Malaria is a major infectious disease that is a significant global health concern because of its high morbidity and mortality worldwide [1]. The Securing Ugandans' Right to Essential Medicines (SURE) program of the Uganda Ministry of Health reported in its The Value Chain Newsletter that approximately 31% of adults and 36% of children aged less than 5 years in Uganda had been diagnosed with malaria in 2010 [2]. Although many medical service organizations and health centers are working to prevent and control malaria, they do not have sufficient medical or human resources to accomplish these goals [2]. Therefore, malaria-related mortality in Uganda remains high and without significant reduction in the last decade [1,2]. Because the clinical symptoms of malaria are very similar to those of other febrile illnesses, diagnosis of malaria based on clinical manifestations alone is unreliable [3]. The microscopic examination of thick blood films is the gold-standard method for malaria diagnosis [4,5], but requires expensive and fragile microscopes as well as well-trained microscopists. Furthermore, the diagnostic reliability of microscopy can be questionable, especially in samples with low parasitemia or with mixed species infections [6,7,8]. To overcome these diagnostic problems, detection methods such as rapid diagnostic tests (RDTs) and nucleic acid tests (NATs) have been developed for malaria diagnosis [9,10,11,12]. Because RDTs are easy to use, fast to assay, and require no special instruments, these tests have been widely used in recent years to detect malaria infections. Several antigens, such as Plasmodium histidine-rich protein 2 (HRP2) [13], lactate dehydrogenase (pLDH) [14], aldolase [15], and glutamate dehydrogenase [16], have been identified and used in RDTs to detect malaria infections. Among them, HRP2 and pLDH are targets used in most currently deployed RDTs [17,18,19,20].

The Asan EasyTest™ Malaria Pf/Pan Ag is an RDT that detects parasite HRP2 and pLDHs and has been approved by the Korean Food and Drug Administration (KFDA). This RDT is widely used in many clinic centers and hospitals in Korea and India, but further evaluation of its diagnostic accuracy is required, especially in endemic countries on other continents. In this study, we assessed the diagnostic accuracy of the Asan EasyTest™ Malaria Pf/Pan Ag by comparing the sensitivity, specificity, and positive and negative predictive values of this RDT with those of microscopic examinations in symptomatic malaria patients in Uganda.

Twice field tests were carried out in 6 villages of Kiyuni in Uganda at April and at September of 2011. Among 185 individuals with suspected malaria whose age ranged from 1 to 53 years, 110 individuals (59.5%) were positive for malaria by microscopic examinations and PCR analysis (Table 1). The mean±SD age of the malaria patients was 10.4±11.0 years; 27.2% were ≤3 years, 37.3% were 4-10 years, 18.2% were 11-20 years, 13.6% were 21-40 years, and 3.6% were ≥40 years. The ratio of male:female patients was 0.55:1. The parasitemia of the 110 positive samples ranged from 39 to 40,888 parasites/µl. Microscopic examinations of blood smears from 110 malaria-positive patients revealed that P. falciparum infection was most prevalent: P. falciparum infection (n=71; 64.6%), non-falciparum malaria infection (n=36; 32.7%), and mixed infection of P. falciparum and non-falciparum malaria (n=3; 2.7%) (Table 1).

To evaluate the diagnostic performance of EasyTest™ Pf/Pan Ag, the 185 blood samples were also examined using this RDT. Compared to the results of microscopic examinations, 4 of 71 negative samples were positive for malaria, 2 for P. falciparum and 2 for non-falciparum malaria (Table 1), although the positive response in each sample was very weak. However, 8 of 110 positive samples in microscopic examinations were negative in the Asan EasyTest™ Pf/Pan Ag (Table 1). To confirm the results obtained from microscopic examinations, we also performed genus-specific PCR analysis, which resulted in the same pattern of positivity with microscopic examinations (Table 1). All non-P. falciparum malaria were confirmed to be P. vivax in the PCR analysis.

Based on these results, we analyzed the diagnostic performance of the Asan EasyTest™ Pf/Pan Ag using 95% confidence intervals (CIs). The sensitivity of the RDT was 95.8% for P. falciparum and 83.3% for non-falciparum malaria. The specificity was 97.3% for P. falciparum and 94.4% for non-falciparum malaria; the overall PPV and NPV were 96.3% and 94.9%, respectively. As expected, the sensitivity of the EasyTest™ Pf/Pan Ag test was low for blood samples with low parasitemia. For P. falciparum, the RDT had 100% sensitivity for samples with parasitemia >500 parasites/µl, but only 66.6% when there were <100 parasites/µl in the sample. Similarly, the RDT showed 100% sensitivity for non-falciparum malaria in samples with >500 parasitemia, but it decreased to 50% in samples with <100 parasitemia (Table 2). To determine the detection limit of the RDT, we also examined the analytical sensitivities of the RDT against experimentally diluted blood samples with different parasitemia of P. falciparum or P. vivax. The minimum detection level of the RDT was determined to be 79 parasites/µl for P. falciparum and 125 parasites/µl for P. vivax. The RDT showed similar analytical sensitivity against blood samples with mixed species of P. falciparum and P. vivax (data not shown).

The Asan EasyTest™ Pf/Pan Ag is an RDT approved by KFDA for detection of malaria. In recent years, its use has been increasing in many public and private clinic centers in Korea and several other countries, but its diagnostic performance in field settings had not yet been established. In this study, we evaluated the diagnostic performance of the Asan EasyTest™ Pf/Pan Ag in 6 villages of Kiyuni Parish in Uganda.

According to the WHO recommendations for RDT performance, only 2 RDTs have been approved; these RDTs have a reported sensitivity greater than 95% for samples with parasitemias ≥100 parasites/µl: 96.8% for the CareStart™ Malaria test and 95.2% for the OptiMAL-IT™ test [24]. However, the present study shows that the sensitivity of the Asan EasyTest™ Malaria Pf/Pan Ag is 96.8% (95.8% for P. falciparum and 83.3% for non-P. falciparum) for samples having the same parasite density, similar to the sensitivity reported for the CareStart™ Malaria test. Although we found 4 false-positive results with the RDT, we could not fully exclude the possibility that false-positive results were actually true-positives, which could be missed by microscopic examinations because of sequestration that limited the number of circulating parasites at the time of blood collection [25] or that the blood samples were from individuals with circulating rheumatoid factors [19,26,27]. As shown in Table 1, 3 cases (1.6%) were identified as mixed infections. The rate of malaria co-infection was similar to that reported in previous surveys [28,29]. However, the RDT was unable to detect mixed infections in the presence of P. falciparum because of cross-reactivity with the pan-specific pLDH band.

In conclusion, we evaluated the clinical performance of the Asan EasyTest™ Malaria Pf/Pan Ag using blood samples collected in Uganda. The accuracy of the RDT was similar to that of RDTs recommended by WHO. Therefore, the Asan EasyTest™ Malaria Pf/Pan Ag may be a reliable diagnostic tool to detect P. falciparum and non-P. falciparum malaria infections in Uganda, and can contribute to malaria control efforts in the country. However, we used a limited number of blood samples and a small number of sites in this study; more extensive tests with more samples from various global populations are needed to fully evaluate the diagnostic performance of the Asan EasyTest™ Malaria Pf/Pan Ag RDT.