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

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.

History

1927 First probable observation of P. knowlesi by Giuseppe Franchini

1931–32 Campbell and Napier inoculated P. knowlesi in a rhesusmonkey 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; theynamed 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.

Lt.Col.Robert Knowles

History……

1961 Wharton and Eyles showed that Anopheles hackeri is a naturalvector 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 Kapitdistt.-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.

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.

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.

Natural hosts

Long tailed macaque(M.fascicularis)-

Found in Borneo,Brunei,Singapore,southThailand,south Vietnam,PeninsularMalaysia,java,sumatra

Pig tailed macaque(M.Nemistrina)-

Found in East India,Bangladesh,main land southeast asia,Borneo

Macaque fascicularis

The early trophozoites of P. knowlesimorphologically 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. knowlesito P. falciparum and later erythrocytic stages to P. malariae makes it extremely difficult to identify P. knowlesi infections by microscopy alone.

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 multipletrophozoites 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

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.

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.

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

Vectors

Initially Anopheles hackeri was recognised as vector in Malaysian Borneo.

In 1968 Chin etal.identified A.latens of Leucosphyrusgp. 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,Southeastasia,Indonesia &Phillipines.

Other vectors…

Vietnam-An.Dirus(Leucosphyrus gp)

Sabah-A.Balabecencis

Singapore-An.Kochi(Not in leuco.gp.)

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.

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.

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

Laboratory findings

Thrombocytopenia(Platelets can reduce upto50,000/microltr).

Anemia(Not very common)

Mild hyponatremia

Raised C reactive proteins

Deranged renal & liver function tests in advanced cases.

Median parasite count may go upto1387(Max.20,000)/microltr.

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).

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.

Treatment

Chloroquine as standard regeme.

3 days regeme of Chloroquine and primaquine

Best option for severe cases is intrvenousadministration of Artesunate.

Artesunate leads to faster parasite clearance.

Being used in Sabah since 2008 for severe malaria,irrespective of of species detection.

Conclusions

In Feb.2011 WHO guidelines were given in an informal meeting at Univ,Malaysiaserawk.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.

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.

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.

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 knowlesiinfection. 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.

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