DIAGNOSING INFECTION LEVELS OF FOUR HUMAN MALARIA PARASITE SPECIES BY A POLYMERASE CHAIN REACTION/LIGASE DETECTION REACTION FLUORESCENT MICROSPHERE-BASED ASSAY

DAVID T. MCNAMARA LAURIN J. KASEHAGEN BRIAN T. GRIMBERG JENNIFER COLE-TOBIAN WILLIAM E. COLLINS PETER A. ZIMMERMAN Improving strategies for diagnosing infection by the four human Plasmodium species parasites is important as field-based epidemiologic and clinical studies focused on malaria become more ambitious. Expectations for malaria diagnostic assays include rapid processing with minimal expertise, very high specificity and sensitivity, and quantitative evaluation of parasitemia to be delivered at a very low cost. Toward fulfilling many of these expectations, we have developed a post-polymerase chain reaction (PCR)/ligase detection reaction-fluorescent microsphere assay (LDRFMA). This assay, which uses Luminex FlexMAP microspheres, provides simultaneous, semi-quantitative detection of infection by all four human malaria parasite species at a sensitivity and specificity equal to other PCR-based assays. In blinded studies using P. falciparum-infected blood from in vitro cultures, we identified infected and uninfected samples with 100% concordance. Additionally, in analyses of P. falciparum in vitro cultures and P. vivax-infected monkeys, comparisons between parasitemia and LDR-FMA signal intensity showed very strong positive correlations (r > 0.95). Application of this multiplex Plasmodium species LDR-FMA diagnostic assay will increase the speed, accuracy, and reliability of diagnosing human Plasmodium species infections in epidemiologic studies of complex malaria-endemic settings. - Controlling malaria infection in endemic regions has been an important goal of the World Health Organization since it was founded in 1948.1 Confidence in attaining and surpassing this goal flourished in the 1950s when chloroquine and DDT showed promise of eradicating malaria by killing plasmodia and anopheline species, respectively.2 However, widespread resistance to these agents in parasite and vector populations has complicated malaria control.2 A recent estimate suggests that approximately 2 billion people are exposed to malaria and more than 500 million clinical cases of Plasmodium falciparum malaria occur annually.3 In the context of these current challenges, significant efforts are underway to develop malaria vaccines,4 introduce new strategies to maximize the impact of a limited arsenal of approved, effective and affordable antimalarial drugs,5 and develop more effective mosquito control efforts that reduce transmission of malaria parasites.68 In addition to these new approaches for limiting replication and spread of malaria parasites, advancing technology continues to improve the efficiency of diagnosing infection by the four human malaria parasite species.911 These improved diagnostic strategies will be important in clinical trials of new antimalarial drugs and in vaccine development where estimating post-treatment parasitemia by highly sensitive and specific techniques is necessary for patient safety, reducing diagnostic error, and improving the estimation of drug/vaccine efficacy.12,13 Moreover, it is important to overcome inconsistencies in malaria diagnosis commonly observed with blood smear microscopy, as Ohrt and others have shown how relatively low levels of false positive errors can contribute to substantial underestimation of protective efficacy of drugs in clinical trials.12 In particular, this has resulted in a substantial effort to improve training and expertise in malaria microscopy (Ohrt C, unpublished data). Our earlier studies have applied14 and advanced9,15 previously developed polymerase chain reaction (PCR)based approaches16,17 for diagnosing infection by malaria parasites. Most recently, we have shown how sequence-specific ligase chain reaction techniques9 may be used to perform multiplex assays to simplify diagnosis of P. falciparum, P. vivax, P. malariae, and P. ovale. Here we introduce implementation of oligonucleotide probes bound covalently to microspheres designed to emit unique classification fluorescence to this diagnostic strategy. This modification of our multiplex malaria diagnostic assay simplifies 96-well plate processing. Furthermore, in evaluating the semi-quantitative capacity of this method, we find that this assay is capable of performing simultaneous comparisons of blood-stage parasite levels for all four human malaria parasite species in mixing experiments and in detection of parasites from in vitro cultures and infected animals. MATERIALS AND METHODS Plasmodium falciparum culture. The P. falciparum laboratory strain Mun01 (kindly provided by Alfred Cortes, Papua New Guinea Institute of Medical Research, Mill Hill, London) was propagated in vitro in human red blood cells (RBCs) (blood type O+) following standard malaria parasite culturing techniques.18 Briefly, the culture medium (RPMI 1640 medium, 25 mM HEPES, 0.25% sodium bicarbonate) was supplemented with 0.5% Albumax II, 2.2 mM Lglutamine, 0.08 mg/mL of gentamicin, and 0.2 mM hypoxanthine. Parasites were grown at 37C in an atmosphere of 5% CO2, 1% O2, and 94% N2 to a density of 2% (mixed developmental stage infection) at a 5% hematocrit before harvesting by centrifugation at 1,200 rpm for five minutes. All serial dilutions were performed using leukocyte-depleted whole blood washed three times with equal volumes of RPMI 1640 medium. Thin blood smears were fixed with 100% methanol for 30 seconds, stained with 4% Giemsa for 30 minutes, and examined by microscopy with an oil-immersion objective (100). Parasitemia was based on the number of infected RBCs (IRBCs)/(infected plus uninfected RBCs [n 1,000]). Plasmodium vivax infections. Monkeys (Aotus nancymai) were infected by intravenous inoculation of parasitized RBCs. Beginning one day after inoculation of P. vivaxIRBCs, thick and thin blood films were made by the method of Earle and Perez,19 stained with Giemsa, and examined microscopically. Parasites were recorded per microliter of blood. Blood samples (0.2 mL) from 11 infected monkeys harboring infections with P. vivax (Salvador I, Chesson, and Thai 3 strains) were obtained by venipuncture. Blood samples were collected in K+ EDTA-coated Vacutainer (Becton Dickinson, Franklin Lakes, NJ) tubes and shipped from the Centers for Disease Control and Prevention (CDC) (Atlanta, GA) to Case Western Reserve University (Cleveland, OH) where DNA extraction and ligase detection reaction fluorescent microsphere assay (LDR-FMA)based diagnosis was conducted. Monkeys did not receive anti-malarial drugs during this study. Protocols for infecting monkeys with malaria parasites were reviewed and approved by the CDC Institutional Animal Care and Use Committee according to Public Health Service Policy. Extraction of DNA. DNA was extracted from malariainfected human (parasite cultures) and non-human primate (whole blood) samples (200 L) using the QIAamp DNA blood mini kit (Qiagen, Valencia, CA). Parasitemias of blood samples harboring individual Plasmodium species infections were 5.0 104 IRBCs/ L fo (...truncated)