METHODS: We did an environmentally stratified, population-based, cross-sectional survey across households in the Kudat, Kota Marudu, Pitas, and Ranau districts in northern Sabah, Malaysia, encompassing a range of ecologies. Using blood samples, the transmission intensity of P knowlesi and other malaria species was measured by specific antibody prevalence and infection detected using molecular methods. Proportions and configurations of land types were extracted from maps derived from satellite images; a data-mining approach was used to select variables. A Bayesian hierarchical model for P knowlesi seropositivity was developed, incorporating questionnaire data about individual and household-level risk factors with selected landscape factors.
FINDINGS: Between Sept 17, 2015, and Dec 12, 2015, 10 100 individuals with a median age of 25 years (range 3 months to 105 years) were sampled from 2849 households in 180 villages. 5·1% (95% CI 4·8-5·4) were seropositive for P knowlesi, and marked historical decreases were observed in the transmission of Plasmodium falciparum and Plasmodium vivax. Nine Plasmodium spp infections were detected. Age, male sex, contact with macaques, forest use, and raised house construction were positively associated with P knowlesi exposure, whereas residing at higher geographical elevations and use of insecticide were protective. Agricultural and forest variables, such as proportions and fragmentation of land cover types, predicted exposure at different spatial scales from households.
INTERPRETATION: Although few infections were detected, P knowlesi exposure was observed in all demographic groups and was associated with occupational factors. Results suggest that agricultural expansion and forest fragmentation affect P knowlesi exposure, supporting linkages between land use change and P knowlesi transmission.
FUNDING: UK Medical Research Council, Natural Environment Research Council, Economic and Social Research Council, and Biotechnology and Biosciences Research Council.
METHODS AND FINDINGS: A search using Ovid MEDLINE and Embase was initially conducted to identify studies on severe Plasmodium falciparum malaria that included information on treatment delay, such as fever duration (inception to 22nd September 2017). Studies identified included 5 case-control and 8 other observational clinical studies of SM and UM cases. Risk of bias was assessed using the Newcastle-Ottawa scale, and all studies were ranked as 'Good', scoring ≥7/10. Individual-patient data (IPD) were pooled from 13 studies of 3,989 (94.1% aged <15 years) SM patients and 5,780 (79.6% aged <15 years) UM cases in Benin, Malaysia, Mozambique, Tanzania, The Gambia, Uganda, Yemen, and Zambia. Definitions of SM were standardised across studies to compare treatment delay in patients with UM and different SM phenotypes using age-adjusted mixed-effects regression. The odds of any SM phenotype were significantly higher in children with longer delays between initial symptoms and arrival at the health facility (odds ratio [OR] = 1.33, 95% CI: 1.07-1.64 for a delay of >24 hours versus ≤24 hours; p = 0.009). Reported illness duration was a strong predictor of presenting with severe malarial anaemia (SMA) in children, with an OR of 2.79 (95% CI:1.92-4.06; p < 0.001) for a delay of 2-3 days and 5.46 (95% CI: 3.49-8.53; p < 0.001) for a delay of >7 days, compared with receiving treatment within 24 hours from symptom onset. We estimate that 42.8% of childhood SMA cases and 48.5% of adult SMA cases in the study areas would have been averted if all individuals were able to access treatment within the first day of symptom onset, if the association is fully causal. In studies specifically recording onset of nonsevere symptoms, long treatment delay was moderately associated with other SM phenotypes (OR [95% CI] >3 to ≤4 days versus ≤24 hours: cerebral malaria [CM] = 2.42 [1.24-4.72], p = 0.01; respiratory distress syndrome [RDS] = 4.09 [1.70-9.82], p = 0.002). In addition to unmeasured confounding, which is commonly present in observational studies, a key limitation is that many severe cases and deaths occur outside healthcare facilities in endemic countries, where the effect of delayed or no treatment is difficult to quantify.
CONCLUSIONS: Our results quantify the relationship between rapid access to treatment and reduced risk of severe disease, which was particularly strong for SMA. There was some evidence to suggest that progression to other severe phenotypes may also be prevented by prompt treatment, though the association was not as strong, which may be explained by potential selection bias, sample size issues, or a difference in underlying pathology. These findings may help assess the impact of interventions that improve access to treatment.
