Malaria, a mosquito-borne disease caused by parasites of the Plasmodium genus, exacts a global toll of at least 216 million clinical cases and 655,000 deaths annually of which ~85% are children under the age of five . Currently, diagnosis of malaria is based on four different approaches: microscopy, antigen detection using immuno-chromatographic rapid diagnostic tests (RDTs), malaria antibody detection, and nucleic acid-based assays. Microscopy remains the “gold standard” method for laboratory confirmation of malaria, and involves examination of thick and thin blood films stained with Romanowsky stain (mainly Giemsa or Field stain). However, microscopic diagnosis of malaria is dependent on the personnel performing and interpreting the results, and requires specialized equipment that is difficult to support in remote areas lacking a reference laboratory. Moreover, low parasitaemia, especially in asymptomatic subjects, is not detected by microscopy.
To alleviate some of the difficulties of microscopy-based diagnosis of malaria, RDTs that detect parasite-specific antigens were developed . The most commonly targeted malaria antigens are Plasmodium falciparum histidine-rich protein-2 (pfHRP2) and Plasmodium lactate dehydrogenase (pLDH) [3–6]. RDTs offer ease of operation, a timely diagnosis, and do not require trained personnel or special equipment [2, 7]. However, they are relatively expensive and prone to false-positive responses due to persistence of pfHRP2 antigen in blood for up to two weeks after the parasite is cleared [2, 8]. Also, the relatively low RDT sensitivity is a constraint for endemic regions attempting malaria pre-elimination, where detection and treatment of low-grade reservoir infections is required for effective elimination of infection .
In recent years, a molecular approach has been used to detect Plasmodium nucleic acids circulating in blood, saliva, and other body fluids [10–13]. Polymerase chain reaction (PCR) is more accurate and sensitive than microscopy and RDTs, detects low-grade parasitaemia and is indicative of active infection [14, 15]. Detection and amplification of Plasmodium DNA is generally performed using nested PCR, a two-step procedure in which the product of the initial reaction is amplified a second time with a new pair of “inner” primers that hybridize to the dihydrofolate reductase (DHFR) gene located within the previously amplified region [10, 16]. Nested PCR typically requires transfer of a small amount of product from the first step to serve as template for the second amplification in a second tube. The requirement to transfer PCR-amplified products dramatically increases the risk of carry-over and environmental contamination. Moreover, the two rounds of amplification may require up to six hours to complete. Investigators have attempted to develop single-tube nested or closed-tube nested PCRs to eliminate the transfer procedure and thus minimize contamination, reducing false-positive results, and maintaining high sensitivity . However, to date, despite the high sensitivity and low risk of carry-over contamination associated with a single-tube nested PCR, this technique is susceptible to inhibition by inappropriate sample preparation . Real-time PCR also minimizes contamination, but nesting is needed to optimize the limit of detection (LOD) [18, 19]. Also, the common use of SYBR Green DNA-intercalating dye in many real-time PCR kits, which binds any double strand DNA, makes the assay less specific and prone to false-positive results. Furthermore, the use of specific fluorescent probes for certain types of qPCR, although specific, are expensive which greatly diminishes their routine use .
A generic platform [21–23] was developed to analyse HIV and other pathogen-related antigens, antibody, and nucleic acids in saliva or blood simultaneously by a combination of RT-PCR and an immunoassay with detection by up-converting phosphors (UCP) label and lateral flow technology [21, 24–26]. The UCP reporter converts photons of lower energy infrared light into higher energy visible light and is ultrasensitive since this unique process does not demonstrate autofluoresence . Using a similar approach an uninterrupted, asymmetric, semi-nested PCR providing quantitative detection of a P. falciparum DNA target with a significant reduction in overall assay time and improved robustness with respect to reducing the probability of contamination was developed. The system is ideal for further development to a point-of-care (POC) device.