ZOONOTIC MALARIA PARASITES AMONG NON-HUMAN PRIMATES IN , MALAYSIAN BORNEO Khamisah Bt Abdul Kadir 1, Paul Cliff Simon Divis1, Dayang Shuaisah Bt Awang Mohamad1, Asmad Matusop1,2, Mohd Tajuddin Abdullah1 & Balbir Singh1. 1 Malaria Research Centre, Universiti Sarawak, Malaysia, 2 Sarawak Health Department, Malaysia. INTRODUCTION Human infections with Plasmodium knowlesi, a malaria parasite of long-tailed and pig-tailed macaques (Macaca fascicularis and M. nemestrina respectively), were thought to be extremely rare1 until a large focus of human infections were reported in the division of Sarawak, Malaysian Borneo in 20042. Human infections have since been reported throughout Southeast Asia and P. knowlesi is regarded as the fifth Plasmodium species causing human malaria3. Macaques are the natural hosts of P. knowlesi, P. cynomolgi (Pcy), P. inui (Pin), P. coatneyi (Pco) and P. fieldi (Pfi)1. Under experimental conditions Pcy and Pin can infect humans1 and are potentially zoonotic. Macaques in the harbour all 5 Plasmodium species4 but the prevalence of malaria among non-human primates in other regions in Sarawak is unknown.

OBJECTIVE To determine the prevalence and distribution of simian malaria parasites among non-human primates in Sarawak.

MATERIALS & METHODS From 7 of 9 administrative divisions in Sarawak, blood samples were obtained from 84 non-human primates: Macaca fascicularis (n=45), M. nemestrina (n=28), Presbytis cristata (n=2), Nasalis larvatus (n=2), Macaca arctoides (n=3), Hylobates muelleri (n=4). Following DNA extraction, they were screened with nested PCR assays using primers specific for Pk, Pcy, Pin, Pco and Pfi: Details of primer sequences and PCR parameters are provided in Reference number 4.

RESULTS Only long-tailed and pig-tailed macaques were infected with malaria parasites. A total of 32 (43.8%) macaques (20 M. fascicularis and 12 M. nemestrina) were malaria-positive. All 5 Plasmodium species were detected, with macaques harbouring either single (n=12), double (n=9), triple (n=7) or quadruple (n=4) infections (Fig. 1). The zoonotic malaria parasites (Pk, Pcy and Pin) were detected either singly or in combination in all the 32 malaria-positive macaques. These macaques were sampled from 6 of the 7 divisions in Sarawak that were studied. Number detected by PCR assays Number detected by PCR assays Macaque Total Malaria-Positive Positive Macaque Total Neg Neg Pk + Pin + Pct Total malaria Sarawak Limbang Pcy Pin Pk + Pcy + Pcy + Pk + Pcy + Total Pin Pin Pin + Pct Pin + Pct malaria Long-tailed 2 1 1 3 Long-tailed 11 0 0 1 0 0 1 2 13 Pig-tailed 2 0 0 2 Pig-tailed 5 1 6 1 1 1 0 10 15 Total 4 1 1 5 Total 16 1 6 2 1 1 1 12 28 Miri Number detected by PCR assays Number detected by PCR assays Sibu Sarikei Macaque Positive Positive Total Macaque Total Kapit Neg Pcy Pk Pcy + Pk + Pcy + Pk + Pcy + Total Neg Pin + Pct Pk + Pcy + Total Pin + Pct malaria Pin Pcy + Pct Pin + Pct Pin + Pct malaria Long-tailed 2 0 0 1 1 3 1 6 8 Samarahan Sri Aman Long-tailed 2 3 2 5 7 Pig-tailed 2 1 1 0 0 0 0 2 4 Total 2 3 2 5 7 Total 4 1 1 1 1 3 1 8 12 Kuching Samarahan Sri Aman Number detected by PCR assays Macaque Number detected by PCR assays Number detected by PCR assays Total Macaque Macaque Positive Total Neg Total malaria Positive Total Neg Neg Pct Pin + Pct Pcy + Pct Pk + Pin + Pct Total malaria Long-tailed 1 0 1 Pk Pin Total malaria Long-tailed 6 1 1 1 1 4 10 Pig-tailed 3 0 3 Long-tailed 1 1 1 2 3 Total 6 1 1 1 1 4 10 Total 4 0 4 Total 1 1 1 2 3

Fig. 1. PCR results and map showing administrative divisions of Sarawak, with inset map showing location of Sarawak on the island of Borneo.

CONCLUSION The simian malaria parasites that can infect humans (Pk, Pcy and Pin) naturally occur in macaques that are widely distributed in Sarawak. Travelers

and people living and working in forested and forest-fringe areas in Sarawak and similar areas in Southeast Asia should be made aware of the potential risk of acquiring simian malaria. Similarly, physicians treating returning travelers with fever from such areas should consider zoonotic malaria.

REFERENCES 1. Coatney, GR. et al (1971) The Primate Malarias. U.S. Department of Health, Education and Welfare, Bethesda. 2. Singh, B. et al. (2004) A large focus of naturally acquired Plasmodium knowlesi infections in human beings. The Lancet, 363, 1017-1024. 3. Singh, B. & Daneshvar, C. (2013) Human infections and detection of Plasmodium knowlesi. Clinical Microbiology Reviews, 26, 165-184. 4. Lee, K-S et al (2011) Plasmodium knowlesi: Reservoir hosts and tracking the emergence in macaques and humans. PLoS Pathogens 7(4),

e1002015.

tailed macaque © Anthony Sebastian Anthony © macaque tailed - ACKNOWLEDGEMENTS This study was supported by research grants from Universiti Malaysia Sarawak. We thank the Sarawak Forestry Department for permission to obtain blood samples from non-human primates. Travel funds were provided by the U.S. Defense Threat Reduction Agency’s Cooperative Biological

Engagement Program. Photograph of long of Photograph