Purification was carried out using nickel nitrilotriacetic (Ni-NTA) columns (Qiagen GmbH, Germany)

Purification was carried out using nickel nitrilotriacetic (Ni-NTA) columns (Qiagen GmbH, Germany). 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. Conclusion The increased reactivity of Sudanese VL sera with the Gimap5 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. Author Summary Visceral leishmaniasis (VL) is an infectious disease caused by the complex including in East Africa and India and by in the Mediterranean area and Latin America. Clinical diagnosis of VL in East Africa is difficult as maladies with similar symptoms are endemic. For this reason, reliable diagnosis of VL is extremely important. However, tests based on antibody reaction with rK39 are not sensitive in East Africa most likely due to the genetic diversity of different species. In this study, we cloned and expressed a new antigenic protein (rKLO8) of strain originating from Sudan. Sequence analysis confirmed that KLO8 differs from other kinesin proteins of (in East Africa and the Indian subcontinent, in Europe and North Africa and in Latin America [1], [2]. However, recent molecular and enzymatic studies revealed that is synonymous with amastigotes in tissue aspirates is still used for confirmation of the disease in Sudan although it is invasive and of low sensitivity. Diagnosis is further hindered, as the disease XL-147 (Pilaralisib) sometimes appears with atypical clinical pictures [7] which need confirmation by laboratory tests. Due to high fatality and toxicity of commonly used drugs [8], [9], diagnostic tests have to be of high accuracy. Commercially available rapid tests are either based on the rK39 of (synonym. have shown similarly high sensitivity and specificity compared to the classically used rK39-based tests in India [11], [21]. A multiregional study with five different rapid tests based on either rK39 or rKE16 demonstrated equal performance with high sensitivity (92.8C100%) in India [20]. However, sensitivity was significantly lower (36.8C92%) in Brazil and East Africa [20]. The direct agglutination test (DAT), which detects antibodies against whole promastigotes, has proven XL-147 (Pilaralisib) to be a useful tool for diagnosis of VL in several countries including Sudan [22]C[27]. The stability of DAT was improved by using freeze-dried and glycerol preserved antigens which does not require storage at 4C thus making the test suitable for field application [28], [29]. However, the test procedure and the need for overnight incubation give limitations for the field use. Here, we report identification, expression and testing of a new immunodominant protein (rKLo8) from an autochthonous strain in Sudan. For detection of VL antibodies in patients and controls from Sudan, an rKLo8 ELISA was developed and compared with the rK39 ELISA and two commercial XL-147 (Pilaralisib) kits. Methods Parasite and culture The reference strain Lo8 was kindly provided by Prof. Bernhard Fleischer, Bernhard Nocht Institute for Tropical Medicine (BNITM), Hamburg. The strain was originally isolated in Sudan from a confirmed case of visceral leishmaniasis. The parasite was maintained in RPMI-1640 supplemented with L-glutamine, NaHCO3 (Sigma-Aldrich) and 10% (v/v) fetal calf serum (Sigma-Aldrich). Ethical statement 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. Tests and experiments with patients’ sera and strains were anonymized. The study was approved by the Ethical Review Committee of the Federal Ministry of Health in Sudan and by the Regierungspr?sidium Gie?en, Germany. Human sera A total number of 183 human serum samples were obtained from the serum bank at the Biomedical Research Laboratory, Ahfad University (Omdurman-Sudan). The majority of samples (106) were from VL patients with confirmed lymph-node aspiration, 30 from healthy individuals resident at Doka village (an endemic area for VL) and.