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Field Adaptable Tests for Kala-Azar
Methods in Medicine
Field Adaptable Tests for Kala-Azar Lt Col VK Agrawal* MJAFI 2006; 62 : 178-179 Key Words : Strip test; Kala-Azar
Introduction eishmaniasis is endemic in the tropical and subtropical regions. 12 million cases occur worldwide with 1 to 1.5 million cutaneous and 500,000 visceral leishmaniasis (kala-azar, VL) occuring fresh every year. LeishmaniaHIV coinfection is emerging in southern Europe where 25 to 70% of adults with VL have AIDS as well. In the Indian subcontinent, the disease is almost exclusively caused by L donovani. Diagnosis of kala azar (VL) is by demonstration of amastigotes in splenic or bone marrow smears or by culture of leishmania promastigotes from clinical specimen. While the sensitivity of splenic smears could be as high as > 95%, it carries the risk of severe/fatal haemorrhage. Bone marrow aspiration is painful, cumbersome and has a low sensitivity (60-85%). Culture can not be used for routine clinical diagnosis as it requires expensive equipment and expertise [1].
L
Antibody detection VL methods based on rise in immunoglobulin levels (Napier’s formol gel or aldehyde test and the Chopra antimony test) are unreliable because of low sensitivity and specificity. Conventional methods for antibody detection for diagnosis of VL are gel diffusion, complement fixation test, indirect hemagglutination (IHA), indirect immunofluorescence (IFA) and countercurrent immunoelectrophoresis. Direct agglutination test (DAT) based on agglutination of the trypsinized whole promastigotes is 91 to 100% sensitive and 72 to 100% specific. Difficult field conditions, the fragility of aqueous antigen, lack of cold chain, variations in antigen batches and lack of objectivity in test readings have limited its applicability in India [2].ELISA is highly sensitive, but its specificity depends upon the antigen used. An amastigote-specific recombinant 39 amino acid antigen (rk39) derived from L. chagasi has been found to be 100% sensitive and 100% specific in the diagnosis of VL [3].
A promising ready-to-use immunochromatographic strip test based on rk39 antigen has been developed as a rapid test for use in difficult field conditions. In this test, either serum or blood is smeared at the tip of the strip and anti k-39 antibodies leads to development of a red colour band 1 cm below a similar control band .This test is read with naked eye and the strips can be stored between 25-40oC for at least a year. The test has a sensitivity of 100 % ( CI 98-100) and specificity of 98% (CI 95-100) [4]. However a study which evaluated the performances of IHA, IFA and strip test for diagnosis of imported cases of kala azar in Kuwait, where the disease is not endemic showed that IHA is the most sensitive (100%), followed by IFA (86.6%) and the strip-test (80.0%). The strip-test was most specific (100%), followed by IFA (93.0%) and IHA (86.0%) [5]. Sensitivity of strip test was markedly less in Kuwait than in India (100%). The reason for this is unclear; but differences in the antibody responses ethnic background, the severity of infection and time since infection may be relevant. IFA is more subjective and requires fluorescence microscope and a darkroom facility and is costlier than IHA or strip test. Although IHA is sensitive, fragility of aqueous antigen, lack of cold chain, variations in the antigen batches have limited its applicability in India. High specificity of strip test is explained by the high degree of specificity of anti-K39 antibodies for active VL (the strip-test uses the specific K39 antigen) unlike IHA and IFA, which use crude parasite lysates or undefined antigens. Limitation of this test is the presence of antibodies in healthy controls hailing from the endemic region. Utility of this rapid test in predicting cure or diagnosing relapses is limited as anti-rk39 antibody/strip test remain positive for years after successful cure. Antigen detection Antigen detection is more specific than antibodybased techniques. This method is also useful in the
*Reader, Department of Preventive and Social Medicine, Armed Forces Medical College, Pune-411040. Received : 30.03.2005; Accepted : 04.07.2005
Field Adaptable Tests for Kala-Azar
179
diagnosis of disease in cases where there is deficient antibody production (as in AIDS patients). A new latex agglutination test (KATEX) using monoclonal antibodies for detecting leishmanial antigen in urine of patients with VL has shown sensitivities between 68 and 100% and a specificity of 100% in preliminary trials [6]. The antigen is detected quite early during the infection, and the results of animal experiments suggest that the amount of detectable antigen tends to decline rapidly following chemotherapy. These antigens are also detectable in blood. Conclusion Though many noninvasive tests are available for the diagnosis of Kala-azar, none have become popular in areas of endemicity. They are expensive, require skilled personnel, electricity and are technically demanding. Parasite diagnosis by splenic and marrow smear examination remain the “gold standard”. Strip test (rk39) has the potential to be used under field conditions.
