title: Retrospective PCR-based species identification ofLeishmaniain two patients with visceral leishmaniasis in Serbia.
abstract: BACKGROUND
  Retrospective molecular identification ofLeishmaniaparasites in two patients with visceral leishmaniasis (VL) previously treated in Serbia was carried out. DNA was isolated from unstained bone marrow smears (BMSs) kept for 11 and 8 years. Genus-specific real-time PCR was combined with conventional PCR and sequencing for detection and species identification.
  
  
  METHODS
  In 2003, a 40-year-old Serbian male was admitted to the Clinical Centre of Serbia (CCS) with fever, sweating, fatigue and splenomegaly, which developed over a period of 7 weeks. He had frequently travelled around Europe. VL was confirmed by microscopy of Giemsa-stained BMS. Treatment by pentavalent antimonials was successfully completed. Two years later, the patient developed post-kala-azar dermal leishmaniasis. Treatment resulted in symptom resolution. Later on,Leishmania infantumwas identified as the causative agent of the VL by sequencing of the ITS (internal transcribed spacer) region; mixedLeishmaniaspp. infection could not be excluded. In 2006, a 33-year-old female from Vojvodina, Serbia, with pre-existing diabetes mellitus and chronic meningoencephalitis and a history of frequent visits to the Montenegrin seacoast, was admitted to the CCS with fever, pancytopenia and moderate hepatosplenomegaly. A stained BMS revealed abundantLeishmaniaamastigotes. Indirect haemagglutination analysis was positive with a titre of 1 : 2048, and a rapid dipstick rK39 test was also positive. Treatment by liposomal amphotericin B was successful; however, shortly after, the patient developed neural infection and pneumonia and died. The causative agent was identified asL. infantum.
  
  
  CONCLUSIONS
  Molecular diagnosis of VL and species delineation using DNA from unstained BMSs stored for several years is possible.
authors.affiliation: Parasitological Laboratory, Department of Microbiology, Clinical Centre of Serbia, Bulevar Oslobodjenja 16 , 11000 Belgrade , Serbia.
authors.forename: Zorica
authors.initials: Z
authors.lastname: Dakić
authors.affiliation: Laboratory of Parasitology, Department of Microbiology and Infection Control, Division of Diagnostics and Infection Control, Statens Serum Institute, 5 Artillerivej , DK-2300 Copenhagen , Denmark.
authors.forename: Henrik Vedel
authors.initials: HV
authors.lastname: Nielsen
authors.affiliation: Medical Faculty, University of Belgrade, Dr Subotića 8, 11000 Belgrade, Serbia; Clinic for Infectious and Tropical Diseases, Clinical Centre of Serbia, Bulevar Oslobodjenja 16, 11000 Belgrade, Serbia.
authors.forename: Milorad
authors.initials: M
authors.lastname: Pavlović
authors.affiliation: Medical Faculty, University of Belgrade, Dr Subotića 8, 11000 Belgrade, Serbia; Clinic for Infectious and Tropical Diseases, Clinical Centre of Serbia, Bulevar Oslobodjenja 16, 11000 Belgrade, Serbia.
authors.forename: Jasmina
authors.initials: J
authors.lastname: Poluga
authors.affiliation: Medical Faculty, University of Belgrade, Dr Subotića 8, 11000 Belgrade, Serbia; Clinic for Infectious and Tropical Diseases, Clinical Centre of Serbia, Bulevar Oslobodjenja 16, 11000 Belgrade, Serbia.
authors.forename: Goran
authors.initials: G
authors.lastname: Stevanović
authors.affiliation: Medical Faculty, University of Belgrade, Dr Subotića 8, 11000 Belgrade, Serbia; Clinic for Infectious and Tropical Diseases, Clinical Centre of Serbia, Bulevar Oslobodjenja 16, 11000 Belgrade, Serbia.
authors.forename: Lidija
authors.initials: L
authors.lastname: Lavadinović
authors.affiliation: Medical Faculty, University of Belgrade, Dr Subotića 8, 11000 Belgrade, Serbia; Clinic for Infectious and Tropical Diseases, Clinical Centre of Serbia, Bulevar Oslobodjenja 16, 11000 Belgrade, Serbia.
authors.forename: Branko
authors.initials: B
authors.lastname: Milošević
authors.affiliation: Medical Faculty, University of Belgrade, Dr Subotića 8, 11000 Belgrade, Serbia; Clinic for Infectious and Tropical Diseases, Clinical Centre of Serbia, Bulevar Oslobodjenja 16, 11000 Belgrade, Serbia.
authors.forename: Mijomir
authors.initials: M
authors.lastname: Pelemiš
authors.affiliation: Medical Faculty, University of Belgrade, Dr Subotića 8, 11000 Belgrade, Serbia; Clinic for Infectious and Tropical Diseases, Clinical Centre of Serbia, Bulevar Oslobodjenja 16, 11000 Belgrade, Serbia.
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Urošević
authors.affiliation: Parasitological Laboratory, Department of Microbiology, Clinical Centre of Serbia, Bulevar Oslobodjenja 16 , 11000 Belgrade , Serbia.
authors.forename: Snežana
authors.initials: S
authors.lastname: Jovanović
authors.affiliation: Laboratory of Parasitology, Department of Microbiology and Infection Control, Division of Diagnostics and Infection Control, Statens Serum Institute, 5 Artillerivej , DK-2300 Copenhagen , Denmark.
authors.forename: Christen Rune
authors.initials: CR
authors.lastname: Stensvold
keywords: Leishmania infantum; PKDL; amastigotes; molecular diagnostic; sequencing; visceral leishmaniasis