Detection, quantification, and concentration of PfHRPII in urine of malaria patients: implications for urine-based Plasmodium falciparum diagnostics.
Assumpcao, Nicole Rimoli
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Plasmodium falciparum remains a major source of global mortality, causing nearly 500,000 deaths per year. Although diagnostics have made major improvements in recent years, microscopy on blood smears remains the gold standard, with blood-based rapid diagnostic tests (RDTs) as the other major player in field tests. However, there remain social challenges as well as health concerns associated with taking blood, and discovery of an alternate body fluid diagnostic has been an area of major research. Previous attempts at urine-based diagnostics have generally suffered from impaired sensitivity, particularly among afebrile patients. This is likely to be due to a decreased quantity of the antigen being detected (PfHRPII) in urine as compared to plasma. This work uses urine and plasma samples collected from patients at a health post in Kpone on Sea, Ghana, of which 64% were malaria-positive by microscopy, to examine this dilution factor and other correlates of urine PfHRPII detection. The first aim was to detect PfHRPII in the urine using Western Blot, dot blot, and ImmunoPCR, and compare these to a buffered urine-specific RDT. Of these, ImmunoPCR was the most sensitive, and in each subset there was a higher proportion of positive results in febrile patients. The next aim was to quantitatively examine the ratio of PfHRPII in plasma versus urine using both ELISA and ImmunoPCR techniques, which yielded a geometric mean ratio of 69.0 and 137.6 respectively. The implication for urine-based diagnostics is that they will require an almost 100-fold (or more) increase in sensitivity or sample volume to be viable and comparable to the sensitivity of blood-based diagnostics. Thus, our final aim was to attempt to rectify this by applying Nanotrap nanoparticle technology to sequester and concentrate PfHRPII in 500 microliter aliquots of urine. Although the nanoparticles were able to concentrate the protein five or six fold, this was insufficient to counteract the dilution from plasma and was much below the theoretical 31-fold increase in protein content corresponding to a 31-fold increase in sample volume. Further, this technology ii was suboptimal for detection in actual patient samples, showing concentration in only one sample. Although there is room for increasing sample size and altering composition of nanoparticles, to surmount the obstacle of decreased PfHRPII concentration in urine. Additionally, due to the high variability in PfHRPII detection in the urine, any adequate diagnostic will likely have to go well above and beyond the 100-fold benchmark elucidated in this work in order to capture a meaningful proportion of malaria-infected patients.