Disease Progression in Plasmodium knowlesi Malaria Is Linked to Variation in Invasion Gene Family Members

et al. (2014) Disease Progression in Plasmodium knowlesi Malaria Is Linked to Variation in Invasion Gene Family Members. PLoS Negl Trop Dis 8(8): e3086. doi:10.1371/journal.pntd.0003086 Disease Progression in Plasmodium knowlesi Malaria Is Linked to Variation in Invasion Gene Family Members Atique M. Ahmed 0 Miguel M. Pinheiro 0 Paul C. Divis 0 Angela Siner 0 Ramlah Zainudin 0 Ing Tien Wong 0 Chan Woon Lu 0 Sarina K. Singh-Khaira 0 Scott B. Millar 0 Sean Lynch 0 Matthias Willmann 0 Balbir Singh 0 Sanjeev Krishna 0 Janet Cox-Singh 0 Kenji Hirayama, Institute of Tropical Medicine (NEKKEN), Japan 0 1 Malaria Research Centre, University Malaysia Sarawak , Kuching, Sarawak , Malaysia , 2 School of Medicine, University of St Andrews , St Andrews , United Kingdom , 3 Faculty of Resource Science and Technology, University Malaysia Sarawak , Kuching, Sarawak , Malaysia , 4 Sibu Hospital , Sibu, Sarawak , Malaysia , 5 Sarikei Hospital , Sarikei, Sarawak , Malaysia , 6 Division of Clinical Sciences, St. George's, University of London , London , United Kingdom , 7 Clinical Blood Sciences, St. George's, University of London , London , United Kingdom , 8 Institute of Medical Microbiology and Hygiene, University of Tu bingen , Tu bingen , Germany Emerging pathogens undermine initiatives to control the global health impact of infectious diseases. Zoonotic malaria is no exception. Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, has entered the human population. P. knowlesi, like Plasmodium falciparum, can reach high parasitaemia in human infections, and the World Health Organization guidelines for severe malaria list hyperparasitaemia among the measures of severe malaria in both infections. Not all patients with P. knowlesi infections develop hyperparasitaemia, and it is important to determine why. Between isolate variability in erythrocyte invasion, efficiency seems key. Here we investigate the idea that particular alleles of two P. knowlesi erythrocyte invasion genes, P. knowlesi normocyte binding protein Pknbpxa and Pknbpxb, influence parasitaemia and human disease progression. Pknbpxa and Pknbpxb reference DNA sequences were generated from five geographically and temporally distinct P. knowlesi patient isolates. Polymorphic regions of each gene (approximately 800 bp) were identified by haplotyping 147 patient isolates at each locus. Parasitaemia in the study cohort was associated with markers of disease severity including liver and renal dysfunction, haemoglobin, platelets and lactate, (r = $0.34, p = ,0.0001 for all). Seventyfive and 51 Pknbpxa and Pknbpxb haplotypes were resolved in 138 (94%) and 134 (92%) patient isolates respectively. The haplotypes formed twelve Pknbpxa and two Pknbpxb allelic groups. Patients infected with parasites with particular Pknbpxa and Pknbpxb alleles within the groups had significantly higher parasitaemia and other markers of disease severity. Our study strongly suggests that P. knowlesi invasion gene variants contribute to parasite virulence. We focused on two invasion genes, and we anticipate that additional virulent loci will be identified in pathogen genome-wide studies. The multiple sustained entries of this diverse pathogen into the human population must give cause for concern to malaria elimination strategists in the Southeast Asian region. - Funding: This study was funded by The Medical Research Council (MRC) UK; Grant number G0801971, http://www.mrc.ac.uk. MMP is supported by The Wellcome Trust (ISSF 097831/Z/11/Z), http://www.wellcome.ac.uk. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Plasmodium knowlesi malaria is widespread in Southeast Asia (SEA). Descriptions of the aetiology of knowlesi malaria support a zoonotic origin of infection [1] and highlight variability in disease severity between those at risk across the region [2]. For example, very young children living in a forested area of Southern Vietnam have asymptomatic mixed Plasmodium species infections that include P. knowlesi [3]. Adults and children in Malaysian Borneo experience symptomatic single species P. knowlesi infections that are severe in .10% of patients and can be fatal [4,5]. P. knowlesi transmission is restricted to the Leucosphyrus group of mosquito vectors found in forested areas of Southeast Asia [6,7]. The vector group is diverse and capable of simultaneous transmission of human and non-human primate adapted Plasmodium species [8]. The majority of reported cases of P. knowlesi malaria are associated with time spent in the jungle or jungle fringe areas where the ranges of the natural vertebrate hosts, the long and pig tailed macaques (Macaca fascicularis and Macaca nemestrina) and leucosphyrus vectors overlap [9,10]. However, a change in pattern has recently emerged in Malaysian Borneo, where children living in a deforested area are infected [11]. This new pattern may signal a change in vector or vector habitat preference and a move towards human-tohuman transmission. Restricted spread of P. knowlesi within human populations is attributed to non-urban vector habitat. Also human-host adapted Plasmodium species, where prevalent, may present a biological barrier to the entry of P. knowlesi into human populations concurrently at risk from human adapted and zoonotic species infections. On a backdrop of vector, human and parasite diversity, Plasmodium knowlesi, a parasite of Southeast Asian macaques, has entered the human population. Approximately 10% of P. knowlesi infections are severe, 12% are fatal, in Sarawak, Malaysian Borneo. Increase in parasitaemia is associated with disease severity, but little is known about parasite virulence in this newly described human pathogen. Here we present the results of a study on P. knowlesi parasites collected from 147 patients. We use the isolates to produce DNA sequences from a polymorphic (genetically variable) region of two P. knowlesi genes, Pknbpxa and Pknbpxb, that are involved in parasite entry into host red blood cells. We addressed the question that some parasite genotypes may have an invasion advantage leading to severe disease in human infections. We analysed the DNA sequences with matched clinical and laboratory data from the patient cohort (n = 147). We found that specific DNA sequences (Pknbpxa and Pknbpxb alleles) clustered with high parasitaemia and markers of disease severity. Here, for the first time, we provide evidence that variant alleles of the Plasmodium Reticulocyte Binding-Like Protein invasion gene family can influence disease progression in patients with malaria. The biological characteristics of the variants will be studied to aid our understanding of malaria pathophysiology and to inform intervention strategies. it would be folly for malaria elimination stra (...truncated)