APMEN III Annual Business and Technical meeting Kota Kinabalu Malaysia
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1 APMEN III Annual Business and Technical meeting Kota Kinabalu Malaysia May , 2011
2 MALAYSIA A A
3 Microscopy-confirmed malaria cases in 1999 Sarawak Kapit P. vivax 2,030 (64%) 414 (76%) P. falciparum 786 (25%) 20 ( 4%) P. malariae 270 ( 9%) 108 (20%) Mixed 69 ( 2%) 5 ( 1%) Total 3, Bintulu BRUNEI Limbang Miri Sarikei Sibu Kapit Kuching Samarahan Sri Aman INDONESIA
4 General characteristics P. malariae infections Chronic and asymptomatic Low parasitaemias (< 5,000 parasites/µl blood) Affects all ages Kapit Division 98% cases detected at hospital & clinics 19% cases > 5,000 parasites/ µl blood 95% adults Sandosham (1959); Garnham (1966)
5 Four microscopy-positive P. malariae blood samples from Kapit PCR Positive for: Plasmodium PCR Negative for: P. falciparum P. vivax P. ovale P. malariae Variant P. malariae? Newly emergent Plasmodium species?
6 Molecular Characterisation DNA extraction from 8 Pm blood samples PCR amplification of SSU rrna & csp genes Cloning and Sequencing Analysis of DNA Analysis of DNA sequence data
7 KH 35 KH 115 KH 33 KH 131 KH 43 KH 50 KH 107 P. knowlesi 100 KH 96 P. knowlesi W1 P. knowlesi Nuri P. fragile P. vivax 99 P. cynomolgi P. cynomolgi P. berghei P. ovale 1 P. ovale 2 P. malariae 1 P. malariae 2 P. malariae 100 P. falciparum P. reichenowi i P. gallinaceum P. lophurae P. knowlesi Neighbour-joining tree of the A-type SSU rrna gene
8 P. malariae P. malariae KH 131 KH KH 107 KH 43 KH 115 KH 96 KH 35 KH 33 P. knowlesi H P. knowlesi Nuri P. cynomolgi P. cynomolgi P. simiovale P. vivax P. simium 100 P. berghei P. yoelii P. gallinaceum P. falciparum 100 P. falciparum P. reichenowi P. knowlesi 0.05 Neighbour-joining tree of the csp gene (non-repeat region)
9 Development of P. knowlesi-specific p primers Human Plasmodium DNA Monkey Plasmodium DNA Plasmodium- specific primers P. knowlesi -specific primers
10 Kapit Hospital malaria study ( ) Nested PCR assays: BRUNEI Limbang 0 P. malariae 106 P. knowlesi, 8 Pk+Pv, 6 Pk+Pf 48 P. vivax 32 P. falciparum 8Pf+PvorPovale P. ovale Bintulu Miri Sarikei Sibu Kuching Samarahan Sri Aman Kapit 208 (Microscopy:141Pm, 42Pv, 25Pf)
11 Morphological Characteristics P. knowlesi Trophozoites Schizonts Gametocytes P. falciparum P. malariae
12 Plasmodium knowlesi Isolated 1931 in India Long-tailed macaque from Singapore Blood into Singaporean long-tailed macaques Low parasite counts Blood into Indian rhesus macaques High parasite counts: All died d Monkey blood into 2 humans Malaria with 24 hr fever peak (Pv, Pf, Po:48 hrs, Pm:72 hrs) Treatment of patients with neurosyphillis ( ) Napier & Campbell (1932) Ind Med Gaz; Knowles & Das Gupta (1932) Ind Med Gaz Sinton & Mulligan (1932) Rec Mal Survey India; Ciuca et al (1955) Ed Ac Rep Pop Roumaine
13 Human P. knowlesi infections by injection of blood since Human infections by mosquito bite?
14 Naturally-acquired human P. knowlesi infection 1965 (Chin et al, Science) American army surveyor, worked in Peninsular Malaysia Diagnosed P. falciparum & then P. malariae in USA (Silver Spring & Bethesda, Maryland) Blood injected into human volunteers (Atlanta, Georgia) Daily fever peaks (Pm = 72 hrs) Confirmed by blood into 3 rhesus monkeys Coatney, Collins, Warren & Contacos (The Primate Malarias,1971): y,, (, ).was working by night and sleeping by day.
