SIMIAN MALARIA IN SIMIAN MALARIA IN MALAYSIA WITH SPECIAL MALAYSIA WITH SPECIAL

REFERENCE TOREFERENCE TOPLASMODIUM KNOWLESIPLASMODIUM KNOWLESI

Indra Vythilingam, Parasitology Unit, Institute for Medical Research,

Kuala Lumpur. Malaysia

INTRODUCTIONINTRODUCTION

1st natural infection of P. knowlesi in humans was reported from Pahang –1965

2nd case was reported from Johore in 1971.

Plasmodium knowlesi

Macaca fascicularis , M. nemestrina, Pryesbytis melalophos

Vectors – Peninsular MalaysiaAnopheles hackeri•natural vector of P. knowlesi•highly zoophagic

Anopheles cracens (as An. balabacensis)•vector of P. inui and P. cynomolgi

Anopheles latens (as An. leucosphyrus)

REF: Wharton et al 1961, 1962; Cheong et al 1965

• vector of P. inui

INTRDOUCTIONINTRDOUCTION

• In 2004 Singh et al reported a large focus of P. knowlesi in Sarawak Malaysian Borneo

(Lancet 2004)

Is P. knowlesioccurring only in Malaysian Borneo ?

GENERAL OBJECTIVESGENERAL OBJECTIVES

To study simian malaria in humans and non human primates

To elucidate the vectors of simian malaria

MATERIALS & METHODSMATERIALS & METHODS

Samples received since July 2005

Extraction of DNA from whole blood or blood film

Nested PCR following protocol of Singh et al 2004

Species specific primers used in the Species specific primers used in the NestedNested--PCR AssayPCR Assay

rPLU1&5rPLU1&5

Human malaria Simian malaria

P. falciparum P. vivaxP. malariae

P. knowlesi

Table 1 Results of blood samples of malaria obtained by microscopy and PCRJuly 2005- December 2007

Cases detected by microscopy Cases detected by PCR

PCR results

Pf Pv Pm Pf+Pm Pm+Pv

Pf 4 1 1 6Pv 6 3 1 1 11Pm 6 6Pk 2 1 58 61Pf+Pm 1 1Pf+Pk 1 1Pv+Pk 1 1 2Pk+Pm 2 2Total 6 9 73 1 1 90

CASES BY MICROSCOPY AND PCR JULY 2005-DEC. 2007

MALARIA CASES BY MICROSCOPY

PCR Pf Pv Pm Pf+Pv Pm+Pv Pf+Pm Negative Cases by PCR

Pf 2 2 1 5

Pv 4 12 1 5 22

Pm 2 2

Pk 3 40 2 1 46Pf+Pv 1 1

Pf+Pk 1 2 1 1 5

Pv+Pk 12 1 1 1 15

Pm+Pk 1 1

Pv+Pf+Pk

1 1

Pf+Pm+Pk

1 1

Total 6 4 71 5 2 4 7 99

2008 Jan- Aug.

CASES OF P. KNOWLESI IN P. MALAYSIA

NON HUMAN PRIMATESNON HUMAN PRIMATES

Species specific primers used in theNested-PCR Assay

rPLU1&5

Simian malaria

P. knowlesiP. coatneyiP. inuiP. fieldiP. cynomolgiP. fragile

RESULTS OF MONKEY RESULTS OF MONKEY SAMPLESSAMPLES

Locality Samples collected

BFMPPositive (%)

PCR PositiveGenus (%)

Selangor 60 0 0 (0)Kuala Lipis

104 102 (98.1) 95 (91.3)

Temerloh 11 11 (100) 11 (100)Kuala Lumpur

29 2 (6.9) 2 (6.9)

Total 214 115 (56.7) 108 (53.2)

Malaria Species in Monkeys by Malaria Species in Monkeys by PCRPCR

0

5

10

15

20

25

P.knowlesi P.cynomolgi P.coatneyi P.inui P.k+P.cy P.k+P.cot P.cot+P.cy P.k+P.cot+P.cy

% P

ositi

ve

MOSQUITO COLLECTIONMOSQUITO COLLECTIONSTUDY SITESTUDY SITE

RESULTSRESULTSPredominant Anopheles species in study site Aug 2007-Aug 2008

Anopheles speciesAnopheles species MelaMela Sg Sg UlarUlar

Total (%)Total (%)

BLCBLC MBTMBT BLCBLC

An.aconitusAn.aconitus 00 00 66 6 (0.46)6 (0.46)

An. barbirostris An. barbirostris grgr 1010 1111 44 25 (1.93)25 (1.93)

An. cracens 186 77 584 847 (65.55)An. hyrcanus An. hyrcanus grgr 34 15 1 50(3.86)

An. kochi 2 51 1 54 (4.17)

An. maculatus 99 1 163 263 (20.35)An. An. phillippinenssisphillippinenssis

66 00 66 12 (0.92)12 (0.92)

