Molecular epidemiology of the emerging human malaria parasite "Plasmodium knowlesi".

Malaria is the most important parasitic disease with global concern. Plasmodium knowlesi recently has emerged from its natural simian host as a significant cause of human malaria, particularly in Malaysian Borneo. Therefore, it has been added as the fifth human Plasmodium specie which is widely distributed in Southeast Asia. Recent developments of new molecular tools enhanced our understanding about the key features of this malaria parasite. Here, we review some of the ways in which molecular approaches might be used for epidemiology of P. knowlesi and finally lead to an efficient control of malaria.

INTRODUCTION

Despite more than a century of efforts to control and eradicate human malaria, it still remains as a major public health problem in the world. The global malaria deaths increased from 995,000 in 1980 to a peak of 1,817,000 in 2004 and declined to 1,238,000 in 2010 because of the scaling up of the control measures especially in Africa.[1] Five species of the genus Plasmodium namely Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi, are believed to contribute to the human malaria. Zoonotic malaria caused by P. knowlesi has recently been revealed to be a widespread infection in Southeast Asia.[2] To date, over 20 species of Plasmodium are known to be capable of infecting monkeys in which five of them have zoonotic potential including Plasmodium simium, Plasmodium brasilianum, Plasmodium cynomolgi, Plasmodium inui and P. knowlesi.[3] The P. knowlesi-infection has emerged as a common and potentially fatal cause of human malaria in Malaysian Borneo.[2]

The nuclear genome sequence of P. knowlesi is known[4] with close phylogenetic relationship to P. vivax; however, there are important phenotypic differences between them such as length of asexual cycle, host blood cell preference and absence of hypnozoite in P. knowlesi.[5] The 23.5 Mb nuclear genome of P. knowlesi encodes for 5188 proteins which approximately 80% of them has ortholog in P. falciparum and P. vivax.[4] The availability of genomic sequence of several Plasmodium species allows performing comparative genomic analysis and providing insights into the evolution of Plasmodium genes and gene families.[6] Molecular epidemiology is the application of molecular techniques to study pathogen genotypes and gene expression and infer them to the occurrence of infection in human population.[7] Recent new genomic information allows more extensive molecular epidemiological studies for a better control of malaria.

GEOGRAPHICAL DISTRIBUTION OF P. KNOWLESI

P. knowlesi was isolated for the 1st time from a long-tailed macaque monkey (Macaca fascicularis)[8] and the first experimental transmission of this monkey malaria to human was reported by Knowles and Das Gupta in 1932.[9] In 1965, the first natural infection of P. knowlesi in human was reported in an American traveler returning from Peninsular Malaysia.[10] Initially, it was uncertain that the natural human infections could take place and thus P. knowlesi could have zoonotic importance. But later on, experimental mosquito transmission to human and transmission from monkey to human approved the zoonotic potential of P. knowlesi.[11] Until recently, it was believed that natural human cases of P. knowlesi are rare; however, a study by Singh et al. showed that this parasite is a major cause of malaria in Malaysia.[12] Recent molecular studies in several Southeast Asian countries showed the presence of this parasite in human population in Malaysia,[1213] Thailand,[14] Singapore,[15] Palawan Island in the Philippines,[16] Vietnam,[17] southern Myanmar near the border of China,[18] Indonesian Borneo[19] and Cambodia.[20] There has been an increasing number of imported knowlesi malaria cases in travelers to Southeast Asian countries from the non-endemic countries in Europe,[2122232425] USA,[26] New Zealand,[27] Taiwan[28] and Japan.[29]

The long-tailed macaque (M. fascicularis), pig-tailed macaque (Macaca nemestrina) and banded leaf monkeys (Presbytis malalophos) in Southeast Asia are the principal natural hosts of P. knowlesi, although other monkey species are also capable of carrying the parasite.[13] The Anopheles species belong to the leucosphyrus group that have been incriminated as vectors of the parasite, are inhabited in forested areas in Southeast Asia.[30] The geographical range of P. knowlesi corresponds to the overlapping distribution of the vector mosquitoes and the macaque hosts and it defines the risky areas for knowlesi malaria transmission.[13]

APPLICATION OF MOLECULAR METHODS TO DETECT P. KNOWLESI

Conventional microscopy is the primary method for detection and species differentiation of malaria parasites. However, even most skillful microscopists may misdiagnose Plasmodium species in mixed infections especially with morphologically similar malaria parasites.[31] The early trophozoites of P. knowlesi are similar to those of P. falciparum while the mature asexual stages morphologically resemble those of P. malariae underlining the difficulty of identifying knowlesi malaria on the basis of morphology alone and misdiagnosing as P. malariae.[31]