15 P. knowlesi experiments in USA ( ) An. balabacensis Chin et al (1968) Am J Trop Med Hyg
16 Investigations of malaria as a zoonosis 1966: Pahang, Peninsular Malaysia. 1,117 human blood samples (only 28+ve: 13Pv, 11Pf, 4 unidentifiable) * Into 135 rhesus monkeys at IMR. No rhesus monkey with Pk or Pcy infections. Local LT macaques with Pk. Conclusion: Zoonotic infections extremely rare. Warren et al. (1970) Am J Trop Med Hyg
17 P. knowlesi morphology in humans Knowles & Das Gupta (1932) In general, the infection in man resembles one with P. malariae in its morphology. Garnham (1966) A P. knowlesi infection in a human being could easily pass unrecognized as such in routine laboratories, where it probably be diagnosed as P. malariae, or if rings only were present, as P. falciparum. Chin et al (1968) In the detection of human infections under usual field conditions, evaluation of blood films or clinical symptoms adequate for differentiation between P. knowlesi and the normal parasites of man would be impossible.
18 Plasmodium knowlesi : Historical Aspects & Epidemiology Introduction Discovery of human P. knowlesi infections in Kapit Division, Sarawak ( ) History of P. knowlesi Human P. knowlesi infections Distribution Differences between P. knowlesi & P. malariae
19 Nested PCR assay results for Kapit ( ) Total P. knowlesi (Total Plasmodium spp.) LIMBANG LAWAS 385 (765) BETONG Singh et al (2004) Lancet; Cox-Singh et al (2008) Clin Infect Dis; Daneshvar et al (2009) Clin Infect Dis
20 Nested PCR assay results for Sarawak ( ) Total P. knowlesi (Total Plasmodium spp.) LIMBANG LAWAS 99 (157) 193 (248) 84 (116) 5 (273) 385 (765) BETONG Singh et al (2004) Lancet; Cox-Singh et al (2008) Clin Infect Dis; Daneshvar et al (2009) Clin Infect Dis
21 Nested PCR assay results for Sarawak ( ) Total P. knowlesi (Total Plasmodium spp. tested) 63 (256) 5 (9) LIMBANG LAWAS 17 (22) 10 (36) 879 Pk (2016 Plasmodium spp) 5 (273) 99 (157) 84 (116) 3 (117) 193 (248) 385 (765) BETONG 6 P. malariae by PCR: 0 (72) 3 (139) 12 (19) All imported: 4 PNG&2 Irian Jaya Singh et al (2004) Lancet; Cox-Singh et al (2008) Clin Infect Dis; Daneshvar et al (2009) Clin Infect Dis
22 Reported human P. knowlesi infections Jiang et al, 2010 ** Zhu et al., 2006 * Putaporntip et al, 2009 ** Jongwutiwes et al, 2004 Berry et al, 2011 * * * * * * * van den Eede et al, 2010 ** * Luchavez et al, 2008 Cox Singh et al, 2008 BRUNEI Cox Singh et al, 2008; Anderios et al, 2008; Vythilingam et al 2008 * Barber et al, 2011 * Single case * Chin et al, 1965 Singh et al 2004; Cox Singh et al 2008; Daneshvar Fong et al, 1971 * et al 2009; Singh & Daneshvar, 2010 * 3-5 cases * 32 cases (Pre PCR) ** ** ** ** 77 cases 230 cases 879 cases Ng et al, 2008 Ong et al, 2009 Wong et al, 2010 ** ** Figtree et al, 2010; Sulistyaningsih et al, 2010
23 Anopheles leucosphyrus group distribution Distribution of human P. knowlesi cases? **? * * * * *? * * * * *?? BRUNEI * **? * *? * **?? M. fascicularis distribution
24 Introduction Plasmodium knowlesi : Historical i Aspects & Epidemiology i Human P. knowlesi infections in the Kapit Division ( ) 2003) History of P. knowlesi Human P. knowlesi infections Distribution Zoological, l Entomological l & Molecular l studies
25 Natural Hosts of P. knowlesi Macaca fascicularis (Long-tailed macaque) Macaca nemestrina (Pig-tailed macaque) Presbytis melalophos (Banded leaf monkey) Peninsular Malaysia Eyles, et al (1962) Med J Malaya
