Malaria antigen based tests were initially pioneered by the development of immunoflorescent antibody and enzyme linked immunosorbent assays (IFA and ELISA respectively) with commercial application remaining scarce and use largely relegated to research purposes (275- 277).
Immunochromatographic assays, commonly known as malaria rapid diagnostic tests (mRDTs) were developed based on bound antibodies detecting specific malaria antigens such as histidine rich protein 2 (HRP – 2), plasmodium lactate dehydrogenase (pLDH) or aldolase which would elicit a chromatographic response, in the form of a colored line, if the specific antigen is present in the blood sample (278). The WHO sets a required sensitivity for rapid diagnostic tests used for active case management of malaria of at least 95% at a parasite density of 100 parasites per microliter (279, 280).
The efficiency of RDTs is largely dependent on the intensity of transmission that dictates the mean parasite density associated with clinical symptoms as well as the prevalence of infections that has an impact on the sensitivity of the test (281, 282). Evidence from performance analysis in children show that RDTs demonstrate a drastic decline in sensitivity at parasite densities below 200 parasites/μL (283). Target antigen expression is crucial in determining the calibre of RDTs appropriate in particular settings. In areas in Latin America and India, the existence of HRP2-deleted
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strains of Plasmodium have been identified (284-286) and as such HRP2 based RDT would have dismal performance in these areas and would largely be inappropriate. Polymorphism of HRP2 and 3 surface antigens may impact negatively on HRP-based RDTs in areas with HRP-expressing parasites (287), though a previous study that conducted a pooled analysis of HRP2 antigens of
P.falciparum isolates from 38 countries did not demonstrate the sequence variation as a cause for
sensitivity variation. However, performance of HRP-2 based RDTs may be further compromised by high titres of HRP-2 antibodies (288).
Further complicating the reliability of malaria RDTs is the observation of cross reactivity of autoimmune factors and bacterial antigens leading to false positives and consequentially an overestimation of parasitaemia and treatment. Iqbal and colleagues demonstrated that P.falciparum HRP2 tests gave a false positive result in 26% of patients with rheumatoid factor but were aparasitaemic by microscopy and PCR (289). In tropical settings where both malaria and bacterial infections, individually or as co-infections, abound, false positivity due to cross reactivity possesses a considerable concern. Non-Typhiodal Salmonella bacteraemia has been associated with increased RDT positivity in the presence of a negative gold standard malaria test (290) and a recent case report documented a positive malaria rapid diagnostic tests with Salmonella typhi without evidence of co- malaria infection (291).
A plethora of studies have been conducted evaluating the performance of several rapid malaria diagnostic tests in field settings and controlled laboratory environments. The evidence is well summed in a systematic review and meta-analysis by Abba et al in 2011 and a later publication by Maltha et al in 2013. Abba and colleagues categorized RDTs based on antigen combinations incorporated in the test for diagnosis of malaria with classes 1 – 3 having HRP-2 alone or in combination with other antigens and types 4 and 5 being anchored by pLDH (168). Of 74 studies incorporated in the analysis, 73% were types 1 – 3. The pooled performance estimates of the HRP-2 based tests demonstrated high sensitivity and specificity of 95.0 % (95% C.I, 93.5% - 96.2%) and
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95.2% (95% C.I, 93.4 % - 99.4 %). The meta-analytical averages for type 4 and 5 tests had lower sensitivity but higher specificity, at 93.2% (95% C.I, 88.0 % – 96.2%) and 98.5% (95% C.I, 96.7 % - 99.4%) respectively. Conclusively, HRP-2 assays would miss less cases but pLDH tests would correctly diagnose more cases.
Figure 2.4 A positive rapid malaria diagnostic test
Adapted from “Malaria Diagnosis – Rapid Diagnostic Test”. Centers for Disease Control and Prevention, 2014. (292)
The majority of RDTs on the market are tailored toward the diagnosis of P.falciparum and
P.vivax, demonstrating poor detection of P.ovale and P.malariae (293, 294). RDTs targeting
conventional malaria antigens expressed by P.falciparum and P.vivax have been utilized for the detection of the newest identified species of Plasmodium causing malaria in humans, P.knowlesi. Some RDTs have been able to diagnose P.knowlesi infection (295) but with sensitivities markedly below the WHO recommended cut off of 95%. RDTs with greater diagnostic reliability, preferably
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based on an antibody specific for P.knowlesi, present a pressing niche in product development in light of the proliferation of P.knowlesi malaria disease in south-east Asia (296, 297).
In a review by Maltha et al (298), the reliability of RDTs was concluded to be mainly compromised by poor detection at low parasitaemia due to design limitations. Paradoxically, tests were also susceptible to the hook/prozone effect, precipitating false negative results in instances of extremely high parasitaemia. Deficiencies in the expression of target antigens such as HRP-2 or the cross reactivity with other antigens has rendered test performance vulnerable to either poor or exaggerated performance indicators respectively. Other factors such as end user errors and product packaging were also faulted in contributing to poor field performance of RDTs.
The WHO, in conjunction with the Centers for Disease Control and Prevention (CDC), TDR and FIND, conduct routine rounds of test performance for commercially available RDTs manufactured under ISO 13485:2003 quality system standard. The most recent round of RDT performance testing, Round 5, was conducted in 2013 (299). The panel detection score (PDS) is the main test score used to evaluate RDT performance. Of note is the great emphasis that has been placed on greater expectation of robust test performance at lower parasite concentrations, reflecting the growing need for more sensitive diagnostics at lower parasite densities (< 200 parasites/μL) that may still render clinically significant disease owing to reductions in parasite transmission in light of current eradication efforts in many endemic countries.
Results from the fifth WHO round of RDT performance evaluations showed that HRP2 based tests had the highest detection rates with pLDH only tests registering some of the poorest results. This was particularly evident at parasite densities of under 200 parasites/μL, with all evaluated tests having high detection at parasite densities greater than 2,000 parasites/μL. The proportion of tests that achieved a PDS greater than 75% at 200 parasites/μL were 78.6% and 42.4% for
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reliable tests for P.vivax (300), the implications of which could result in difficult administration of parasitaemia confirmed treatment guidelines in areas endemic to vivax malaria. Most of the evaluated tests demonstrated heat stability, an important factor in the confidence in test results as they are mostly used in tropical climates which register high temperatures, and are easy to use with the majority of tests being in cassette form.