Definitive diagnosis and confirmation of disease status is the cornerstone of evidence-based medicine. Many malaria-endemic areas of the world lack sufficient capacity and resources to accurately diagnose the infection and where reliance on the presentation of clinical signs and symptoms alone are inadequate and imprecise indicators of specific disease. The WHO recommendations for procurement of malaria RDTs are currently based on the attainment of a set of minimum performance criteria (e.g., detection rate/panel detection score, specificity, invalid rate, etc.) in the WHO Malaria RDT Product Testing Program [16] and recommendations established by the WHO Malaria Policy Advisory Committee in 2012 [20]. Products that fail to meet the full set of minimum performance criteria are not eligible for procurement by WHO. To some degree, many other organizations and government procurement authorities also follow the WHO guidelines for product selection. Based on published findings [10,16], the XW-P07 RDT has not been tested by the standardized, laboratory-based WHO program; therefore, an evaluation regarding its performance for detection of malaria compared to microscopy and qPCR in a point-of-care operational setting was deemed prudent and essential.
The RDT cassettes were easy to use and provided distinct, easy to interpret test lines. In only 3 tests did the RDT fail to show a control line, producing an ‘invalid rate’ of 0.72%, well within the acceptable limit (<5%) established by WHO [16]. The RDT showed sensitivity and specificity values of >90%, PLR >10, NLR <0.1, and Kappa >0.8 for detecting P. falciparum infections when compared with microscopy and qPCR. On the other hand, for P. vivax, the RDT showed the same specificity >90% and PLR >10; while the overall sensitivity was much lower when compared with microscopy and qPCR (73% and 66% respectively), with NLR >0.1 and Kappa score slightly <0.8. The test specificity for both parasite species easily met the WHO recommended minimum performance criteria of >90% (i.e., less than 10% false positive rate) detection at 200 parasite/μl [20].
An unpublished product evaluation by the XW-P07 manufacturer reported findings from 251 samples compared to microscopy (SPR 16.3%) showing Sn and Sp of 100% for both P. falciparum and P. vivax [8]. A study performed in Lampung Province, Sumatra, with 400 samples (SPR 36%) showed Sn 91% (85-97%), Sp 99% (98-100%), PPV 98% (95-100%), NPV 97% (95-99%) for P. falciparum; and Sn 84% (75-92%), Sp 100%, PPV 100%, NPV 96% (94-98%) for P. vivax based on comparisons with matched microscopy [9]. The study in Mimika demonstrated different predictive values for both parasites compared to previous investigations which may have been influenced by the different disease prevalence in each study [21]. Any direct comparison between studies on RDT performance may be compromised by other factors related to location, sample population, background malaria exposure, and degree of acquired partial immunity in the target populations. This study showed that RDT sensitivity is clearly influenced by parasite density, not an unexpected finding based on testing of other products [10,16]. RDTs have been shown to produce lower sensitivity in areas with more frequent low parasite densities [22,23]. In this study, body temperature was significantly associated with parasite density as measured in peripheral blood – normal temperatures at time of exam produce lower infection densities while corroborating other observations that a rise in body temperature is correlated with an increase in parasite density [24,25].
This product evaluation was not without some underlying limitations. Firstly, this study only used RDT cassettes from a single lot number; thus, possible inter-lot variability in performance between product production periods was not assessed. This study was conducted in a reasonably controlled setting with trained laboratory technicians; therefore, extrapolation of findings to areas under more demanding environmental conditions and clinical expertise (e.g., remote primary health care clinics) should be made with caution. Albeit relatively uncommon infections, P. ovale and P. malariae were not specifically included in the panel assay (only as a pan-specific pLDH for all Plasmodium spp.); however, the majority of these infections in Papua are often coincident (mixed) with other plasmodial species. In this study, three P. malariae infections were either mixed with P. falciparum (two cases) or P. vivax (one case). Lastly, a set minimum of 100 high magnification fields were used for blood examination of films which possibly contributed to the relatively high (15.4%) discordant results between the first 2 microscopists. The detection accuracy would have likely been enhanced had each reader examined a minimum of 200 fields.
Various host and parasite factors are possible reasons for varying RDT performance values between different malaria endemic populations [10,26-31]. Greater sensitivity is a desired attribute and maybe more important compared to test specificity to ensure malaria infections are correctly diagnosed and promptly treated to avoid development of disease complications and more severe infections when left untreated. Undetected cases due to false negative results also contribute a continuing source of gametocyte carriers (reservoirs) for sustaining malaria transmission in an area [31]. Conversely, higher test sensitivity may result in lower test specificity (higher false positive results), thereby increasing unnecessary malaria treatments [32]. Typically, P. falciparum infections have been regarded as the only human plasmodial species responsible for common causes of severe morbidity and mortality; however, that general perception has changed and been challenged by a number of recent studies showing that P. vivax can produce substantially greater morbidity manifesting as severe infections, causing acute and chronic anaemia, and ultimately resulting in death [33-36]. It is because of these heightened concerns regards the higher likelihood of more severe outcomes caused by P. vivax infections, that accurate diagnosis of this species becomes an even greater priority to ensure early and effective treatment. An RDT that lacks the necessary sensitivity for detecting P. vivax (>90% preferred) and poor exclusion power (a negative diagnostic likelihood ratio of >0.1) to adequately rule out infection presents a distinct disadvantage to both patient and health care provider in areas where the parasite is common.
