title: Clinical use of polymerase chain reaction performed on peripheral blood and bone marrow samples for the diagnosis and monitoring of visceral leishmaniasis in HIV-infected and HIV-uninfected patients: a single-center, 8-year experience in Italy and review of the literature.
abstract: BACKGROUND
  To overcome some of the limitations of conventional microbiologic techniques, polymerase chain reaction (PCR)-based assays are proposed as useful tools for the diagnosis of visceral leishmaniasis.
  
  
  METHODS
  A comparative study using conventional microbiologic techniques (i.e., serologic testing, microscopic examination, and culture) and a Leishmania species-specific PCR assay, using peripheral blood and bone marrow aspirate samples as templates, was conducted during an 8-year period. The study cohort consisted of 594 Italian immunocompetent (adult and pediatric) and immunocompromised (adult) patients experiencing febrile syndromes associated with hematologic alterations and/or hepatosplenomegaly. Identification of the infecting protozoa at the species level was directly obtained by PCR of peripheral blood samples, followed by restriction fragment-length polymorphism analysis of the amplified products, and the results were compared with those of isoenzyme typing of Leishmania species strains from patients, which were isolated in vitro.
  
  
  RESULTS
  Sixty-eight patients (11.4%) had a confirmed diagnosis of visceral leishmaniasis. Eleven cases were observed in human immunodeficiency virus (HIV)-uninfected adults, 20 cases were observed in HIV-infected adults, and the remaining 37 cases were diagnosed in HIV-uninfected children. In the diagnosis of primary visceral leishmaniasis, the sensitivities of the Leishmania species-specific PCR were 95.7% for bone marrow aspirate samples and 98.5% for peripheral blood samples versus sensitivities of 76.2%, 85.5%, and 90.2% for bone marrow aspirate isolation, serologic testing, and microscopic examination of bone marrow biopsy specimens, respectively. None of 229 healthy blood donors or 25 patients with imported malaria who were used as negative control subjects had PCR results positive for Leishmania species in peripheral blood samples (i.e., specificity of Leishmania species-specific PCR, 100%). PCR and restriction fragment-length polymorphism analysis for Leishmania species identification revealed 100% concordance with isoenzyme typing in the 19 patients for whom the latter data were available.
  
  
  CONCLUSIONS
  PCR assay is a highly sensitive and specific tool for the diagnosis of visceral leishmaniasis in both immunocompetent and immunocompromised patients and can be reliably used for rapid parasite identification at the species level.
authors.affiliation: Dipartimento di Scienze Cliniche L. Sacco, Sezione di Malattie Infettive e Tropicali, Università di Milano, Milan, Italy. spinello.antinori@unimi.it
authors.forename: Spinello
authors.initials: S
authors.lastname: Antinori
authors.forename: Sara
authors.initials: S
authors.lastname: Calattini
authors.forename: Erika
authors.initials: E
authors.lastname: Longhi
authors.forename: Giovanna
authors.initials: G
authors.lastname: Bestetti
authors.forename: Roberta
authors.initials: R
authors.lastname: Piolini
authors.forename: Carlo
authors.initials: C
authors.lastname: Magni
authors.forename: Giovanna
authors.initials: G
authors.lastname: Orlando
authors.forename: Marina
authors.initials: M
authors.lastname: Gramiccia
authors.forename: Veronica
authors.initials: V
authors.lastname: Acquaviva
authors.forename: Antonella
authors.initials: A
authors.lastname: Foschi
authors.forename: Stefano
authors.initials: S
authors.lastname: Corvasce
authors.forename: Claudia
authors.initials: C
authors.lastname: Colomba
authors.forename: Lucina
authors.initials: L
authors.lastname: Titone
authors.forename: Carlo
authors.initials: C
authors.lastname: Parravicini
authors.forename: Antonio
authors.initials: A
authors.lastname: Cascio
authors.forename: Mario
authors.initials: M
authors.lastname: Corbellino
mesh_terms: D017088:AIDS-Related Opportunistic Infections; D000328:Adult; D000368:Aged; D000465:Algorithms; D000818:Animals; D001853:Bone Marrow / Q000469:parasitology*; D002648:Child; D002675:Child, Preschool; D005260:Female; D015658:HIV Infections / Q000150:complications; D006801:Humans; D016867:Immunocompromised Host; D007223:Infant; D007558:Italy; D007891:Leishmania / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D007898:Leishmaniasis, Visceral / Q000097:blood / Q000175:diagnosis* / Q000235:genetics / Q000821:virology; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods*