β-Mercaptoethanol-modified ELISA for diagnosis of visceral leishmaniasis

Abstract

Following antigen preparation procedures similar to those of the direct agglutination test (DAT), an IgG ELISA employing intact b-mercaptoethanol (b-ME)-treated Leishmania donovani promastigotes was developed. The performance of the b-ME ELISA thus developed was assessed in patients with confirmed visceral leishmaniasis (VL), revealing slightly lower sensitivity (39/40=97?5 %) than that of the DAT (40/40=100 %). When challenged with sera of individuals with non-VL conditions, including leukaemia and African trypanosomiasis, the specificity of the b-ME ELISA was 100 % (158/158), compared to 98?8 % (156/158) for DAT. In an endemic population (n=145) manifesting a clinical suspicion of VL, results obtained with the b-ME ELISA were highly concordant with those of DAT, both in the seropositive (65/68=95?6 %) and seronegative (77/80=96?3 %) groups. Furthermore, the incorporated intact antigen demonstrated higher sensitivity in ELISA (16/18=88?9 %) than the water-soluble equivalent (13/18=72?2 %). The stability of the formaldehyde-fixed antigen (2 months at 4 6C) in b-ME ELISA, as well as the option for direct testing of whole-blood samples and visual reading of results (within 2 h, compared to 18 h for DAT), advocate the simultaneous application of the technique with DAT for confirmation of VL in laboratories with limited facilities.

rKLO8, a Novel Leishmania donovani – Derived Recombinant Immunodominant Protein for Sensitive Detection of Visceral Leishmaniasis in Sudan

“…Sera used in this study were collected in the rural hospital of Doka, Eastern State of Sudan [30], [31]. All patients and controls have given consent for participation in the study.…”

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

“…Further evaluation of the rK39 strip test in eastern and southern Sudan revealed significantly lower sensitivity (90.0%) and specificity (70.0%) than those reported for the DAT (respectively, 99% and 85%) (12,14). In our opinion, the high specificity demonstrated for ␤-ME ELISA and DAT in this and a previous study (1) can be attributed only to the favorable effect brought about by ␤-ME on the promastigote membrane, whereby cross-reacting molecules were drastically reduced to elicit reaction against non-VL sera. However, as has been found with other versions of the ELISA, determination of the cutoff level in this ␤-ME ELISA can be influenced by the level of VL endemicity in the study area, the causative Leishmania subspecies involved, and the reagent lot employed.…”

“…The reaction was measured 15 min after addition of the substrate (5-aminosalicylic acid), at a 450-nm wavelength on a Titertek Multiscan. ␤-ME ELISA absorbance values of Ն0.27 were considered indicative of VL infection (1).…”

“…The antigen processing for ␤-ME ELISA was carried out as described for DAT with the exception of a Coomassie brilliant blue staining step (1). Following ␤-ME treatment and repeated washing in citrate saline solution, promastigotes were fixed as a stock suspension (1.5 ϫ 10 8 /ml) in a 1.2% (vol/vol) formaldehyde citrate-saline solution.…”

“…By combining the use of a ␤-mercaptoethanol-modified antigen similar to that used in the DAT and an anti-human immunoglobulin G (IgG) conjugate for targeting specific IgG antibodies, a hybrid enzyme-linked immunosorbent assay (␤-ME ELISA) was developed and successfully evaluated in a panel of reference VL and non-VL sera (1). The purpose of this study was to determine the reliability of the ␤-ME ELISA for detecting VL in patients suspected of having the disease presenting at a rural hospital in eastern Sudan.…”