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

2005 ◽  
Vol 12 (12) ◽  
pp. 1410-1415 ◽  
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.

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

2002 ◽  
Vol 9 (4) ◽  
pp. 789-794 ◽  
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.

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

2007 ◽  
Vol 14 (12) ◽  
pp. 1592-1595 ◽  
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.

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

2002 ◽  
Vol 9 (2) ◽  
pp. 370-373 ◽  
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.

scholarly journals Predicting Kala-Azar Disease Manifestations in Asymptomatic Patients with Latent Leishmania donovani Infection by Detection of Antibody against Recombinant K39 Antigen

2002 ◽  
Vol 9 (3) ◽  
pp. 568-572 ◽  
Author(s):  
Sarman Singh ◽  
Veena Kumari ◽  
Niti Singh
Keyword(s):  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Clinical Manifestations ◽  
Latex Agglutination ◽  
Enzyme Linked Immunosorbent Assay ◽  
Kala Azar ◽  
Asymptomatic Patients ◽  
Iga Antibodies ◽  
Latently Infected ◽  
Scanty Information

ABSTRACT Clinically visceral leishmaniasis is suspected in only a fraction of infected persons, as the majority of these may not have clinical manifestations and remain asymptomatic. There is scanty information on diagnosing latent infections and predicting disease in asymptomatic persons. We therefore carried out a study on asymptomatic contacts of patients with visceral leishmaniasis and post-kala-azar dermal leishmaniasis by using methods for detection of antibody to recombinant K39 (rK39) antigen. A total of 240 patients with leishmaniasis and 150 asymptomatic contacts were tested for anti-rK39 immunoglobulin G (IgG) and IgA antibodies. Fifty-five asymptomatic persons were found to be seropositive. These individuals were monitored every 3 months for 1 year. On follow-up, 43.9% of the asymptomatic seropositive contacts developed kala-azar within the first 3 months, and a cumulative total of 69% developed kala-azar within 1 year. The rest remained asymptomatic and self-healed the infection. The sensitivity and specificity of rK39 enzyme-linked immunosorbent assay (ELISA) and dipstick tests were 100%, while an in-house-developed latex agglutination test had 80% sensitivity. The antibody profile showed that the IgG anti-rK39 antibodies reached a titer of up to 10−6 within 6 months of infection, started declining thereafter, and completely disappeared in 2 to 3 years in successfully treated cases. Significant titers of IgA antibodies were detectable a little earlier than those of IgG antibodies and were undetectable after 6 months. The study showed that mass screening of family members and contacts by using anti-rK39 ELISA could be a highly reliable tool for early diagnosis and to plan prophylactic treatment of latently infected asymptomatic carriers to eradicate kala-azar.

scholarly journals Persistence ofLeishmania donovaniAntibodies in Past Visceral Leishmaniasis Cases in India

2010 ◽  
Vol 18 (2) ◽  
pp. 346-348 ◽  
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.

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

1998 ◽  
Vol 5 (5) ◽  
pp. 717-720 ◽  
Author(s):  
E. E. Zijlstra ◽  
N. S. Daifalla ◽  
P. A. Kager ◽  
E. A. G. Khalil ◽  
A. M. El-Hassan ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Leishmania Donovani ◽  
Epidemiological Studies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Direct Agglutination Test ◽  
Subclinical Infection ◽  
Kala Azar ◽  
Leishmanin Skin Test ◽  
Parasite Replication ◽  
Negative Controls

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.

scholarly journals Immunoglobulin Subclass Distribution and Diagnostic Value of Leishmania donovani Antigen-Specific Immunoglobulin G3 in Indian Kala-Azar Patients

1999 ◽  
Vol 6 (2) ◽  
pp. 231-235 ◽  
Author(s):  
Khairul Anam ◽  
Farhat Afrin ◽  
Dwijadas Banerjee ◽  
Netai Pramanik ◽  
Subhasis K. Guha ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Specific Antibody ◽  
Leishmania Donovani ◽  
Diagnostic Value ◽  
Igg Subclasses ◽  
Antibody Responses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Kala Azar ◽  
Specific Immunoglobulin ◽  
Specific Igg

