METHODS: Thick and thin blood films were made prior to administration of anti-malarial treatment in patients who were subsequently confirmed as having single species knowlesi infections by PCR assays. Giemsa-stained blood films, prepared from 10 randomly selected patients with a parasitaemia ranging from 610 to 236,000 parasites per microl blood, were examined.
RESULTS: The P. knowlesi infection was highly synchronous in only one patient, where 97% of the parasites were at the late trophozoite stage. Early, late and mature trophozoites and schizonts were observed in films from all patients except three; where schizonts and early trophozoites were absent in two and one patient, respectively. Gametocytes were observed in four patients, comprising only between 1.2 to 2.8% of infected erythrocytes. The early trophozoites of P. knowlesi morphologically resemble those of P. falciparum. The late and mature trophozoites, schizonts and gametocytes appear very similar to those of P. malariae. Careful examinations revealed that some minor morphological differences existed between P. knowlesi and P. malariae. These include trophozoites of knowlesi with double chromatin dots and at times with two or three parasites per erythrocyte and mature schizonts of P. knowlesi having 16 merozoites, compared with 12 for P. malariae.
CONCLUSION: Plasmodium knowlesi infections in humans are not highly synchronous. The morphological resemblance of early trophozoites of P. knowlesi to P. falciparum and later erythrocytic stages to P. malariae makes it extremely difficult to identify P. knowlesi infections by microscopy alone.
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: A systematic search was conducted for articles published from January 1990 to December 2009 in PubMed/MEDLINE using terms for malaria and 11 target countries (Bhutan, China, North Korea, Indonesia, Malaysia, Philippines, Solomon Islands, South Korea, Sri Lanka, Thailand and Vanuatu). The references were collated and categorized according to subject, Plasmodium species, and whether they contained original or derivative data.
RESULTS: 2,700 articles published between 1990 and 2009 related to malaria in the target countries. The annual output of malaria-related papers increased linearly whereas the overall biomedical output from these countries grew exponentially. The percentage of malaria-related publications was nearly 3% (111/3741) of all biomedical publications in 1992 and decreased to less than 1% (118/12171; p < 0.001) in 2009. Thailand had the highest absolute output of malaria-related papers (n = 1211), followed by China (n = 609) and Indonesia (n = 346). Solomon Islands and Vanuatu had lower absolute numbers of publications, but both countries had the highest number of publications per capita (1.3 and 2.5 papers/1,000 population). The largest percentage of papers concerned the epidemiology and control of malaria (53%) followed by studies of drugs and drug resistance (47%). There was an increase in the proportion of articles relating to epidemiology, entomology, biology, molecular biology, pathophysiology and diagnostics from the first to the second decade, whereas the percentage of papers on drugs, clinical aspects of malaria, immunology, and social sciences decreased.
CONCLUSIONS: The proportion of malaria-related publications out of the overall biomedical output from the 11 target Asian-Pacific countries is decreasing. The discovery and evaluation of new, safe and effective drugs and vaccines is paramount. In addition the elimination of malaria will require operational research to implement and scale up interventions.
METHODS: Blood was drawn from patients infected with P. falciparum in seven sentinel provinces along Thai international borders with Cambodia, Myanmar, Laos, and Malaysia. In vitro parasite sensitivity was tested using the World Health Organization's microtest (mark III) (between 1994 and 2002) and the histidine-rich protein-2 (HRP2)-based enzyme-linked immunosorbent assay (in 2010). Following World Health Organization protocol, at least 30 isolates were collected for each province and year represented in this study. Where possible, t-tests were used to test for significant differences.
RESULTS: There appears to be little variation across study sites with regard to parasite sensitivity to chloroquine. Quinine resistance appears to have been rising prior to 1997, but has subsequently decreased. Mefloquine sensitivity appears high across the provinces, especially along the north-western border with Myanmar and the eastern border with Cambodia. Finally, the data suggest that parasite sensitivity to artemisinin and its derivatives is significantly higher in provinces along the north-western border with Myanmar.
CONCLUSIONS: Parasite sensitivity to anti-malarials in Thailand is highly variable over time and largely mirrors official drug use policy. The findings with regard to reduced sensitivity to artemisinin derivatives are supported by recent reports of reduced parasite clearance associated with artemisinin. This trend is alarming since artemisinin is considered the last defence against malaria. Continued surveillance in Thailand, along with increased collaboration and surveillance across the entire Greater Mekong sub-region, is clearly warranted.
METHODS: A total of 189 whole blood samples were collected from Telupid Health Clinic, Sabah, Malaysia, from 2008 to 2011. All patients who participated in the study were microscopically malaria positive before recruitment. Complete demographic details and haematological profiles were obtained from 85 patients (13 females and 72 males). Identification of Plasmodium species was conducted using PlasmoNex™ targeting the 18S ssu rRNA gene.
RESULTS: A total of 178 samples were positive for Plasmodium species by using PlasmoNex™. Plasmodium falciparum was identified in 68 samples (38.2%) followed by 64 cases (36.0%) of Plasmodium vivax, 42 (23.6%) cases of P. knowlesi, two (1.1%) cases of Plasmodium malariae and two (1.1%) mixed-species infections (i e, P. vivax/P. falciparum). Thirty-five PlasmoNex™ positive P. knowlesi samples were misdiagnosed as P. malariae by microscopy. Plasmodium knowlesi was detected in all four districts of Sandakan division with the highest incidence in the Kinabatangan district. Thrombocytopaenia and anaemia showed to be the most frequent malaria-associated haematological complications in this study.
CONCLUSIONS: The discovery of P. knowlesi in Sandakan division showed that prospective studies on the epidemiological risk factors and transmission dynamics of P. knowlesi in these areas are crucial in order to develop strategies for effective malaria control. The availability of advanced diagnostic tool PlasmoNex™ enhanced the accuracy and accelerated the speed in the diagnosis of malaria.