rK39 Enzyme-Linked Immunosorbent Assay for Diagnosis of Leishmania donovani Infection

ABSTRACT The rK39 enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for Leishmania donovani infection in the Sudan. rK39 ELISA proved more sensitive than DAT in diagnosis of kala-azar (93 and 80%, respectively); both tests may remain positive up to 24 months after treatment. For patients with post-kala-azar dermal leishmaniasis and individuals with subclinical infection, rK39 ELISA performed as well as DAT but could detect infection 6 months earlier in ∼40% of patients. Conversion in DAT and rK39 ELISA also occurred in leishmanin skin test (LST)-positive individuals, suggesting active parasite replication (rK39 is an amastigote antigen) in these presumably immune individuals. In contrast to DAT, rK39 ELISA also detected infection in randomly selected LST-positive individuals (in four of six) and endemicity (LST-negative) controls (in one of five). rK39 ELISA appears more sensitive than DAT and may prove an important tool in epidemiological studies.

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Use of a Newly Developed β-Mercaptoethanol Enzyme-Linked Immunosorbent Assay To Diagnose Visceral Leishmaniasis in Patients in Eastern Sudan

Clinical and Vaccine Immunology ◽

10.1128/cvi.00313-07 ◽

2007 ◽

Vol 14 (12) ◽

pp. 1592-1595 ◽

Cited By ~ 5

Author(s):

Durria Mansour ◽

Elfadil M. Abass ◽

Mohamed el Mutasim ◽

Abdelhafeiz Mahamoud ◽

Abdallah el Harith

Keyword(s):

Visceral Leishmaniasis ◽

Immunosorbent Assay ◽

Leishmania Donovani ◽

Clinical Evidence ◽

High Reliability ◽

Agglutination Test ◽

Enzyme Linked Immunosorbent Assay ◽

Direct Agglutination Test ◽

Symptomatic Group ◽

Eastern Sudan

ABSTRACT Corroboration of serology results is essential for restricting the risk of inappropriate antileishmanial prescription. A direct agglutination test (DAT) and a recently developed β-mercaptoethanol-modified enzyme-linked immunosorbent assay (β-ME ELISA) based on the use of antigen prepared as described for the DAT were applied to 416 sera from two Sudanese populations with and without clinical evidence of visceral leishmaniasis (VL). Of 285 sera with the lowest antileishmanial DAT titers (≤1:100 to 1:1,600), 270 (94.7%) scored comparable minimum β-ME ELISA absorbance values (≤0.1 to 0.26). In 117 sera that demonstrated the highest DAT titers (1:12,800 to ≥1:25,600), 86 (73.5%) scored maximum (0.81 to ≥1.35) and 30 (25.6%) medium (0.27 to 0.80) β-ME ELISA absorbance values. VL diagnosis was established for 142 (44.1%) patients in the VL-symptomatic group (n = 322), based on positive microscopy for Leishmania donovani in lymph node aspirates or positive DAT (titer, ≥1:3,200). Of the 125 sera from the symptomatic patients for whom microscopy was positive for VL, 111 (88.8%) had comparable positive DAT and β-ME ELISA readings. In all 17 sera from the symptomatic DAT-positive patients for whom leishmaniasis was not established by microscopy but who responded favorably to antileishmanial therapy, absorbance values (≥0.27) indicative of VL were obtained by β-ME ELISA. Of 197 symptomatic patients for whom microscopy was negative for VL, 172 (87.3%) tested negative in β-ME ELISA and 180 (91.4%) in DAT. Based on the high reliability demonstrated here for VL detection, β-ME ELISA fulfills the requirement of confirming DAT results in patients manifesting suspected VL.

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Evaluation of Enzyme-Linked Immunosorbent Assay for Diagnosis of Post-Kala-Azar Dermal Leishmaniasis with Crude or Recombinant k39 Antigen

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.2.370-373.2002 ◽

2002 ◽

Vol 9 (2) ◽

pp. 370-373 ◽

Cited By ~ 1

Author(s):

P. Salotra ◽

G. Sreenivas ◽

A. A. Nasim ◽

B. V. Subba Raju ◽

V. Ramesh

Keyword(s):

