RAPID IDENTIFICATION OF LEISHMANIA COMPLEXES BY A REAL-TIME PCR ASSAY GLENN WORTMANN,* LISA HOCHBERG, HUO-HSU HOUNG, COLLEEN SWEENEY, MICHAEL ZAPOR, NAOMI ARONSON, PETER WEINA, AND CHRISTIAN F. OCKENHOUSE Infectious Disease Service, Walter Reed Army Medical Center, Washington, DC; Department of Entomology, Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Viral Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland; Infectious Disease Division, Uniformed Services University of the Health Sciences, Bethesda, Maryland; Department of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland; Department of Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland Abstract. A real-time PCR assay for the detection of four Leishmania complexes (L. Viannia, L. mexicana, L. donovani/infantum, and L. major) was developed and evaluated. The assay was developed to detect the glucosephos- phate isomerase gene and capitalizes on DNA sequence variability within that gene for Leishmania complex identifi- cation. Primer/probe sets were created and tested against a panel of 21 known negative controls and on DNA extracted from cultured promastigotes or from tissue biopsies from patients with cutaneous leishmaniasis. The assay was highly specific, as no amplification products were detected in the negative control samples while simultaneously retaining a high degree of complex-specific diagnostic accuracy for cultured organisms and patient clinical samples. Real-time PCR offers rapid (within hours) identification of Leishmania to the complex level and provides a useful molecular tool to assist both epidemiologists and clinicians. INTRODUCTION Leishmaniasis is a protozoan infection that is endemic throughout the tropical and subtropical regions. 1 The World Health Organization estimates that 1.5 million to 2 million new cases occur each year. 2 Twenty-two species of Leishma- nia have been reported to cause human infections. 3 In the Old World, cutaneous leishmaniasis is predominantly due to L. major, L. tropica, L. aethiopica, and L. infantum, whereas L. donovani and L. infantum are responsible for visceral disease. In the New World, members of the L. Viannia subgenus (in- cluding L. V. braziliensis and L. V. panamensis) and the L. Leishmania mexicana complex cause the majority of cutane- ous disease, whereas L. L. chagasi is associated with visceral disease. Identification of the infecting parasite to the complex or species level is important for prognostic, epidemiologic, and therapeutic reasons. 4 The classical method used for this task is isoenzyme analysis, a procedure that is slow, laborious, and requires the growth of cultured promastigote parasites. 5 This method of identification is impractical for real-time patient treatment decisions, as the turn-around period from biopsy to successful growth of culture to isoenzyme identification can take weeks. Newer methods of identification that could po- tentially classify Leishmania directly from patient specimens (abrogating the requirement for culture) include monoclonal antibodies and numerous DNA-based assays including mul- tiplex polymerase chain reaction (PCR), PCR plus sequenc- ing, and restriction fragment length polymorphism (RFLP) analysis. 6–11 Most recently, real-time PCR, a platform that can process a sample in less than an hour, has been reported to rapidly differentiate single nucleotide mutations within a target DNA sequence. 12,13 We have previously developed and field tested a real-time PCR assay to diagnose cutaneous and visceral leishmaniasis to the genus level and have used this assay to assess the absence or presence of infection in both vertebrate (human hosts) and invertebrate sand fly vec- tors. 14,15 We now describe the development and testing of a second-generation real-time PCR assay to classify samples to the level of Leishmania complexes and species. This assay has the potential to provide the treating clinician with a rapid identification of the infecting Leishmania species, which sub- sequently offers the potential for targeted treatment strate- gies. MATERIALS AND METHODS Parasites. Cultured Leishmania reference strains were ob- tained from the strain collections of the Walter Reed Army Institute of Research (Silver Spring, MD). The identification of all strains had been accomplished by isoenzyme analysis. The strains were cultured at 24°C in Schneider’s Drosophila Medium (Gibco, Grand Island, NY) containing 20% fetal calf serum. 16 Parasites were harvested at a density of 2 × 10 7 parasites/mL, pelleted at 500 × g for 8 minutes at 4°C, washed three times in phosphate-buffered balanced salt solution (PBSS) pH 7.4, and then resuspended in 500 L PBSS. Clinical samples. Skin biopsy samples were obtained from patients with suspected cutaneous leishmaniasis who pre- sented to the Walter Reed Army Medical Center (Washing- ton, DC). Samples were placed in Schneider’s Drosophila Medium for culture and 70–100% ethanol for PCR process- ing. The maximum time from sampling to PCR processing was 7 days. All cultures were identified by isoenzyme analysis of cultured promastigotes. Negative control samples. Negative control DNA was ex- tracted from paraffin-embedded clinical samples representing a wide range of dermatological and tropical diseases. In ad- dition, DNA was extracted from cultures of Trypanosoma rangelli (ATCC 30032) and Crithidia fasiculata (ATCC 11745), two organisms closely related to Leishmania. DNA extraction. DNA purification was performed by col- umn chromatography (QIAamp Blood and Blood Products Kit for the cultured promastigotes and QIAamp Tissue Kit for clinical samples; Qiagen, Chatsworth, CA) following the manufacturer’s instructions. For the paraffin-embedded clini- * Address correspondence to Glenn Wortmann, 6900 Georgia Ave., NW, Walter Reed Army Medical Center, Washington, DC 20307- 5001. E-mail: glenn.wortmann@na.amedd.army.mil Am. J. Trop. Med. Hyg., 73(6), 2005, pp. 999–1004 Copyright © 2005 by The American Society of Tropical Medicine and Hygiene 999