The use of urine and saliva as a non-invasive procedure to detect malaria parasites that avoid the use of blood with no need of special equipment and may be suitable for societies with blood taboos [12, 17]. In surveillance studies, blood sampling and in some cases repeated blood sampling often dissuade many study subjects from involving themselves in clinical and epidemiological studies [18, 19]. Research findings also confirm that patients are usually willing to donate any other samples than blood towards biomedical studies . Based on these, if analytical procedures are modified to use other human samples instead of blood and other invasive procedures, patronage of biomedical studies will increase with its attendant benefits of reduction in transmission of blood borne infections.
Previous studies attempted to evaluate the use of non-blood samples to detect malaria antigens. In India, the sensitivities of urine in two different studies were found to be 38%  and 86.67% . Like this study, rapid diagnostic test kits intended to be used for detecting malaria antigens in blood were used. In another studies, diagnostic kits specifically meant for detecting malaria antigens in urine were used. Oyigbo et al.  reported sensitivity of 85% while Oguonu et al.  also reported sensitivity of 84%. However, these previous studies failed to report the conditions under which malaria antigens were detected in non-blood samples. Urine has been widely evaluated as an alternative specimen for malaria diagnosis. Few studies have evaluated the use of saliva. In this study, saliva, with or without blood contamination, was found to be of higher sensitivity compared to urine as an alternative sample to diagnose malaria. This study has provided some conditions that could enhance the detection of malaria antigens in urine and saliva. The identified conditions were sample contamination with blood, malaria parasitaemia > 60,000 parasites/μL, moderate to severe anaemia and hyperthermia (temperature > 38.7 °C). Considering the low sensitivities observed in this study compared to previous studies, it could be due to lower concentrations of the antigens in random samples and/or degradation of malaria antigens in urine samples from time of collection to analysis of specimens .
In spite of these limitations, higher detection rate of P. falciparum specific antigens was observed in saliva compared to urine. This could probably be because, in uncomplicated malaria where fever is characterized by body temperatures above 37.5 °C, vasodilation of vessels supplying the buccal cavity, the skin and other body openings is a possibility. This will ensure the capillaries underneath the skin and other mucous membranes are filled with blood if the body gets too hot. Subsequently, blood is brought closer to surfaces so more heat can be lost. Moreover, gingivitis is common in malaria so with the least trauma to the gum, blood is released into the mouth which can contaminate saliva for subsequent detection of P. falciparum specific antigens. This study reports 57.0% detection rate of P. falciparum specific antigens in saliva whilst previously P. falciparum DNA has been detected in human saliva (73%) [11, 25]. Again similar to this study, pLDH was detected in saliva in previous study in Nigeria. That study reported 77.9% sensitivity of detected pLDH in saliva  while in this study, the detection rate of pLDH was 89.4% (17/19). Again, P. falciparum HRP2 antigens were detected in saliva of malaria patients using ELISA technique, and even though the sensitivity was low (43%) with comparatively long turnaround time (approx. 2 h and 15 min) , this report together with findings in this study showed the potential of a non-invasive approach for malaria diagnosis using saliva. High detection rate of pLDH confirms the usefulness of saliva in detecting active malaria where concentration of pLDH is very high and short-lived. The presence of pLDH has been associated with malaria parasite viability in some studies [26, 27].
Again, in this study, urine samples of 35.2% of acute malaria patients were found to contain PfHRP2 and pLDH proteins. It has been previously reported that kidney involvement is not uncommon in falciparum and malariae malaria . Haemodynamic dysfunction and immune response are the main mechanism of malaria associated kidney pathology . Furthermore, malaria has been reported as the first parasitic infection to be clearly associated with glomerular diseases in tropical areas  with subsequent detection of Plasmodium antigens in the glomeruli . Proteinuria and microalbuminuria associated with kidney involvement in malaria  made it possible for malaria specific proteins to be detected in urine, as was observed in this study. The molecular weight of albumin is 69 kDa  whilst that of PfHRP2 is 30 kDa , and pLDH is 32 kDa . Thus, the respective molecular weight of PfHRP2 and pLDH is less than half the molecular weight of albumin, so if albumin is excreted in urine in uncomplicated malaria, then PfHRP2 and pLDH could be freely excreted as well.
In P. falciparum infected patients, over 85% of salivary and urinary PfHRP2 and pLDH were identified in haemoglobin concentration less than 9.9 g/dL, body temperature greater than 38.1 °C, occult blood in saliva and urine and parasitaemia of > 60,000 parasites/µL of blood.
Plasmodium falciparum is an obligatory intraerythrocytic parasite so destruction of red cells containing parasites and uninfected red cells resulting in anaemia is very common . In severe malaria, hemoglobin (molecular weight = 65 kDa)  in urine has been reported as well as nephrotic syndrome especially in P. malariae infections . Based on these separate reports, association of malaria antigenuria with anaemia is not surprising. Moreover, the smaller molecular sizes of these malaria antigens and their water solubility make them easily excreted in urine, hence their detection. Additionally, anaemia in malaria is further worsened by discharge of blood in body fluids. Malaria antigens were detected in all haematuric infected patients, and in 95% of malaria patients with positive occult blood in saliva. Meanwhile, PfHRP2 proteins are found on the surface of infected red blood cells , so once red cells are detected in urine and saliva, detection malaria antigens in these specimens is highly possible.
It has been reported that glomerular capillary dilatation, haemorrhage into the interstitium, in small and large renal vessels occur during pathogenesis of fever , also acute gingival bleeding has been reported in fever especially in dengue hemorrhagic fever . Vasodilation of the renal vasculature and gingival bleeding as a result of hyperthermia enhanced the detection of malaria antigens in urine and saliva in this study.
Finally, even though detection of malaria antigens improved with increased parasitaemia, Gbotosho et al.  found otherwise. In their study, RDT failed to detect parasite antigen in some saliva samples, despite high parasitaemia (2,571–334,298 parasite/µL blood) and positive RDT in matching whole-blood samples from the same patients. The reasons for this disparity could be due to the difference in the sensitivities of the malaria RDT used in these two studies (SD Bioline was used in this study, while Gbotosho et al.  used OptiMAL-IT dipstick). Again, in their study, patients with concomitant illness were excluded while in this study, patients with gingivitis (evidenced by occult blood in saliva) and haematuria were included. Also, this study did not exclude any patient with possible concomitant infections (other infectious disease markers were not screened). So the differences in the study patients could also account for the differences observed with respect to the detection threshold of parasitaemia that influenced detection of malaria antigens in saliva. In addition, Gbotosho et al.  performed RDT strictly according to manufacturer’s instruction as validated for blood sample testing while in this study, the detection of malaria proteins was optimized to increase improve sensitivity.