References 1. Sundar S, Rai M. Laboratory diagnosis of visceral leishmaniasis. Clin Diag Lab Immunol 2002; 9 : 951-8. 2. Sundar S, Singh GS, Singh VP, Singla N, Kumar K, Vinayak VK. Comparative evaluation of DAT, IFAT and micro-ELISA in the serodiagnosis of Indian kala-azar. J Parasitic Dis 1996; 20 : 41- 3. 3. Kumar R, Pai K, Pathak K, Sundar S. Enzyme-linked immunosorbent assay for recombinant K39 antigen in diagnosis and prognosis of Indian visceral leishmaniasis. Clin Diagn Lab Immunol 2001; 8 : 1220-4. 4. Bern C, Jha SN, Joshi AB, Thakur GD, Bista MB. Use of the recombinant K39 dipstick test and the direct agglutination test in a setting endemic for visceral leishmaniasis in Nepal. Am J Trop Med Hyg 2000; 63 : 153-7. 5. Iqbal J, Hira P R, Saroj G, Philip R, Faiza Al-Ali, Patrick J. et al. Imported Visceral Leishmaniasis: Diagnostic Dilemmas and Comparative Analysis of Three Assays. Journal of Clinical Microbiology 2002; 40(2) : 475-9. 6. Attar ZJ, Chance ML, el-Safi S, et al. Latex agglutination test for the detection of urinary antigens in visceral leishmaniasis. Acta Trop 2001; 78 : 11-6.
ANNOUNCEMENT BEST REFEREE AWARD : MJAFI The Best Referee Award has been instituted with effect from 2006 to appreciate the contribution made by the referees in publication of the articles in MJAFI. The award would be given during the AFMRC annually. The criteria for selection of Best Referee would be: (a)
Usage of email in all correspondence
(b)
First response within two weeks of receipt of article
(c)
Subsequent responses within one week
(d)
Most constructive comments
MJAFI, Vol. 62, No. 2, 2006
Field Adaptable Tests for Kala-Azar Lt Col VK Agrawal* MJAFI 2006; 62 : 178-179 Key Words : Strip test; Kala-Azar
Introduction eishmaniasis is endemic in the tropical and subtropical regions. 12 million cases occur worldwide with 1 to 1.5 million cutaneous and 500,000 visceral leishmaniasis (kala-azar, VL) occuring fresh every year. LeishmaniaHIV coinfection is emerging in southern Europe where 25 to 70% of adults with VL have AIDS as well. In the Indian subcontinent, the disease is almost exclusively caused by L donovani. Diagnosis of kala azar (VL) is by demonstration of amastigotes in splenic or bone marrow smears or by culture of leishmania promastigotes from clinical specimen. While the sensitivity of splenic smears could be as high as > 95%, it carries the risk of severe/fatal haemorrhage. Bone marrow aspiration is painful, cumbersome and has a low sensitivity (60-85%). Culture can not be used for routine clinical diagnosis as it requires expensive equipment and expertise [1].
L
Antibody detection VL methods based on rise in immunoglobulin levels (Napier’s formol gel or aldehyde test and the Chopra antimony test) are unreliable because of low sensitivity and specificity. Conventional methods for antibody detection for diagnosis of VL are gel diffusion, complement fixation test, indirect hemagglutination (IHA), indirect immunofluorescence (IFA) and countercurrent immunoelectrophoresis. Direct agglutination test (DAT) based on agglutination of the trypsinized whole promastigotes is 91 to 100% sensitive and 72 to 100% specific. Difficult field conditions, the fragility of aqueous antigen, lack of cold chain, variations in antigen batches and lack of objectivity in test readings have limited its applicability in India [2].ELISA is highly sensitive, but its specificity depends upon the antigen used. An amastigote-specific recombinant 39 amino acid antigen (rk39) derived from L. chagasi has been found to be 100% sensitive and 100% specific in the diagnosis of VL [3].
