title: An approach for interlaboratory comparison of conventional and real-time PCR assays for diagnosis of human leishmaniasis.
abstract: Protozoa of the Leishmania genus are transmitted to humans by the bite of infected sandflies, and are the causative agents of leishmaniasis which ranges from cutaneous to visceral clinical forms. The definitive diagnosis of leishmaniasis has relied traditionally on parasite demonstration, either by microscopy or culture; in the last years, diagnosis based on PCR methods has overcome some drawbacks of traditional methods, increasing sensitivity and allowing using less invasive sampling for diagnosis. However, there are not defined protocols and almost each laboratory applies its own in-house method. Although there are several studies comparing the performance of different methods within the same laboratory, those addressing interlaboratory comparison are scarce, in spite of the growing number of collaborative projects between partners from different leishmaniasis endemic and non-endemic countries. In this work we propose a protocol for interlaboratory comparison of conventional and real-time PCR methods involving four participant laboratories from four different endemic regions in four continents; the protocol includes a quality control step and reduces the variability among the samples tested by each participant. A panel of 77 samples from human origin and 9 from different parasite strains was blindly tested by the participants, aiming to assess the sensitivity of the different methods as well as their usefulness for species identification. Real-time PCR methods targeting the kDNA minicircles returned the highest sensitivity, while both PCR targeting ITS-1 and further HaeIII digestion and a combined algorithm including hsp70 PCR and restriction fragment length polymorphism analysis were the most appropriate approaches for species identification.
authors.affiliation: WHO Collaborating Center for Leishmaniasis, Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra. Majadahonda-Pozuelo, Km2, 28220 Madrid, Spain. cruzi@isciii.es
authors.forename: Israel
authors.initials: I
authors.lastname: Cruz
authors.forename: Aurélie
authors.initials: A
authors.lastname: Millet
authors.forename: Eugenia
authors.initials: E
authors.lastname: Carrillo
authors.forename: Mehdi
authors.initials: M
authors.lastname: Chenik
authors.forename: Poonam
authors.initials: P
authors.lastname: Salotra
authors.forename: Sandeep
authors.initials: S
authors.lastname: Verma
authors.forename: Nicolás
authors.initials: N
authors.lastname: Veland
authors.forename: Marlene
authors.initials: M
authors.lastname: Jara
authors.forename: Vanessa
authors.initials: V
authors.lastname: Adaui
authors.forename: Carlos
authors.initials: C
authors.lastname: Castrillón
authors.forename: Jorge
authors.initials: J
authors.lastname: Arévalo
authors.forename: Javier
authors.initials: J
authors.lastname: Moreno
authors.forename: Carmen
authors.initials: C
authors.lastname: Cañavate
mesh_terms: D016022:Case-Control Studies; D004247:DNA / Q000097:blood / Q000737:chemistry; D018105:DNA, Kinetoplast / Q000032:analysis; D016054:DNA, Protozoan / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D007891:Leishmania / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D007896:Leishmaniasis / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000592:standards; D012150:Polymorphism, Restriction Fragment Length; D011786:Quality Control; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D017422:Sequence Analysis, DNA / Q000379:methods / Q000592:standards; D013045:Species Specificity
chemical_list: D018105:DNA, Kinetoplast; D016054:DNA, Protozoan; D004247:DNA