Research note Detection of malaria liver-stages in mice infected through the bite of a single Anopheles mosquito using a highly sensitive real-time PCR Oscar Brun ˜a-Romero, Julius C.R. Hafalla, Gloria Gonza ´lez-Aseguinolaza, Gen-ichiro Sano, Moriya Tsuji, Fidel Zavala * Department of Medical and Molecular Parasitology, New York University School of Medicine, New York, NY 10010, USA Received 23 April 2001; received in revised form 7 June 2001; accepted 7 June 2001 Abstract We describe a highly sensitive real-time PCR to detect and measure the development of the liver-stages of malaria parasites in mice infected with sporozoites ranging in number from 25 to more than 164,000, using the same reaction conditions. Furthermore, this assay detects and measures parasite loads in the livers of mice exposed to the bite of a single malaria-infected Anopheles mosquito. This unique method should greatly facilitate studies aimed at evaluating very precisely the efficacy of anti-malarial experimental drug treatments and vaccination regimens in conditions of infection resembling those found in the field. q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Malaria; Plasmodium; Liver-stages; Real-time PCR The liver stage is a critical phase in the life cycle of species of Plasmodium infecting mammals, marking the transition of these malaria parasites from the invertebrate to a vertebrate host. During this intra-hepatocytic stage, the parasite undergoes major adaptive changes in phenotype, gene expression, and biological properties. Recent investi- gations underscore the importance of the liver stage as a target for vaccine development (Nardin et al., 1999), as the inhibition of parasite growth in hepatocytes can result in the reduction or complete ablation of the erythrocytic stages, thus attenuating or eliminating the symptoms and the pathology of the disease. This stage is also an important target for drug development as some species of Plasmodium generate hypnozoites (Krotoski, 1985), dormant liver forms that can initiate new cycles of erythrocytic infection inde- pendently of vector transmission. In spite of their obvious biological and epidemiological importance, the intra-hepa- tocytic forms are still poorly characterised and remain the least studied of the plasmodial stages in the vertebrate host. The liver stages of malaria parasites have been traditionally studied using histopathological methods (Yoeli et al., 1965; Khan et al., 1992), which involve labour-intensive and time- consuming procedures. The development of methods such as reverse transcription-PCR (Briones et al., 1996; Vernick et al., 1996), which can detect malaria-specific nucleotide sequences, partially overcame these limitations. However, despite the significant improvement achieved with these new techniques, their use is limited as they are only semi- quantitative and require the injection of thousands of para- sites into experimental animals (Briones et al., 1996). More recently, a highly sensitive technique for the contin- uous on-line monitoring of PCR-amplified products has been developed for the quantification of nucleic acids (Higuchi et al., 1993; Bassler et al., 1995; Morrison et al., 1998, 1999; Abe et al., 1999; Donovan et al., 2000). It is based on the detection and measurement in ‘real-time’ of fluorescence emitted proportionally to the synthesis of the PCR product. The fluorescent signals required for detection can be obtained by labelling the PCR product with either a fluorescent dye (Morrison et al., 1998, 1999) or a fluoro- genic probe (Bassler et al., 1995; Abe et al., 1999; Donovan et al., 2000). Fluorescent dyes (i.e. SYBR Green I) bind non- specifically to any PCR product generated, while the fluoro- genic probes are designed to release fluorescence after they are annealed to the specific target sequence. Thus, fluoro- genic probes can be considered sequence-specific detection reagents. We adapted the real-time PCR to develop an assay for the detection and quantification of the liver stages of Plasmo- dium yoelii parasites. Two specific primers were designed to amplify the parasite 18S rRNA sequence in addition to a fluorogenic probe for the detection of the PCR product generated after amplification. Both the primers and the International Journal for Parasitology 31 (2001) 1499–1502 0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S0020-7519(01)00265-X www.parasitology-online.com * Corresponding author. Tel.: 11-212-263 6760; fax: 11-212-263 8116. E-mail address: fz5@is2.nyu.edu (F. Zavala).