This study showed that XW-P07 has a significantly lower detection rate for P. vivax than for P. falciparum, even when excluding low density infections below 100 parasites per μl/peripheral blood. Published WHO product evaluation on different malaria RDT products submitted for testing has shown that targeting P. falciparum HRP2 has the highest and most consistent detection rate [16]. However, this conflicts with other findings in which HRP2-based RDTs have shown a lower performance value than products using pLDH for detecting P. falciparum, as the pLDH capture system is not affected by a possible ‘prozone’ effect, parasite antigen polymorphisms or gene deletions [37-41]. In Myanmar, a study comparing a commonly used RDT utilizing HRP2 and pan pLDH compared with microscopy demonstrated P. vivax and P. malariae were detected to a far lesser extent (lower sensitivity, NPV, and NLR) than P. falciparum [42]. For P. vivax detection, separate aldolase and pLDH targeting RDTs have been shown to perform differently depending on the samples tested, increasing the risk of misdiagnosis and therefore suggesting that test sensitivity for P. vivax can be improved by using a combination of both aldolase and pLDH in a single RDT [43]. The RDT evaluated in this study only utilizes pLDH for detection of P. vivax, thus one possible explanation for the inferior sensitivity seen. The XW-P07 showed considerably lower sensitivity and a poor negative likelihood ratio, both measures falling below general acceptance criteria for detecting P. vivax infections. This is especially problematic for infections presenting with lower parasitaemia; thus, RDT results with such limitations must be interpreted with caution if the test is the sole method of diagnosis, particularly given the importance and high prevalence of vivax malaria in the Mimika area.
Furthermore, as a four-band RDT with one control line and three test lines (Figure 1), the XW-P07 relies on more antigen/antibody reactions using a single buffer compared to other RDTs with only three indicator bands. In areas where the prevalence of P. ovale and P. malariae is relatively low, a three-band RDT with better overall performance and able to differentiate P. falciparum and P.vivax, or possibly a P. falciparum/Pan-malaria test, may be a better format. Whenever possible, it would also be prudent and strongly advised to back-up all RDT diagnosis, regardless of RDT performance rating, with matched blood films and proficient microscopic examination.
A recent study in Flores, Indonesia, found that qPCR revealed almost eight times more Plasmodium infections when compared with microscopy, taking into account the high number of sub-microscopic infections in a relatively low transmission area [44]. Molecular methods are universally accepted as more sensitive than microscopy alone. However, PCR (e.g., multiplex, real-time or conventional) requires a sophisticated laboratory setting, trained technicians, entails a longer diagnosis time and higher costs to support the system; thereby precluding its routine use in most malaria endemic areas of the world - Indonesia and Mimika included. New or improved diagnostic methods are in development [45-48] that may vastly improve diagnostic capabilities and accuracy in challenging locations and basic health care settings. However, until superior, easy-to-use alternatives are available, both the RDT and microscopy, alone or in combination, will remain the mainstays for routine point-of-care malaria diagnosis.
In areas with high prevalence of P. vivax infection, from a cost-effectiveness point of view, standard expert microscopy should continue to be used as the reference gold standard for malaria diagnosis despite the likelihood of missing some low density parasitemia and sub-microscopic infections. With skilled technicians and experienced health care providers, microscopy has more than sufficient, if not excellent, diagnostic capacity in most instances. All public-funded health facilities and private clinics in the Mimika Regency must either begin, or ensure the continuation of microscopy, as their primary means of malaria diagnosis. Microscopy should be used for routine confirmation of all RDTs performed in clinical settings whenever possible. Lastly, in many circumstances without external funding to support procurement and routine access to RDTs, the sustainability for maintaining these devices in all clinics is vulnerable to supply disruptions without adequate safeguards and reliable logistical support in place. The use of microscopy, even absent the aid of electricity in the most remote areas, is a sustainable approach and within the supportive framework of the Indonesian health care system. Moreover, providing basic electrical power using efficient solar capture devices and battery storage for a microscope and basic clinical equipment is well within the means of most local health budgets. The availability and routine use of microscopy also enables a facility to diagnose other important endemic diseases (e.g., tuberculosis, lymphatic filariasis, intestinal helminths and protozoa) and hematological conditions and indicators without the need of more sophisticated and costly techniques and medical instrumentation.
As malaria represents one of the leading and arguably most important health concern in the Mimika area, various health program stakeholders should continue or adopt a policy of investment in the procurement and maintenance of quality microscopes, the recruitment of additional trained laboratory technicians, and organize regular refresher training on microscopy and RDT proficiency. Nevertheless, for logistical and operational rationale, the use of RDTs will continue to play a valuable and important role in remote areas for first line diagnosis of malaria. Health care facilities in remote locations with limited laboratory capacity should continue to use high quality malaria RDTs combined with evidence from good clinical observations until microscopy can be introduced.
