METHODS AND ANALYSIS: ACT KNOW, the first randomised controlled trial ever performed in knowlesi malaria, is a two-arm open-label trial with enrolments over a 2-year period at three district sites in Sabah, powered to show a difference in proportion of patients negative for malaria by microscopy at 24 h between treatment arms (clinicaltrials.gov #NCT01708876). Enrolments started in December 2012, with completion expected by September 2014. A total sample size of 228 is required to give 90% power (α 0.05) to determine the primary end point using intention-to-treat analysis. Secondary end points include parasite clearance time, rates of recurrent infection/treatment failure to day 42, gametocyte carriage throughout follow-up and rates of anaemia at day 28, as determined by survival analysis.
ETHICS AND DISSEMINATION: This study has been approved by relevant institutional ethics committees in Malaysia and Australia. Results will be disseminated to inform knowlesi malaria treatment policy in this region through peer-reviewed publications and academic presentations.
TRIAL REGISTRATION NUMBER: NCT01708876.
METHODS: A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples.
RESULTS: Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/μL and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA.
CONCLUSIONS: This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.
METHODS: Gerbils, 5-7 weeks old were infected by PbA via intraperitoneal injection of 1 × 106 (0.2 mL) infected red blood cells. Parasitemia, weight gain/loss, hemoglobin concentration, red blood cell count and body temperature changes in both control and infected groups were monitored over a duration of 13 days. RNA was extracted from the brain, spleen and whole blood to assess the immune response to PbA infection. Organs including the brain, spleen, heart, liver, kidneys and lungs were removed aseptically for histopathology.
RESULTS: Gerbils were susceptible to PbA infection, showing significant decreases in the hemoglobin concentration, RBC counts, body weights and body temperature, over the course of the infection. There were no neurological signs observed. Both pro-inflammatory (IFNγ and TNF) and anti-inflammatory (IL-10) cytokines were significantly elevated. Splenomegaly and hepatomegaly were also observed. PbA parasitized RBCs were observed in the organs, using routine light microscopy and in situ hybridization.
CONCLUSION: Gerbils may serve as a good model for severe malaria to further understand its pathogenesis.
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.