The main finding from this study is that we have a cheaper homemade formulation f 1 which can stabilize parasite DNA in saliva even after 12 months of RT storage. Comparing PCR-f 1 /PCR-S0, we showed that the same samples were positive in each group after a 12-month storage at RT. The sensitivity was 100% and Kappa value was 0.814. Moreover, for PCR-f 1 /MIC, sensitivity was 100% and Kappa value was 0.641. The sensitivity and Kappa values found by [16] were 94% and 0.907 respectively. Valid concerns about this study could include the method of extraction of DNA and parasite density. However, since we treated both samples in the same way after collection, these aspects are non-consequential to our finds. Though, Fung and collaborators [17] suggested that saliva samples preserved in ethanol yielded superior positive PCR results when compared to samples kept on ice. However, such preservation cannot last for up to 12 months and besides, the absence of ethanol preservation in this study does not appear to have negatively affected PCR amplification. A very good agreement (κ = 0.814) was observed for DNA derived from saliva using the Chelex extraction method compared to DNA purified from blood samples. Besides, this method is very cheap ~$ 0.5 compared to other saliva extraction methods which cost ~ $4.10/ saliva sample [18].
f which can stabilize parasite DNA in saliva even after 12 months of RT storage. Comparing PCR-10f/MIC, sensitivity was 100% and Kappa value was 0.641. The sensitivity and Kappa values found by [] were 94% and 0.907 respectively. Valid concerns about this study could include the method of extraction of DNA and parasite density. However, since we treated both samples in the same way after collection, these aspects are non-consequential to our finds. Though, Fung and collaborators [] suggested that saliva samples preserved in ethanol yielded superior positive PCR results when compared to samples kept on ice. However, such preservation cannot last for up to 12 months and besides, the absence of ethanol preservation in this study does not appear to have negatively affected PCR amplification. A very good agreement (κ = 0.814) was observed for DNA derived from saliva using the Chelex extraction method compared to DNA purified from blood samples. Besides, this method is very cheap ~$ 0.5 compared to other saliva extraction methods which cost ~ $4.10/ saliva sample [].
A study in Gambia found that the sensitivity of nested PCR increased from 73–82% in samples with a parasite density of > 1000 parasites/µl [19]. Although the study by [20] found that the positive rates of nested PCR of saliva samples increased with parasite density for P. falciparum, this was not the case with our results.
Storage conditions have been shown to be a factor that may potentially influence antigen detection in spun saliva samples [21, 22]. A study showed 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 [16]. Although this study and many others [23, 24] clearly show the potential to use saliva as a non-invasive body fluid for rapid diagnosis of malaria, there are still many challenges in establishing it as a reference. Prominent among these challenges are failure of RDT to detect parasite antigen in some whole-saliva samples despite high parasitemia of > 1000 parasite/µL blood[25]. The reasons for this disparity are unclear. Quantitative PCR showed up to ~ 600-fold greater DNA quantity in blood compared to saliva samples from infected patients, and a statistically significant correlation between parasite density and amount of parasite DNA in saliva was observed [19]. We will understand these disparities better when we have a clearer mechanism of the biological processes leading to the release of parasite antigen in saliva. Nonetheless, saliva still has its merits as we and others [26] have shown that it is capable of identifying submicroscopic parasitemia in both clinical and nonclinical settings even with archived saliva samples.
Also, it is known that malarial products such as P. falciparum histidine-rich protein II (PfHRP-II) or P. falciparum lactate dehydrogenase (pLDH) released upon schizont rupture into circulation may get into saliva through pericellular ultrafiltration from the surrounding vasculature[17]. The study by [20] detected PfHRP-II in whole saliva at 43% sensitivity, while [17] achieved a sensitivity of 100%. The difference in the sensitivities was probably due to the method of storage and stabilization of the samples [20]. Unfortunately, only DNA downstream analysis are possible in saliva samples stored in our buffer.
The study by [27] detected both P. falciparum and P. vivax in urine and saliva albeit with a low sensitivity compared blood through nested PCR of all three types of samples. Similar results were later reported by [28]. However, reports of the presence of P. ovale and P. malariae in non-blood samples like saliva and urine are infrequent. We show the presence of P. ovale and P. malariae in saliva preserved in both our homemade kit and the OMNIgene®ORAL (OM-501) kit (DNA Genotek, Ottawa, Ontario, Canada) commercial kit, although the band for P. malariae was almost invisible after 12 months of room temperature storage in our kit.