Development of an Immunochromatographic Test for Diagnosis of Visceral Leishmaniasis Based on Detection of a Circulating Antigen

RESEARCH ARTICLE Development of an Immunochromatographic Test for Diagnosis of Visceral Leishmaniasis Based on Detection of a Circulating Antigen Chun-hua Gao1, Yue-tao Yang1, Feng Shi1, Jun-yun Wang1*, Dietmar Steverding2*, Xia Wang2 a11111 1 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Laboratory of Parasite and Vector, Ministry of Public Health, National Centre for International Research on Tropical Diseases, WHO Collaborating Center for Malaria, Schistosomiasis and Filariasis, Shanghai, China, 2 BioMedical Research Centre, Norwich Medical School, Norwich Research Park, University of East Anglia, Norwich, United Kingdom * (JyW); (DS) Abstract OPEN ACCESS Citation: Gao C-h, Yang Y-t, Shi F, Wang J-y, Steverding D, Wang X (2015) Development of an Immunochromatographic Test for Diagnosis of Visceral Leishmaniasis Based on Detection of a Circulating Antigen. PLoS Negl Trop Dis 9(6): e0003902. doi:10.1371/journal.pntd.0003902 Editor: Henk D. F. H. Schallig, Royal Tropical Institute, NETHERLANDS Received: May 6, 2015 Background Visceral leishmaniasis (VL) is a life-threatening disease caused by protozoan parasites of the Leishmania donovani complex. Early case detection followed by adequate treatment is essential to the control of VL. However, the available diagnostic tests are either invasive and require considerable expertise (parasitological demonstration of the parasite in tissue smears) or unable to distinguish between past and active infection (serological methods). Therefore, we aimed to develop a lateral flow assay in the form of an immunochromatographic test (ICT) device based on the detection of a circulating Leishmania antigen using monoclonal antibodies (mAbs). Accepted: June 11, 2015 Published: June 30, 2015 Methodology/Principal Findings Copyright: © 2015 Gao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. mAbs were produced by fusion of murine myeloma cells with splenocytes isolated from a mouse immunized with L. donovani soluble crude antigen. Out of 12 cloned hybridoma cell lines, two secreted mAbs recognizing the same leishmanial protein. These mAbs were used to produce an ICT as a sandwich assay for the detection of circulating antigen in serum and blood samples. The ICT was evaluated with 213 serum samples from VL patients living in VL endemic areas in China, and with 156 serum samples from patients with other diseases as well as 78 serum samples from healthy donors. Sensitivity, specificity and diagnostic efficiency of the new ICT was 95.8%, 98.7% and 97.3%, respectively. Compared with a commercially available antibody detecting ICT, our antigen-based ICT performed slightly better. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by National S & T Major Program (http://www.nmp.gov.cn), grant No.: 2008ZX10004-011, 2012-ZX10004-201 and 2012ZX10004-220. The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Conclusion/Significance The newly developed ICT is an easy to use and more accurate diagnostic tool which fulfils the performance and operational characteristics required for VL case detection under field PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0003902 June 30, 2015 1 / 12 Antigen-Based ICT for Diagnosis of Visceral Leishmaniasis and laboratory conditions. As our ICT detects a circulating antigen, it will also be useful in monitoring treatment success and diagnosing VL in immunocompromised patients. Author Summary Visceral leishmaniasis is a neglected disease caused by different species of protozoan parasites of the genus Leishmania. The disease is endemic in 61 countries, and in many of them it poses a serious public health issue. As visceral leishmaniasis is fatal if left untreated, early diagnosis is essential for treatment and control of the disease. Current available diagnostic tests are either difficult to carry out under field conditions or insufficiently accurate. In this study we developed a new diagnostic test which detects a circulation parasite-derived antigen in serum or blood samples of patients with visceral leishmaniasis. We found that our test performed better than most other tests based on the detection of parasite-specific antibodies. In addition, as our test is a device for the detection of an acute infection, it will be useful to validate treatment and in the diagnosis of the disease in patients with deficient antibody production (as in AIDS patients). Introduction Visceral leishmaniasis (VL), or kala-azar, is a vector-borne disease caused by protozoan parasites belonging to the Leishmania donovani complex, which includes L. donovani, L. infantum and L. chagasi. The parasites infect and multiply preferentially in macrophages of the spleen, liver, bone marrow, and lymph nodes of their mammalian host. VL is a systemic disease and is fatal if left untreated. The disease is endemic in large areas of the tropics, subtropics and the Mediterranean Basin affecting 61 countries. It is estimated that approximately 200,000 to 400,000 cases of VL occur annually and that about 20,000 to 40,000 people die each year from the disease [1]. VL is also an important public health problem in China being endemic in six provinces or autonomous regions in western China including Xinjiang, Gansu, Sichuan, Shaanxi, Shanxi and Inner Mongolia [2–4]. The causative agents of VL in China are L. donovani and L. infantum [4]. Since the clinical features of VL mimic several other common diseases, accurate and early diagnosis is crucial for treatment and control of VL as the drugs currently used for chemotherapy have significant toxic side effects [5, 6]. Parasitological detection remains the gold standard for diagnosis of VL because of its high specificity [7]. However, as for all microscopic procedures, parasitological VL diagnosis is affected by variability in detection sensitivity (e.g. the sensitivity of bone marrow smears varies between 60% to 85% while that of splenic aspirates can exceed 95% [7]) and by the expertise of the microscopist. In addition, invasive bone marrow and spleen aspiration are painful and risky techniques. Culturing the parasite can improve the sensitivity of VL diagnosis but can be affected by contamination of bacteria or yeast species and are time-consuming [7]. Since a strong humoral response is generally induced in VL patients, serodiagnosis is an alternative to detection of the parasite in tissue samples. Serological tests for diagnosis of VL (e.g. enzyme-linked immunosorbent assay (ELISA), indirect fluorescence an (...truncated)