In this study we assessed the field performance of the PT and BN RDT in a rural Ghanaian hospital laboratory using conventional Giemsa stain thick and thin blood films of pediatric specimens as reference standard. Only few studies in other endemic areas have been conducted specifically in children so far [12–16]. In one study an increased sensitivity of a HRP-2 assay in children compared to adults was demonstrated. This was attributed to lower immunity and possibly less interference by antibodies . Despite this, there is concern that the benefits of parasitological confirmation in children under 5 years may be outweighed by the risks of not treating children with false negative tests .
Studies conducted earlier on BN RDT showed a low sensitivity (61.5%) for the detection of pure P. malariae and P. ovale infections . We found false negative results of BN RDT for only two non-P. falciparum infections with low parasite counts (Pm: 24 parasites/μl and Po: 16 parasites/μl) and thus this might be inadequate to confirm or refute this point. The BN RDT however, has been shown to have a very good sensitivity (100%) for the detection of pure P. falciparum infection . P. falciparum accounts for almost 99% of malaria infections in the study area (unpublished data, Biostatistics Dept, Agogo Presbyterian Hospital) and, therefore, the low sensitivity of the BN RDT for non-P. falciparum malaria may not be a serious cause for concern in this area and most parts of sub-Saharan Africa where malaria infection is predominantly caused by P. falciparum . A slight cause for concern might be the number of steps (about 6 steps) involved in performing the test as compared to the four-step test of other RDT formats . However, with adequate training this concern can be addressed. The BN RDT detects the presence of plasmodial antigens HRP-2 and pan-Plasmodium aldolase . The detection of 10 false-positive results could be attributed to one main observation made in earlier studies. It is well documented that the HRP-2 antigen can persist up to 28 days after treatment [22, 23]. Even though only untreated patients were enrolled in this study, reliability of information obtained from mothers/guardians on this might be questionable. This assumption could have contributed to the increased numbers of false positive tests. In addition this assay appears to be an unsuitable tool for monitoring treatment of malaria as previously discussed by Murray et al . The BN RDT has also been shown to detect parasite levels > 20 parasites/μl for P. falciparum and ≤ 100 parasites/μl for P. vivax . False negative results could be attributed to the fact that parasite antigen levels were too low to be detected as a result of very low parasitaemia or by semi-immune parasite carriers with low parasitaemia.
Overall the test is simple to perform, rapid (< 15 minutes), easy to interpret, requires less training, needs no laboratory setup and thus it is applicable for field conditions [8, 9, 21]. The test has also been shown to be heat stable and performs very well at temperatures up to 45°C . On the other hand, it has a poor detection rate for non-P. falciparum infections and has a higher false positive rate due to the persistence of the antigen target HRP 2 [8, 9, 26].
PT uses a fluorescent dye 4'-6-Diamidino-2-phenylindole (DAPI) which detects intracellular double stranded DNA which is present within Plasmodium-infected erythrocytes. The bright shiny dots within infected erythrocytes under UV light is extremely characteristic for malaria and has a very high PPV in areas mainly endemic with P. falciparum . One major limitation of PT is the disability of specific identification of Plasmodia and the differentiation of the species. Compared to GM, PT exhibited four false-positive results which have not been further investigated. PT could also be more sensitive than GM in detecting low number of parasites . The presence of artifacts such as non-specific aggregated DAPI, immature erythrocytes or bacterial cells might have been misinterpreted as plasmodial DNA. The performance characteristics of the tests were very similar as indicated in their sensitivities, specificities, PPV and NPV with very good agreements to the GM reference standard (Table 3). Our study findings correlate well with the results of a study conducted in Sudan for adults using the Partec CyScope® when compared only with conventional Giemsa stained microscopy  as well as with our first report comparing this malaria test in another patient's collective . In this first study we could attest to the fact that the PT has a high sensitivity and specificity by referring to the highly sensitive gold standard RT-PCR which, however, cannot be applied in field studies in contrast to the PT assay . Both studies [4, 27] confirmed that PT requires very little training and has a short turnaround time of averagely 5 minutes per test. In addition our findings regarding PT performance characteristics and applicability under field conditions were confirmed by a study conducted in Uganda . This study also underlines the disability of specific identification and differentiation of Plasmodia species as the disadvantage of PT. However, the characteristic fluorescence of infected erythrocytes has a very high PPV in areas mainly endemic with P. falciparum.
Thus we value PT analog to GM thick film as a useful screening method where positive and doubtful results need to be completed by GM thin film examination for confirmation and accurate identification of species later. This strategy takes also into account that Giemsa staining and microscopy is a very elaborate and challenging procedure when performed accurately in a high quality level.
Parasitaemia is used to guide treatment, thus underestimation could have significant ramifications for malaria patients. Parasite counts obtained from PT were significantly lower than those obtained from GM. It is unlikely that parasites are hidden during Giemsa stain microscopy, since adequate preparation of the slide ensures visibility through all planes of focus. The lysis of the red blood cells during the staining process reveals the parasites and ease the detection . The Partec Rapid Malaria Test® employs fluorescence in which red blood cells are not completely lysed, therefore, they may lie on each other or overlap with each other thereby preventing parasites in red blood cells that may be lying beneath other cells from being identified and counted.
The World Health Organization Sexually Transmitted Diseases Diagnostics Initiative (SDI) has developed the ASSURED criteria as a benchmark to decide if tests address disease control needs: Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users . Even though this criteria was developed as a benchmark for the evaluation of RDTs for sexually transmitted infections such as syphilis, it can also be used as a benchmark for the evaluation of RDTs for other diseases such as malaria (Table 4). In rural endemic areas where majority of the people are poor, these tests are affordable enough to be accessed in the health centers and homes.
The accepted level of sensitivity for a rapid diagnostic test in diagnosing malaria is a sensitivity of 95% . Compared to the reference standard, both methods were sensitive and specific enough to be used as diagnostic tools for the diagnosis of malaria in endemic areas.
Both methods require little training and the BN RDT can withstand temperatures up to 45°C , making it quite useful for tropical conditions and does not require any equipment. The PT is battery operated, portable and can thus be carried for field work and to places where there may be no regular electricity.
There is the need therefore to expand malaria diagnostic services as part of a greater framework of health system strengthening within resource-limited settings. Increasingly, countries and implementing partners have identified that limited diagnostic capacity represents a major barrier to implementation and sustainability of prevention, treatment and care programs for malaria . The PT and BN RDT, therefore, present as very good tools in the prevention, treatment and care programs for malaria.