IDENTIFICATION OF VECTORS OF Plasmodium knowlesi AND OTHER MALARIA PARASITES, AND STUDIES ON THEIR

BIONOMICS IN KAPIT, SARAWAK, MALAYSIA

TAN CHEONG HUAT

A thesis submitted in fulfillment of the requirements for the degree of Master of Science

Faculty of Medicine and Health Sciences UNIVERSITI MALAYSIA SARAWAK

2008

Dedicated to Mdm. Lau Kah Gan ...

... my first teacher

... my inspiration

... my best friend

... my mother

ACKNOWLEDGEMENTS

I owe a debt of gratitude to Prof. Balbir Singh, my supervisor; and Dr. Indra Vythilingam, my co- supervisor; for enabling me to partake in the search for the vector of P. knowlesi in Sarawak... their individual expertise and excellent supervisions during the research phase of my study have allowed me to overcome numerous obstacles in the field of medical entomology and molecular malariology. I would also like to acknowledge their patience, guidance and relevant suggestions during the preparation of this manuscript.

I am also indebted to the following people, without them this work would not have been possible or otherwise be meaningless...

... Mr. Asmad Matusop of the Sarawak State Department of Health and his staff at HQ and at the

Kapit Vector Control Office for their cooperation and providing the necessary logistics support that ensured the success of the entomological field survey.

... Prof. Janet Cox-Singh, for her comments and suggestions during my research work conducted at

the Malaria Research Centre, her persistent reminder in pursuing academic and research excellence has taught me to become a more conscientious student of science...

... Sunita, David, Angela, Hui Chong, Siaw Shi, Siti, Paul, Grace my colleagues at the MRC, and Yi

Ching and Adela my colleague at IMR for their infectious friendship, warmness and camaraderie which have allowed me to cope with the stress and loneliness of being away from my family, their willingness to share their scientific ideas and prowess have enabled me to grasp new skills and knowledge especially in the field of molecular biology.

... Uncle Chan and Uncle Maniam, for unselfishly imparting their invaluable techniques learned from

decades of experience in various aspects of medical entomology, their fatherly advice has taught me humility and patience in my chosen field.

... Avi, for her devotion as a loving and patient wife, and for enduring the difficulty of the dual roles of

being a father and a mother to our children during the period of my pursuit to obtain further knowledge.

... Mary Anne and Allen Michael, my two kulets... my enhancers and source of strength, their

youthful innocence yet matured understanding, that their papi cannot be with them as often as they

want, has inspired me to work hard.

nn)' Mother, for her resolute understanding, unconditional support and unfaltering love has motivate

me to strive harder in order to achieve my goals... and for which this hard work is dedicated to.

And lastly I would like to express my sincere gratitude and devotion...

... to HIM, for his constant presence and guidance and for granting me the strength and wisdom to learn and täce new challenges.

This research was funded by the Malaysian Ministry of Health Research Grant. Norman Medical

Foundation Research Grant, The Wellcome Trust Collaborative Research Initiative Grant and

studentship from the Malaysian Ministry of Science and Inovation (MOSTI). I would also like to

express my sincere appreciation to Mr. Julian Lim of Medigene Sdn. Blid. fir his kindness and

uenerosit v.

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TABLE OF CONTENTS

ACKNOWLEDEMENTS TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES ABBREVIATIONS ABSTRACT ABSTRAK

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111

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CHAPTER ONE: General Introduction and Literature Review 1.1 Malaria 1

1.1.1 Life cycle of Plasmodium 3 1.2 Non-human primate malaria parasites 7 1.3 Simian malaria infections in man 7 1.4 Vectors of simian malaria parasites 10 1.5 Vectors of human malaria parasites in Sarawak 12 1.6 Detection and identification of malaria parasites in Anopheles vectors 18 1.7 Objectives of the Study 20

CHAPTER TWO: Malaria entomological survey in the Kapit Division of Sarawak 2.1 Introduction 23 2.2 Methodology 24

2.2.1 Brief description of Kapit Division 24 2.2.2 Mosquito collection sites and methods for collecting adult mosquitoes 25

2.2.2.1 Human bare-leg catch method 27 2.2.2.2 Monkey-baited net trap 27

2.2.3 Processing of mosquitoes collected 31 2.2.3.1 Identification of Anopheles mosquito based on external 31

morphology 2.2.3.2 Dissection of mosquitoes 31

2.2.3.2.1 Dissection of the stomach and ovaries of anopheline 34

mosquitoes 2.2.3.2.2 Dissection of the salivary glands of anopheline 36

mosquitoes 2.2.4 Determination of entomological indicators in relation to disease 41

transmission 2.2.4.1 Man-biting rate (MBR) 2.2.4.2 Parity rate (PR) 2.2.4.3 Sporozoite rate (S) 2.2.4.4 Annual Entomological Inoculation Rate (EIR) 2.2.4.5 Longevity and infectivity 2.2.4.6 Vectorial capacity (VC)

2.2.5 Statistical analyses

41 42 42 43 43 44 44

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2.3 Results 45 2.3.1 Spatial distribution of Anopheles species collected in different ecotypes in 45

