Detecting Malaria Parasites outside the Blood

In 1880, Charles Laveran first visualized the protozoan parasite that causes malaria in a smear of blood under a microscope, and from the early decades of the 20th century to date, microscopy has been the mainstay of malaria detection. In laboratories throughout the world, routine diagnosis is carried out by microscopic examination of Giemsa-stained blood films for the intraerythrocytic asexual stages of the Plasmodium parasite that are indicative of active malaria infection. Clinical trials of antimalarial interventions also rely on good-quality slide reading for endpoint determination. In recent years, molecular methods for detecting the DNA of Plasmodium species that infect humans have been incorporated into some trial protocols to increase the sensitivity of detection (polymerase chain reaction [PCR] is typically 1–2 orders of magnitude more sensitive than microscopy), to distinguish genetically distinct parasite lineages, and to answer additional questions, such as the prevalence of mutations associated with drug resistance [1, 2]. A major constraint for such studies is the need to obtain blood samples repeatedly during posttreatment follow-up; these samples are often collected from infants, young children, and pregnant women, who bear the brunt of the malaria disease burden and are therefore the targets of most antimalarial interventions. Blood sampling, with its requirement for trained personnel, an attendant risk of infection and, in some countries, associated taboos can result in poor compliance when repeated testing is needed. In this issue of the Journal, Nwakanma et al. [3] have comprehensively evaluated the potential of saliva and urine samples as alternative sources of parasite DNA from Plasmodium falciparum, the species that causes the majority of malaria deaths worldwide and that predominates in subSaharan Africa. After obtaining blood, saliva, and urine samples from Gambian patients with suspected malaria infections, skilled microscopists carried out standard diagnosis and DNA was extracted from all samples for analysis by 2 methods: an established conventional nested PCR [1] was used to provide a benchmark for diagnostic sensitivity and specificity, and a quantitative PCR (qPCR) was used to estimate the amount of parasite DNA in these biological fluids. The study demonstrated that nested PCR, using DNA extracted from saliva samples, had encouragingly high sensitivity of 73% and specificity of 97%, compared with microscopic examination of peripheral blood samples obtained simultaneously. Moreover, a significant positive correlation was observed between parasite counts established by microscopy and estimates of parasite DNA in saliva by qPCR (r 0.58; P .001). When DNA extracted from urine was used as a template, the sensitivity of nested PCR compared with microscopy of blood was much lower (32%), and the correlation between counts established by microscopy and qPCR results was not significant. This suggests that either insufficient parasite DNA is excreted in the urine for these samples to provide a useful amplification template or that novel approaches to isolation, purification, and concentration are needed for DNA obtained from urine samples. In addition to diagnostic applications, the possibility that DNA from saliva samples could be used to distinguish variant parasite genotypes in samples from patients with malaria was raised by Mharakurwa et al. [4], who attempted to amplify a polymorphic region of the P. falciparum msp2 gene from blood, saliva, and urine samples obtained from individuals in Zambia who tested positive for malaria by microscopy. These authors also found that urine rarely yielded a PCR product, whereas the msp2 genotypes amplified from saliva samples regularly matched those found in blood samples obtained from the same person. The chance of amplifying parasite DNA from saliva samples improved as parasite density in the blood Received 7 January 2009; accepted 7 January 2009; electronically published 7 May 2009. Potential conflicts of interest: none reported. Financial support: European Union FP7 MALACTRES Project (R.H.); United Kingdom Health Protection Agency (C.J.S). Reprints or correspondence: Dr. Colin J. Sutherland, Dept. of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel St., London WC1E 7HT, United Kingdom (colin.sutherland@lshtm.ac.uk). The Journal of Infectious Diseases 2009; 199:1561–3 © 2009 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2009/19911-0001$15.00 DOI: 10.1086/598857 E D I T O R I A L C O M M E N T A R Y