An. pujutensisAn. pujutensis 00 11 00 1 (0.07)1 (0.07)

An. separatusAn. separatus 22 11 55 8 (0.61)8 (0.61)

An. tesselatusAn. tesselatus 77 1010 55 22 (1.70)22 (1.70)

An. umbrosusAn. umbrosus 11 11 00 2 (0.15)2 (0.15)

An. vagusAn. vagus 11 11 00 2 (0.15)2 (0.15)

TotalTotal 348348 169169 775775 12921292

RESULTSRESULTS

To verify species of Plasmodium found, cloning and sequencing of the circumsporozoite protein (csp) genes were conducted…

MKEL3 EU687467

Mo62 2 EU821335

M197 EU821336

KH115 AH013337

KH43 AH013333

KH50 AH013334

KH107 AH013336

MPG45 EU708437

MPHG38 EU687468

KH35 AH013332

MPRK13 EU687469

Pknowlesi K00822

MSel26 EU687470

Pknowlesi M11031

Pcoatneyi AY135360

Pcynomolgi M15104

Psimiovale U09765

Psimium L05068

Pvivax M34697

Pmalariae U09766

Pmalariae J03992

Pfalciparum K02194

Pvinckei lentum AF162331

100

98

91

81

85

71

99

96

70

100

100

81

0 05

Phylogenetic tree based on the non-repeat region of the circumsporozoite (csp) genes of malaria parasites produced by the neighbor-joining method. Figures on the branches are bootstrap percentages based on 1000 replicates and only those 70 and aboveshown.

Vythilingam et al 2008Parasites & Vectors 1:26

MPG45 EU708437KH50 AH013334KH115 AH013337KH107 AH013336KH43 AH013333M197 EU821336MKEL3 EU687467Mo62 2 EU821335

MPHG38 EU687468KH35 AH013332Pknowlesi K00822MSel26 EU687470Pknowlesi M11031

MPRK13 EU687469Pcoatneyi AY135360Pinui

Psimiovale U09765Pcynomolgi M15104

Psimium L05068Pvivax M34697

Pmalariae J03992Pmalariae U09766

Pfalciparum K02194Pvinckei lentum AF162331

100

100

100

100

100

100

96

100

100

100

96

72

82

100

100

0 05

Phylogenetic tree based on the non-repeat region of the circumsporozoite (csp) genes of malaria parasites produced by the Bayesian method. Figures on the branches are the posterior probabilities from the Bayesian analysis.

Vythilingam et al 2008Parasites & Vectors 1:26

DISCUSSIONDISCUSSION

KnowlesiKnowlesi malaria occurs in this regionmalaria occurs in this region(Jongwutiwes et al 2004, Zhu et al 2006, (Jongwutiwes et al 2004, Zhu et al 2006, Cox Cox ––Singh Singh et alet al 2008, Ng 2008, Ng et alet al 2008, 2008, Luchavez Luchavez et alet al 2008, Vythilingam 2008, Vythilingam et alet al2008)2008)With better molecular techniques one can With better molecular techniques one can differentiate between differentiate between P. malariaeP. malariae and and P. P. knowlesiknowlesi

DISCUSSIONDISCUSSION

Chin Chin et alet al 1968: Experimental transmission 1968: Experimental transmission studiesstudies

Due to declining antiDue to declining anti--plasmodial immunity plasmodial immunity in humansin humans

Most cases occurring in malaria free areas Most cases occurring in malaria free areas not subject to control activities not subject to control activities

DISCUSSIONDISCUSSION

This study has shown a link between This study has shown a link between AnAn. . cracenscracens, humans and monkeys, humans and monkeys

Early workers in Malaysia felt that simian Early workers in Malaysia felt that simian malaria transmission to humans will be malaria transmission to humans will be remoteremote

Development and deforestationDevelopment and deforestation--macaques close to human habitationmacaques close to human habitation

DISCUSSIONDISCUSSION

In the rain forests of Southeast Asia In the rain forests of Southeast Asia natural hosts of natural hosts of P. knowlesiP. knowlesi –– macauqes macauqes abound so does the abound so does the An. leucosphyrusAn. leucosphyrusgroup of mosquitoesgroup of mosquitoes

Current control strategies are not going to Current control strategies are not going to work. Zoonotic transmission has to be work. Zoonotic transmission has to be widely publicized widely publicized

CONCLUSIONCONCLUSION

Fifth human malaria parasite has been established and thus new strategies for malaria control should be considered especially in the elimination of malaria

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

Grant from the NIH, MOH MalaysiaGrant from the NIH, MOH Malaysia

Staff from Vector Borne Disease Control Staff from Vector Borne Disease Control Program Kuala Lipis, PahangProgram Kuala Lipis, Pahang

Wildlife Department, MalaysiaWildlife Department, Malaysia