Kapit 2.3.2 Attractiveness of Anopheles species to different primate hosts 47 2.3.3 MBR of Anopheles species caught in different ecotypes in Kapit 51 2.3.4 Monthly distribution of major anophelines caught in three different 53

ecological sites in Kapit 2.3.5 Biting cycles of major anophelines caught in three different ecological 58

sites in Kapit 2.3.6 Infection rates of Anopheles collected in Kapit 58 2.3.7 Parous rates, probability of daily survival, probability of vectorial capacity 65

of An. latens, An. watsonii and An. donaldi 2.4 Discussion 70

CHAPTER THREE: Detection and identification of malaria parasites from infected Anopheles by the nested polymerase chain reaction (PCR)

assay 3.1 Introduction 89 3.2 Materials and methods 90

3.2.1 Extraction of DNA from the salivary glands and midguts of anophelines 90 positive for the malaria parasites

3.2.2 Extraction of DNA from the salivary glands of anophelines negative for 91 malaria parasites by dissection

3.2.3 Detection of malaria parasites by nested PCR assay 92 3.2.3.1 Primers used for nested PCR assay 92 3.2.3.2 Nest 1 amplification reaction and PCR cycling parameters 92 3.2.3.3 Nest 2 amplification reaction and PCR cycling parameters 95 3.2.3.4 Preparation of agarose gel 95 3.2.3.5 Running of samples and DNA marker 97 3.2.3.6 Visualization of PCR products 97

3.2.4 Statistical analyses 97 3.3 Results 98

3.3.1 Detection of malaria parasites in An. lutens by nested PCR assay 98

3.3.2 Detection of malaria parasites in An. watsonii by nested PCR assay 100

3.3.3 Detection of malaria parasites in An. vunus, An. kokhani, An. muculutus 103

s. I. and An. tesselutu. s by nested PCR assay 3.4 Discussion 103

CHAPTER FOUR: Characterization of the circumsporozoite protein (csp) gene of simian malaria parasites from Anopheles latens

4.1 Introduction 109 4.2 Materials and methods 111

4.2.1 Extraction of DNA from the salivary glands and midguts of anophelines 111

positive for the malaria parasites 4.2.2 PCR amplification of the csp genes 112 4.2.3 Cloning of PCR products 112 4.2.4 Preparation of glycerol stocks 114

IV

4.2.5 Extraction of plasmid DNA 116 4.2.6 EcoR I digestion of Plasmid DNA 117 4.2.7 Sequencing of the csp gene 117 4.2.8 Analysis of DNA 118 4.2.9 Phylogenetic Analysis 118

4.3 Results 122 4.3.1 Amplification, cloning and sequencing of the csp genes 122 4.3.2 Phylogenetic analyses of the csp genes 122 4.3.3 Polymorphisms of the non-repetitive regions of the csp gene 130

4.3.3.1 P. knowlesi clones 130 4.3.3.2 P. inui clones 133 4.3.3.3 P. inui-like clones 133 4.3.3.4 P. coatnevi clones 137 4.3.3.5 P. fieldi and P. simiovale clones 141

4.3.4 Polymorphisms within the Region I, Region Il-plus and the central tandem 141 repeat region

4.3.4.1 P. knowlesi clones 141 4.3.4.2 P. inui clones 145 4.3.4.3 P. inui-like clones 149 4.3.4.4 P. coatnevi clones 157 4.3.4.5 P. fieldiand P. simiovale clones 159

4.4 Discussion 159

CHAPTER FIVE: Summary and indications for future research

5.1 Summary 5.2 Indications for future research

170 176

REFERENCES 179

APPENDICES 194

A: The total number of each anopheline species caught at the three different 195

study sites (farm, longhouse and forest) in Kapit, Sarawak from May 2005 to April 2006. The total number parous and nulliparous mosquitoes, those that were not dissected but were subjected to the nested-PCR assay (head and thorax) and those that were pinned for taxonomic references are also shown

B: Monthly biting cycles (1800h to 0600h) of each anophelines species caught 219

at the three study sites (farm, longhouse and forest) in Kapit, Sarawak from May 2005 to April 2006.

C: DNA sequences of the circurnsporzoite gene 245

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LIST OF FIGURES

Figure 1.1 Map of Sarawak 4

Figure 1.2 Life cycle of human malaria parasite 5

Figure 2.1 Satellite image of Kapit District showing the different 26 mosquito collection sites and Kapit town

Figure 2.2 Human bare-leg catch, picture taken at the farm 28

Figure 2.3 Glass vial used in mosquito collection 29

Figure 2.4 Monkey baited-net trap consists of a large modified mosquito net, 30 enclosing a metal cage holding 2 monkeys as bait.