METHODS: On Ambae Island, blood samples were collected from 231 and 282 individuals in 2003 and 2007, respectively. Parasite prevalence was determined by microscopy. Antibodies to three Plasmodium falciparum (PfSE, PfMSP-119, and PfAMA-1) and three Plasmodium vivax (PvSE, PvMSP-119, and PvAMA-1) antigens, as well as the Anopheles-specific salivary antigen gSG6, were detected by ELISA. Age-specific seroprevalence was analysed using a reverse catalytic modelling approach to estimate seroconversion rates (SCRs).
RESULTS: Parasite rate decreased significantly (P malaria prevalence on Ambae Island between 2003 and 2007. The recombinant gSG6 antigen originally developed to indicate exposure to the Afrotropical vector An. gambiae may be used in the Pacific to complement the traditional measure of entomological inoculation rate (EIR).
OBJECTIVE: To evaluate immune-hematological profiles among HIV infected patients compared to HIV/malaria co-infected for ART management improvement.
METHODS: This was a cross sectional study conducted at Infectious Disease Hospital, Kano. A total of 761 consenting adults attending ART clinic were randomly selected and recruited between June and December 2015. Participants' characteristics and clinical details including two previous CD4 counts were collected. Venous blood sample (4ml) was collected in EDTA tube for malaria parasite diagnosis by rapid test and confirmed with microscopy. Hematological profiles were analyzed by Sysmex XP-300 and CD4 count by Cyflow cytometry. Data was analyzed with SPSS 22.0 using Chi-Square test for association between HIV/malaria parasites co-infection with age groups, gender, ART, cotrimoxazole and usage of treated bed nets. Mean hematological profiles by HIV/malaria co-infection and HIV only were compared using independent t-test and mean CD4 count tested by mixed design repeated measures ANOVA. Statistical significant difference at probability of <0.05 was considered for all variables.
RESULTS: Of the 761 HIV infected, 64% were females, with a mean age of ± (SD) 37.30 (10.4) years. Prevalence of HIV/malaria co-infection was 27.7% with Plasmodium falciparum specie accounting for 99.1%. No statistical significant difference was observed between HIV/malaria co-infection in association to age (p = 0.498) and gender (p = 0.789). A significantly (p = 0.026) higher prevalence (35.2%) of co-infection was observed among non-ART patients compared to (26%) ART patients. Prevalence of co-infection was significantly lower (20.0%) among cotrimoxazole users compared to those not on cotrimoxazole (37%). The same significantly lower co-infection prevalence (22.5%) was observed among treated bed net users compared to those not using treated bed nets (42.9%) (p = 0.001). Out of 16 hematology profiles evaluated, six showed significant difference between the two groups (i) packed cell volume (p = <0.001), (ii) mean cell volume (p = 0.005), (iii) mean cell hemoglobin concentration (p = 0.011), (iv) absolute lymphocyte count (p = 0.022), (v) neutrophil percentage count (p = 0.020) and (vi) platelets distribution width (p = <0.001). Current mean CD4 count cell/μl (349±12) was significantly higher in HIV infected only compared to co-infected (306±17), (p = 0.035). A significantly lower mean CD4 count (234.6 ± 6.9) was observed among respondents on ART compared to non-ART (372.5 ± 13.2), p<0.001, mean difference = -137.9).
CONCLUSION: The study revealed a high burden of HIV and malaria co-infection among the studied population. Co-infection was significantly lower among patients who use treated bed nets as well as cotrimoxazole chemotherapy and ART. Six hematological indices differed significantly between the two groups. Malaria and HIV co-infection significantly reduces CD4 count. In general, to achieve better management of all HIV patients in this setting, diagnosing malaria, prompt antiretroviral therapy, monitoring CD4 and some hematology indices on regular basis is critical.
METHODS: Blood samples from 1874 patients were tested for Plasmodium species by microscopy and nested polymerase chain reaction. P. knowlesi was characterized by sequencing the merozoite surface protein 1 gene (msp-1).
RESULTS: Of all Plasmodium species identified, P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi contributed 43.52%, 68.08%, 1.37%, 1.03%, and 0.57%, respectively. Mixed-species infections were more common in northwestern and southwestern regions bordering Myanmar (23%-24%) than in eastern and southern areas (3%-5%). In northwestern and southwestern regions, mixed-species infections had a significantly higher prevalence in dry than in rainy seasons (P < .001). P. knowlesi was found in 10 patients, mostly from southern and southwestern areas-9 were coinfected with either P. falciparum or P. vivax. Most of the P. knowlesi Thai isolates were more closely related to isolates from macaques than to isolates from Sarawak patients. The msp-1 sequences of isolates from the same area of endemicity differed and possessed novel sequences, indicating genetic polymorphism in P. knowlesi infecting humans.
