Elsevier

The Lancet

Volume 363, Issue 9414, 27 March 2004, Pages 1017-1024
The Lancet

Articles
A large focus of naturally acquired Plasmodium knowlesi infections in human beings

https://doi.org/10.1016/S0140-6736(04)15836-4Get rights and content

Summary

Background

About a fifth of malaria cases in 1999 for the Kapit division of Malaysian Borneo had routinely been identified by microscopy as Plasmodium malariae, although these infections appeared atypical and a nested PCR assay failed to identify P malariae DNA. We aimed to investigate whether such infections could be attributable to a variant form of P malariae or a newly emergent Plasmodium species.

Methods

We took blood samples from 208 people with malaria in the Kapit division between March, 2000, and November, 2002. The small subunit ribosomal RNA and the circumsporozoite protein genes were sequenced for eight isolates that had been microscopically identified as P malariae. All blood samples were characterised with a genus-specific and species-specific nested PCR assay together with newly designed P knowlesi-specific primers.

Findings

All DNA sequences were phylogenetically indistinguishable from those of P knowlesi, a malaria parasite of long-tailed macaque monkeys, but were significantly different from other malaria parasite species. By PCR assay, 120 (58%) of 208 people with malaria tested positive for P knowlesi, whereas none was positive for P malariae. P knowlesi parasites in human erythrocytes were difficult to distinguish from P malariae by microscopy. Most of the P knowlesi infections were in adults and we did not note any clustering of cases within communities. P knowlesi infections were successfully treated with chloroquine and primaquine.

Interpretation

Naturally acquired P knowlesi infections, misdiagnosed by microscopy mainly as P malariae, accounted for over half of all malaria cases in our study. Morphological similarities between P knowlesi and P malariae necessitate the use of molecular methods for correct identification. Further work is needed to determine whether human P knowlesi infections in the Kapit division are acquired from macaque monkeys or whether a host switch to human beings has occurred.

Introduction

Estimates of the prevalence and distribution of Plasmodium species that infect human populations in Malaysia are based on microscopy-confirmed cases from government health clinics and hospitals.1 Between 1998 and 2002, the yearly incidence of malaria in Sarawak, Malaysian Borneo was between 2496 and 3155 cases. In Sarawak, P vivax is the predominant species (69·1% of all cases) followed by P falciparum (19·7%), parasites identified as P malariae (9·4%), and mixed species infections (1·8%) with no reported cases of P ovale. Before our study began in March, 2000, we noted major differences in the relative frequencies of Plasmodium species reported within Sarawak. P vivax was widely distributed in the nine administrative divisions, while P falciparum was predominant in the southern divisions of the state and parasites identified as P malariae cases were mainly reported in the central divisions of Kapit and Miri. In the Kapit division, parasites identified by microscopy as P malariae accounted for about a fifth of all malaria cases in 1999.

There were features of the infections identified by microscopy as P malariae that were atypical. Usually, P malariae infections are chronic and asymptomatic with low parasitaemias, seldom exceeding 5000 parasites per μL blood.2, 3 However, almost all (97·4%) of the 108 cases reported for 1999 in the Kapit division were in people with clinical signs and symptoms who sought treatment at Kapit Hospital, the Kapit polyclinic, or rural health clinics. Furthermore, these patients had 48–66640 parasites per μL blood, with 20 (18·5%) having a parasitaemia greater than 5000 parasites per μL blood.

In a preliminary examination, we used a nested PCR malaria detection assay4 to study five isolates identified as P malariae by microscopy, and noted that they contained Plasmodium spp DNA but were negative for all four human malaria parasite species, including P malariae. These findings suggested to us that a variant form of P malariae, previously reported in Asia,5 or a newly emergent Plasmodium species was responsible for the infections identified by microscopy as P malariae, in the Kapit division. Therefore, we undertook a detailed study to identify the Plasmodium species causing these infections.

Section snippets

Study area and population

The Kapit division, with a population of 90697 people and an area of 38934 km2, is the largest of the administrative divisions in Sarawak. The region is hilly and largely covered by primary and secondary rainforest. Inhabitants of the division are mainly rural indigenous people; most belong to the Iban ethnic group. Each rural community occupies a longhouse that typically houses between 17 and 300 people. Longhouses are situated along the Rejang and Balleh rivers and their tributaries, and boat

SSU rRNA and csp DNA sequence and phylogenetic analysis

Results of phylogenetic analysis showed that the eight Kapit “P malariae” isolates sequenced were indistinguishable from P knowlesi, but clearly distinct from all other previously sequenced Plasmodium species in humans, nonhuman primates, rodents, and birds (figure 1). This result was obtained independently with the SSU rRNA gene sequences and the csp gene sequences, with 100% support by bootstrap analysis in each case. Phylogenetic trees of both genes were also constructed with the maximum

Discussion

Emerging infectious disease agents in human beings are increasingly important in public health, and need to be made a high priority at an international level as well as in the primary epidemiological context. We report a large number of cases of malaria in humans caused by an unexpected parasite species in Malaysian Borneo. Naturally acquired P knowlesi infections, misdiagnosed by microscopy mainly as P malariae, accounted for over half of all cases of malaria in our study. Phylogenetic

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