Rapid Diagnosis of Plasmodium Knowlesi

Singh et al have recently described over 70 cases of human malaria utilizing polymerase chain reaction (PCR ) that were caused by P.knowlesi thought to originate from a crab eating Macaque (Macaca fascicularis) of Borneo, and originally mis-diagnosed as P.malariae by microscopy (1). White reviewed these finding and suggested that P.knowlesi malaria be considered the Fifth Human Malaria Parasite (2).

Utilizing, a rapid malaria test (RMT) that detects plasmodium lactate dehydrogenase (pLDH) we were rapidly able to diagnose P. knowlesi. Depending upon the selection of monoclonal antibodies (mabs) to pLDH we could differentiate P. knowlesi from the other four forms of human malarias.

Figure 1 illustrates the principle of our pLDH assay using human blood from a finger prick that contains both P. vivax and P.falciparum.








Figure 2 demonstrates a series of actual immunochromatographic (dip) strips. These strips were stripped with 5 antibodies. The uppermost antibody is a control line containing goat anti mouse (G) antibody. It will react with all mouse mabs. The four other lines: 9C1, 10D, 11D, and 17E, contain mabs designed to be specific to: P.falciparum, P.vivax, P,malariae and P.ovale. Observe that : P.falciparum reacts only with mab 17; P.vivax with mab 11D; P.ovale with mab 10; P. malariae with mab 9 ; and P.knowlesi reacts with 13 and 17. P.knowlesi reacts with 11D and 17E. P. knowlesi reacts with no other mabs.





Figure 3 is an analysis of the the reaction of P. knowlesi first utilizing the Sequence of LDH from P. knowlesi deduced from genomic DNA fragments sequenced by the Sanger malaria genome project. LDH isoforms from Plasmodium vivax, malariae, ovale, berghei, yoelli, and falciparum were compared with that of knowlesi. Residues that were unique to both P. knowlesi and P. vivax are shown in blue, while residues unique to both P. knowlesi and P. falciparum are shown in red. The upper right side of this figure also demonstrates the binding specificity of many different anti-pLDH antibodies. Shown are the reactivities of the indicated monoclonal antibodies to the pLDH from 7 Plasmodium species. These include human, primate, avian, and murine malaria. P. vivax LDH is immobilized only by 11D9 (13H11) and P. falciparum LDH was only immobilized by 17E4(7G9). P. knowlesi LDH was immobilized by both 11D9(13H11) and 17E4(7G9).

The lower part of the figure is a model of P. knowlesi LDH and specific epitopes. A three dimensional model for P. knowlesi LDH was calculated using Modeller V9 and the P. falciparum and P. vivax crystal structures (PDB: 2A94 and 2AA3). Shown is the monomer as well as the assembled tetramer. The NAD co-factor analog 3-acetyl pyridine adenine dinucleotide is shown in black. Residues important for substrate binding and
catalysis are shown in yellow. P. knowlesi residues shared only with P. vivax are shown in blue and indicate where the 11D9/13H11 epitopes could be. P. knowlesi residues shared only with P. falciparum are shown in redand indicate a critical determinant of the 17E4/7G9 epitopes.


We conclude that the pLDH RMT is able to speciate all 5 forms of human malaria. This diagnostic assay takes 10-20 minutes to complete. The one limitation of the pLDH RMT is that it is currently unable to distinguish between a mixed infection with P.vivax and P.falciparum. However, in these infections, unlike the P. knowlesi infection there is often a much greater difference in the intensity of staining of the two bands, consequently together with light microscopy, this combination of tests should provide adequate diagnosis of this serious condition.


References
1.Cox-Singh J, Davis TM, Lee KS, Shamsul SS, Matusop A, Ratnam S, Rahman HA, Conway DJ, Singh B. Plasmodium knowlesi malaria in humans is widely distributed and potentially life threatening. Clin Infect Dis. 2008 Jan 15;46(2):165-71
2. White NJ. Plasmodium knowlesi: The Fifth Human Malaria Parasite Clin Infect Dis. 2008 Jan 15:46(2): 172-73
Authors
Michael T. Makler, MD Flow Inc. Portland Or 97239: 503 481 7994
Robert C. Piper, PhD , Phys, U of Iowa, Iowa City, IA 52242: 319 335 7842