Detection of Leishmania Antigen from Buccal Swabs in Kala-azar Patients Using KATEX Method

Background: Visceral leishmaniasis (VL) or kala-azar is a chronic protozoan infection in humans that is fatal unless treated and is associated with significant global morbidity and mortality. Confirmation of visceral leishmaniasis (VL) diagnosis requires microscopic examination to visualize the causative agent Leishmania Donovani usually in spleen or bone marrow aspirate. Tissue aspiration is invasive, potentially risky and require skilled personnel. KATEX (Kalon biologicals) antigen test for VL in buccal swabs has not been evaluated locally and could offer a significant advantage in screening patients suspected of VL.  Method: A cross sectional study was conducted after receiving approval from KEMRI SERU. We obtained buccal swabs from patients presenting at Kimalel Health Centre, Baringo County, Kenya from VL cases and controls. VL was defined as patients meeting VL case definition with positive splenic aspirate microscopy while endemic controls were defined as patients presenting to the health center with no fever and no prior history of Kala azar but living in VL endemic. Latex agglutination based test (KATEX) was used to detect parasite antigen in buccal swabs. It was a proof of principle study carried out to explore the ability to use KATEX, a simple non-invasive diagnostic test to detect leishmania antigens in buccal swabs, determine the ability of the kit to detect leishmania antigens in buccal cells of kala-azar patients and compare the sensitivity and specificity of KATEX - buccal assay using microscopy as the gold standard. Results: 88 patients were analyzed, including 44 VL and 44 non-VL patients. The median age of VL patients was 18 years with predominance of males (68.2%). None of the tested VL patients were co-infected with HIV. KATEX kit was able to detect visceral leishmaniasis antigens from the buccal swabs giving a sensitivity of (81.8%; 95%CI: 67.3% to 91.8%, specificity of (79.5%; 95%CI: 64.7 –90.2%), Positive predictive value n= 36(80.0%); 95%CI: 65.4% to 90.4% and Negative predictive values n = 35(81.4%); 95% CI: 66.6% to 91.6%. Conclusion and Recommendation: Buccal swab test assay using KATEX is an easy test to perform and promising non-invasive based antigen detection test which may be useful for screening kala-azar patients and could be applied in the diagnosis of VL. It is a functional assay that warrants a larger study with a larger sample size for the purpose of evaluating the utility of the test in diagnosing visceral leishmaniasis. There is dire need to identify non-invasive, less risky and field adapted point of care diagnostics for VL. Keywords: Kala-azar; Visceral Leishmaniasis; Diagnosis; KATEX; Latex; Buccal swab antigen detection

A panel of recombinant Leishmania donovani cell surface and secreted proteins identifies LdBPK_323600.1 as a serological marker of symptomatic infection

Visceral leishmaniasis is a deadly infectious disease and is one of the world’s major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK_323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK_323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease. IMPORTANCE Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK_323600.1 that could accurately diagnose visceral leishmaniasis infections in humans

Induction of transformation of leishmania through lipophosphoglycan by serum lectins

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

C-reactive protein-mediated phagocytosis of Leishmania donovani promastigotes does not alter parasite survival or macrophage responses

C-reactive protein (CRP) is an acute phase protein that binds to surface structures of a number of different organisms. Leishmania donovani express CRP ligand when first entering the mammalian host and CRP has been shown to alter macrophage function. The aim of this study was to investigate the functional significance of CRP-mediated uptake of L. donovani on survival of the parasite within human macrophages and macrophage cell responses to the infection. CRP opsonized L. donovani uptake was inhibitable by including excess CRP in the fluid phase, suggesting Fc receptor usage rather than indirect complement-mediated uptake. Comparing equivalent initial infection loads, parasite survival over 72 h within peripheral blood derived macrophages (PBMs) and differentiated U937 cells was unaltered by CRP. Whereas CRP increased macrophage responses to phosphorylcholine coated erythrocytes, no significant alteration in tumour necrosis factor-alpha, interleukin (IL)-10 or IL-12 production from PBMs was observed between CRP opsonized or unopsonized L. donovani promastigotes. Thus, in contrast to other systems, where CRP opsonization results in macrophage activation, Leishmania can use CRP to improve infection without inducing detrimental macrophage activation

Assessment of hepatitis B vaccination status and hepatitis B surface antibody titres among health care workers in selected public health hospitals in Kenya.