ABSTRACT Visceral leishmaniasis, or kala-azar, a fatal tropical disease, remains problematic, as early diagnosis is difficult and treatment often results in drug resistance and relapse. We have developed a sensitive enzyme-linked immunosorbent assay (ELISA), using leishmanial membrane antigenic extracts (LAg) to detect specific antibody responses in 25 untreated Indian visceral leishmaniasis patients. To investigate the pathogenetic significance of isotype markers in kala-azar, relative levels of specific immunoglobulin G (IgG), IgM, IgA, IgE, and IgG subclasses were analyzed under clinically established diseased conditions. Since LAg showed higher sensitivity for specific IgG than lysate, the immunoglobulin isotype responses were evaluated, with LAg as antigen. Compared to 60 controls, which included patients with malaria, tuberculosis, leprosy, and typhoid and healthy subjects, visceral leishmaniasis patients showed significantly higher IgG (100% sensitivity, 85% specificity), IgM (48% sensitivity, 100% specificity), and IgE (44% sensitivity, 98.3% specificity) responses. Low levels of IgA in visceral leishmaniasis patients contrasted with a 13-fold-higher reactivity in sera from patients with leprosy. Among IgG subclasses, IgG1, -3, and -4 responses were significantly higher in visceral leishmaniasis patients than in the controls. IgG2 response, however, was significantly higher (twofold) in leprosy than even visceral leishmaniasis patients. The rank orders for sensitivity (IgG = IgG1 = IgG3 = IgG4 > IgG2 > IgM > IgE > IgA) and specificity (IgM = IgG3 > IgE > IgG4 > IgG2 > IgG > IgG1 > IgA) for LAg-specific antibody responses suggest the potentiality of IgG3 as a diagnostic marker for visceral leishmaniasis.

Comparative evaluation of enzyme-linked immunosorbent assay based on crude and purified antigen in the diagnosis of canine visceral leishmaniasis in symptomatic and oligosymptomatic dogs

2008 ◽  
Vol 157 (3-4) ◽  
pp. 175-181 ◽  
Author(s):  
Teresinha Cristina Cândido ◽  
Sílvia Helena Venturoli Perri ◽  
Tatiana de Oliveira Gerzoschkwitz ◽  
Maria Cecília Rui Luvizotto ◽  
Valéria Marçal Felix de Lima
Keyword(s):  
Visceral Leishmaniasis ◽  
Immunosorbent Assay ◽  
Comparative Evaluation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Canine Visceral Leishmaniasis ◽  
Purified Antigen

Enzyme-Linked Immunosorbent Assay for Microcystins in Blue-Green Algal Blooms

1990 ◽  
Vol 73 (3) ◽  
pp. 451-456 ◽  
Author(s):  
Fun S Chu ◽  
Xuan Huang ◽  
R D Wei
Keyword(s):  
Liquid Chromatography ◽  
Immunosorbent Assay ◽  
Algal Blooms ◽  
Ammonium Bicarbonate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Standard Curve ◽  
Green Algal ◽  
Recovery Study ◽  
Minimum Detection Level ◽  
Elisa Plate

Abstract A direct competitive enzyme-linked immunosorbent assay (ELISA) for the freshwater blue-green algal toxin mlcrocystln (MCYST) In algae and water was developed. The assay Involves coating antl-MCYST-variant leuclne-arglnine (LR) antibody to the ELISA plate and the use of MCYST-LRperoxidase as the enzyme marker. The linear portion of the standard curve for MCYST in phosphate buffer containing saline (PBS) was 0.5-10.0 ng/mL (25-500 pg/assay). The minimum detection level for MCYST-LR was 0.20 ng/mL (10 pg/assay). Contaminated water could be directly used In the ELISA. The overall analytical recoveries for MCYST-LR added to water at levels of 1-20 ng/mL was 83.4%. For analysis of cellular MCYST, the toxin was first extracted from the algae with 0.1M ammonium bicarbonate, diluted with PBS to less than 0.5 mg dried algae/mL (&lt;5.0 mg wet welght/mL) and directly used in the ELISA. C-18 reverse-phase Sep-Pak cartridges effectively adsorbed MCYST from the toxln-containlng solutions. The toxin could be recovered from the cartridge by elutlng with 60% methanol. Using this approach, an algae extract that was relatively free of MCYST was prepared and was used in a recovery study. The overall analytical recovery of MCYST added to the algae extract In the range of 0.25-20 ppm was 83% with a coefficient of variation of 11.9%. The detection limit for MCYST In dried algae was about 0.25-0.5 pg/g (0.25-0.5 ppm) lyophlllzed algae sample. This method was applied for the analysis of several naturally occurring algal blooms. Limited samples were also analyzed for MYCST by liquid chromatography. ELISA data were in general agreement with those obtainedby liquid chromatography. MCYST concentrations from 0.006 to 2.9 fig/g (6 to 2900 ppb) and from 26 to 5200 /ig/g (26 ppm to 5200 ppm) were found In water and algae (dried weight), respectively

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