Identification of a Novel Recombinant Protein for Improved Diagnosis of Visceral Leishmaniasis in Sudan

Die effiziente Kontrolle der viszeralen Leishmaniose (VL) in Ostafrika hängt ganz besonders von einer schnellen und sensitiven Diagnostik ab. Gegenwärtige Testsysteme sind für die Diagnostik der VL im Sudan leider nicht besonders gut geeignet. Ziel dieses Projektes war die Identifikation und Testung neuer Antigene eines aus dem Sudan stammenden Leishmania donovani-Stammes zur Verbesserung der VL-Diagnose in Ostafrika. Es wurde ein neues Antigen aus Leishmania donovani identifiziert und kloniert (rKLO8), das eine hohe Sequenzübereinstimmung mit dem immundominaten Kinesinprotein verschiedener Leishmania-Stämme aufweist. Die Immun-reaktivität des aufgereinigten rekombinanten Proteins wurde durch Westernblot und ELISA getestet und bestätigt. Es zeigte sich, dass rKLO8 nur mit Seren von VL- Patienten, nicht jedoch gesunden Individuen reagiert. Zusätzlich wurde ein auf dem rKLO8-Protein basierter Test (ELISA) etabliert und mit Patientenseren aus dem Sudan, Indien und Frankreich evaluiert. Eine vergleichende Studie zeigte, dass das diagnostische Potential des neu entwickelten rKLO8 Tests im Sudan und Indien gegenüber dem derzeit verwendeten Testantigen, rK39, deutlich besser ist. Weiterhin wurde das diagnostische Potential der rKLO8 - und rK39 ELISA mit verschiedenen kommerziellen Tests, den rK39- und rKE16-Schnelltests und dem direkten Agglutinationstest (DAT) verglichen. Alle Tests zeigten bei Patienten aus Indien ähnlich gute Ergebnisse, bei VL-Patienten aus anderen Ländern jedoch zeigten der rKLO8- und rK39-ELISA die höchste Sensitivität. Ein weiterer Befund war, dass die Koinfektion mit dem HI-Virus die Sensitivität aller Testsysteme beträchtlich reduzierte. Zuletzt wurde der neu entwickelte rKLO8-Test auch mit Seren VL-infizierter Hunde aus Portugal, Kroatien und Brasilien getestet. Der ELISA war in seiner diagnostischen Potenz ähnlich dem DAT, im Vergleich zum routinemäßig eingesetzten immunofluoreszenz-basierten Antikörpertest (IFAT) jedoch deutlich sensitiver. Zusammengefasst stellt rKLO8 aufgrund seiner erhöhten Reaktivität mit Patientenseren aus dem Sudan ein potentielles Antigen dar, mit dem die VL- Diagnostik im Sudan und anderen Leishmania donovani endemischen Regionen Ostafrikas verbessert werden kann

Indoor Navigation Algorithm For Mobile Robot

Recently there has been increasing research on the development of localization and navigation systems. Whereas most of the proposed approaches are suitable for outdoor operation, only a few techniques have been designed for indoor environments. This paper details the development of an indoor navigation system. It presents a general system consisting of sensors and algorithms for localization and navigation which enables to operate indoors. This is done by using trilateration method which has been successfully applied on complex nature of indoor environments. Set of experiments presented to validate our system using MATLAB program. Testing verified that good accuracy, sufficient for navigation, was achieved. This technique shows promise for future handheld indoor navigation systems that can be used in malls, museums, hospitals, and college campuses

Comparison of Different Serological Tests in Three Endemic Regions

Diagnostic tests for visceral leishmaniasis that are based on antigens of a single Leishmania strain can have low diagnostic performance in regions where heterologous parasites predominate. The aim of this study was to investigate and compare the performance of five serological tests, based on different Leishmania antigens, in three endemic countries for visceral leishmaniasis. A total number of 231 sera of symptomatic and asymptomatic cases and controls from three endemic regions of visceral leishmaniasis in East Sudan, North India and South France were evaluated by following serological tests: rKLO8- and rK39 ELISA, DAT (ITMA-DAT) and two rapid tests of rK39 (IT LEISH) and rKE16 (Signal-KA). Overall, rKLO8- and rK39 ELISA were most sensitive in immunocompetent patients from all endemic regions (96–100%) and the sensitivity was reduced to 81.8% in HIV co-infected patients from France. Sera of patients from India demonstrated significantly higher antibody responses to rKLO8 and rK39 compared with sera from Sudan (p<0.0001) and France (p<0.0037). Further, some Indian and Sudanese patients reacted better with rKLO8 than rK39. Sensitivity of DAT (ITMA-DAT) was high in Sudan (94%) and India (92.3%) but low in France being 88.5% and 54.5% for VL and VL/HIV patients, respectively. In contrast, rapid tests displayed high sensitivity only in patients from India (96.2%) but not Sudan (64–88%) and France (73.1–88.5% and 63.6–81.8% in VL and VL/HIV patients, respectively). While the sensitivity varied, all tests showed high specificity in Sudan (96.7–100%) and India (96.6%).Heterogeneity of Leishmania parasites which is common in many endemic regions complicates the diagnosis of visceral leishmaniasis. Therefore, tests based on homologous Leishmania antigens are required for particular endemic regions to detect cases which are difficult to be diagnosed with currently available tests