Healthcare workers (HCWs) have a significant occupational risk of hepatitis B virus (HBV) infection. Vaccination remains the most effective measure recommended to avert the risk. However, there's limited information on hepatitis B vaccine uptake rates and the seroprotection status of HCWs, especially in sub-Saharan Africa. This study aimed to assess hepatitis B vaccination status and also seroprotection status of HCWs in three selected public hospitals in Kenya. This was a cross-sectional study carried out among HCWs at Kenyatta National Hospital (KNH), Naivasha and Mbagathi County hospitals. Data on participants' demographics and hepatitis B vaccination status was collected using an interviewer-guided questionnaire. Blood samples were collected and tested for hepatitis B surface antigen (HBsAg), hepatitis B surface antibodies (anti-HBs), and hepatitis B core antibodies (anti-HBc) using Enzyme Linked Immuno Sorbent Assay technique. Data were analyzed using Statistical Package for the Social Sciences (SPSS) and Graph pad prism. Of the 145 eligible HCWs, 120 (82.8%) were vaccinated, with 77 (53.1%) having received the recommended three doses. Three quarters (108/145) of the vaccinated HCWs were seroprotected (titres ≥10 mIU/ml) against HBV infection, while 16.6% were non-responders (titres <10 mIU/ml). Vaccination with more than two doses and HBV exposure were significantly associated with anti-HBs titre levels (P<0.05). HCWs who received less than 2 doses of the vaccine were 70% less likely to have high anti-HBs titre levels (aOR, 0.3; 95% CI, 0.1-0.8; P = 0.013). Nearly all HCWs were vaccinated against hepatitis B virus. The majority of all HCWs were seroprotected against hepatitis B virus but a number of them had an insufficient immunity to the virus despite vaccination or prior exposure. There's need to sensitize HCWs and enforce mandatory full vaccination as per the recommended vaccination schedule

Classification of immune responses based on anti-HBs titres, vaccination and HBV exposure status among HCW at KNH, Mbagathi County and Naivasha sub-county hospitals.

Classification of immune responses based on anti-HBs titres, vaccination and HBV exposure status among HCW at KNH, Mbagathi County and Naivasha sub-county hospitals.</p

Demographic characteristics and hepatitis B vaccination status of participating HCW at KNH, Mbagathi County and Naivasha sub-county hospitals (N = 145).

Demographic characteristics and hepatitis B vaccination status of participating HCW at KNH, Mbagathi County and Naivasha sub-county hospitals (N = 145).</p

Interviewer-guided questionnaire.

Healthcare workers (HCWs) have a significant occupational risk of hepatitis B virus (HBV) infection. Vaccination remains the most effective measure recommended to avert the risk. However, there’s limited information on hepatitis B vaccine uptake rates and the seroprotection status of HCWs, especially in sub-Saharan Africa. This study aimed to assess hepatitis B vaccination status and also seroprotection status of HCWs in three selected public hospitals in Kenya. This was a cross-sectional study carried out among HCWs at Kenyatta National Hospital (KNH), Naivasha and Mbagathi County hospitals. Data on participants’ demographics and hepatitis B vaccination status was collected using an interviewer-guided questionnaire. Blood samples were collected and tested for hepatitis B surface antigen (HBsAg), hepatitis B surface antibodies (anti–HBs), and hepatitis B core antibodies (anti–HBc) using Enzyme Linked Immuno Sorbent Assay technique. Data were analyzed using Statistical Package for the Social Sciences (SPSS) and Graph pad prism. Of the 145 eligible HCWs, 120 (82.8%) were vaccinated, with 77 (53.1%) having received the recommended three doses. Three quarters (108/145) of the vaccinated HCWs were seroprotected (titres ≥10 mIU/ml) against HBV infection, while 16.6% were non–responders (titres PP = 0.013). Nearly all HCWs were vaccinated against hepatitis B virus. The majority of all HCWs were seroprotected against hepatitis B virus but a number of them had an insufficient immunity to the virus despite vaccination or prior exposure. There’s need to sensitize HCWs and enforce mandatory full vaccination as per the recommended vaccination schedule.</div