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Evaluation an in–house IgM-ELISA for the diagnosis ofhuman leptospirosis

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Author(s): Hamidreza Honarmand | Motahare Nezafat Tabalvandi | Abtin Heidarzadeh | Bahram Soltani | Ebrahim Mirzajani | Mahdi Asmar

Journal: Koomesh
ISSN 1608-7046

Volume: 09;
Issue: 04;
Start page: 309;
Date: 2008;
Original page

Keywords: Leptospirosis | IgM-ELISA | Sensitivity | Specificity

ABSTRACT
Background: Leptospirosisis a very common zoonosis in the world. Culture is low sensitive withhigh rate false negative. So, serological assays are best alternative way for its diagnosis. Microscopicagglutination test (MAT) is gold standard but performing it requires a panel of some standard strainsand need periodic subculturing of them, and also requires double sera with at least two weeks intervalto investigate seroconversion. Furthermore, other serological methods should be investigated. The aimof this study was to evaluate an in-house IgM-ELISA developed by using antigen extracted fromendemic isolates.Material and Method: I4 endemic isolates belonged to the serogroups: Icterohaemorrahgia,Pomona, Hardjo, and Gripotyphosa, were inoculated in EMJH to take well grown cultures. Wholeantigen was extracted from each culture by Freezing-Thawing method in distilled water. Same amountof extraction of each culture with same OD number in 550nm were mixed together and were used forcoating Elisa plates. Antihuman IgM conjugated with alkaline phosphatase were used in this assay. Weused a commercial quantitative IgM-ELISA (SERION ELISA classic) for cut off determination. MATwas used for confirmation positive and negative cases. Sera with titer ≥ 1:100 in MAT and positivecriteria in commercial quantitative IgM-ELISA were considered as positive cases.Results: 98 positive cases and 54 negative cases were chosen by screening 200 sera of patientssuspected to leptospirosis by using MAT and commercial quantitative IgM-ELISA. We also used 30sera of patients affected by hepatitis B, salmonelosis, and brucellosis as control cases. 88 of 98 positivecases were positive (false negative=10), 1 of 54 negative and all control case were negative (falsepositive =1) in the test. Sensitivity, specificity, PPV, NPV, and accuracy of the test were evaluated:99.0% , 89.1% , 90.75 , 98.8% , and 94.25 , respectively.Conclusions: ELISA for measuring specific IgM to leptospires antigen(s) could be a goodalternative to MAT, which is not a routine diagnostic assay to perform in clinical diagnosticlaboratories and only is reliable when there is paired sera. Sensitivity and specificity of the assay isdependent to several factors, especially to the type of antigen coated on plates, quality of assaymaterials, and also to the time of sampling. Sera of days ≥ 6 of the disease has enough antibodies tomeasure and a common antigen extracted from several common pathogenic leptospires, especiallyfrom endemic isolates, could be more helpful to increase accuracy of the assay.
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