Disease Progression in Plasmodium knowlesi Malaria Is Linked to Variation in Invasion Gene Family Members Atique M. Ahmed 1. , Miguel M. Pinheiro 2. , Paul C. Divis 1 , Angela Siner 1 , Ramlah Zainudin 1,3 , Ing Tien Wong 4 , Chan Woon Lu 5 , Sarina K. Singh-Khaira 6 , Scott B. Millar 2 , Sean Lynch 7 , Matthias Willmann 8 , Balbir Singh 1 , Sanjeev Krishna 1,6 , Janet Cox-Singh 1,2,6 * 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 Abstract 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). Seventy- five 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. Citation: Ahmed AM, Pinheiro MM, Divis PC, Siner A, Zainudin R, 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 Editor: Kenji Hirayama, Institute of Tropical Medicine (NEKKEN), Japan Received October 21, 2013; Accepted June 30, 2014; Published August 14, 2014 Copyright: ß 2014 Ahmed et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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. * Email: jcs26@st-andrews.ac.uk . These authors contributed equally to this work. Introduction 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-to- human 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, PLOS Neglected Tropical Diseases | www.plosntds.org 1 August 2014 | Volume 8 | Issue 8 | e3086