Serological and molecular tools to diagnose visceral leishmaniasis: 2-years’ experience of a single center in Northern Italy

PLOS ONE, Nov 2019

The diagnosis of visceral leishmaniasis (VL) remains challenging, due to the limited sensitivity of microscopy, the poor performance of serological methods in immunocompromised patients and the lack of standardization of molecular tests. The aim of this study was to implement a combined diagnostic workflow by integrating serological and molecular tests with standardized clinical criteria. Between July 2013 and June 2015, the proposed workflow was applied to specimens obtained from 94 in-patients with clinical suspicion of VL in the Emilia-Romagna region, Northern Italy. Serological tests and molecular techniques were employed. Twenty-one adult patients (22%) had a confirmed diagnosis of VL by clinical criteria, serology and/or real-time polymerase chain reaction; 4 of these patients were HIV-positive. Molecular tests exhibited higher sensitivity than serological tests for the diagnosis of VL. In our experience, the rK39 immunochromatographic test was insufficiently sensitive for use as a screening test for the diagnosis of VL caused by L. infantum in Italy. However, as molecular tests are yet not standardized, further studies are required to identify an optimal screening test for Mediterranean VL.

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Serological and molecular tools to diagnose visceral leishmaniasis: 2-years’ experience of a single center in Northern Italy

August Serological and molecular tools to diagnose visceral leishmaniasis: 2-years' experience of a single center in Northern Italy Stefania Varani 0 1 2 Margherita Ortalli 0 2 Luciano Attard 0 2 Elisa Vanino 0 2 Paolo Gaibani 0 1 2 Caterina Vocale 0 1 2 Giada Rossini 0 1 2 Roberto Cagarelli 0 2 Anna Pierro 0 2 Patrizia Billi 0 2 Antonio Mastroianni 0 2 Simona Di Cesare 0 2 Mauro Codeluppi 0 2 Erica Franceschini 0 2 Fraia Melchionda 0 2 Marina Gramiccia 0 2 Aldo Scalone 0 2 Giovanna A. Gentilomi 0 2 Maria P. Landini 0 2 0 Editor: Farhat Afrin, Taibah University , SAUDI ARABIA 1 Unit of Clinical Microbiology, Regional Reference Centre for Microbiological Emergencies (CRREM), St. Orsola-Malpighi University Hospital , Bologna , Italy , 2 Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna , Bologna , Italy , 3 Infectious Diseases Unit, Department of Medical and Surgical Sciences, University of Bologna , Bologna , Italy , 4 Public Health Authority Emilia Romagna , Bologna , Italy , 5 Unit of Microbiology, The Romagna Hub Laboratory , Pievesestina (Fc) , Italy , 6 Infectious Diseases Unit, G.B. Morgagni Hospital , ForlÁõ , Italy , 7 Infectious Diseases Unit, Azienda Ospedaliero- Universitaria Policlinico Modena , Modena , Italy , 8 Pediatric Hematology and Oncology Unit, St. Orsola- Malpighi University Hospital , Bologna , Italy , 9 Unit of Vector-Borne Diseases and International Health, Infectious Diseases Department, Istituto Superiore di Sanità , Rome , Italy , 10 Department of Pharmacy and Biotechnology, University of Bologna , Bologna , Italy , 11 Department of Research, Innovation and Technology, Istituto di Ricovero e Cura a carattere scientifico, Istituto Ortopedico Rizzoli , Bologna , Italy 2 Citation: Varani S , Ortalli M, Attard L, Vanino E The diagnosis of visceral leishmaniasis (VL) remains challenging, due to the limited sensitivity of microscopy, the poor performance of serological methods in immunocompromised patients and the lack of standardization of molecular tests. The aim of this study was to implement a combined diagnostic workflow by integrating serological and molecular tests with standardized clinical criteria. Between July 2013 and June 2015, the proposed workflow was applied to specimens obtained from 94 in-patients with clinical suspicion of VL in the Emilia-Romagna region, Northern Italy. Serological tests and molecular techniques were employed. Twenty-one adult patients (22%) had a confirmed diagnosis of VL by clinical criteria, serology and/or real-time polymerase chain reaction; 4 of these patients were HIVpositive. Molecular tests exhibited higher sensitivity than serological tests for the diagnosis of VL. In our experience, the rK39 immunochromatographic test was insufficiently sensitive for use as a screening test for the diagnosis of VL caused by L. infantum in Italy. However, as molecular tests are yet not standardized, further studies are required to identify an opti- - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Lab P3 funds from the Emilia-Romagna Region (to MPL) and by RFO funds (to SV and MPL) from the University of Bologna. The funders had no role in 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. mal screening test for Mediterranean VL. Introduction Visceral leishmaniasis (VL) is a serious disease caused by obligate intracellular protozoa belonging to the Leishmania donovani complex and associated with considerable morbidity and mortality. Leishmania is transmitted by the bite of sandflies of the genus Phlebotomus and Lutzomyia and is endemic in around 100 countries including Southern Europe, Asia, Africa, and Latin America [ 1, 2 ]. In Mediterranean Europe, VL is caused by Leishmania infantum, which is mainly transmitted by Phlebotomus perniciosus, P. perfiliewi, P. ariasi, P. neglectus and P. tobbi [ 3 ]. VL is endemic in South-Eastern and Western Europe, with about 500 new autochthonous cases reported annually [ 4 ]. The incidence of leishmaniasis has increased since the early 1990s; this phenomenon occurred particularly in Spain, France and Italy, the latter facing a spread in the North of the country [5±9]. Because of its high endemicity and the recent outbreaks in low endemic areas, leishmaniasis is becoming a public health concern in Mediterranean Europe and improved diagnostic methods are needed to identify this disease [ 10 ]. The diagnosis of VL was traditionally based on serologic testing and the direct demonstration of Leishmania by microscopic examination of bone marrow aspirates, but limits have to be considered, including low sensitivity of serologic testing in immunosuppressed patients [ 11 ], the necessity to use invasive procedures to obtain bone marrow samples, the lo (...truncated)


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Stefania Varani, Margherita Ortalli, Luciano Attard, Elisa Vanino, Paolo Gaibani, Caterina Vocale, Giada Rossini, Roberto Cagarelli, Anna Pierro, Patrizia Billi, Antonio Mastroianni, Simona Di Cesare, Mauro Codeluppi, Erica Franceschini, Fraia Melchionda, Marina Gramiccia, Aldo Scalone, Giovanna A. Gentilomi, Maria P. Landini. Serological and molecular tools to diagnose visceral leishmaniasis: 2-years’ experience of a single center in Northern Italy, PLOS ONE, 2017, Volume 12, Issue 8, DOI: 10.1371/journal.pone.0183699