The ideal procedure for obtaining samples for the diagnosis of malaria should be simple, non-invasive, painless, and practicable by all categories of health workers. In addition, presence of parasites or its product should be readily demonstrable in samples obtained by the procedure. Obtaining saliva samples seems to have fulfilled many of these criteria. Rapid detection of pLDH antigen in whole saliva of children with P. falciparum
infection using OptiMAL-IT dipsticks was shown for the first time in this study with a test sensitivity of 77.9%. This finding raises hope of the potential for the use of non-invasive, non-microscopic yet rapid techniques in the diagnosis of malaria. The present observation supports the limited reports describing potential to diagnose malaria using saliva samples.8–10
The high sensitivity (detection threshold > 2,000 parasites/μL of blood) and specificity of the Optimal-IT dipsticks in patient blood at presentation as observed in this study are consistent with previous reports.14–16
Results from the present study show that spinning saliva samples before analysis significantly decreased the sensitivity for antigen detection by the RDT (sensitivity = 48.3%). This finding was consistent with a previous report describing detection of pfHRP-II in supernatant of spun saliva by plate assay at a sensitivity of 43%.10
One possible explanation for this finding is the fact that centrifugation of saliva samples may have resulted in the sedimentation of parasite proteins and thus, concentrations of parasite antigen detectable in supernatant may have been low. Reports have shown that the apparent accuracy of any RDT in detecting malaria parasites is dependent on various factors, which include concentration of target antigen in host blood, dynamics of antigen–antibody flow along the nitrocellulose strip, and availability of target epitopes to bind antibodies in the test.17
Storage also seems to be a factor that may have influenced antigen detection in spun saliva samples. Bell and Peeling17
reported that stored blood can lose antigen activity, and early lysis and protein coagulation can inhibit flow, thus influencing the results of RDT-based malaria diagnosis.
The approach for analysis of whole-saliva samples from patients as described in this study is a less complex and more sensitive alternative that can be easily applied in malaria-endemic countries of Africa. This approach contrasts with the technique described by Wilson and others,10
which is cumbersome, time-consuming, and more expensive because it requires centrifugation and two incubation steps as well as the use of a spectrophotometric plate reader; this hinders its routine use in resource-depleted countries where malaria is endemic. In the present study, the enhanced detection of parasite antigen in fresh whole-saliva samples shows potential superiority of whole saliva over spun saliva for rapid detection of malaria infection.
Although this study clearly shows the potential to use saliva as a non-invasive body fluid for rapid diagnosis of malaria, there are still many challenges in establishing saliva as a reference fluid for diagnosis. For instance, in this study, RDT failed to detect parasite antigen in some whole-saliva samples, despite high parasitemia (2,571–334,298 parasite/μL blood) and positive RDT in matching whole-blood samples from the same patients. The reasons for this disparity are unclear. However, it is possible that differences in concentration of parasite antigen in whole blood and saliva may be responsible for the lack of detection of parasite antigen in saliva, despite high parasite densities and positive RDT in matching blood samples. In a recently reported study using quantitative PCR analysis, it was shown that the amount of parasite DNA quantified in peripheral blood samples from infected patients was ~600-fold greater than in saliva samples, although a statistically significant correlation between parasite density and amount of parasite DNA in saliva was observed.9
A full understanding of biological processes leading to the release of parasite antigen in saliva would provide more insight into these disparities. Furthermore, matched blood and saliva RDTs of four patients with microscopically confirmed P. falciparum
malaria were negative despite high parasite densities. The exact reason for this is not immediately known, although the possibility of gene deletion may explain, in part, the reason for the false-negative RDT. Gene deletion has been postulated in isolates that do not express pfHRP-II and yield false-negative results with Parasight-F dipsticks.18,19
Further investigation for these and other non-expressed antigens need to be considered.
The additional challenge with the use of saliva as a diagnostic fluid is the fact that the sensitivity of the RDT in whole saliva reported in our study is still below optimum. The World Health Organization recommends a minimum standard of 95% sensitivity at parasite densities of 100/μL.17
Obviously, one of the reasons for the low sensitivity of detection using Optimal-IT dipsticks in saliva may be the fact that this RDT was commercially designed to detect high levels of lactate dehydrogenase in whole blood and not in saliva. Therefore, proteomic analysis and quantification of pLDH or other potential Plasmodium
antigens circulating in saliva is needed to develop an RDT specifically for saliva samples.
The results from this study also confirm detection of P. falciparum
DNA by PCR in saliva of patients with P. falciparum
malaria.8,9 P. falciparum
population structure in most saliva samples was identical to that found in corresponding finger-prick blood samples of the same individual. The polyclonal nature of most infections in both saliva and blood samples is consistent with previous reports12,13
from the same study site. Nested PCR with RFLP techniques are frequently used to detect polymorphisms in parasite genes that serve as molecular markers of antimalarial drug resistance.20–22
This approach was used in saliva samples to detect K76T polymorphism on the pfcrt
gene that is associated with chloroquine resistance. Similar K76T pfcrt
genotypes were observed in the parasite genomic DNA extracted from filter-paper blood samples and saliva samples. The high prevalence of the mutant pfcrt76T
allele in both saliva (57%) and blood (60%) specimens is consistent with previous reports20–22
from genomic DNA extracted from blood sample.
Overall, this study has shown the rapid detection of malaria parasite antigen in saliva. Improvement of the sensitivity of this non-invasive method would provide a potentially valuable adjunct to microscopy, especially at the time of emergency for rapid diagnosis of malaria. However, expert microscopy will still be required for species identification and confirmation of a negative blood and saliva RDT result. Furthermore, there is a need to evaluate the value of using RDTs in detecting parasite antigen in saliva during longitudinal studies to monitor and evaluate the efficacy of antimalarial drugs with lengthy follow-up periods and low levels of parasitemia. Such studies would provide an opportunity to study the time course of appearance and dissipation of parasite antigen in saliva.