Malaria diagnostic tests should be easy to perform and the interpretation of the results by examiners needs to be simple, however, in many RDTs sensitivities and specificities fluctuate in various field settings. The evaluations of pLDH-based RDTs for P. vivax indicate that quality and performance are not satisfactory due to low sensitivity. Poor manufacturing conditions or inadequate precautions in transportation and storage of RDTs result in low sensitivity and reproducibility [11, 12].
Even though patient parasitaemia levels are the most important determining factor for sensitivity of RDTs, several exceptional cases could not be explained by parasitaemia factors only. Traditionally, the performances of malaria RDTs are analysed by comparing the value with the parasitaemia levels from microscopic examinations. Microscopy-based evaluations reveal frequent limitations due to incorrect interpretation results dependent on examiners’ training and experience. Hence, the measured value of pLDH by ELISA could be more objective than the parasitaemia value, and the value of pLDH could provide more reliable analysis of selecting adequate malaria RDTs.
Without adequate baseline data of pLDH levels from clinically isolated P. vivax, it is difficult to correct the interpretation of results to make better decisions for selecting reliable malaria RDTs. Since none of the malaria RDTs detect the parasite itself, the secreted biomarkers became the diagnostic targets. To date, the level of target biomarkers using a standardized protocol for clinically isolated malaria samples are rarely reported .
Here, In this study, the distribution of pLDH in clinically isolated P. vivax samples, and the relationships between levels of pLDH and parasitaemia and between levels of pLDH and sensitivities of pLDH-based RDTs are assessed. In WHO/FIND malaria RDT testing round 4 report, Humasis Malaria P.F/Pan Antigen test (AMAL-7025) showed panel detection score (PDS) of 0% at both 200 and 2000 parasites/μL of P. vivax. In addition, false positive rate of Humasis kit was 97.8%. While the performance parameter in this study was not PDS, but clinical sensitivity, Humasis kit in this study demonstrated the clinical sensitivity of 55.5 ~ 100.0% at similar parasitaemia (Table 1). Next, specificity of Humasis kit for P. vivax was 100% with no observed false positivity. As for OptiMAL-IT kit (710024), round 4 report presented PDS of 97.1 and 68.6% at 200 and 2,000 ~ 5,000 parasites/μL of P. vivax, respectively. On the other hand, OptiMAL-IT showed increased clinical sensitivity from 52.7 to 100%, as the P. vivax parasitaemia increased. As for the SD Bioline Malaria Ag P.f/Pan kit (05 F60), round 4 report showed PDS of 97.1 and 100.0% at 200 and 2,000 ~ 5,000 parasites/μL of P. vivax, respectively. However, SD Bioline demonstrated slightly decreased clinical sensitivity of 87.5 ~ 100.0% (Table 1). In this study, lower sensitivities in all three RDTs were observed from groups with lower pLDH antigen levels (Table 1). From the study for determining the limit of detection, lower levels pLDH associated with lower sensitivities in all three RDTs (Table 2). These direct correlations were similar to the levels of parasitaemia in all three RDTs. Above results suggested the possibility of pLDH, as a performance parameter for malaria RDTs. This information on currently available RDTs is valuable in revealing detection limits against pLDH. Therefore, the current results could provide one more reasonable protocol for comparing various malaria RDTs and their practical guidelines in developing sensitive malaria RDTs.
pLDH antigen ELISA system showed higher sensitivity (95.4%) in comparison with three RDTs (85.5-86.8%), which might be associated with the fact that ELISA uses 50 μL of blood, while RDTs used only 5–10 μL of blood. RDTs are fast and convenient diagnostic tools. Parasitaemia levels of P. vivax in ROK ranged 2–25,000/μL with mean level of 3,954.1 ± 3712.5/μL, while pLDH levels of P. vivax ranged between 0–22,387 ng/mL with mean value of 3,971.5 ± 6120.9 ng/mL. pLDH levels showed moderate correlation with parasite density of P. vivax, as determined by microscopy (r = 0.4, p < 0.05).
From three tested RDTs, pLDH levels and parasitaemia levels of three RDTs, positive cases were higher than two RDT positive cases or all RDT negative cases. One positive sample from two RDT tests (n = 3) showed disparity between parasitaemia and pLDH levels, where the pLDH and parasitaemia levels were 15 ng/mL and 2,300/μL, respectively. These levels of parasitaemia most likely produce clinical symptoms. RDTs are fast and convenient diagnostic tools. Using only RDTs, malaria diagnosis might be difficult and patients might be left untreated, leading to severe complications. Since the evaluation of RDTs is currently based on patients’ parasitaemia levels, with little information on targeted biomarkers in clinical isolates, the current study provides additional information for selecting adequate malaria RDT in fields.