For the first time, P. knowlesi was detected using molecular diagnostic tools, including polymerase chain reaction (PCR) using specific primers for the small subunit ribosomal ribonucleic acid (SSU rRNA), circumsporozoite protein gene and deoxyribonucleic acid (DNA) sequencing [Table 1].[12] Surprisingly, they found that more than half of the P. malariae infection confirmed cases by microscopy were actually P. knowlesi infection and these findings initiated a series of studies in the other Southeast Asian countries to determine the epidemiological distribution of knowlesi malaria.[14151617181920] However, Imwong et al. showed that the primers targeting P. knowlesi SSU rRNA cross-react with P. vivax genomic DNA.[32] Therefore, they designed a new set of primers[32] which was used in a semi-nested multiplex PCR identifying the five human Plasmodium species in a three-step reaction.[33] A TaqMan real-time PCR was developed targeting a specific DNA sequence within SSU rRNA of P. knowlesi with the sensitivity of three parasites/μl in the blood and 100% specificity.[34] Moreover, it was proven to be a powerful diagnostic tool for early detection of imported malaria in non-endemic areas.[35] Targeting the same gene, Chew et al. developed a single-step hexaplex PCR system that was able to detect all five human Plasmodium species simultaneously as well as mixed infections up to two-species level.[36] This multiplex PCR was further applied in Sandakan division, Sabah, Malaysia and was able to accurately discover P. knowlesi in the region.[37] In a recent study, a new single-step PCR assay was developed using bioinformatics approach and target multi-copy sequences of P. knowlesi which was able to detect one parasite/μl with 100% specificity.[38]

Table 1

Summary of molecular methods for specific detection of P. knowlesi

Two different loop-mediated isothermal amplification methods (LAMP) have been developed targeting species-specific β-tubulin gene[39] and apical membrane antigen-1 gene of P. knowlesi [Table 1].[40] It was shown that both LAMPs were specific and more sensitive than the nested-PCR targeting SSU rRNA.[3940] The high sensitivity and specificity together with the high speed of LAMP and no need of sophisticated instruments like PCR makes LAMP a promising tool for diagnosing this human malaria parasite in the remote areas.

Beside PCR and LAMP, rapid diagnostic tests (RDTs) are very useful for epidemiological purposes in endemic areas where many people can be screened in a short period of time. However, RDTs are still not available to detect P. knowlesi. Commercial RDTs for malaria diagnosis use monoclonal antibodies that target one of three antigens namely histidine-rich protein 2 (HRP-2), Plasmodium lactate dehydrogenase (pLDH) and aldolase.[41] McCutchan et al. showed that P. knowlesi can react with the monoclonal antibodies targeting pLDH of P. falciparum and P. vivax possibly due to the highly similar amino acid sequences among these malaria parasites.[42] However, P. knowlesi did not react with HRP-2-based RDT, since HRP-2 is only expressed in P. falciparum.[43] Targeting other antigens which are abundant and well-conserved among human Plasmodium species may improve prompt detection of knowlesi malaria.[44]

APPLICATION OF MOLECULAR METHODS FOR EVALUATION OF DRUG RESISTANCE AND ITS SPREAD

After chloroquine resistance development in P. falciparum, several studies have been done to find the molecular background of the resistance with regard to gene mutation and copy number.[454647] Recently, there have been some studies to evaluate antimalarial susceptibility of P. knowlesi.[484950] Ex vivo studies on P. knowlesi isolates in Malaysian Borneo showed that these parasites are sensitive to artemisinins and chloroquine while they are less sensitive to mefloquine.[49] In another study, chloroquine resistance transporter and dihydrofolate reductase sequences of P. knowlesi clinical isolates in the Andaman and Nicobar Islands, India, were all found to be wild type[51] which these findings are consistent with Faith's findings.[49] Increased copy number of multidrug resistant gene 1 (mdr1) in P. falciparum is the most important determinant of resistance to mefloquine.[52] Although the copy number of P. knowlesi mdr1 was not reported in Faith's study,[49] less sensitivity of P. knowlesi to mefloquine may indicate an innate tolerance[49] or acquired tolerance under drug pressure since this parasite is not newly emerged[13] and has caused malaria in a great number of patients since 1996.[53]

Since P. knowlesi is a major cause of malaria in Malaysia[12] and large human population in Southeast Asia may be at risk of infection, it is worthy to continuously screen the drug susceptibility of this human malaria parasite especially in the mixed infections to better formulate an appropriate drug policy. The recent successful culture of P. knowlesi in human erythrocytes[54] pave the way for such drug screening and other in vitro studies on this human malaria parasite.

CONCLUSION

In the post-genomic era where the genome sequences of several Plasmodium species are available, molecular biological studies and genetic investigations should be combined effectively to epidemiological and clinical studies. Comparative genomics, molecular evolutionary analysis, population genetics and phylogenetic studies are some of the new research fields which were developed after the recent advances in Plasmodium genome. Human and mosquito vector genetics can influence the epidemiology of malaria in many potential aspects of ecological and evolutionary interaction.[5556] There have been some studies to find the correlation between polymorphisms in specific genes and P. falciparum virulence.[57585960] In order to expand our knowledge on the pathogenesis of P. knowlesi which also can be fatal,[61] similar studies could be worthy.