Figure 2.5 Monkey baited-net traps set-up at different elevations 32

Figure 2.6 Collection of mosquitoes found resting on the net 33

Figure 2.7 Dissection of the midgut and ovary of anopheline mosquito 35

Figure 2.8 Dissected stomach of An. latens 37

Figure 2.9 Dried Anopheles ovary 38

Figure 2.10 Dissection of the salivary glands of anopheline mosquito 39

Figure 2.11 Dissected salivary glands of an anopheline mosquito 40

Figure 2.12 The monthly biting rate of An. latens and An. watssonii in the forest 54 from May 2005 until April 2006

Figure 2.13 The monthly biting rate of An. latens, An. donuldi, An. wnus, and 55 An. kokhani in the farm from May 2005 until April 2006

Figure 2.14 The monthly biting rate of An. latens, An. donaldi and An. vanus at 56

the longhouse from May 2005 until April 2006

Figure 2.15 Nocturnal biting cycles of An. latens and An. tiiatsonii caught in the 59 forest

Figure 2.16 Nocturnal biting cycles of An. latens, An. donaldi, An. i'anus, and 60 An. kokhani in the farm

Figure 2.17 Nocturnal biting cycles ofAn. la/ens, An. donahli and An. vanus at 61 the longhousc (outdoor)

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Figure 2.18 Nocturnal biting cycles of An. latens, An. donaldi and An. vanus at 62 the longhouse (indoor)

Figure 2.19 Sporozoites from the salivary glands of An. latens mosquito 63

Figure 2.20 Numerous oocysts (arrow) found on the midgut of An. watsonii 64 mosquito

Figure 2.21 The presence of a broad white band covering the tibio-tarsal joint of 72 hind legs are the distinguishing characteristics of the members of the Leucosphyrus group

Figure 2.22 Map showing the current distribution of known members of the 73 Leucosphyurus group

Figure 4.1 Schematic diagram of the circumsporozoite protein (csp) gene of 110 malaria parasites

Figure 4.2 Size variations in the csp gene of Plasmodium species in different 123 clones derived from isolate M-H8

Figure 4.3 Phylogenetic tree based on the non-repeat region of the csp genes of 125 Plasmodium sp. produced by the neighbour-joining method.

Figure 4.4 Figure 4.4: Phylogenetic tree based on the non-repeat region of the 128

csp genes of Plasmodium sp. produced by the maximum-parsimony method with heuristic search method.

Figure 4.5 Phylogenetic tree based on the non-repeat region of the csp genes of 129 Plasmodium sp. produced by the maximumlikelihood method

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LIST OF TABLES

Table 1.1 Lists of primate malaria parasites, its natural hosts, periodicity, and 2 distributions

Table 1.2 Lists of potential indigenous vectors that are found susceptible to transmit 13 different species of simian malaria parasites under experimental conditions

Table 2.1 Anopheline mosquitoes collected in different collection sites in Kapit 46 District.

Table 2.2 Species of anopheline mosquitoes attracted to either human or monkey 48 hosts in the forest.

Table 2.3 Numbers of An. latens caught at different height level in relation to the 49 time of collection in the MBT in the forest.

Table 2.4 Numbers of An. watsonii caught at different height level in relation to the 50 time of collection in the MBT in the forest.

Table 2.5 Mean MBR of various anopheline species in different ecotypes in Kapit 52 District (May 2005 to April 2006).

Table 2.6 List of An. latens, An. watsonii and An. donaldi that were found positive 66 by dissection

Table 2.7 Sporozoite and oocyst rates of anophelines caught in the forest and farm 67

ecotypes in Kapit

Table 2.8 Parous rate, probability of daily survival, life expectancy in days, 69 probability of transmission of malaria parasite, and the vectorial capacity of An. latens, An. watsonii and An. donaldi caught in different ecological sites at Kapit.

Table 3.1 Oligonucleotide sequences used in the amplifications of the SSUrRNA 93

genes of Plusinodiu, n sp.

Table 3.2

Table 3.3

Table 3.4

Table 3.5

Components of a single PCR amplification reaction (nest 1) 94

Components of a single PCR amplification reaction (nest 2). 96

Malaria parasites detected in An. lutens by nested PCR assay 99

Plu. Sinotlium species specific sporozoite from An. latens caught in the 101 forested and farming ecotypes in the District of Kapit, Kapit Division, Sarawak.

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Table 3.6 Malaria parasites detected in An. watsoni (W) and An. donaldi (D) by 102 nested PCR assay

Table 3.7 Total number of nested PCR assays performed for An. vanus, An. kokhani, 104 An. maculatus s. 1. and An. tesselatus.

Table 4.1 Components of a single PCR amplification reaction of the csp gene 113

Table 4.2 Components of a single PCR amplification reaction for the analysis of 115 transformants

Table 4.3 Oligonucleotides sequence used in the sequencing of csp genes of P. 119 knowlesi, P. inui, P. inui-like, P. coutnevi, and P. fieldi-P. simiovale

Table 4.4 Sequences of the csp gene of various Plasmodium species used in the 121 phylogenetic analyses

Table 4.5 Summary of number of E. coli transformants analyzed by PCR, 124 approximate insert sizes and codes for selected complete sequences of Plasmodium csp clones isolated from 12 An. latens from the Division of Kapit

Table 4.6 Gene polymorphisms based on the 453 nucleotide residues encoding the 131 non-repeat regions of the esp gene of P. knowlesi malaria parasites obtained from An. latens (M, in bold), humans (KH) and long-tailed (LT) macaques caught in the Kapit Division

Table 4.7 Percent divergence of the non-repeat regions of the csp sequence between 132

of P. knowlesi clones isolated from An. latens (M), long-tailed (LT) macaques and humans (KH) of the Kapit Division calculated with the Kimura 2 parameter method using transitions and traversions

