Malaria:

Emerging problems of Plasmodium knowlesi

Balbir Singh

Malaria Research Centre

Universiti Malaysia Sarawak

Species of Plasmodium

P. falciparum, P. vivax, P. ovale,

P. malariae

P. pitheci, P. silvaticum

P. knowlesi, P. inui, P. cynomolgi,

P. coatneyi, P. fieldi, P. simiovale

>200 species in mammals, birds & reptiles

Malaria life cycle

Pf, Pv & Po: 48 hrs

Pm: 72 hrs

Man Mosquito

Pf, Pv, Po:2 days

Pm:3 days

Man Mosquito

1st

Day

2nd

Day

3rd

Day

4th

Day

Fever peaks

Pf

Pv

Po

Pm

Kapit

Miri

Sibu

Bintulu

Sri Aman

Sarikei

Limbang

KuchingSamarahan

Notified malaria cases (1999)

P. vivax 2,030P. falciparum 786P. malariae 270Mixed 69

Total 3,155

Kapit41420

1085

547

INDONESIA

BRUNEISarawak

P. malariae infections

General characteristics

• 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)

4 P. malariae samples

PCR +ve for: Plasmodium

-ve for : P. falciparum

P. vivax

P. ovale

P. malariae

Variant Pm? New Plasmodium??

DNA extraction from 8

P+ve blood samples

PCR amplification of

SSU rRNA genes

Cloning and

Sequencing

Analysis of DNA

sequence data

Molecular Characterisation

DNA sequence = Plasmodium knowlesi

Not P. malariae

KH 35

KH 115

KH 33

KH 131

KH 43

KH 50

KH 107

P. knowlesi

KH 96

P. knowlesi W1

P. knowlesi Nuri

P. fragile

P. vivax

P. cynomolgi

P. cynomolgi

P. berghei

P. ovale 1

P. ovale 2

P. malariae 1

P. malariae 2

P. malariae

P. falciparum

P. reichenowi

P. gallinaceum

P. lophurae

100

100

95

100

100

80

9999

89

100

100

0.01

Neighbour-joining tree of the SSU rRNA gene

P. knowlesi

KH 131

KH 50

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

P. malariae

P. malariae

P. berghei

P. yoelii

P. gallinaceum

P. falciparum

P. falciparum

P. reichenowi100

100

100

90

100

99

100

100

83

92

0.05

Neighbour-joining tree of the csp gene

P. knowlesi

Human Plasmodium

DNA

Monkey Plasmodium

DNA

Development of P. knowlesi-specific primers

Plasmodium-

specific

primers

P. knowlesi

-specific

primers

Kapit

Miri

Sibu

Bintulu

Sri Aman

Sarikei

Limbang

KuchingSamarahan

BRUNEI

Kapit Hospital (2000-2002)

.

208 malaria patients

PCR assays:

0 Pm

Microscopy: 141 Pm, 42 Pv, 25 Pf

120 (58%) Pk

88 Pf, Pv & Po

Singh et al. (2004) Lancet

Morphological Characteristics

Trophozoites Schizonts Gametocytes

P. knowlesi

P. malariaeP. falciparum

Emerging problems of P. knowlesi

• Introduction

• Human P. knowlesi infections in Kapit Division,

Sarawak (2000-2003)

• History of P. knowlesi

Calcutta School of Tropical Medicine

Plasmodium knowlesi

India (1931)

LT macaque from Singapore

Blood into Indian rhesus macaques

High parasite counts: death

Monkey blood into 2 humans

Napier & Campbell (1932) Ind Med Gaz; Knowles & Das Gupta (1932) Ind Med Gaz

Sinton & Mulligan (1932) Rec Mal Survey India

Blood into Singaporean LT macaques

Low parasite counts

R. Knowles

BM Das Gupta

Malaria with 24 hr fever peak (Pv, Pf, Po: 48 hrs, Pm: 72 hrs)

Malaria & neurosyphilis

Pre-1950’s - no antibiotics

1917: Wagner-Juaregg used P. vivax

- fever peak 48 hrs

1935 - 1955: Ciuca & others used P.

knowlesi - fever peak 24 hrs

1927: Nobel prize for ‘malaria therapy’

Ciuca et al (1955) Ed Ac Rep Pop Roumaine

Human P. knowlesi infections by

injection of blood since 1931.

Human infections by mosquito bite?

Naturally-acquired human P. knowlesi infection

1965 (Chin et al, Science)

– American ‘army surveyor’, worked in Pahang, Pen. Malaysia.

– Diagnosed P. falciparum & then P. malariaein USA

– Blood injected into ‘human volunteers’

– Daily fever peaks (Pm = 72 hrs)

– Blood into 3 rhesus monkeys ; all died

*

‘……spent 5 days working

alone in the bush…………….

working by night & sleeping by

day.’

