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1. A P D R . R A J E N D R A C H O U B E Y
R O Y A L C O L L E G E O F M E D I C I N E
U N I V E R S I T Y K U A L A L U M P U R
I P O H , P E R A K
M A L A Y S I A
Fifth human malarial
parasite”Plasmodium knowlesi”:An
emerging challange

2. Introduction
 Pl.knowlesi was identified in 30s as a natural
Plasmodium of Macaca fascicularis monkey also
infecting humans.
 Now it has established as a primate malaria endemic
in some southeast asian macacqes.
 It infects humans with other plasmodium infections
most prevelant across Malaysian Borneo.
 It can be termed as a zoonotic malaria acquired by
humans when they enter the habitat of macaques
and anopheline mosquito vectors in the forest.

3. History
 1927 First probable observation of P. knowlesi by Giuseppe
Franchini
 1931–32 Campbell and Napier inoculated P. knowlesi in a rhesus
monkey with description of a fulminant disease
 1932 Robert Knowles and Biraj Das Gupta, working in
Calcutta, describe the blood forms of the parasite; they
experimentally demonstrated the transmission to
humans
 1932 Sinton and Mulligan described the 24-h schizogonic cycle; they
named the plasmodium as P. knowlesi in honour of Knowlese.
 1935 Van Rooyen and Pile used for the first time P.
knowlesi to treat neurosyphilis but stopped it in 1955 as it
caused severe malaria.

4. Lt.Col.Robert Knowles

5. History……
 1961 Wharton and Eyles showed that Anopheles hackeri is a natural
vector in Malaysia
 1965 First description by Chin and co-workers of a
naturally acquired infection in a US soldier deployed in
Pahang, Peninsular,Malaysia.
 1965 Observation of antigenic variation in P. knowlesi infection
 1975 Duffy binding proteins identified as the receptors that mediate
P. knowlesi invasion of erythrocytes
 2004 Balbir Singh and his co-workers report a large focus
of human malaria caused by P. knowlesi in the Kapit
distt.-Malaysian Borneo.
 2008 The entire genome of P. knowlesi is sequenced and published
 2008 Cox-Singh and her co-workers show that P. knowlesi
can cause severe and fatal malaria.

6. Characterstick features of PK
 It phytogenetically resembles P.Vivax and
morphologically resembles P.falciparum&P.malarae.
 It exclusively has 24 hr.asexual blood stage development.
Its latent liver stage is absent.This leads to heavy
parasitaemia.
 Similar to P.vivax it uses duffy bld gp.antigen as a
receptor to invade erythrocytes.
 This is facilitated through duffy binding protein in its
micronemes.

7. Contd..
 The genome PK is made of 14 chromosomes containing a
total of 5188 proteins-encoding genes.80% can be
identified with P.vivax&P.fal.
 Exclusively there are two variant gene families(Sica var
&Kir).
 A special feature- molecular mimicry of Kir proteins with
host CD99 T cells in immunoregulatory mechanism.
 Sica var genes associated with virulence of PK.

8. Natural hosts
 Long tailed macaque(M.fascicularis)-
Found in Borneo,Brunei,Singapore,south
Thailand,south Vietnam,Peninsular
Malaysia,java,sumatra
 Pig tailed macaque(M.Nemistrina)-
Found in East India,Bangladesh,main land
southeast asia,Borneo

9. Macaque fascicularis

10. The early trophozoites of P. knowlesi
morphologically resemble those of P.
falciparum….The late and mature trophozoites,
schizonts and gametocytes appear very similar to
those of P. malariae….The morphological
resemblance of early trophozoites of P. knowlesi
to P. falciparum and later erythrocytic stages to
P. malariae makes it extremely difficult to
identify P. knowlesi infections by microscopy
alone.

