Among the RDTs tested in this study, the OptiMAL-IT test was the most sensitive for P. knowlesi-infected blood samples. However, the test was not specific and the majority of P. knowlesi samples were identified as P. falciparum by this test due to antibody cross-reactivity, as noted in previous studies [19–22]. BinaxNOW® Malaria was found to be the least sensitive of the three RDTs assessed, but there was no cross-reactivity observed between the P. falciparum antibody for P. falciparum-HRP-2 and P. knowlesi samples. In this study, all positive results attained using this test with P. knowlesi-confirmed samples correctly indicated a non-P. falciparum malaria infection. The Paramax-3 test showed low sensitivity and cross-reactivity between the P. vivax LDH-detecting antibody and P. knowlesi. This observation has been noted in several other single case reports using different tests which also contain a P. vivax LDH-detecting antibody [19, 23, 24].
The sensitivity of detecting P. knowlesi in blood samples with all three RDTs assessed in this study was significantly lower than that reported for other Plasmodium species. For example, the sensitivity of detection of P. falciparum using OptiMAL-IT has been reported as 95.3% (100% for >500 parasites/μl and 72% for 50 parasites/μl) and 96% for P. vivax malaria infections . For BinaxNOW® Malaria, the sensitivity of detection for P. falciparum has been reported as 95.3% (99.7% for >5,000 parasites/μl and 53.9% for 100 parasites/μl or fewer) and 68.9% for P. vivax malaria infections . For Paramax-3, the sensitivity and specificity of detection for both P. falciparum and P. vivax malaria infections is reported as 100% in an in-house study of 251 samples . Although the number of positive controls conducted in this study was relatively low, none of the RDTs used in this study detected P. vivax or P. falciparum infections in fresh blood samples with parasitaemias less than 2,000 parasites/μl.
One limitation of this study is the relatively low numbers of fresh samples tested. To strengthen the results from fresh samples, frozen blood samples were also included. The sensitivity of detection of knowlesi malaria infections with the RDTs tested were fairly similar using fresh versus frozen samples. Freeze-thawing and the storage of blood at low temperatures can accelerate deterioration of antigen activity, although it is also possible that target antigens are more accessible in freeze-thawed samples and may actually improve the sensitivity of RDTs .
A recently published paper from Sabah, Malaysian Borneo presents the use of two different RDTs, First Response™, which detects pan-Plasmodium LDH and Pf-specific HRP-2, and ParaHIT™, which detects pan-Plasmodium aldolase and Pf-specific HRP-2 . A total of 129 P. knowlesi patient samples were studied, only 34 of whom were enrolled in the study prior to treatment, while the remainder were referred from district hospitals where they had already received anti-malarial treatment . The findings of this study indicated a sensitivity of 74% for the pLDH component of the First Response™ RDT, which is similar to that observed in the current study with OptiMAL-IT (71 and 73% sensitivity with fresh and frozen samples, respectively), and higher than that observed with the Paramax-3 test (40 and 32% sensitivity), both of which also detect pLDH. In the current study as well as the one in Sabah, the RDTs with the pan-aldolase component had the lowest sensitivity of detection of P. knowlesi samples; 29% with the ParaHIT™ test , and 29% with fresh and 24% with frozen samples using the BinaxNOW® Malaria RDT.
RDTs cost between 10 and 15 Malaysian Ringgit (US$3.17-4.80) per test when purchased at a dispensary in Malaysia. Although when purchased in bulk for malaria control programs this cost tends to be significantly reduced, microscopy is still the most affordable diagnostic tool and costs just one Ringgit (US$0.30) per patient to screen for malaria. The cost of nested PCR assay is comparable to RDTs per patient sample, and although PCR assay is significantly more sensitive and specific than microscopy, this technique requires specialized equipment, electricity supply and training, and is not suitable for resource-poor settings. RDTs confer the advantages of speed (all types used in this study took 20 minutes or less to conduct), minimal training and ease of use, and do not require electricity or any specific hardware. However, currently available RDTs lack sensitivity and specificity compared to microscopy and PCR-based methods for all Plasmodium species infections, especially P. knowlesi. The development of loop-mediated isothermal amplification (LAMP) assays combine the sensitivity and specificity of PCR with low cost, low technology and rapid results. LAMP-based tests for malaria diagnosis that include reagents specific for P. knowlesi are under development and may be useful for resorce-poor settings [31, 32].
In areas with relatively low malaria prevalence such as Sarawak, the cost of RDTs, even if sensitive and specific, would likely outweigh the benefit. To understand this in practical terms, consider, for example, the case of Julau Health Clinic, which is a small, rural health clinic in Sarawak surveyed as part of the current study. The prevalence of malaria at this clinic during a five-month study period in which 108 febrile patients whose clinical presentations were suggestive of malaria were screened using nested PCR assay was 0.2% (Foster et al., unpublished data). As such, it would have cost between MYR 1,080 and 1,620 (US$342-832) to perform RDTs for these 108 query malaria patients and only two were positive.
This study confirms that the RDTs evaluated are not adequately sensitive for use in the diagnosis of P. knowlesi. Also, P. knowlesi cross-reacted with P. falciparum and P. vivax LDH antibodies used in two of the three commercially available RDTs tested, resulting in misdiagnosis of malaria species in an area where human P. knowlesi infections are prevalent. Since not all species of malaria warrant the same level of medical care, misidentification can result in mismanagement, especially when the potentially severe knowlesi malaria is misdiagnosed as vivax malaria. Because P. knowlesi is morphologically similar to P. malariae and P. falciparum, it is also misdiagnosed by microscopy [10, 23, 24]. However microscopy should not be replaced by RDTs in areas where P. knowlesi occurs until the sensitivity, specificity and costs are comparable.