Chin et al (1965): Surveyor for US Army Map Service

P. knowlesi experiments in USA (1965 -1967)

An. balabacensis

Chin et al (1968) Am J Trop Med Hyg

P. knowlesi H strain

11 volunteers

Simian Malaria Studies at IMR (1960-1967)

Don Eyles, McWilson Warren, Elizabeth Guinn, Gordon Bennet,

R.H. Wharton, Yap Loy Fong, A.A. Sandosham, Cheong Weng

Hooi, C.P. Ramachandran & J.C.C. Hoo.

Investigations of malaria as a zoonosis

1,117 human blood samples

Into 135 rhesus monkeys at IMR.

none with Pk or Pcy infections.

Warren et al. (1970) Am J Trop Med Hyg

*

1966: Pahang, Peninsular Malaysia.

Local LT macaques with Pk.

Conclusion: Zoonotic infections extremely rare.

(only 28+ve: 13Pv, 11Pf, 4 unidentifiable)

Emerging problems of P. knowlesi

• Introduction

• Human P. knowlesi infections in the Kapit Division (2000-2003)

• History of P. knowlesi

• Human P. knowlesi infections• Distribution in SEAsia

PCR results for Sarawak (2000-04; 2006-08; 2012-14; 2015-17)

3 (117)

0 (72)3 (139)

84 (116)101 (159)

1,251 (1,809)201 (256)

63 (256)

140 (155)

5 (9)

BETONG

10 (36)

LIMBANG

•

•

5 (273)

•LAWAS44 (51)

Singh et al (2004) Lancet; Cox-Singh et al (2008) Clin Infect Dis; Daneshvar et al (2009) Clin Infect Dis

Total P. knowlesi (Total patients tested)

PCR results for Sarawak (2000 - 2017)

BETONG

LIMBANG

•

• •LAWAS1,910 P. knowlesi (3,448 patients)

14 P. malariae

All imported: 4 PNG, 2 Irian Jaya, 8 Africa

Reported/PCR confirmed human Pk infections

1,910

BRUNEI

*

*

* *6

427

*5

*

**

**

*

*

**

*

**

38

7

37

45

4

1,402

10

53

Nicobar &

Andaman IsLANDS.

463

10

**

*

????

Reported Pk cases

Unreported Pk cases

Misdiagnosed as Pm, Pv, Pf or mixed

Asymptomatic Pk cases

Undetected (microscopy unavailable)

Reported malaria cases (Peninsular Malaysia)2009–2018

Pf

Pk/Pm

Pv

Pf

0

100

200

300

400

500

600

700

800

900

To

tal

no

of

cases

Year

Pv

Pf

Pk/Pm

0

10

20

30

40

50

60

70

80

90

% o

f to

tal

mala

ria

cases

Year

Pf

Pk/Pm

Pv

Data source: MoH, Malaysia

Reported malaria cases (Sabah & Sarawak) 2000–2018

Pf

0

10

20

30

40

50

60

70

80

90

20

00

20

01

20

02

20

03

20

04

20

05

20

06

20

07

20

08

20

09

20

10

20

11

20

12

20

13

20

14

20

15

20

16

20

17

20

18

% o

f to

tal m

alar

ia c

ase

s

Year

Pv

Pk/Pm

Pf0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

20

00

20

01

20

02

20

03

20

04

20

05

20

06

20

07

20

08

20

09

20

10

20

11

20

12

20

13

20

14

20

15

20

16

20

17

20

18

To

tal n

o.o

f c

as

es

Year

Pv

Pk/Pm

Pf

Data source: MoH, Malaysia

Why was there a need to

differentiate Pk from Pm

(when Pm treatment was CQ + PQ)???

Mosquito

Differences: P. knowlesi & P. malariae

Pm: 72 hrs

Pk: 24 hrs

Pm:15-30 days

Pk: 9-12 days

Man Mosquito

Parasitaemia

Pm: seldom >5,000 parasites/µl

Pk: 30% > 5,000 parasites/µl

32% < 500 parasites/µl

Potentially fatal

Pm: No

Pk: Yes

Confirmed

fatal cases of P. knowlesi

Brunei : 1 (2003)