CONCLUSIONS: This survey highlights the widespread distribution of P. knowlesi in Thailand, albeit at low prevalence and mostly occurring as cryptic infections.
METHODS: A total of 3002 blood samples on filter paper were collected from 555 inhabitants of 8 longhouses with recently reported knowlesi malaria cases in the Betong Division of Sarawak, Malaysian Borneo. Each longhouse was visited bimonthly for a total of 10 times during a 21-month study period (Jan 2014-Oct 2015). DNA extracted from blood spots were examined by a nested PCR assay for Plasmodium and positive samples were then examined by nested PCR assays for Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, Plasmodium knowlesi, Plasmodium cynomolgi and Plasmodium inui. Blood films of samples positive by PCR were also examined by microscopy.
RESULTS: Genus-specific PCR assay detected Plasmodium DNA in 9 out of 3002 samples. Species-specific PCR identified 7 P. knowlesi and one P. vivax. Malaria parasites were observed in 5 thick blood films of the PCR positive samples. No parasites were observed in blood films from one knowlesi-, one vivax- and the genus-positive samples. Only one of 7 P. knowlesi-infected individual was febrile and had sought medical treatment at Betong Hospital the day after sampling. The 6 knowlesi-, one vivax- and one Plasmodium-infected individuals were afebrile and did not seek any medical treatment.
CONCLUSIONS: Asymptomatic human P. knowlesi and P. vivax malaria infections, but not P. cynomolgi and P. inui infections, are occurring within communities affected with malaria.
METHODOLOGY/PRINCIPAL FINDINGS: A total of 439 records of P. knowlesi infections in humans, macaque reservoir and vector species were collated. To predict spatial variation in disease risk, a model was fitted using records from countries where the infection data coverage is high. Predictions were then made throughout Southeast Asia, including regions where infection data are sparse. The resulting map predicts areas of high risk for P. knowlesi infection in a number of countries that are forecast to be malaria-free by 2025 (Malaysia, Cambodia, Thailand and Vietnam) as well as countries projected to be eliminating malaria (Myanmar, Laos, Indonesia and the Philippines).
CONCLUSIONS/SIGNIFICANCE: We have produced the first map of P. knowlesi malaria risk, at a fine-scale resolution, to identify priority areas for surveillance based on regions with sparse data and high estimated risk. Our map provides an initial evidence base to better understand the spatial distribution of this disease and its potential wider contribution to malaria incidence. Considering malaria elimination goals, areas for prioritised surveillance are identified.
METHODOLOGY/PRINCIPAL FINDINGS: Using in silico tools, we designed and expressed four novel P. knowlesi protein products to address the distinct lack of suitable serosurveillance tools: PkSERA3 antigens 1 and 2, PkSSP2/TRAP and PkTSERA2 antigen 1. Antibody prevalence to these antigens was determined by ELISA for three time-points post-treatment from a hospital-based clinical treatment trial in Sabah, East Malaysia (n = 97 individuals; 241 total samples for all time points). Higher responses were observed for the PkSERA3 antigen 2 (67%, 65/97) across all time-points (day 0: 36.9% 34/92; day 7: 63.8% 46/72; day 28: 58.4% 45/77) with significant differences between the clinical cases and controls (n = 55, mean plus 3 SD) (day 0 p<0.0001; day 7 p<0.0001; day 28 p<0.0001). Using boosted regression trees, we developed models to classify P. knowlesi exposure (cross-validated AUC 88.9%; IQR 86.1-91.3%) and identified the most predictive antibody responses.
CONCLUSIONS/SIGNIFICANCE: The PkSERA3 antigen 2 had the highest relative variable importance in all models. Further validation of these antigens is underway to determine the specificity of these tools in the context of multi-species infections at the population level.
