recombinant antigens such as the above mentioned rK9, rK16, rK26, rK28 and rk39 have been evaluated for its potential use in rapid diagnostic tests in field conditions.

Of all commercial RDTs, the rK39 is the most widely used. The rK39 rapid immunechromatographic dipstick test was developed to meet the need for diagnosis in field conditions. Among the recombinant antigens, this one showed promising diagnosis and has been extensively tested in the last 5 years with IC tests in several leishmaniasis endemic areas [60, 122], being associated with several commercial applications (Table 2). It is an easy and qualitative test able to detect anti-Leishmania circulating antibodies, not requiring scientifically trained personnel. The test procedure involves adding the patient's blood or serum with diluent buffer on the strip [110]. These rK39 ICTs give an immediate result (typically between 10 and 20 minutes) and give a binary reading (positive or negative) [110]. This test has been approved by the Food and Drug Administration and tested by the World Health Organization in many endemic countries. The rapid rK39 immunochromatographic dipstick test is both

Application Product name Bound antigen Manufacturer Human DiaMed-IT LEISH rK39 Bio-Rad Laboratories

> OnSite Leishmania Ab Rapid Test rK39 CTK Biotech, Inc Crystal KA (Kala azar) rKE16 Span Diagnostics, Lda Kalazar Detect™ rK39 InBios International Inc

Speed Leish K Kinesin Capture Complex Virbac Animal Health DPP® Canine Leishmaniasis rK39 Bio-Manguinhos/Fiocruz ImmunoRun Na Biogal, Galed Labs. Canine Leishmania Antibody Test HRPO Conjugate Quicking Biotech Leishmania Ab na EcoDiagnóstica

Dog Canine Visceral Leishmaniasis Dipstick\* rK39 InBios International Inc Anigen Rapid Leishmania Ab Test Kit Na Vtrade Anigen Rapid Leishmania Ab Test Kit Na BIONOTE

limited number of commercially available RDTs for VL [110] (Table 2).

Latex Agglutination Kit (KATex) is a simple, noninvasive, rapid, reliable and easily executable extensively used in diagnosis of L. donovani in endemic areas as Sudan, India and Nepal. It is not commonly used in L. infantum endemic areas [125]. Latex beads coated with anti-Leishmania antibodies detect the presence of a low molecular weight, heat-stable glycoconjugate antigen in urine [126]. This antigen is present in both promastigote and amastigote forms of VL patients with active infection. It has a high specificity (from 82 to 100%) and a sensitivity that varies from 47 to 95%. Although, for immunocompromised patients, the sensitivity and specificity have been reported at 85–100% and at 96–100%, respectively. The antigen may be detectable from one to 6 months after treatment. The method is appropriate for the diagnosis of primary VL, for monitoring the efficacy of treatment and for the detection of sub-clinical infection. KAtex test was also tested with oral fluids, which demonstrated the usefulness of oral-fluid collection in the detection of both Leishmania antibodies and DNA [125]. Sensitivity was higher in saliva that in urine, but specificity was lower. The immunoassay probably detects the same antigen present in urine [125].