Table 4.8 Gene polymorphisms based on the 453 nucleotide residues encoding the 134

non-repeat regions of the csp gene of P. inui malaria parasites obtained from An. lawns (M, in bold) and pig-tailed (PTK) macaque caught in the Kapit Division

Table 4.9 Percent divergence of the non-repeat regions of the csp sequence between 135

of P. inui clones isolated from An. latens (M), and pig-tailed (PTK)

macaque of the Kapit Division calculated with the Kimura 2 parameter method using transitions and traversions

Table 4.10 Gene polymorphisms based on the 453 nucleotide residues encoding the 136

non-repeat regions of the csjp gene of P. inui-like malaria parasites obtained from An, laiens (M, in bold) and long-tailed (LT) macaques caught in the Kapit Division.

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Table 4.11 Percent divergence of the non-repeat regions of the csp sequence between 138 of P. inui-like clones isolated from An. latens (M), and long-tailed (LT) macaques of the Kapit Division calculated with the Kimura 2 parameter method using transitions and traversions.

Table 4.12 Gene polymorphisms based on the 453 nucleotide residues encoding the 139 non-repeat regions of the csp gene of P. coatneyi malaria parasites obtained from An. latens (M, in bold) and long-tailed (LT) macaques caught in the Kapit Division

Table 4.13 Percent divergence of the non-repeat regions of the csp sequence between 140 of P. coatneyi clones isolated from An. latens (M), and long-tailed (LT) macaques of the Kapit Division calculated with the Kimura 2 parameter method using transitions and traversions

Table 4.14 Gene polymorphisms based on the 453 nucleotide residues encoding the 142 non-repeat regions of the csp gene of P. fieldi and P. simiovale malaria parasites obtained from An. latens (M, in bold) and long-tailed (LT) macaque caught in the Kapit Division

Table 4.15 Percent divergence of the non-repeat regions of the csp sequence between 143

of P. _fieldi

and P. simiovale clones isolated from An. latens (M), and long-tailed (LT) macaques of the Kapit Division calculated with the Kimura 2 parameter method using transitions and traversions

Table 4.16 Comparison of the amino acid sequences of region I and the region Il-plus 144

of the csp gene for P. knowlesi H and Nuri strain and isolates from An. latens (M), humans (KH), long-tailed macaques (LT).

Table 4.17 Comparison of the amino acid motifs, the sequence of the motifs and sizes 146

of tandem repeat regions and total length of the csp genes for P. knowlesi isolates from An. latens (M), humans (KH), long-tailed macaques (LT), strains H and Nuri

Table 4.18 Comparison of the amino acid motifs, the sequence of the motifs and sizes 150

of tandem repeat regions and total length of the csp genes for P. inui isolates from An. latens (M), and pig-tailed macaque (PT)

Table 4.19 Comparison of the amino acid sequences of region I and the region 11-plus 153

of the c.,; p genes for P. inui and P. inui-like isolates from An. latens (M),

and long-tailed macaques (LT)

Table 4.20 Comparison of the amino acid motifs, the sequence of the motifs and sizes 154

of tandem repeat regions and total length of the csp genes for P. inui-like isolates from An. lawns (M), and long-tailed macaques (LT)

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Table 4.21 Comparison of amino acid sequences of region I and region Il-plus of the 158 csp gene for P. coatneyi Hackeri strain and isolates from An. latens (M), long-tailed macaques (LT)

Table 4.22 Comparison of the amino acid motifs, the sequence of the motifs and sizes 160 of tandem repeat regions and total length of the csp genes for P. coatneyi isolates from An. latens (M), and long-tailed macaques (LT)

Table 4.23 Comparison of amino acid sequences of region I and region II-plus of the 161 csp genes for P.. fieldi, P. simiovale isolates and those isolated from An. latens (M), long-tailed macaques (LT), and P. fieldi

Table 4.24 Comparison of the amino acid motifs, the sequence of the motifs and sizes 162 of tandem repeat regions and total length of the csp genes for P.

,f eldii isolates from An. latens (M), and long-tailed macaques (LT)

Table 4.25 Comparison of malaria parasites from twelve An. latens identified by the 164 nested PCR assay and characterization of the csp gene

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ABBREVIATIONS

An Anopheles bp base pair csp circumsporozoite protein DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate EDTA ethylenediaminetetraacetic acid EIR entomological inoculation rate GC gonotrophic cycle LE life expectancy MgCl2 magnesium chloride min minute ml millilitre mm millimolar ML maximum likelihood MP maximum parsimony NJ neighbour-joining PR parous rate p probability of daily survival Pct Plasmodium coatnevi Pcy Plasmodium cvnomologi Pf Plasmodium, falciparum Ptld Plasmodium fieldi Pfr Plasmodium fragile Pi Plasmodium inui Pk Plasmodium knowlesi Pm Plasmodium malariae Po Plassmdoium ovule Pv Plasmodium vivax PCR polymerase chain reaction Rpm round per minute S sporozoite rate SSU rRNA small sub-unit ribosomal ribonucleic acid sec second TBE tris-borate EDTA VC vectorial capacity WHO World Health Organization

