Development and evaluation ofLeishmania infantumrK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran

SUMMARYThe purpose of this study was to prepare recombinant K26 antigen fromLeishmania infantumand evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96·8%), whereas 85 sera were positive with DAT (sensitivity=91·4%) and 89 sera were positive with whole cell ELISA (sensitivity=95·7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89·2%) and whole cell ELISA was negative in 114 cases (specificity=87·7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused byLeishmania infantum.

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The rK39 Antigen from an Iranian Strain of Leishmania infantum: Detection of Anti-Leishmania Antibodies in Humans and Dogs

Iranian Journal of Parasitology ◽

10.18502/ijpa.v15i1.2526 ◽

2020 ◽

Author(s):

Bibi Razieh HOSSEINI FARASH ◽

Mehdi MOHEBALI ◽

Bahram KAZEMI ◽

Abdolmajid FATA ◽

Reza RAOOFIAN ◽

...

Keyword(s):

Leishmania Infantum ◽

Field Studies ◽

Severe Form ◽

Recombinant Antigen ◽

Agglutination Test ◽

Enzyme Linked Immunosorbent Assay ◽

Healthy Controls ◽

Direct Agglutination Test ◽

Human Sera ◽

Extended Field

Background: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis in Iran with high mortality rates in the case of inaccurate diagnosis and treatment. This study aimed to prepare and evaluate a new rk39 recombinant antigen from an Iranian strain of Leishmania infantum for diagnosis of VL in humans and dogs. Methods: rK39-based enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for the detection of anti L. infantum antibodies. We screened 84 human sera and 87 dog sera from clinical cases in the endemic area of Meshkin-Shahr, Iran along with 176 sera from healthy controls (collected from 86 humans and 90 dogs) during 2013 -2016. Results: Using the rK39 ELISA, a sensitivity of 85.7% (95% CI, 95-99%) and a specificity of 86.0% (95% CI, 95%-99%) were detected in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 96.6% (95% CI, 95%-99%) and specificity of 94.4% (95% CI, 95%-99%) were found at a 1:80 (cut-off) titer compared with DAT. Kappa analysis indicated agreement between the rK39 ELISA and DAT (0.718) when using human sera at a 1:800 (cut-off) titer as well as (0.910) at a 1:80 (cut-off) titer when using dog sera (P<0.05). Conclusion: New rk39 recombinant antigen from an Iranian strain of Leishmania infantum seems to be used for diagnosis of VL in humans and dogs. Further extended field studies are recommended

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Performance of an indigenous β-mercaptoethanol-modified antigen in comparison with a commercial reference in direct agglutination test for detection of canine visceral leishmaniasis

Journal of Medical Microbiology ◽

10.1099/jmm.0.063891-0 ◽

2014 ◽

Vol 63 (1) ◽

pp. 106-110

Author(s):

Saul José Semião-Santos ◽

Laura Barral Veloso ◽

Paulo Paes de Andrade ◽

Marcia Almeida de Melo ◽

Luis Miguel Lourenço Martins ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Leishmania Infantum ◽

Leishmania Donovani ◽

Agglutination Test ◽

Canine Visceral Leishmaniasis ◽

Direct Agglutination Test ◽

Serum Samples

We compared the performance of a locally produced β-mercaptoethanol-modified promastigote antigen (β-ME-Ag) of an indigenous Leishmania infantum strain against that of a trypsinized Leishmania donovani reference (REF-Ag) in the direct agglutination test (DAT) for detection of canine visceral leishmaniasis (CVL). One hundred and fifty-one serum samples collected from dogs belonging to four groups with different conditions were included. At a DAT titre of 1 : 320, statistically determined as optimal cut-off value for β-ME-Ag, and 1 : 160 for REF-Ag, a sensitivity and a specificity of 100 % were estimated for β-ME-Ag in comparison with 96.6 % and 100 %, respectively, for REF-Ag. Overall, levels of agglutination titres recorded for the two antigens were highly concordant (Cohen’s κ = 0.879) in both the CVL and non-CVL groups. Based on current results, and ease experienced in processing the antigen and reading the test outcome, we recommend incorporation of β-ME-Ag in DAT for confirmation or exclusion of suspected CVL in dogs.

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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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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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DIRECT AGGLUTINATION TEST WITH URINE SAMPLES FOR THE DIAGNOSIS OF VISCERAL LEISHMANIASIS

American Journal of Tropical Medicine and Hygiene ◽

10.4269/ajtmh.2004.70.78 ◽

2004 ◽

Vol 70 (1) ◽

pp. 78-82 ◽

Cited By ~ 25

Author(s):

MOHAMMAD ZAHIDUL ISLAM ◽

IFTIKHAR AHMED ◽

ABDUL HALIM SARDER ◽

RUSELLA MIRZA ◽

EISAKU KIMURA ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Agglutination Test ◽

Urine Samples ◽

Direct Agglutination Test

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Diagnosis of Leishmania infantum using Direct Agglutination Test and rKE16 Dipstick Rapid Test in Domestic Dogs from Ardabil Province, Iran

Research Journal of Parasitology ◽

10.3923/jp.2015.102.110 ◽

2015 ◽

Vol 10 (3) ◽

pp. 102-110

Author(s):

H. Nahrevania ◽

P. Ghaffarine ◽

M. Farahmand ◽

M. Mohebali ◽

F. Zaboli ◽

...

Keyword(s):

Leishmania Infantum ◽

Rapid Test ◽

Agglutination Test ◽

Domestic Dogs ◽

Direct Agglutination Test

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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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Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

Clinical and Vaccine Immunology ◽

10.1128/cvi.00583-13 ◽

2013 ◽

Vol 21 (1) ◽

pp. 96-106 ◽

Cited By ~ 18

Author(s):

Lourena E. Costa ◽

Mayara I. S. Lima ◽

Miguel A. Chávez-Fumagalli ◽

Daniel Menezes-Souza ◽

Vivian T. Martins ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Phage Display ◽

Leishmania Infantum ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Phage Display Technology ◽

Predictive Values ◽

Content Type ◽

Phage Elisa

ABSTRACTVisceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy andTrypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected withLeishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n= 31) compared to those from vaccinated dogs (n= 21), experimentally infected dogs with cross-reactive parasites (n= 23), and healthy controls (n= 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity withT. cruzi- orEhrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes ofL. infantumantigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.

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