Sabah : 11 (2007-2013)*

Sarawak : 13 (2005-2009)**

**Cox-Singh et al (2008) Clin Infect Dis

**Daneshvar et al (2009) Clin Infect Dis

*Cox-Singh et al (2010) Malaria J

*William et al (2011) Emerg Inf Dis

*William et al (2014) Malaria J

```````````````````````````````````

Fatal Pk infection

WBC

WBC

WBC

WBC

WBC

WBC

Severe knowlesi malaria (n=86)

Singh & Daneshvar (2013)

Clin Microbiol Rev

Emerging problems of P. knowlesi

• Introduction

• Human P. knowlesi infections in the Kapit Division

• History of P. knowlesi

• Human P. knowlesi infections• Distribution in SEAsia

• Differences between Pm & Pk

• Entomological, Zoological & Molecular studies

Vector 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

An. balabacensis

An. latens

An. cracensAn. hackeri

An. dirus

P. knowlesi vectors in SEAsia

Wharton & Eyles (1961) Science; Jiram et al (2012) Malar J; Tan et al (2008) Malar J; Nakazawa et al (2009) Int J Parasitol

Detailed studies on feeding behavior of mosquitoes and

vectors of Pk required

Natural HostsMacaca fascicularis

(Long-tailed macaque)

Macaca nemestrina

(Pig-tailed macaque)

Presbytis melalophos

(Banded leaf monkey)

Peninsular Malaysia

Eyles et al (1962) Med J Malaya Trachypithecus obscurus

(Dusky leaf monkey)

Thailand

Putaporntip et al (2010) Mol Ecol

http://en.wikipedia.org/wiki/File:Trachypithecus_obscurus_-_Kaeng_Krachan.jpg

2010

1931

2009

1953

1960s

P. knowlesi in long-tailed macaques

2011

P. knowlesi in pig-tailed macaques

2010 , 1960s

Macaques from Kapit, Sarawak

71 (86.6%) of 82 long-tailed Pk +ve

13 (50%) of 26 pig-tailed Pk +ve

101 of 108 (93.5%) malaria +ve:

6 Plasmodium species (Pk, Pfi, Pcy, Pin, Pco, Psi)

Lee et al (2011) PLoS Pathogens

Whole genome sequencing

Cluster 1 & 2: Human isolates (n=48)

Assefa et al (2015) PNAS

(Sarawak)

(Sarawak)

(Pen. M’sia)

Cluster 3: Monkey (n=4) & human isolates (n=1)

Microsatellite typing: P. knowlesi from Sarawakk

Humans (n=167)

Divis et al (2015) PLoS Pathogens

Long-tailed

macaques (n=34)

Pig-tailed

macaques (n=

Cluster 1 Cluster 2

10)

Cluster 1 Cluster 2

Microsatellite typing: P. knowlesi from Sarawakk

Humans (n=167)

Divis et al (2015) PLoS Pathogens

Long-tailed

macaques (n=34)

Pig-tailed

macaques (n=

Cluster 1 Cluster 2

10)

Cluster 1 Cluster 2

P. knowlesi transmission in Malaysian Borneo

Cluster 1 Cluster 2

LT macaques PT macaques

Who are at risk?

Farmers, hunters, timber camp workers, army

personnel & travelers to forest/forest fringe

Habitat of macaques and mosquito vectors

Pk in 17 travelers since 2009 who visited:

Pen. Malaysia (fm Finland, Japan, Sri Lanka & USA)

M’sian Borneo (fm Sweden, Netherlands, UK & NZ )

Thailand (fm France & Germany)

Philippines (fm Taiwan, USA & Japan)

Indonesia (fm Australia)

Brunei (fm UK)

Cramer (2015) Curr Inf Dis Rep

P. knowlesi malaria

Widely distributed in South East Asia (incidence??)

Misdiagnosed since Pk = Pm

Ancient Zoonosis – macaques reservoir hosts

3 Pk sub-populations

Spectrum of disease (potentially fatal)

Susceptible to antimalarials

Challenge for malaria control & elimination

Huge reservoir of Pk in macaques in forests

Malaria prevention, control & elimination

Insecticide impregnated bednets.

Indoor residual spraying.

Elimination of breeding sites /

reduction of vector population.

Repellants/appropriate clothing

Early detection & treatment.

Chemoprophylaxis

Human-to-human

transmission

Monkey-to-human

transmission

X

X

Y ?

Y

X/Y

Y

Future of P. knowlesi??

Humans as preferred hosts??

Changes in ecology (logging/deforestation) + vector

species/behaviour + increase in human population:

Malaria Research Centre

UNIMAS

Cyrus Daneshvar

Janet Cox-Singh

Lee Kim Sung

Angela Siner

Paul Divis

Sunita Shamsul

Roynston

Julin

Siti Khatijah

Lau Hui Chong

Sophia LauTan Cheong Huat

‘Malaria Detectives’ @ MRC, UNIMAS

Dyg Shuaisah

Khamisah

Ismandy

Angela

Liew Sze Tze

Thamayanth

i

Khatijah

Joshua

Hu Ting

Huey

David Conway

King Abdullah University of

Science & Technology

Arnab Pain,

Manoj Duraisingh

Daniel Neafsey

Reuben

Sharma

Shamilah HisamClemens Kocken,

Alan Thomas

Jeffrin

Rovie-Ryan

Fread Anderois

Sarawak

Health

Departmen

t

Asmad

Matusop

Tim Davis

Acknowledgements

Kevin Palmer

Staff of District Hospitals, Polyclinics & Malaria

Control Units in Sarawak.

Funding

Wellcome Trust, UK.

Malaysian Ministry of Science & Technology.

Universiti Malaysia Sarawak.

bsingh@unimas.my