Contribution of Plasmodium knowlesi to Multispecies Human Malaria Infections in North Sumatera, Indonesia

Abstract

Background: As Indonesia works toward the goal of malaria elimination, information is lacking on malaria epidemiology from some western provinces. As a basis for studies of antimalarial efficacy, we set out to survey parasite carriage in 3 communities in North Sumatera Province.

Methods: A combination of active and passive detection of infection was carried out among communities in Batubara, Langkat, and South Nias regencies. Finger-prick blood samples from consenting individuals of all ages provided blood films for microscopic examination and blood spots on filter paper. Plasmodium species were identified using nested polymerase chain reaction (PCR) of ribosomal RNA genes and a novel assay that amplifies a conserved sequence specific for the sicavar gene family of Plasmodium knowlesi.

Results: Of 3731 participants, 614 (16.5%) were positive for malaria parasites by microscopy. PCR detected parasite DNA in samples from 1169 individuals (31.3%). In total, 377 participants (11.8%) harbored P. knowlesi. Also present were Plasmodium vivax (14.3%), Plasmodium falciparum (10.5%) and Plasmodium malariae (3.4%).

Conclusions: Amplification of sicavar is a specific and sensitive test for the presence of P. knowlesi DNA in humans. Subpatent and asymptomatic multispecies parasitemia is relatively common in North Sumatera, so PCR-based surveillance is required to support control and elimination activities.

Keywords: Indonesia; Malaria; Plasmodium knowlesi..

Figure 1.

Map of North Sumatera province, Indonesia. The 3 studied regencies (Batubara, Langkat, and South Nias) are indicated.

Figure 2.

Validation of Plasmodium knowlesi primers targeting sicavar against human and simian malaria parasites reported from Southeast Asia. Control DNA of human malaria Plasmodium species were from imported cases in the United Kingdom (courtesy of the Public Health England Malaria Reference Laboratory); 2 isolates each are shown for Plasmodium falciparum, Plasmodium vivax, and Plasmodium malariae. Genus-specific primers for the cytochrome B gene (cytb) were used to confirm presence of detectable Plasmodium DNA in each sample, indicated by plus signs; bp, base pairs.

Figure 3.

Sequence alignment of representative 120 and 141 nucleotide sequences of sicavar amplicons from the peripheral blood DNA of 4 participants from Batubara (BB) and 2 each from Langkat (LK) and South Nias (NS). Amplicons were produced by heminested polymerase chain reaction, as described in Materials and Methods. Sample order is determined by the alignment. Representative amplification products were chosen for this sample and sequenced directly using amplification primers to prime forward and reverse sequencing reactions. Sequences shown were confirmed in both directions. Two loci from the Plasmodium knowlesi strain H reference genome (PKNH_0932000, type I schizont-infected cell agglutination variant antigen (SICAvar), chromosome 9, centrally located; PKNH_0118500, type II SICAvar, chromosome 1, subtelomeric) are shown for comparison. Double peaking was seen in some samples; only the peak with highest amplitude was read for this analysis.

Figure 4.

Proportion of Plasmodium species and multiplicity of infections by regency. The denominator for each site (total number of individuals tested) is given under the regency name, and the number of parasite-positive individuals is shown at the top-left of each bar graph. The horizontal axis represents the proportion of the total number of infections in each bar. Colored bars denote species; gray bars denote proportions of mixed-species infections identified in each site. Abbreviations: Pf , Plasmodium falciparum; Pk, Plasmodium knowlesi; Pm, Plasmodium malariae; Pv, Plasmodium vivax.