The important finding of this study was that the CS kit had sensitivity of 89.68% and specificity of 98.26% compared to the gold standard microscopy method for detection of malaria. The CS sensitivity for the detection of P. falciparum was 98.3% and 99.3% in samples with parasite densities above 100/μl and 1,000/μl respectively whereas the sensitivity for P. vivax was 97.6% for parasite densities above 500/μl. No positive result occurred among the Plasmodium-negative samples, which is consistent with the recent study of CS kit in Ethiopia . RDTs detecting P. vivax-specific pLDH CS kit based on specific monoclonal antibody, detected histidine-rich protein 2(HRP-2, test line 1) and four kinds of lactic acid dehydrogenase of Plasmodium in human blood (pLDH, test line 2) in a card detection device, aseptic and single packing. The comparison of the sensitivity and specificity of the CS-RDTs versus microscopy corrected by PCR assay (Table 3) indicated that the detection rate was relatively similar for P. vivax and P. falciparum, indicating that the microscopic training performed had a high efficiency on the performance microscopists. This kit showed no need of sophisticated equipment and facilities, and was easy to operate. Its applicability and rapidity in diagnosis could be of additional value for both falciparum and vivax malaria detection and prompt case management as well as cross borders malaria monitoring in the China-Myanmar endemic areas in the National Malaria Elimination programme.
In total, 241 kits, and 20 kits for quality assurance and control, were used in this study without the invalid case, specificity and sensitivity of this batch kit was 100%, which implied the quality was stable. However, the false negative rate of (10.4%) 13/126 documented in the study can be explained only partly by the CS threshold level of >100 parasites/μl and probably the CS storage conditions and duration in the field resulted in loss of sensitivity, since the quality assurance and control testing was 100%.
As shown in Tables 2 and 3, the diagnostic standards of kit were highly specific and sensitive, indicating that kit was valuable for malaria diagnosis.
The CareStart™ P.f/P.v Combo (detecting HRP-2 and P. vivax-specific pLDH) test has been evaluated in Ethiopia [11–25] compared to this study which reported higher sensitivity for P. falciparum (99.4%). No clear reason could be given for increasing sensitivity and possibly included exclusively P. falciparum samples with parasite densities above 100/μl, which is above the detection threshold of most RDTs . Also, CareStart™ malaria pLDH (Pf/pan) combo test has been evaluated in a field study in Madagascar which reported sensitivities for P. falciparum low values at parasite densities <100/μl (60.0%) and increasing sensitivity at higher parasite densities (100% at >500/μl) .
The two products (CareStart™ malaria pLDH (Pf/pan) combo test) and CareStart™ brand (malaria pLDH (pan)), evaluated in Myanmar, reported sensitivities for the detection of P. vivax were significantly higher than those found in Ethiopia with pLDH (pan) (91.0%), but results were similar for the CareStart™ malaria pLDH (pan/Pf) [13–25].
A study in The Philippines compared the results of the ICT malaria P.f/P.v RDT with microscopy and found a high discordant rate involving cases positive for P. falciparum by the RDT, but negative by microscopy .
The CareStart™ malaria pLDH (Pf/pan) combo test field study in Madagascar reported sensitivities for P. falciparum that are comparable to the present study, but there were only nine P. vivax samples included, making comparison difficult . Moreover, the CareStart™ brand (malaria pLDH (pan)) has also been evaluated in Myanmar with similar results obtained across the China-Myanmar malaria endemic borders .
When PCR was the reference method, the microscopic method showed a low specificity (88.2%). The P. vivax and P. falciparum positive and negative predictive values of CS were 100% and 94.96% compared to 100% and 94.17% respectively. In addition, P. vivax diagnostic accuracy of CS was 96.67% compared to 94.89% for P. falciparum. The differential explanation could be the lack of differentiation of other species of Plasmodium species as reported in Thailand . However, the two blood slides from the samples could not show malaria parasites microscopically. The negative predictive values of CS observed could not be directly explained but may be attributed to possible genetic heterogeneity of HRP2 as well as possible geographic variations in malaria antigens.
This study recorded two misdiagnosed microscopic cases, corrected using PCR assay, whereas 13 cases were not detected by CS kit. The reported sensitivities for the detection of P. vivax were significantly higher than those found in the present study in case of the CareStart™ malaria pLDH (Pf/pan) (88.52%), but for the CareStart™ malaria pLDH (Pv/pan) they were in line with the previous findings (90.77%) [10, 11].
Studies evaluating other RDTs in non-endemic countries report similar sensitivities as those found for the CareStart™ malaria HRP-2/pLDH (Pf/pan) combo test in this study for P. falciparum where they ranged from 87.5-99.0%, with one exception of 76.2%, whereas for P. vivax, RDTs detecting pan-pLDH showed sensitivities of 33.5% and 62.0%-95.0% [6–9]. The detection rates as demonstrated by WHO/FIND are slightly higher as compared to the sensitivities found in this study, but the differences were not statistically significant .
The specificity and sensitivity of CS kit were 89.68% and 98.26%, a difference not statistically significant than previous CS results in southwest Ethiopia (see Table 2) . The sensitivity and specificity documented in this study can be accounted for by the long historical trends of anti-malarial usage, counterfeit, substandard and counter prescription contributing to the progression of selective pressure of malaria parasites in the blood stream and hence low parasitaemia below the CS kit detection threshold. The findings showed that CS usefulness in detection and case management of malaria in remote areas and on motile populations across borders has implications in the search for more efficient and sensitive diagnostic tools for parasite detection and case management in areas of low endemicity, as well as targeting gametocyte detection towards blocking the transmission, and monitoring malaria elimination.