868 Am. J. Trop. Med. Hyg., 59(6), 1998, pp. 868–871 Copyright q 1998 by The American Society of Tropical Medicine and Hygiene MALARIA DIAGNOSIS BY DIPSTICK ASSAY IN A HONDURAN POPULATION WITH COENDEMIC PLASMODIUM FALCIPARUM AND PLASMODIUM VIVAX MIGUEL QUINTANA, ROBERT PIPER, HELEN-LOUISE BOLING, MICHAEL MAKLER, CATALINA SHERMAN, ENRIQUE GILL, EDUARDO FERNANDEZ, AND SAMUEL MARTIN Department of Entomology, Division of Communicable Disease and Immunology, Walter Reed Army Institute of Research, Washington, District of Columbia; Flow, Incorporated, Portland, Oregon; Joint Task Force-Bravo Soto Cono Air Base, Soto Cono, Honduras; Division of Vector Borne Diseases, Ministry of Health, Tegucigalpa, Honduras Abstract. A Plasmodium lactate dehydrogenase dipstick designed to separately detect P. falciparum and P. vivax malaria was evaluated in two Honduran populations where both species are endemic. The dipstick was compared to thick film microscopy; the polymerase chain reaction (PCR) was used to analyze discordant results. The dipstick had a sensitivity of 100% and a specificity of 95% compared with microscopy in the diagnosis of Plasmodium infections in a hospital population; the mean parasite density was approximately 590/mm 3 . In a field sample of mostly asymp- tomatic volunteers, the sensitivity of the dipstick for Plasmodium infection varied with parasite density. Additionally, the sensitivity and specificity of the dipstick was similar to thick film microscopy in the diagnosis of vivax malaria compared with the PCR. The dipstick was unable to detect P. vivax in the presence of P. falciparum because of cross-reactivity in the pan-specific band. Accurate species identification in mixed infections remains a problem in malaria diagnosis. The advent of immunochromatographic wicking dipstick assays for malaria diagnosis has provided a rapid, user- friendly technique useable in remote areas. 1–4 However, for dipsticks to effectively replace microscopy, they must pro- vide reliable species diagnosis. Of the four Plasmodium spe- cies that naturally infect humans, P. falciparum causes the most severe disease. Despite its obligate requirement for re- ticulocytes, 5 P. vivax, does not always cause benign infec- tions. Reports of significant morbidity, mortality and drug resistance in P. vivax infections are generating new interest in this Plasmodium. 6,7 The need to clear the hepatic hypno- zoite stage of patients with P. vivax infections is another reason to document vivax infections. Most routinely admin- istered antimalarials are ineffective against the exo-erythro- cytic stages of Plasmodia. Thus, a tissue schizontocidal drug must be added to the treatment regimen for vivax malaria to preclude an unpredictable relapse of the disease. 8 Plasmodium species diagnosis in mixed infections can be difficult to make. 9,10 Discrimination by microscopy is based on morphologic characteristics of the various asexual stages and the erythrocytes that they inhabit. Experienced micro- scopists are increasingly rare, especially at hospitals and clinics in endemic areas. The uncommon presence of mature gametocytes on a stained blood film is diagnostic for falci- parum malaria. Moreover, P. falciparum asexual parasites un- dergo deep tissue schizogony and unlike P. vivax, only the ring stage parasite is found on the blood smear. Primers exist for the reliable identification of the human malarias by poly- merase chain reaction (PCR) analysis; 11,12 however, this is largely a research tool unsuited for routine clinical labora- tory or field use. The Plasmodium lactate dehydrogenase (pLDH) assay de- rives from the unique ability of Plasmodia to use 3-acetyl- pyridine adenine dinucleotide (APAD) instead of NAD as a cofactor in the interconversion of lactate to pyruvate. 13,14 The pLDH dipstick assay has a dipstick format (OptiMALt) with a falciparum-specific band and a pan-specific band. It pur- ports to distinguish between P. falciparum and P. vivax in- fections. This assay captures pLDH as an antigen and does not measure enzyme activity. The effectiveness of Opti- MALt was evaluated in Honduras. MATERIALS AND METHODS The pLDH dipstick assay was evaluated in a Honduran hospital and field population. Malaria is a cause of morbidity and mortality in both populations. The study protocol used was approved by the Walter Reed Army Institute of Re- search Human Use Committee and the Honduran Ministry of Health (HMOH). The hospital study was conducted in May 1997 at the HMOH hospital in Trujillo, a port town located approximately 180 km east of La Ceiba, Altantida. The clinical laboratory at the hospital in Trujillo evaluates between 80 and 200 patients a week for malaria using thick film microscopy. Approximately 25% of these patients have positive blood smears. Consenting patients referred to the hospital laboratory with a presumptive diagnosis of malaria were recruited into the study. In the field study, blood samples were collected from res- idents of La Ceibita, a small village located approximately 10 km south of Tocoa, Colon. Every home in the village was visited in November 1996, and blood taken from all consenting individuals, regardless of symptoms. A previous study in June 1996 found 18% of the 550 residents of La Ceibita infected with P. vivax and 5% with P. falciparum (Quintana M, unpublished data). Blood was obtained from the same fingerstick for the thick blood film, pLDH dipstick assay, and PCR analysis. Heparinized blood for the pLDH dipstick assay was stored on wet ice until the assay was performed later that same day. Thick film microscopy. Thick films were coded and stained with Giemsa. Parasitemias were determined by counting the number of parasites in 100 oil-immersion fields (100 high-powered fields) 1,0003 magnification. Parasite- mias were then expressed as the number of parasites counted per/mm 3 . 15 Dipstick pLDH assay. The immunochromatographic test referred to as OptiMALt was provided by Flow, Inc. (Port- land, OR). This assay detects the presence of pLDH antigen in lysed whole blood. Aside from a control antibody reaction zone at the top of the test strip, the OptiMALt assay con- tains two test lines or reaction zones. The first encountered