12. Resemblance of Pl.knowlesi with
Pl.faciparum and Pl.Malarae
(a and b) early
trophozoites; (c) late
trophozoite (d–f)
schizonts . It worth
noting that the “early
trophozoites” (ring
forms) can be
misidentified as P.
falciparum especially
when multiple
trophozoites with an
“accolè” form is seen
such as in b. The mature
trophozoites in c and d
may be confused with
the band form of P.
malariae

16. Important study conducted
 In 2004 Balbir singh etal.using a PCR assay specific
for 185 ribosomal genes of PK found that 58% of208
malaria pts.were infected with PK.
 In Kapit distt. Of Malaysian Borneo116 of
141(82%)pts. diagnosed as Pl.malarae by microscopy
were actually P.Knowlesi pts. As detected by PCR.

17. Contd……….
 Balbirsingh etal also did a retrospective study on
archived blood films (1996) from seven distt. Of
Serawak(Mal.).
 They discovered that 97%of pts.diagnosed by
microscopy as P.Malarae were actually PK as
detected by PCR.
 In 2008 complete genome of PK (H
strain,Pk1(A+)clone)was sequenced and published.

18. Latest studies
 PK infection has been described as a asymptomatic to
fatal infection with severe hepatorenal dysfunction and
ARDS.
 Total 19 pts.have died(3.4%)
 These are common outcomes of latest studies:
 Singh etal2004
 Cox Singh etal2008
 Daneshwar etal 2009,Bronner etal2009
 Cox-Singh etal2010
 Hoosen &shaw 2011
 Link etal 2012,Rajharam etal.2012

19. Vectors
 Initially Anopheles hackeri was recognised as vector
in Malaysian Borneo.
 In 1968 Chin etal.identified A.latens of Leucosphyrus
gp. in Kapit(Mal.Borneo) as the main natural vector
for humans and monkeys.
 An.leucosphyrus is distributed in south west
India,south China,Taiwan, Taiwan,Southeast
asia,Indonesia &Phillipines.

20. Other vectors…
 Vietnam-An.Dirus(Leucosphyrus gp)
 Sabah-A.Balabecencis
 Singapore-An.Kochi(Not in leuco.gp.)

21. Clinical features
 Two possible modes of transmission to humans have
been proposed: either from an infected monkey to a
human or from an infected human to another
human.
 Symptoms typically begin approximately 11 days
after an infected mosquito has bitten a person and
the parasites can be seen in the blood between 10 –
12 days after infection. The parasite may multiply
rapidly resulting in very high parasite densities that
may be fatal.

22. Clinical features……
 The parasite count amy reach upto 20,000/microleter.
 The malaria is nonrelapsing type as there is lack of
hypnozoites in its exoerythrocytic stage.
 Common symptoms-Fever with chills&rigors-100%,
headache-32%,
cough-18%,
vomiting-16%,
nausea-06%
 Complications-Resp.distress,altered LFTs with
jaundice,Renal failure.

23. Geographic distribution of P.knowlesi
 Kapit distt.of Serawak(Malaysia)
 Pahang(Peninsular Malaysia)
 Palawan island of Phillipines
 China Myanmar border
 Khan phu area of south central vietnam
 Island of ko Payam-Thialand
 Forested area of Lim chu kang in Singapore
 Pailin province in Cambodia

25. Laboratory findings
 Thrombocytopenia(Platelets can reduce upto
50,000/microltr).
 Anemia(Not very common)
 Mild hyponatremia
 Raised C reactive proteins
 Deranged renal & liver function tests in advanced
cases.
 Median parasite count may go upto
1387(Max.20,000)/microltr.

26. Diagnosis
 Peripheral smear examination by expert
microscopist to detect morphology-Usually confused
with Pl.malarae.
 Many rapid diagnostic tests were tried but their
specificity was not authentic.
 A realtime PCR which binds specifically with a 30
base pair of variable regions of PK showed 100%
specificity and high sensitivity(detecting 3
parasites/microltr).