Immunosorbent Assay ◽

Leishmania Donovani ◽

Skin Lesions ◽

Elisa Test ◽

Recombinant Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Invasive Test ◽

Kala Azar ◽

Axenic Amastigotes ◽

Higher Sensitivity

ABSTRACT The diagnosis of post-kala-azar dermal leishmaniasis (PKDL), a dermatosis that provides the only known reservoir for the parasite Leishmania donovani in India, remains a problem. Timely recognition and treatment of PKDL would contribute significantly to the control of kala-azar. We evaluated here the potential of the enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for PKDL. Antigen prepared from promastigotes and axenic amastigotes with parasite isolates that were derived from skin lesions of a PKDL patient gave sensitivities of 86.36 and 92%, respectively, in the 88 PKDL cases examined. The specificity of the ELISA test was examined by testing groups of patients with other skin disorders (leprosy and vitiligo) or coendemic infections (malaria and tuberculosis), as well as healthy controls from areas where this disease is endemic or is not endemic. A false-positive reaction was obtained in 14 of 144 (9.8%) of the controls with the promastigote antigen and in 14 of 145 (9.7%) of the controls with the amastigote antigen. Evaluation of the serodiagnostic potential of recombinant k39 by ELISA revealed a higher sensitivity (94.5%) and specificity (93.7%) compared to the other two antigens used. The data demonstrate that ELISA with crude or recombinant antigen k39 provides a relatively simple and less-invasive test for the reliable diagnosis of PKDL.

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Persistence ofLeishmania donovaniAntibodies in Past Visceral Leishmaniasis Cases in India

Clinical and Vaccine Immunology ◽

10.1128/cvi.00473-10 ◽

2010 ◽

Vol 18 (2) ◽

pp. 346-348 ◽

Cited By ~ 39

Author(s):

Kamlesh Gidwani ◽

Albert Picado ◽

Bart Ostyn ◽

Shri Prakash Singh ◽

Rajiv Kumar ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Immunosorbent Assay ◽

Leishmania Donovani ◽

Extended Period ◽

Agglutination Test ◽

Enzyme Linked Immunosorbent Assay ◽

Direct Agglutination Test ◽

History Of ◽

Antibody Titers

ABSTRACTThe persistence of anti-Leishmania donovaniantibodies in past visceral leishmaniasis (VL) cases was retrospectively assessed by means of the direct agglutination test (DAT) and the rK39 enzyme-linked immunosorbent assay (ELISA). Antibody titers remained high for an extended period of time in past cases of VL. These results highlight the need to carefully elicit the history of patients with VL symptoms.

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Application of an Improved Method for the Recombinant K39 Enzyme-Linked Immunosorbent Assay To Detect Visceral Leishmaniasis Disease and Infection in Bangladesh

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.12.12.1410-1415.2005 ◽

2005 ◽

Vol 12 (12) ◽

pp. 1410-1415 ◽

Cited By ~ 15

Author(s):

K. M. Kurkjian ◽

L. E. Vaz ◽

R. Haque ◽

C. Cetre-Sossah ◽

S. Akhter ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Immunosorbent Assay ◽

Leishmania Donovani ◽

Asymptomatic Infection ◽

Negative Control ◽

Original Method ◽

Cutoff Point ◽

Enzyme Linked Immunosorbent Assay ◽

Standard Curve ◽

Kala Azar

ABSTRACT Several serology-based immunoassays are used to diagnose visceral leishmaniasis (VL), a chronic protozoan parasitic disease caused by the Leishmania donovani complex. These tests are primarily designed to diagnose the most severe clinical form of VL, known as kala-azar. However, leishmanial infection is frequently asymptomatic and may manifest only as a positive serologic response or positive leishmanin skin test. We modified a previously described enzyme-linked immunosorbent assay (ELISA) that detects patient antibodies reactive with the recombinant Leishmania protein K39 (rK39) to confirm suspected kala-azar and to detect asymptomatic infection in a community study in Bangladesh. With the inclusion of a standard curve on each ELISA plate, the rK39 ELISA was more repeatable (kappa coefficient of agreement = 0.970) and more reliable compared to the original method (kappa = 0.587, P < 0.001). The cutoff point for a positive antibody response was chosen based on the 99th percentile of the ELISA distribution for the negative-control sera. However, we found that sera from all patients with active kala-azar yielded values more than twice the magnitude of this cutoff. Using receiver-operator characteristic curves, we determined a second cutoff value predictive of kala-azar. Using these criteria, the sensitivity and specificity of the modified ELISA for kala-azar were 97.0% and 98.9%, respectively, for sera from our study population. We hypothesize that individuals with antibody levels greater than the 99th percentile of the negative controls but less than the cutoff point for kala-azar have asymptomatic leishmanial infections.