26 P. knowlesi in macaques Pig-tailed 1960s Long-tailed 1953, 1960s, , 1961 Long-tailed 1931, 2011 Knowles & Das Gupta (1932) Ind Med Gaz; Garnham (1966) Malaria Parasites; Vythilingam et al (2010) Parasites & Vectors; Wong et al (2011) Vector-Borne Zoon Dis Lambrecht et al (1961) Nature; Tsukamoto et al, (1978) Nagasaki Uni
27 108 wild macaques: 82 long-tailed 26pg pig-tailed aed 26 locations in the Kapit Division PCR detection for P. knowlesi, P. inui, P. coatneyi, y, P. cynomolgi, g, & P. fieldi Lee et al (2011) PLoS Pathogens
28 Malaria parasites of macaques from Kapit 101 of 108 (93.5%) wild macaques malaria +ve: 5 Plasmodium species (Pk, Pfi, Pcy, Pin, Pco) 71 (86.6%) of 82 long-tailed Pk +ve 13 (50%) of 26 pig-tailed Pk +ve Lee et al (2011) PLoS Pathogens
29 Mitochondrial (mt) genome of P. knowlesi bp Genes: cox III cox I cyt b LSU rrna SSU rrna 37 different mt sequences Humans (n=25) : 17 different sequences Monkeys (n=11) : 23 different sequences Lee et al (2011) PLoS Pathogens
30 P. knowlesi mt DNA haplotype network Lee et al (2011) PLoS Pathogens
31 Demographic history of P. knowlesi Population expansion 30,000-40,000 yrs ago TMRCA: P. knowlesi 98, ,000 yrs ago* P. vivax 53, ,000 yrs ago** P. falciparum 50, ,000 yrs ago*** Ancestral parasite Macaques colonised Asia >5 million yrs ago # *Lee et al (2011) PLoS Pathogens **Escalante et al (2005) PNAS; Mu et al (2005) Mol Biol Evol ***Joy et al (2003) Science; Krief et al (2010) PLoS Pathogens # Ziegler et al (2007) Mol Phylogenet Evol
32 Vector of P. knowlesi in Kapit: Anopheles latens Feed outdoors on humans & macaques. Feed after dusk. Forest/forest fringe mosquito. Vythilingam et al (2006) Trans Roy Soc Trop Med Hyg; Tan et al (2008) Malaria J
33 P. knowlesi transmission in Kapit Mosquito behaviour High Pk prevalence. Low incidence. id Mainly adults. No clustering of cases. Csp alleles & Mt haplotypes shared between monkeys & humans. No mtdna lineage associated with either host.
34 P. knowlesi malaria in Kapit, Sarawak Zoonosis since ancient times
35 Who are at risk? Farmers, hunters, logging camp workers, army personnel & travelers to forest/forest fringe Habitat of macaques and mosquito vectors
36 Human knowlesi malaria Widely distributed in South East Asia (incidence??) Ancestral parasite, misdiagnosed since microscopically P. knowlesi = P. malariae. Ancient Zoonosis macaques reservoir hosts. Vector restricted (An. leucosphyrus group). Spectrum of disease (potentially fatal).
37 Challenge for malaria elimination: Huge reservoir of P. knowlesi in macaques in forests
38 Future of P. knowlesi?? Changes in ecology (logging), vector types/ vector behaviour + increase in human population: Humans as preferred hosts??
39 Malaria Research Centre UNIMAS Tan Cheong Huat Angela Siner Sophia Lau Siti Khatijah Paul Divis Sunita Shamsul Lau Hui Chong Roynston Julin Lee Kim Sung Cyrus Daneshvar Janet Cox-Singh
40 Collaborators ato (Epidemiology ogy Studies) London School of Hygiene & Tropical Medicine, UK. Sarawak Health Dept., Malaysia. Sabah Health Dept., Malaysia. Pahang Health Dept., Malaysia. Inst. Medical Research, Malaysia. Dave Conway Asmad Matusop Shanmuga Ratnam Hasan Abdul Rahman Indra Vythilingam BPRC, The Netherlands. Alan Thomas
41 Acknowledgements Kevin Palmer. Staff of District Hospitals, Polyclinics & Malaria Control Units in Sarawak. Sarawak Forestry Department & Sarawak a a Biodiversity Council Funds Wellcome Trust, United Kingdom. Malaysian Ministry of fs Science & Technology. Universiti Malaysia Sarawak.
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