Identification of a Novel Recombinant Protein for Improved Diagnosis of Visceral Leishmaniasis in Sudan

Die effiziente Kontrolle der viszeralen Leishmaniose (VL) in Ostafrika hängt ganz besonders von einer schnellen und sensitiven Diagnostik ab. Gegenwärtige Testsysteme sind für die Diagnostik der VL im Sudan leider nicht besonders gut geeignet. Ziel dieses Projektes war die Identifikation und Testung neuer Antigene eines aus dem Sudan stammenden Leishmania donovani-Stammes zur Verbesserung der VL-Diagnose in Ostafrika. Es wurde ein neues Antigen aus Leishmania donovani identifiziert und kloniert (rKLO8), das eine hohe Sequenzübereinstimmung mit dem immundominaten Kinesinprotein verschiedener Leishmania-Stämme aufweist. Die Immun-reaktivität des aufgereinigten rekombinanten Proteins wurde durch Westernblot und ELISA getestet und bestätigt. Es zeigte sich, dass rKLO8 nur mit Seren von VL- Patienten, nicht jedoch gesunden Individuen reagiert. Zusätzlich wurde ein auf dem rKLO8-Protein basierter Test (ELISA) etabliert und mit Patientenseren aus dem Sudan, Indien und Frankreich evaluiert. Eine vergleichende Studie zeigte, dass das diagnostische Potential des neu entwickelten rKLO8 Tests im Sudan und Indien gegenüber dem derzeit verwendeten Testantigen, rK39, deutlich besser ist. Weiterhin wurde das diagnostische Potential der rKLO8 - und rK39 ELISA mit verschiedenen kommerziellen Tests, den rK39- und rKE16-Schnelltests und dem direkten Agglutinationstest (DAT) verglichen. Alle Tests zeigten bei Patienten aus Indien ähnlich gute Ergebnisse, bei VL-Patienten aus anderen Ländern jedoch zeigten der rKLO8- und rK39-ELISA die höchste Sensitivität. Ein weiterer Befund war, dass die Koinfektion mit dem HI-Virus die Sensitivität aller Testsysteme beträchtlich reduzierte. Zuletzt wurde der neu entwickelte rKLO8-Test auch mit Seren VL-infizierter Hunde aus Portugal, Kroatien und Brasilien getestet. Der ELISA war in seiner diagnostischen Potenz ähnlich dem DAT, im Vergleich zum routinemäßig eingesetzten immunofluoreszenz-basierten Antikörpertest (IFAT) jedoch deutlich sensitiver. Zusammengefasst stellt rKLO8 aufgrund seiner erhöhten Reaktivität mit Patientenseren aus dem Sudan ein potentielles Antigen dar, mit dem die VL- Diagnostik im Sudan und anderen Leishmania donovani endemischen Regionen Ostafrikas verbessert werden kann

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

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

Pseudopregnancy in goats: Sonographic prevalence and associated risk factors in Khartoum State, Sudan

Aim: This study was conducted to estimate the prevalence of pseudopregnancy in goats and to investigate potential risk factors associated with the condition in Khartoum State. Materials and Methods: A cross-sectional study was carried out from March 2015 to February 2016. A total of 378 female goats which presented to the Veterinary Teaching Hospital, College of Veterinary Medicine, Sudan University of Science and Technology, for routine ultrasonographic pregnancy diagnosis were examined. Ultrasound scanning was performed using a real-time scanner equipped with dual-frequency (3.5-5 MHz) curvilinear transducer. Results: The results showed that the prevalence of pseudopregnancy in goats in Khartoum State was 10.6%. Risk factors such as general body condition (χ2=5.974; p=0.05), age (χ2=11.760; p=0.0129), type of estrus (χ2=12.794; p=0.000), and previous reproductive performance (χ2=13.397; p=0.020) showed significant association (p≤0.05) with the occurrence of pseudopregnancy in the univariate analysis. Breed (χ2=12.627; p=0.082), milk yield (χ2=5.951; p=0.114), type of feeding (χ2=1.721; p=0.190), season (χ2=2.661; p=0.264), locality (χ2=7.66; p=0.264), parity number (χ2=0.451; p=0.767), and rearing system (χ2=1.593; p=0.451) were not significantly associated with pseudopregnancy. Conclusion: The prevalence of pseudopregnancy in goats in Khartoum State was 10.6%. Pseudopregnancy in goats is significantly associated with age, type of estrus, general body condition, and previous reproductive performance. This study showed for the first time that pseudopregnancy is a real reproductive problem in goats in Khartoum State