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Comparison of Enzyme-Linked Immunosorbent Assay, Indirect Fluorescent Antibody Test, and Direct Agglutination Test for Detecting Toxoplasma Gondii Antibodies in Naturally Aborted Ovine Fetuses

Journal of Veterinary Diagnostic Investigation ◽

10.1177/104063878900100206 ◽

1989 ◽

Vol 1 (2) ◽

pp. 124-127 ◽

Cited By ~ 23

Author(s):

Sheryl L. Seefeldt ◽

Clyde A. Kirkbride ◽

Jitender P. Dubey

Keyword(s):

Toxoplasma Gondii ◽

Immunosorbent Assay ◽

Indirect Fluorescent Antibody Test ◽

Fluorescent Antibody ◽

Antibody Test ◽

Agglutination Test ◽

Enzyme Linked Immunosorbent Assay ◽

Direct Agglutination Test ◽

Indirect Fluorescent Antibody ◽

Special Equipment

Results obtained in an enzyme-linked immunosorbent assay (ELISA), an indirect fluorescent antibody test (IFA), and a modified direct agglutination test (MAT) for Toxoplasma gondii antibodies from examination of fetal fluids from 377 aborted ovine fetuses were compared. Sixty-seven samples were positive by MAT (titers 1:16 to > 1:65,536), 58 were positive by ELISA, and 62 were positive by immunoglobulin G-IFA. The MAT was preferred because it required less time, labor, and special equipment. It was simple to run, could be done on serum from any species without modification, and it was more effective than the IFA for detecting toxoplasma antibodies in severely autolyzed fetuses. No advantage was found in determining immunoglobulin M antibodies in ovine fetal sera.

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An enzyme-linked immunosorbent assay for the detection of IgG antibodies against Babesia equi in horses

Ciência Rural ◽

10.1590/s0103-84782004000500031 ◽

2004 ◽

Vol 34 (5) ◽

pp. 1525-1529 ◽

Cited By ~ 12

Author(s):

Cristiane Divan Baldani ◽

Rosangela Zacarias Machado ◽

Paulo de Tarso Landgraf Botteon ◽

Felipe Santoro Takakura ◽

Carlos Luiz Massard

Keyword(s):

Immunosorbent Assay ◽

Serological Test ◽

Epidemiological Studies ◽

Sao Paulo ◽

Enzyme Linked Immunosorbent Assay ◽

São Paulo ◽

Igg Antibodies ◽

Serum Samples ◽

Babesia Equi ◽

Specificity And Sensitivity

A crude antigenic preparation of Babesia equi was used to develop and establish the suitability of an enzyme-linked immunosorbent assay (ELISA) for the detection of parasite carriers. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkboard titrations. The specificity and sensitivity of the ELISA were 100 %. A total of 90 serum samples were taken from horses from the Northeast region of São Paulo State and examined for diagnosis of equine B. equi infection by ELISA. Approximately 75% (n=67) of all the horses tested were found serologically positive for B. equi. These results suggest that the ELISA described may prove to be an appropriate serological test for epidemiological studies on B. equi infections in the field and that equine piroplasmosis is a cause for serious concern in the State of São Paulo, Brazil.

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Reproductive isolation between different forms of Lutzomyia longipalpis (Lutz & Neiva), (Diptera: Psychodidae), the vector of Leishmania donovani chagasi Cunha & Chagas and its significance to kala-azar distribution in South America

Memórias do Instituto Oswaldo Cruz ◽

10.1590/s0074-02761983000300005 ◽

1983 ◽

Vol 78 (3) ◽

pp. 269-280 ◽

Cited By ~ 76

Author(s):

Richard D. Ward ◽

Armando L. Ribeiro ◽

Paul D. Ready ◽

Angela Murtagh

Keyword(s):

South America ◽

Reproductive Isolation ◽

Species Complex ◽

Leishmania Donovani ◽

Epidemiological Studies ◽

The Other ◽

Lutzomyia Longipalpis ◽

Single Pair ◽

Kala Azar

The males of the sandfly Lutzomyia longipalpis occur in two forms, one which bears a single pair of pale spots on tergite 4 and another in which an additional pair of spots characterizes tergite 3. In crosses between laboratory reared stocks of the two forms originating from allopatric and sympatric sites in Brazil nearly all males of one form fail to inseminate females of the other. In addition, insemination failure between some allopatric populaytions of Lu. longipalpis with similar tergal spot patterns is recorded, indicating the existence of additional forms in an apparent species complex. The possibility that Lu. longipalpis sensu latu represents more than a single taxon is discussed and the relevance of these findings to future epidemiological studies on kala-azar is considered.