A promising ready-to-use immunochromatographic strip test based on rk39 antigen has been developed as a rapid test for use in difficult field conditions. In this test, either serum or blood is smeared at the tip of the strip and anti k-39 antibodies leads to development of a red colour band 1 cm below a similar control band .This test is read with naked eye and the strips can be stored between 25-40oC for at least a year. The test has a sensitivity of 100 % ( CI 98-100) and specificity of 98% (CI 95-100) [4]. However a study which evaluated the performances of IHA, IFA and strip test for diagnosis of imported cases of kala azar in Kuwait, where the disease is not endemic showed that IHA is the most sensitive (100%), followed by IFA (86.6%) and the strip-test (80.0%). The strip-test was most specific (100%), followed by IFA (93.0%) and IHA (86.0%) [5]. Sensitivity of strip test was markedly less in Kuwait than in India (100%). The reason for this is unclear; but differences in the antibody responses ethnic background, the severity of infection and time since infection may be relevant. IFA is more subjective and requires fluorescence microscope and a darkroom facility and is costlier than IHA or strip test. Although IHA is sensitive, fragility of aqueous antigen, lack of cold chain, variations in the antigen batches have limited its applicability in India. High specificity of strip test is explained by the high degree of specificity of anti-K39 antibodies for active VL (the strip-test uses the specific K39 antigen) unlike IHA and IFA, which use crude parasite lysates or undefined antigens. Limitation of this test is the presence of antibodies in healthy controls hailing from the endemic region. Utility of this rapid test in predicting cure or diagnosing relapses is limited as anti-rk39 antibody/strip test remain positive for years after successful cure. Antigen detection Antigen detection is more specific than antibodybased techniques. This method is also useful in the
*Reader, Department of Preventive and Social Medicine, Armed Forces Medical College, Pune-411040. Received : 30.03.2005; Accepted : 04.07.2005
Field Adaptable Tests for Kala-Azar
179
diagnosis of disease in cases where there is deficient antibody production (as in AIDS patients). A new latex agglutination test (KATEX) using monoclonal antibodies for detecting leishmanial antigen in urine of patients with VL has shown sensitivities between 68 and 100% and a specificity of 100% in preliminary trials [6]. The antigen is detected quite early during the infection, and the results of animal experiments suggest that the amount of detectable antigen tends to decline rapidly following chemotherapy. These antigens are also detectable in blood. Conclusion Though many noninvasive tests are available for the diagnosis of Kala-azar, none have become popular in areas of endemicity. They are expensive, require skilled personnel, electricity and are technically demanding. Parasite diagnosis by splenic and marrow smear examination remain the “gold standard”. Strip test (rk39) has the potential to be used under field conditions.
References 1. Sundar S, Rai M. Laboratory diagnosis of visceral leishmaniasis. Clin Diag Lab Immunol 2002; 9 : 951-8. 2. Sundar S, Singh GS, Singh VP, Singla N, Kumar K, Vinayak VK. Comparative evaluation of DAT, IFAT and micro-ELISA in the serodiagnosis of Indian kala-azar. J Parasitic Dis 1996; 20 : 41- 3. 3. Kumar R, Pai K, Pathak K, Sundar S. Enzyme-linked immunosorbent assay for recombinant K39 antigen in diagnosis and prognosis of Indian visceral leishmaniasis. Clin Diagn Lab Immunol 2001; 8 : 1220-4. 4. Bern C, Jha SN, Joshi AB, Thakur GD, Bista MB. Use of the recombinant K39 dipstick test and the direct agglutination test in a setting endemic for visceral leishmaniasis in Nepal. Am J Trop Med Hyg 2000; 63 : 153-7. 5. Iqbal J, Hira P R, Saroj G, Philip R, Faiza Al-Ali, Patrick J. et al. Imported Visceral Leishmaniasis: Diagnostic Dilemmas and Comparative Analysis of Three Assays. Journal of Clinical Microbiology 2002; 40(2) : 475-9. 6. Attar ZJ, Chance ML, el-Safi S, et al. Latex agglutination test for the detection of urinary antigens in visceral leishmaniasis. Acta Trop 2001; 78 : 11-6.
ANNOUNCEMENT BEST REFEREE AWARD : MJAFI The Best Referee Award has been instituted with effect from 2006 to appreciate the contribution made by the referees in publication of the articles in MJAFI. The award would be given during the AFMRC annually. The criteria for selection of Best Referee would be: (a)
Usage of email in all correspondence
(b)
First response within two weeks of receipt of article
(c)
Subsequent responses within one week
(d)
Most constructive comments
MJAFI, Vol. 62, No. 2, 2006