27. Diagnosis…..
 USA researchers have developed a new single –step
PCR assay that targets a multicopy sequences for PK
detecting even 1parasite/microltr.with 100%
specificity.
 Two grps.of researchers developed loop-mediated
isothermal amplification(LAMP).
 LAMP method is sensitive assay based and cost
effective but needs to be validated by large no. of
studies.

28. Treatment
 Chloroquine as standard regeme.
 3 days regeme of Chloroquine and primaquine
 Best option for severe cases is intrvenous
administration of Artesunate.
 Artesunate leads to faster parasite clearance.
 Being used in Sabah since 2008 for severe
malaria,irrespective of of species detection.

29. Conclusions
 In Feb.2011 WHO guidelines were given in an informal meeting at Univ,Malaysia
serawk.These guidelines should be followed.
 Human infection with P.Knowlesi,commonly misidentified as more benign
P.Malarae, are widely distributed across Malaysian Borneo , peninsular Malaysia &
other southeast asian countries.
 Large parasite burden and compounding effects of a 24 hr.asexual replication cycle
strongly suggests that P.Knowlesi is a potentially life threatening pathogen.
 In absence of specific diagnostic tests,people who travel around south east asia and
who have reccieved a “hyperparasetimic P.Malarae” diagnosis should be managed
with artesunate to avoid any serious consequences.
 PCR and LAMP methods must help to increase epedemiological informations and
improve diagnosis.
 More studies should be done to asses exact pathophysiology of plasmodium
knowlesi infection.

30. References
 S. Antinori et al. / Acta Tropica 125 (2013) 191– 201Chin,
W., Contacos, P.G., Coatney, G.R., Kimball, H.R., 1965. A
naturally acquired quotidian-type malaria in man
transferable to monkeys. Science 149, 865.
 Singh, B., Sung, L.K., Matusop, A., Radhakrishnan, A.,
Shamsul, S.S., Cox-Singh, J.,et al., 2004. A large focus of
naturally acquired Plasmodium knowlesi infections in
human beings. Lancet 363, 1017–1024.
 Chin, W., Contacos, P.G., Collins, W.E., Jeter, M.H.,
Alpert, E., 1968. Experimentalmosquito-transmission of
Plasmodium knowlesi to man and monkey.
AmericanJournal of Tropical Medicine and Hygiene 17,
355–358.

31. Ref………..
 Cox-Singh, J., Davis, T.M., Lee, K.S., Shamsul, S.S., Matusop, A.,
Ratnam, S., et al., 2008. Plasmodium knowlesi malaria in humans
is widely distributed and potentially life threatening. Clinical
Infectious Diseases 46, 165–171.
 Cox-Singh, J., Singh, B., 2008. Knowlesi malaria: newly emergent
and of public healthimportance? Trends in Parasitology 24, 406–
410.
 Lee, K-S., Cox-Singh, J., Brooke, G., Matusop, A., Singh, B., 2009a.
Plasmodium knowlesi from archival blood films: further evidence
that human infections are widelydistributed and not newly
emergent in Malaysian Borneo. International Journal of
Parasitology 39, 1125–1128.
 Cox-Singh, J., Hiu, J., Lucas, S.B., Divis, P.C., Zulkarnaen, M.,
Chandran, P., et al., 2010. Severe malaria—a case of fatal
Plasmodium knowlesi infection with post-mortem findings: a case
report. Malaria Journal 9, 10.

32. Ref…….
 Daneshwar, C., Davis, T.M.E., Cox-Singh, J., Rafa’ee,
M., Zakaria, S., Divis, P., et al.,2009. Clinical and
laboratory features of human Plasmodium knowlesi
infection. Clinical Infectious Diseases 49, 852–860.
 Daneshwar, C., Davis, T.M.E., Cox-Singh, J., Rafa’ee,
M., Zakaria, S., Divis, P., et al., 2010. Clinical and
parasitological response to oral chloroquine and
primaquine in uncomplicated human Plasmodium
knowlesi infections. Malaria Journal 9, 238.

33. T H A N K Y O U ! !