µl microliter µM micromolar

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ABSTRACT

The aims of this project were to identify the vectors of P. knowlesi and other malaria parasites

in Kapit and to study their bionomics. A one year entomological study was undertaken with

three areas chosen as adult mosquito collection sites; a longhouse, where indoor and outdoor

collections were undertaken, a farm and the forest, where an elevated platform for monkey-

bait trap was also built. The methods of collection were the bare-leg catch and the monkey

baited-net trap. Four thousand eighty one mosquitoes were caught, comprising 2,709

anophelines and 1,383 culicines. The 12 anopheline species caught were Anopheles latens

(43.8% of total caught), An. donaldi (28.9%), An. mutsonii (10.2%), An. vanus (7.1%), An.

kokhani (5.3%), An. tesselatus (2.4%), An. maculatus s. l. (1.0%), An. pujutensis (0.8%), An.

mucarthuri (0.8%), An. barbirostris (0.3%), An. introlatus (0.1%), and An. roperi (0.1%).

The number of An. latens caught at the three sites varied with the most (49.9%) being caught

in the forest, followed by the farm (39.3%) and the longhouse (10.8%). Of the 129 An. latens

caught in the longhouse, 91 (71.1 %) were found biting outdoors while 37 (28.9%) were biting

indoors. Although An. lawns were present all year round, the peak man-biting rate of the

mosquito in the forest was in April, May, October and November, while at the farm it was in

May, June, October, November and December. During these periods the average man-biting

rate was observed to exceed more than five bites/man/night (b/m/n). In the longhouse the

peak man-biting rate was in October (3.4 b/m/n), while for the rest of the months, the man-

biting rate in the longhouse was less than one b/m/n. Anopheles lawns were found to be simio-

anthropophagic mosquitoes, and preference for monkeys was not evident. In the monkey-

baited net trap, significant numbers of An. lutens (202 or 96.6%) were caught at higher

elevations (>3 m) as compared to the ground level (7 or 3.3%). Anopheles lawns were also

found to exhibit spatial behavioural heterogeneity. In the forest, the peak biting time of An.

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latens was between 1800h and 2000h and biting activity stopped from 0200h onward.

However, at both the farm and longhouse, An. latens were found to bite from dusk till dawn,

with peak biting time between 2300h to 0200h. In order to incriminate the vectors of P.

knowlesi, conventional entomological techniques and molecular methods for detection of

malaria parasites in mosquitoes were used. A total of 14 out of 1,115 An. latens that were

dissected were found to be positive for malaria parasites, of which six harboured the

sporozoite stage, five were with the oocyst stage and three had both stages present. There

were also seven An. watsonii and one An. donaldi found positive for the oocyst stage. None

of the anopheline mosquitoes caught in the longhouse were positive by dissection. Using the

Plasmodium genus specific nested-PCR assays, malaria parasite DNA was found in all (22) of

the mosquitoes positive by dissection. In addition, three An. latens were positive by PCR that

were initially sporozoite-negative by dissection. Using human and simian malaria species-

specific PCR primers, four different simian malaria parasites (P. knowlesi, P. coatnevi, P. inui

and P. fieldi) were detected. Five An. latens were positive for P. knowlesi, four with P. inui

and another one had P. coatnevi. One mosquito had both P. inui and P. fieldi parasites and

another with P. inui and P. coatnevi. The malaria parasites in all five An. lutens, seven An.

itutsonii and one An. donaldi that were found to be positive only for the oocyst stage by

dissection, could not be identified with primers specific for P. fulciparum, P. vivax, P.

mulariae, P. ovale, P. knoiilesi, P. coatnevi, P. cvnomolgi, P. fieldi, P. inui and P.. frugile

despite being Plasmodium sp. positive by the nested-PCR assays. The circumsporozoite

protein (csp) genes of Plasmodium sp. from 12 infective An. latens were amplified, cloned

and sequenced. Analyses of the csp gene sequences confirmed the identity of the simian

malaria parasites that were detected by PCR in the mosquitoes and revealed that one of the An.

latens that was PCR-positivc for P. knotin, lesi also had P. fieldi-P. simiovule-like parasites. A

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novel P. inui-like malaria parasite that has recently been discovered in monkeys in Kapit was

also found in three of the infective An. latens. The combined results obtained from the

nested-PCR assays and the cloning and sequencing of Plasmodium sp. csp genes, showed that

four of the An. latens were positive for single P. knowlesi infections. There were also two

mosquitoes found to have single P. inui infections while one had only P. coatneyi and another

one was infected with the P. inui-like malaria parasite. Two An. latens had both P. inui and

the P. inui-like malaria parasites, while one had both P. knowlesi and P. , feldi-P. simiovale-

like parasites and another one had P. inui, P. coatneyi and P. fieldii. Of the five An. latens

positive for P. knoxwlesi, four were caught landing on humans and one from the monkey-

baited net trap. Phylogenetic analyses based on the csp genes of P. knowlesi found in An.

latens showed that they are phylogenetically indistinguishable from P. knowlesi isolated from

humans and monkeys, but genetically distinct from other known malaria parasites infecting

other vertebrate hosts. Although, annual entomological inoculation rate was higher in the farm

(10.95 infective bites per year) as compared to the forest (4.3 infective bites per year), the

computed entomological risk for acquiring P. knowdesi was not significantly different between

both study sites (P>0.05). This means the risks of acquiring P. knotiawlesi in both the farm and

the forest are the same. Humans most probably acquire the infection when their forest-

associated activities exposes them to An. latens. The current entomological findings,

supported by molecular, epidemiological and demographic data, strongly suggest that P.

knowlesi infections in humans in Kapit are a result of a zoonotic transmission rather than

human-to-human transmissions. Intensive entomological studies have to be carried out in

order to identify vectors in other parts of Sarawak where P. knowlwlesi is endemic before any

state-wide vector control strategy is to be set-up to control knowlesi malaria transmission.