METHODOLOGY/PRINCIPAL FINDINGS: We conducted comprehensive surveys in three areas where P. knowlesi transmission is reported: Limbuak, Pulau Banggi and Matunggung, Kudat, Sabah, Malaysia and Bacungan, Palawan, the Philippines. Infection prevalence was low with parasites detected by PCR in only 0.2% (4/2503) of the population. P. knowlesi PkSERA3 ag1 antibody responses were detected in 7.1% (95% CI: 6.2-8.2%) of the population, compared with 16.1% (14.6-17.7%) and 12.6% (11.2-14.1%) for P. falciparum and P. vivax. Sero-prevalence was low in individuals <10 years old for P. falciparum and P. vivax consistent with decreased transmission of non-zoonotic malaria species. Results indicated marked heterogeneity in transmission intensity between sites and P. knowlesi exposure was associated with agricultural work (OR 1.63; 95% CI 1.07-2.48) and higher levels of forest cover (OR 2.40; 95% CI 1.29-4.46) and clearing (OR 2.14; 95% CI 1.35-3.40) around houses. Spatial patterns of P. knowlesi exposure differed from exposure to non-zoonotic malaria and P. knowlesi exposed individuals were younger on average than individuals exposed to non-zoonotic malaria.
CONCLUSIONS/SIGNIFICANCE: This is the first study to describe serological exposure to P. knowlesi and associated risk factors within endemic communities. Results indicate community-level patterns of infection and exposure differ markedly from demographics of reported cases, with higher levels of exposure among women and children. Further work is needed to understand these variations in risk across a wider population and spatial scale.
METHODOLOGY/PRINCIPAL FINDINGS: Anopheles spp. were sampled using human landing catch (HLC) method at Paradason village in Kudat district of Sabah. The collected Anopheles were identified morphologically and then subjected to total DNA extraction and polymerase chain reaction (PCR) to detect Plasmodium parasites in the mosquitoes. Identification of Plasmodium spp. was confirmed by sequencing the SSU rRNA gene with species specific primers. MEGA4 software was then used to analyse the SSU rRNA sequences and bulid the phylogenetic tree for inferring the relationship between simian malaria parasites in Sabah. PCR results showed that only 1.61% (23/1,425) of the screened An. balabacensis were infected with one or two of the five simian Plasmodium spp. found in Sabah, viz. Plasmodium coatneyi, P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Sequence analysis of SSU rRNA of Plasmodium isolates showed high percentage of identity within the same Plasmodium sp. group. The phylogenetic tree based on the consensus sequences of P. knowlesi showed 99.7%-100.0% nucleotide identity among the isolates from An. balabacensis, human patients and a long-tailed macaque from the same locality.
CONCLUSIONS/SIGNIFICANCE: This is the first study showing high molecular identity between the P. knowlesi isolates from An. balabacensis, human patients and a long-tailed macaque in Sabah. The other common simian Plasmodium spp. found in long-tailed macaques and also detected in An. balabacensis were P. coatneyi, P. inui, P. fieldi and P. cynomolgi. The high percentage identity of nucleotide sequences between the P. knowlesi isolates from the long-tailed macaque, An. balabacensis and human patients suggests a close genetic relationship between the parasites from these hosts.
METHODS: Over 3.5 years, we prospectively assessed patients of any age with molecularly-confirmed Plasmodium monoinfection presenting to 3 district hospitals in Sabah, Malaysia.
RESULTS: Of 481 knowlesi, 172 vivax, and 96 falciparum malaria cases enrolled, 44 (9%), 71 (41%), and 31 (32%) children aged ≤12 years. Median parasitemia was lower in knowlesi malaria (2480/μL [interquartile range, 538-8481/μL]) than in falciparum (9600/μL; P < .001) and vivax malaria. In P. knowlesi, World Health Organization-defined anemia was present in 82% (95% confidence interval [CI], 67%-92%) of children vs 36% (95% CI, 31%-41%) of adults. Severe knowlesi malaria occurred in 6.4% (95% CI, 3.9%-8.3%) of adults but not in children; the commenst severity criterion was acute kideny injury. No patient had coma. Age, parasitemia, schizont proportion, abdominal pain, and dyspnea were independently associated with severe knowlesi malaria, with parasitemia >15000/μL the best predictor (adjusted odds ratio, 16.1; negative predictive value, 98.5%; P < .001). Two knowlesi-related adult deaths occurred (fatality rate: 4.2/1000 adults).
CONCLUSIONS: Age distribution and parasitemia differed markedly in knowlesi malaria compared to human-only species, with both uncomplicated and severe disease occurring at low parasitemia. Severe knowlesi malaria occurred only in adults; however, anemia was more common in children despite lower parasitemia. Parasitemia independently predicted knowlesi disease severity: Intravenous artesunate is warranted initially for those with parasitemia >15000/μL.