Characterization of the Plasmodium falciparum histidine-rich protein II to improve malaria diagnostic interpretation and sensitivity
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Date
2019-04-09
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Johns Hopkins University
Abstract
Statement of Problem: The P. falciparum rapid diagnostic test (RDT) is a critical component to malaria control and elimination strategies being implemented to decrease the global malaria burden. However, there are two important challenges to P. falciparum RDTs utilizing PfHRP2 in regards to their sensitivity and specificity that must be addressed. The PfHRP2 protein detected by the RDT persists in the blood, extending the duration of RDT positivity beyond an active infection, leading to false positive results. Additionally, due to lower parasite burdens and less circulating PfHRP2, the RDT poorly detects subclinical, asymptomatic cases of malaria that serve as important transmission reservoirs. We hypothesize that better characterization of the PfHRP2 protein and elucidation of its kinetics and persistence in vivo will aid in the development of novel strategies for enhancing the interpretation, sensitivity and specificity of the PfHRP2- based RDT.
Methods: We optimized three novel model systems- P. falciparum infected patients, splenic and asplenic mice infected with a novel transgenic P. berghei rodent malaria parasite that expresses PfHRP2 (PbPfHRP2), and P. falciparum infected splenectomized monkeys- to measure compartmental kinetics of PfHRP2 in the plasma and erythrocytes (RBCs) and to characterize persistence post-treatment in these two blood fractions. We also investigated the addition of exogenous amino acids on the detection of PfHRP2 by various immunoassays.
Results: PfHRP2 persisted longer in the RBC fraction of whole blood compared to the plasma in both humans infected with P. falciparum and mice infected with PbPfHRP2. We also identified PfHRP2-positive, DNA-negative, once-infected RBCs in patients at 0.1-1% of total RBCs for multiple days post-treatment, even after post-atovaquone-proguanil regimens. Once-infected RBCs positive for PfHRP2 were also detected in our murine model but persistence duration was shorter in mice compared to humans. However, clearance of more than 90% of parasite DNA and RBC localized PfHRP2 in splenic and asplenic mice did not differ. In asplenic monkeys, P. falciparum parasites and parasite genomic DNA persisted longer than in human patients, but a similar clearance rate of PfHRP2 from the RBCs was quantified in both these models. In separate studies, the addition of 10 mM histidine to the primary antibody in an immunoblot increased detection of the PfHRP2 protein by various monoclonal antibodies in an epitope-independent fashion. The presence of whole blood did not inhibit the effects of histidine on detection. Addition of exogenous histidine also increased histidine also increased PfHRP2 detection 2 to 3-fold in the context of low parasite densities (10 to 1000 pg of PfHRP2 protein) not only on an immunoblot but also by the ELISA and RDT formats.
Conclusions: PfHRP2 persistence appears to be a consequence of prolonged clearance of the protein from the RBC fraction of whole blood, further supporting the hypothesis that the extended duration of RDT positivity after parasite clearance is likely due its presence in once-infected RBCs that remain positive for PfHRP2 despite undergoing erythrocyte pitting in the spleen to clear parasites. We also confirmed the importance of the spleen in parasite clearance during a P. falciparum infection. It also appears that pitting of erythrocytes and production of the PfHRP2 positive, parasite negative, once-infected RBCs may not be occurring to the same degree in mice as a result of their altered splenic architecture compared to humans and the increased pliability of P. berghei infected RBCs (iRBCs) compared to P. falciparum iRBCs. Finally, we demonstrated a novel and inexpensive way to potentially increase the sensitivity of the malaria RDT for diagnosing asymptomatic, subclinical malaria that solely involves the simple addition of the amino acid histidine.
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Keywords
histidine-rich protein II, malaria, rapid diagnostic test