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ABSTRAK

Tujuan projek im dijalankan adalah untuk mengenalpasti vektor-vektor P. knowlesi dan parasit

malaria lain di Kapit dan mengkaji bionomik vektor. Kajian entomologi selama setahun telah

dilaksanakan di mana tiga kawasan telah dipilih untuk pengumpulan nyamuk dewasa: rumah-

rumah panjang, dimana pengumpulan di dalam dan di luar rumah dilakukan; kebun; dan di

dalam hutan, di mana pelantaran bertingkat untuk perangkap umpan monyet dibina. Kaedah-

kaedah pengumpulan nyamuk termasuklah penangkapan melalui pendedahan kaki, dan

perangkap jaring berumpan monyet. Sejumlah 4,081 telah di tangkap dan ini termasuk 2,709

anophelines dan 1383 culicines. Dua belas spesis Anopheles yang ditangkap adalah

Anopheles latens (43.8% daripada jumlah keseluruhan tangkapan), An. donaldi (28.9%), An.

watsonii (10.2%), An. l, anus (7.1 %), An. kokhani (5.3%), An. tesselatus (2.4%), An. maculatus

s. l. (1.0%), An. pujutensis (0.8%), An. macarthuri (0.8%), An. barbirostris (0.3%), An.

introlatus (0.1 %), and An. roperi (0.1 %). Jumlah An. latens yang ditangkap di tiga kawasan

tersebut adalah berbeza dengan tangkapan terbanyak (49.9%) di kawasan hutan, diikuti oleh

kawasan kebun (39.3%) dan rumah panjang (10.8%). Daripada 129 An. latens yang ditangkap

di rumah panjang, 91 (71.1 %) didapati mengigit di luar rumah manakala 37 (28.9%) mengigit

di dalam rumah. Walaupun An. latens boleh dijumpai sepanjang tahun, kadar gigitan nyamuk

yang tertinggi di kawasan hutan telah berlaku pada bulan April, Mei, Oktober dan November

menakala di kawasan kebun, kadar gigitan tertinggi berlaku pada bulan Mei, June, Oktober,

November dan December. Sepanjang masa ini, purata kadar gigitan manusia didapati

mclebihi 5 gigitan/manusia/malam (g/m/m). Di rumah panjang kadar gigitan manusia yang

tertinggi diperhatikan pada bulan Oktober (3.4 g/m/m), sementara pada bulan-bulan yang lain,

kadar gigitan manusia adalah kurang daripada satu g/m/m. Anopheles latens dilihat sebagai

nyamuk "simio-anthropophagic" dan tiada keutamaan untuk monyet. Dalam perangkap

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kelambu berumpan monyet, bilangan An. latens (202 atau 96.6%) yang signifikan diperolehi

pada aras yang lebih tinggi (>3.0 m) jika dibandingkan pada aras bawah pelantaran (7 atau

3.3%). Anopheles latens di Kapit juga dilihat mempamerkan tabiat heterogeniti ruangan. Di

dalam hutan, puncak tertinggi masa gigitan nyamuk An. latens adalah di antara jam 1900 dan

jam 2000 dan aktiviti ini berhenti dari jam 0200 dan seterusnya. Namun begitu, pada kedua-

dua kawasan kebun dan rumah panjang, An. latens diperhatikan mengigit dari waktu subuh

hingga senja dengan puncak gigitan tertinggi di antara jam 0100 hingga jam 0200. Dalam

usaha untuk membabitkan vektor P. knovvlesi, teknik entomologi yang biasa dilakukan

bersama dengan kaedah molekular untuk mengesan parasit malaria di dalam nyamuk yang

digunakan. Sejumlah 14 daripada 1,115 An. latens yang dibedah didapati positif untuk parasit

malaria di mana enam ekor mempunyai parasit yang berada dalam peringkat sporozoit, lima

ekor dengan parasit dalam peringkat oosista dan tiga ekor mempunyai kedua-dua peringkat

parasit. Terdapat juga tujuh ekor An. watsonii dan satu An. donaldi yang positif dengan

peringkat oosista. Tiada nyamuk Anopheles yang dikumpul di rumah panjang yang positif

melalui pembedahan. Dengan menjalankan pencerakinan "nested-PCR', DNA parasit malaria

ditemui dalam kesemua (22) nyamuk positif yang telah dibedah. Tambahan pula, tiga An.

lutcns yang pada mulanya dikenalpasti negatif dengan sporozoit melalui pembedahan telah

disahkan positif rnelalui PCR. Dengan menggunakan primer PCR yang spesifik untuk spesis

bagi malaria monyet dan malaria manusia, terdapat 4 jenis parasit malaria monyet yang

dikesan ( P. knoidesi, P. coutnevi, P. inui dan P. fieldi). Lima An. latens adalah positif untuk

P. knotirlcsi, empat dengan P. inui dan satu lagi dengan P. coutnevi. Satu nyamuk mempunyai

kedua-dua P. inui dan I'. heidi dan satu lagi mempunyai kedua-dua P. inui dan P. coutmwi.