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Direct enzyme-linked immunosorbent assay: A simple immunoassay usingleishmania donovani promastigote for diagnosis of kala-azar

Journal of Clinical Laboratory Analysis ◽

10.1002/jcla.1860050413 ◽

1991 ◽

Vol 5 (4) ◽

pp. 299-301 ◽

Cited By ~ 3

Author(s):

Krishna Mukerji ◽

Abhijit Pal ◽

Kshudiram Naskar ◽

Dilip K. Ghosh ◽

Debashish Basu ◽

...

Keyword(s):

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Kala Azar

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Comparison of Two Recombinant Major Outer Membrane Proteins of the Human Granulocytic Ehrlichiosis Agent for Use in an Enzyme-Linked Immunosorbent Assay

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.7.4.652-657.2000 ◽

2000 ◽

Vol 7 (4) ◽

pp. 652-657 ◽

Cited By ~ 8

Author(s):

Tomoko Tajima ◽

Ning Zhi ◽

Quan Lin ◽

Yasuko Rikihisa ◽

Harold W. Horowitz ◽

...

Keyword(s):

Immunosorbent Assay ◽

Outer Membrane Proteins ◽

Cross Reactivity ◽

Immunofluorescence Assay ◽

Babesia Microti ◽

Enzyme Linked Immunosorbent Assay ◽

Granulocytic Ehrlichiosis ◽

Healthy Humans ◽

Human Granulocytic Ehrlichiosis ◽

Negative Controls

ABSTRACT Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated. Sera from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA. Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls. One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA). All IFA-negative sera were negative by both ELISAs. Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive. Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agent as antigen suggests that these four sera were false IFA positive. There was no difference in results with or without the preabsorption of sera with Escherichia coli or with or without the cleavage of the fused protein derived from the vector. There was a significant positive correlation between IFA titers and optical densities of ELISAs. Four Ehrlichia chaffeensis-positive and 10 Borrelia burgdorferi-positive sera were negative by ELISA. However, twoBabesia microti-positive sera showed strong cross-reactivity to the fused vector protein, which was eliminated after cleavage of the protein. Thus, ELISA using rP44 nonfusion protein would provide a simple, specific, and objective HGE serologic test which can be easily automated.

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Diagnosis of Visceral Leishmaniasis by Enzyme-Linked Immunosorbent Assay Using Urine Samples

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.4.789-794.2002 ◽

2002 ◽

Vol 9 (4) ◽

pp. 789-794 ◽

Cited By ~ 3

Author(s):

Mohammad Zahidul Islam ◽

Makoto Itoh ◽

S. M. Shamsuzzaman ◽

Rusella Mirza ◽

Farzana Matin ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Sensitivity And Specificity ◽

Immunosorbent Assay ◽

Leishmania Donovani ◽

Laboratory Diagnosis ◽

Urine Samples ◽

Enzyme Linked Immunosorbent Assay ◽

Healthy Controls ◽

Serum Samples ◽

Crude Antigen

ABSTRACT A diagnostic method has been developed to detect anti-Leishmania donovani immunoglobulin G (IgG) in urine by enzyme-linked immunosorbent assay (ELISA). In measuring anti-L. donovani IgG, IgA, and IgM in urine, the method performed best in the detection of IgG. The sensitivity and specificity of the assay were determined with panels of urine samples from 62 visceral leishmaniasis (VL) patients, 59 healthy controls from areas of endemicity, 53 healthy controls from areas of nonendemicity, 59 malaria patients, 13 tuberculosis patients, 23 cutaneous leishmaniasis patients, and 7 patients with other diseases. Using L. donovani promastigote crude antigen, the test had 93.5% sensitivity (58 positives of 62 VL patient samples) and 89.3% specificity (191 negatives of 214 non-VL patient samples). The ELISA with acetone-treated L. donovani promastigote antigen raised the sensitivity and specificity to 95.0 and 95.3%, respectively. Western blot analysis revealed that most of the samples that cross-reacted with crude antigen in ELISA did not recognize any antigenic component of L. donovani crude antigen. We also checked 40 serum samples from the same group of VL patients for anti-L. donovani IgG and got 90.0% sensitivity with both crude and acetone-treated antigens. As collection of urine is much easier than collection of serum, the detection of anti-L. donovani IgG in urine with acetone-treated antigen will be useful in epidemiological studies. It could be an adjunct of laboratory diagnosis.

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