A Comparative Evaluation of Three Methods for Diagnosis of Visceral Leishmaniasis in Serbia Zorica Dakić1, M. Pelemiš2,3, G. Stevanović2,3, L. Lavadinović2,3, J. Poluga2,3, B. Milošević2,3, N. Indjić4, 2,3 1 2, 3 Clinical Center of Serbia O. Dulović , I. Ofori-Belić , M. Pavlović 1Parasitological Laboratory, Service of Microbiology, Clinical Center of Serbia, Belgrade, Serbia2Faculty of Medicine, University of Belgrade, Serbia; 3Clinic of Infectious and Tropical Diseases, CCS, Belgrade, Serbia4Center of Preventive Medical Care, Belgrade, Serbia Introduction In former Yugoslavia, visceral leishmaniasis was endemic in Macedonia, southern Serbia, Montenegro coast, south Herzegovina and Dalmatia. From 1945- 1955, three epidemic waves of VL were recorded in Serbia. In subsequent three years, 17 cases were reported, the result of eradication of malaria vectors. Rare autochthonous cases were noticed in 1968 and 1969 in Nis. According to epidemiological data, 39 VL cases were reported in Serbia and Montenegro from 1991 to 2000, with only one being imported. Today, the predominant VL risk in Serbian citizens is the stay at the Montenegrian sea-coast, where as many as 10 cases have been diagnosed each year in Bar. A retrospective diagnostic study of VL was carried out from December 2004 to August 2011 and included all patients with suspected VL referred to the Parasitological Laboratory, Cinical Center of Serbia, Belgrade. This study compared efficiency of three methods for the diagnosis of VL. Patients and methods All patients with suspected VL (n=44) were examined by Giemsa-stained bone marrow smears, by the rapid dipstick rK39 test (DiaSys Europe, England) and indirect hemagglutination assay (Siemens, former Behring Diagnostics, Germany). Positive IHA result was defined as titer >1:64. Patients with suspected VL, were defined as patients with a history of fever of ≥14 days with either clinical splenomegaly or wasting syndrome. Clinical suspicion was supported if the patient was from the an endemic area or had travelled to one this area in the recent past. Diagnosis of VL was confirmed on the demonstration of Leishmania amastigotes in Giemsa-stained BM smears. If the initial BM smear was negative but the clinical index of suspicion high, parasitological investigation was repeated, or the diagnosis based on the clinical presentation and positive serology. The control group included 62 patients with other diagnoses (imported malaria and other infectious and non-infectious diseases), who were tested by IHA and strip-test, without BM aspiration. Results VL was diagnosed in 14 patients (8 male and 6 female; age, 11 to 69 years, mean 40). Eleven of them (79%) were treated at the Clinic of Infectious and Tropical Diseases, Belgrade. The infection was contracted in Montenegro (n=8), Herzegovina (n=4), southern Serbia and Portugal (n=1, each). The initial examination of BM smears was successful in 85.7% patients. At the first examination, two patients had negative BM smears. In only one, parasitological investigation was repeated and VL was confirmed. In another patient, diagnosis was based on clinical picture, positive serology and therapeutic effect. Both the strip-test and IHA performed with a sensitivity of 92.9%, specificity 96.7% and a positive predictive value of 92.9%. The density of Leishmania amastigotes and antibody titer by IHA were not always in correlation with each other or with the clinical condition. One patient had positive both the strip-test and IHA (1:256), while parasitological investigation was negative; further examination confirmed liver and spleen multi-focal micro-abscesses. All patients in control group tested negative with both the strip-test and IHA. Patient Age Sex Origin of Incubation Clinical presentation Preexistent IHA Strip-test Dg by BM No infections period diseses titer result smears 1 24 m south Serbia 18 mo Fever with sweating, weight loss, pancytopenia, no 1:1024 + positive 1x hepatosplenomegaly numerous amastigotes 2 68 f Montenegro 3 mo Fever, pancytopenia, hepatosplenomegaly Sarcoidosis 1:256 + positive 1x sea-cost moderate amastigotes 3 27 m Herzegovina unknown, Fever, heavy sweating, bicytopenia, hepatosplenomegaly no 1:1024 + positive 1x resident numerous amastigotes 4 28 f Montenegro 7 mo Fever, weight loss, splenomegaly Ulcerative colitis 1:128 + positive 2x sea-cost rare amastigotes 5 68 f Herzegovina unknown, Fever, pancytopenia, hepatosplenomegaly Diabetes mellitus, 1:2048 + negative 1x resident Anemia ex iuvantibus 6 63 f Montenegro 12 mo Fever, fatigue, cough, weight loss, muscular no 1:16384 + positive 1x pain,hepatosplenomegaly sea-cost numerous amastigotes 7 44 m Montenegro unknown, Trombocytopenia, skin rashes, hepatosplenomegaly no 1:16384 + positive 1x sea-cost resident numerous amastigotes 8 33 f Montenegro unknown, Fever, pancytopenia, moderate hepatosplenomegaly Chronic meningo- 1:2048 + positive 1x sea-cost frequently encephalitis Numerous amastigotes traveling 9 22 m Herzegovina unknown, Fever, nocturnal sweating, cough, fatigue, weight loss, no 1:32± + positive 2x pancytopenia, hepatosplenomegaly resident negative rare amastigotes 10 69 m Montenegro unknown, Fever, headaches, bicytopenia, cough, nocturnal Chronic renal failure, 1:6536 + positive 2x sea-cost frequently sweating, fatigue, muscular pain, artralgia, DM rare amastigotes traveling hepatosplenomegaly 11 34 m Montenegro unknown, Fever, sweating, weight loss, pancytopenia, no 1:4096 + positive 2x hepatosplenomegaly sea-cost resident rare amastigotes 12 44 m Montenegro unknown, Trombocytopenia, skin rashes, hepatomegaly no 1:256 + positive 1x sea-cost frequently numerous amastigotes traveling 13 11 m Herzegovina unknown, Fever, sweating, weight loss, pancytopenia, no 1:64 + positive 1x hepatosplenomegaly resident moderate amastigotes 14 19 f Herzegovina 8 mo Fever, pancytopenia, hepatosplenomegaly, no 1:1024 + positive 2x lymphadenopathy rare amastigotes Conclusions The diagnosis of VL would have been missed in these patients if diagnosis had been solely on one diagnostic method. Inadequate sensitivity of the initial BM smears and rare false-negative reactions of the strip-test and IHA requires introduction of molecular diagnosis..