Parasit-parasit malaria dalarn kesemua lima An. lutens, tujuh An. wutsonii dan satu An.

dunuldi yang didapati positif hanya untuk peringkat oosista melalui pembedahan tidak dapat

Xdl]

dikenalpasti dengan menggunakan primer spesifik untuk P. _f'alciparum,

P. vivax, P. malariae,

P. ovale, P. knowlesi, P. coatneyi, P. cynomolgi, P. fieldi, P. inui dan P. fragile walaupun

didapati positif untuk Plasmodium sp. melalui pencerakinan 'nested-PCR'. Gen protein

sirkumsporozoit (csp) Plasmodium sp. daripada 12 An. latens yang infektif telah

diamplifikasikan, diklonkan dan dirangkaikan secara spesifik. Analisis rangkaian untuk gen

csp mengesahkan identiti parasit-parasit malaria monyet yang dikesan melalui PCR di dalam

nyamuk dan menunjukkan bahawa satu daripada An. latens yang disahkan positif untuk P.

knovilesi melalui PCR juga telah menunjukkan kehadiran parasit serupa P. fi'eldi-P. simiovale.

Parasit malaria baru yang hampir serupa dengan P. inui telah ditemui di dalam monyet di

Kapit baru-baru ini juga telah dijumpai hadir di dalam tiga nyamuk An. latens yang infektif

Gabungan keputusan yang diperolehi melalui pencerakinan 'nested-PCR' dan pengklonan

serta perangkaian gen csp Plasmodium sp. telah menunjukkan bahawa empat ekor An. latens

adalah positif untuk satu jangkitan P. knowlesi. Terdapat juga dua nyamuk yang didapati

mempunyai satu jangkitan P. inui manakala salah satu hanya mempunyai jangkitan P.

coatmwi dan satu lagi dijangkiti dengan parasit malaria yang serupa P. inui. Dua An. latens

dijangkiti kedua-dua P. inui dan parasit malaria serupa P. inui, satu mempunyai kedua-dua

parasit P. knoºtlesi dan P.. ßeldi manakala satu lagi dijangkiti P. inui, P. coatnevi dan P. fieldi.

Daripada lima An. latens yang positif untuk P. knowlesi, empat ditangkap semasa nyamuk

tersebut hinggap pada umpan manusia dan satu daripada perangkap jaring berumpan monyet.

Analisis filogenetik yang berasas kepada gen csp untuk Plasmodium knowlesi yang ditemui di

dalam An. latens menunjukkan bahawa ia tidak dapat dibezakan secara filogenetik daripada P.

knoiilesi yang diasingkan dan manusia dan monyet, namun jika berbeza secara genetik

daripada parasit malaria lain yang diketahui menjangkiti perumah vertebrat yang lain.

Walaupun kadar inokulasi entomologikal tahunan adalah lebih tinggi di kawasan kebun

XVüi

(10.95 ib/y) berbanding di dalam hutan (4.3 ib/y), pengiraan risiko entomologi untuk

dijangkiti P. knoNvlesi tidak berbeza secara signifikan di kedua-dua kawasan (P>0.05). Ini

bermaksud bahawa risiko untuk dijangkiti P. knowlesi di kebun dan di dalam hutan adalah

sama. Manusia mendapat jangkitan apabila aktiviti yang berkaitan dengan hutan

mendedahkan diri mereka kepada An. latens. Penemuan entomologi terkini yang disokong

oleh data molekul, epidemiologi dan demografi, mengusulkan bahawa jangkitan P. knorrlesi

di dalam manusia di Kapit adalah melalui transmisi zoonosis dan bukan transmisi dari

manusia ke manusia. Kajian entomologi yang intensif harus dilaksanakan untuk

mengenalpasti vektor di bahagian lain di Sarawak di mana P. knoivlesi adalah endemik

sebelum sebarang strategi pengawalan vektor untuk seluruh negeri di tubuhkan untuk

mengawal pemindahan malaria P. knovvlesi.

xix

CHAPTER ONE

General Introduction and Literature Review

1.1 Malaria

Malaria still remains a serious public health problem in many of the tropical countries.

Estimates from the World Health Organization (WHO) indicate that 3.2 billion people lived in

areas at risk of malaria transmission and the disease infects up to 500 million people

worldwide leading to millions of deaths each year (WHO, 2005). In fact, malaria due to

Plasmodium falciparum alone causes more than 1 million deaths each year. In 2004, there

were 2.53 million reported malaria cases in the Southeast Asian region with 3,768 attributed

deaths, but the estimated incidence for that year was 18.48 million and 99,600 deaths (WHO,

2007).