Heterogeneity of Leishmania donovani parasites complicates diagnosis of visceral leishmaniasis: comparison of different serological tests in three endemic regions.

Diagnostic tests for visceral leishmaniasis that are based on antigens of a single Leishmania strain can have low diagnostic performance in regions where heterologous parasites predominate. The aim of this study was to investigate and compare the performance of five serological tests, based on different Leishmania antigens, in three endemic countries for visceral leishmaniasis. A total number of 231 sera of symptomatic and asymptomatic cases and controls from three endemic regions of visceral leishmaniasis in East Sudan, North India and South France were evaluated by following serological tests: rKLO8- and rK39 ELISA, DAT (ITMA-DAT) and two rapid tests of rK39 (IT LEISH) and rKE16 (Signal-KA). Overall, rKLO8- and rK39 ELISA were most sensitive in immunocompetent patients from all endemic regions (96-100%) and the sensitivity was reduced to 81.8% in HIV co-infected patients from France. Sera of patients from India demonstrated significantly higher antibody responses to rKLO8 and rK39 compared with sera from Sudan (p<0.0001) and France (p<0.0037). Further, some Indian and Sudanese patients reacted better with rKLO8 than rK39. Sensitivity of DAT (ITMA-DAT) was high in Sudan (94%) and India (92.3%) but low in France being 88.5% and 54.5% for VL and VL/HIV patients, respectively. In contrast, rapid tests displayed high sensitivity only in patients from India (96.2%) but not Sudan (64-88%) and France (73.1-88.5% and 63.6-81.8% in VL and VL/HIV patients, respectively). While the sensitivity varied, all tests showed high specificity in Sudan (96.7-100%) and India (96.6%).Heterogeneity of Leishmania parasites which is common in many endemic regions complicates the diagnosis of visceral leishmaniasis. Therefore, tests based on homologous Leishmania antigens are required for particular endemic regions to detect cases which are difficult to be diagnosed with currently available tests

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

<div><p>Background</p><p>For 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 <i>L. donovani</i> strain in Sudan.</p><p>Methodology and Principle Findings</p><p>We cloned, expressed and purified a novel recombinant protein antigen of <i>L. donovani</i> from Sudan, designated rKLO8, that contains putative conserved domains with significant similarity to the immunodominant kinesin proteins of <i>Leishmania</i>. rKLO8 exhibited 93% and 88% amino acid identity with cloned kinesin proteins of <i>L. infantum</i> (synonymous <i>L. chagasi</i>) (K39) and <i>L. donovani</i> (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.</p><p>Conclusion</p><p>The 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.</p></div

Comparative reactivity of <i>Leishmania</i> antibodies with rKLO8 and rK39.

<p>The rKLO8 or rK39 proteins were used and compared in ELISA using protein concentrations of 5 ng/100 µl in 0.1M sodium carbonate. A panel of sera from VL patients and controls were tested. Visceral leishmaniasis (VL; n = 106), non-VL controls (n = 77) including non-endemic healthy controls (NEC; n = 20), endemic healthy controls (EC; n = 30), malaria (MA; n = 11), tuberculosis (TB; n = 10), or leukaemia (LEU; n = 6). (A) Sera were tested at dilutions of 1∶800 and a cut off value (0.12) was established as means+3 SD of the OD measured for 30 healthy controls from Sudan. (B) VL sera (n = 14) with negative results at 1∶800 were re-tested at a serum dilution of 1∶100 and compared with the controls described in A. Cut off values were recalculated using 20 non-endemic healthy sera and found to be 0.41 and 0.32 for rKLO8 and rK39, respectively. Statistical Analysis was performed by <i>one way ANOVA</i> nonparametric test.</p

The NF-κB transcription factor c-Rel controls host defense against Citrobacter rodentium.

LetterMice lacking CD4+ T cells or B cells are highly susceptible to Citrobacter rodentium infection. In this study, we show that the activity of the transcription factor c-Rel in lymphocytes is crucial for clearance of C. rodentium. Mice deficient for c-Rel fail to generate protective antibodies and to eradicate the pathogen