BackgroundFor effective control of visceral leishmaniasis (VL) in East Africa, new rapid diagnostic tests are required to replace current tests with low sensitivity. The aim of this study is to improve diagnosis of VL in East Africa by testing a new antigen from an autochthonous L. donovani strain in Sudan.Methodology and Principle FindingsWe cloned, expressed and purified a novel recombinant protein antigen of L. donovani from Sudan, designated rKLO8, that contains putative conserved domains with significant similarity to the immunodominant kinesin proteins of Leishmania. rKLO8 exhibited 93% and 88% amino acid identity with cloned kinesin proteins of L. infantum (synonymous L. chagasi) (K39) and L. donovani (KE16), respectively. We evaluated the diagnostic efficiency of the recombinant protein in ELISA for specific detection of VL patients from Sudan. Data were compared with a rK39 ELISA and two commercial kits, the rK39 strip test and the direct agglutination test (DAT). Of 106 parasitologically confirmed VL sera, 104 (98.1%) were tested positive by rKLO8 as compared to 102 (96.2%) by rK39. Importantly, the patients' sera showed increased reactivity with rKLO8 than rK39. Specificity was 96.1% and 94.8% for rKLO8- and rK39 ELISAs, respectively. DAT showed 100% specificity and 94.3% sensitivity while rK39 strip test performed with 81.1% sensitivity and 98.7% specificity.ConclusionThe increased reactivity of Sudanese VL sera with the rKLO8 makes this antigen a potential candidate for diagnosis of visceral leishmaniasis in Sudan. However, the suitability at the field level will depend on its performance in a rapid test format.

Three-hundred and eight patients with suspected visceral leishmaniasis (VL) were received at Doka Hospital (eastern Sudan) during the period September 2004 to October 2005. The sensitivity and specificity of a glycerol-preserved (GP) antigen for VL diagnosis was assessed against the results of repeated lymph node aspiration and readings from a direct agglutination test (DAT) employing standard formaldehyde-fixed (FF) or freeze-dried (FD) antigen. Despite 13 months of storage at ambient temperature (28-47 6C), the GP antigen mean titres obtained from these 308 patients were no different from those that were FD (P=0?945) and stored under similar conditions, but were significantly different (P=0?019) from those that were FF and kept continuously at the optimum temperature for storage (4-8 6C). Taking the parasitological result as the gold standard and using a pre-established titre of 1 : 3200 as the DAT cut-off, the GP antigen revealed a sensitivity (91/105, 86?7 %) and specificity (187/203, 92?1 %) comparable to that of FD antigen (92/105, 87?6 %, and 188/203, 92?6 %, respectively) and FF antigen (94/105, 89?5 %, and 188/203, 92?6 %, respectively). At a titre range of 1 : 400-1 : 800, statistically determined as the optimum cut-off for the three antigens, sensitivities of 92?4, 90?5 and 96?2 % and specificities of 90?6, 90?1 and 88?7 % were achieved for the GP, FD and FF antigens, respectively, at a peripheral hospital. Regardless of the antigen preparation used, DAT results obtained in the peripheral hospital were highly reproducible in the central laboratory in Omdurman (weighted kappa: GP=0?957, FD=0?979 and FF=0?936). With a diagnostic reliability comparable to formaldehyde fixation and stability under ambient conditions similar to freeze drying, glycerol preservation, by virtue of its high potential for reproduction, meets the requirements for the management of VL in developing countries.

The potential of glycerol for long-term preservation of the direct agglutination test (DAT) antigen was evaluated at a fluctuating laboratory temperature of 25-37 degrees C and at constant temperatures of 37 and 45 degrees C for a period of 222 days. DAT titres recorded for the three antigen aliquots preserved in 50% (v/v) glycerol and stored at 25-37, 37 or 45 degrees C at 11 time intervals were within the same range of the control antigen kept at 4 degrees C. Performance of the glycerol-preserved antigen stored at 45 degrees C was compared with that of a freeze-dried version on 24 visceral leishmaniasis (VL) and 54 non-VL patients. For all non-VL patients, a maximum DAT titre of 1/800 was recorded for either of the two antigens. For all VL patients, in comparison with the freeze-dried, the glycerol-preserved antigen always had equal or higher titre; in 16 of the 24 VL sera tested, the latter antigen scored three- to sixfold higher titres. As this glycerol preservation method is economical and easy to perform, it has better potential for wider-scale application than freeze-drying.

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.

Corroboration of serology results is essential for restricting the risk of inappropriate antileishmanial prescription. A direct agglutination test (DAT) and a recently developed ␤-mercaptoethanol-modified enzymelinked immunosorbent assay (␤-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 ␤-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) ␤-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 ␤-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 ␤-ME ELISA. Of 197 symptomatic patients for whom microscopy was negative for VL, 172 (87.3%) tested negative in ␤-ME ELISA and 180 (91.4%) in DAT. Based on the high reliability demonstrated here for VL detection, ␤-ME ELISA fulfills the requirement of confirming DAT results in patients manifesting suspected VL.