New indirect immunofluorescence assay as a confirmatory test for human immunodeficiency virus type 1

Background: Screening of blood and blood products for human immunodeficiency virus (HIV) is routinely performed using the enzyme-linked immunosorbent assay (ELISA), and the results confirmed by Western blot (WB). However, western blot is expensive and mostly performed in developed countries. A technique more superior or comparable to WB and adaptable to developing countries must be sought. In an effort to identify such a technique, this study determined the efficiency of indirect immunofluorescence assay (IFA) to detect antibodies to HIV-1. Objective: To determine the accuracy and sensitivity of an in-house immunofluorescence assay (IFA) to detect antibodies to HIV-1 in plasma. Design: A comparative study to evaluate the performance of indirect immunofluorescence assay (IFA) and western blot (WB) techniques in the detection of antibodies to HIV-1. Setting: Kenya Medical Research Institute, Centre for Virus Research. The study was conducted between June and December 2001. Methods: The evaluation of IFA as a technique for detecting antibodies to HIV-1 utilized a total of 400 samples. For these samples, IFA was compared with ELISA and particle agglutination (PA) (manuscript under preparation). Of the 400 samples, there were discrepant results in the three assays in only 36 samples. IFA was compared with Western blot (WB) to confirm the true HIV-1 serostatus in these 36 plasma specimens. The IFA technique used acetone-fixed HIV-1 infected MOLT-4 cells in one spot on a Teflon coated slide and uninfected MOLT-4 cells alone in a second spot to asses non-specific fluorescence. Western blot was performed according to the instructions of the manufacturer. Results: The sensitivity and specificity of IFA based on 36 plasma specimens tested was 71.4% and 100% respectively. All samples that were HIV seronegative by WB were also HIV seronegative by IFA. However, two (5.6%) samples were HIV seronegative by IFA but seropositive by WB. Conclusion: The data obtained show that IFA can be used as a primary confirmatory test in Kenya. East African Medical Journal Vol. 81 No. 5 May 2004: 222-22

Leishmania major-phlebotomus duboscqi interactions: inhibition of anti-LPG antibodies and characterisation of two proteins with shared epitopes

Objectives: To assess the effect of monoclonal antibodies (MABS) raised against L. major derived LPG on L. major development in vitro and in its natural vector P. duboscqi. Also determine whether LPG molecule and the sand fly the gut Iysates have sharedepitopes. Design: A laboratory based study. Setting: Colony bred P. duboscqi sand flies and all other experiments were done under laboratory conditions. Methods: Laboratory reared sand flies were allowed to feed beneath a blood filled membrane feeder containing 1 x106 amastigotes in 20µl mixed with 0.5 ml of defibrinated rabbit blood with a 1:100 dilution of anti-LPG MABS. Control blood contained a similar number of amastigotes but no MABS. At least five female previously fed sand flies were later dissected on days two, four, and six post-feeding and examined for promastigote forms and parasite loads in the sand fly mid gut. In vitro, the same number of amastigotes in 100µl complete Schneider's Drosophila medium was mixed in a 96 well plate with either 100µl of 1: 100 anti-LPG MABS, 1:1000 anti LPG MABS or undiluted sera from L. major infected mice. The control well contained a similar number of amastigotes but no antibodies added. Following an overnight incubation in a CO2 incubator at 37ºC and growth at 26ºC, parasites were assessed at 3, 6 and 24 hour intervals for changes in their developmental forms. Results: 1:100 dilution of anti-LPG MABS when mixed with amastigotes were effective in reducing L. major development at the early log phase or procyclic stage both in vitro and within the sand fly (

Cell death in Leishmania induced by stress and differentiation: programmed cell death or necrosis?

Unicellular organisms, such as the protozoan parasite Leishmania, can be stimulated to show some morphological and biochemical features characteristic of mammalian apoptosis. This study demonstrates that under a variety of stress conditions such as serum deprivation, heat shock and nitric oxide, cell death can be induced leading to genomic DNA fragmentation into oligonucleosomes. DNA fragmentation was observed, without induction, in the infectious stages of the parasite, and correlated with the presence of internucleosomal nuclease activity, visualisation of 45 to 59 kDa nucleases and detection of TUNEL-positive nuclei. DNA fragmentation was not dependent on active effector downstream caspases nor on the lysosomal cathepsin L-like enzymes CPA and CPB. These data are consistent with the presence of a caspase-independent cell death mechanism in Leishmania, induced by stress and differentiation that differs significantly from metazoa