Plusmoclium, the causative agent of malaria belongs to the Family Plasmodiidae, Suborder

Haemosporidiidae of the Order Cociidia, Phylum Apicomplexa. There are more than 120

species of Plusinodium that can be found in the blood of mammals, reptiles and birds (Sinden

and Gilles, 2002). At present there are 33 species of primate malaria parasites recognized

(Table 1.1), but human malaria is considered to be commonly caused by one or a combination

of the following tour species; P.. fulcipar-um, P. vivux, P. nialuriue and P. ovale.

In Malaysia, although malaria cases have decreased from 51,921 in 1996 to 17,780 in 2001,

malaria continues to he a public health problem in areas such as Sabah, Sarawak and in the

interior parts of Peninsular Malaysia (Tee, 2000, MOH, 2001). The estimated prevalence and

distribution of' local Plusino(li un species are based on microscope confirmed cases from

government health clinics and hospitals. Until 2001, only three species of human

Table 1.1: Lists of primate malaria parasites, its natural hosts, periodicity, and distributionsa.

Plasmodium species Periodicity

P. , falciparum

P. vivax P. malariae P. ovale P. knowlesi P. cvnomolgi P. coatnevi P. fieldi P. inui P.. fi°agile P. simiovale P. shortii P. gonderi P. petersl P. geoýgesi P. hrasilianum P. simiaan P. e vlesi P. hvlohati P. je//ervi P. voungi P. plthecl P. silvat[cunl

P. . cch tiveGi P. reic"henoii"i P. rodhaini P. girardi

P. /olel'l P. coil langesi P. perctgarnhami P. uilenhcrgi P. hucki P. le miu"is

Hosts

Tertian Humans Tertian Humans Quartan Humans Tertian Humans

Quotidian Old world monkeys Tertian Old world monkeys Tertian Old world monkeys Tertian Old world monkeys Quartan Old world monkeys Tertian Old world monkeys Tertian Old world monkeys Quartan Old world monkeys Tertian Old world monkeys

unknown Old world monkeys unknown Old world monkeys Quartan New world monkeys Tertian New world monkeys Tertian Gibbons Tertian Gibbons Tertian Gibbons Tertian Gibbons Tertian Orang utans Tertian Orang utans Tertian Gorrillas, chimpanzees Tertian Chimpanzees Quartan Gorrillas, chimpanzees unknown Lemurs unknown Lemurs unknown Lemurs unknown Lemurs unknown Lemurs unknown Lemurs unknown Lemurs

Distribution

Worldwide Worldwide Worldwide Asia, Africa Southeast Asia Southeast Asia Malaysia, Philippines Malaysia Southeast Asia India, Sri Lanka Sri Lanka India, Sri Lanka Africa Africa Africa South America Brazil Malaysia Malaysia Malaysia Malaysia Malaysia Malaysia Africa Africa Africa Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar Madagascar

''summarized from Coatney et ul., 1971; Garnharn et al., 1972; Gysin, 1998; Cogswell, 2000

I

malaria had been reported in this country. Plasmodium, falciparum was the most common,

followed by P. vivax and P. malariae, although major differences in the relative

frequencies of Plasmodium species can be observed in different geographical areas around

the country (Tee, 2000; Singh and Cox-Singh, 2001). In Sarawak, P. vivax is the

predominant species encountered, followed by P. , falciparum and P. malariae. In 2003, there

were more than 2,600 cases of malaria reported in Sarawak. One thousand six hundred and

sixty two (63.5%) cases were due to vivax malaria, while 647 (24.74%) and 263 (10%) were

due to P.. /alciparum and P. malariae, respectively. With the exception of Limbang Division,

P. viivax is widely distributed throughout the state of Sarawak, while 90% of the cases of

falciparum malaria are found in the Divisions of Kuching, Samarahan, Sri Aman, and Bintulu

(Fig. 1.1). Cases diagnosed by microscopy as P. malariae on the other hand, are commonly

encountered in the Kapit, Limbang, Miri, Sibu and Sarikei Divisions (A. Matusop, personal

communication).

1.1.1 Life cycle of Plasmodium

Despite the different species of the malaria parasites, the life cycle of each basically follows

the same pathway (Fig. 1.2) requiring the presence of two hosts in order to complete their life

cycle; the definitive invertebrate hosts and the intermediate vertebrate hosts. The majority of

malaria parasites are transmitted by mosquitoes, and the parasites of primates are exclusively

transmitted by anophelines (Coatney et al., 1971; Sinden and Gilles, 2002).

The cycle of malaria in vertebrate hosts is initiated by the bite of an infective mosquito,

whereby sporozoites are inoculated into the blood during feeding. Shortly after inoculation,

the sporozoites will invade the liver cells where they will undergo primary schizogony to

3

Sabah

annel

Umbang (7,790.01 krn2 )

South China Sea

Mukah (6,997.61krtt2)

Sibu (8,278.29km2 )

Sarikel (4,332.35km2)

Kuching Betong (4,559.55km2) (4,180.74km2)

Samarahan Sri Aman (4.967.45km2) (5.466.25 km2)

Mid (26. TT7.07krtº2)

Bintulu (12,166.21 km2)

F, Indonesia

Kapft (38,933krr2)

Figure 1.1: Map of Sarawak (http: //www. sarawak. gov. my/content/view/89/1)

4