Age-Related Clinical Spectrum of Plasmodium knowlesi Malaria and Predictors of Severity

Patients with knowlesi malaria were older and had lower parasitemia compared to human-only species. Severe knowlesi malaria occurred only in adults. Anemia was common in children despite lower parasitemia. Parasitemia predicted disease severity: Intravenous artesunate is warranted initially for parasitemia>15000/μL.

in children [2,22], or comparisons between zoonotic knowlesi malaria and locally acquired malaria from the human-only species P. falciparum and P. vivax in district settings.
In this study, we compared the predefined clinical spectrum between children and adults with malaria due to P. knowlesi or other Plasmodium species infection, and evaluated predictors of disease severity in a coendemic primary care setting.

Study Sites and Referral System
This study was conducted in Kudat Division, northwest Sabah, Malaysia, covering an area of 4623 km 2 and with a total growthrate adjusted Malaysian census-estimated population in 2016 of 199 600 people. Each of the 3 districts in this division has a central referral hospital and subdistrict health clinics, consistent with other districts in Sabah. Malaysian Ministry of Health guidelines stipulate that all patients with fever receive microscopic blood slide screening for malaria parasites, with mandatory hospital admission, free treatment, and notification of positive cases [23].

Subjects
Patients of all ages presenting to study hospitals with microscopy-diagnosed malaria were enrolled following written informed consent. Children were predefined as age ≤12 years, consistent with Malaysian Ministry of Health pediatric ward admission. Patients were not included in the final analysis if they were pregnant or had Plasmodium malariae infection on polymerase chain reaction (PCR), if Plasmodium species PCR was not confirmed, or if crosscheck research microscopy was negative. A subset of patients with uncomplicated P. knowlesi and P. vivax malaria was also enrolled in previously reported randomized controlled treatment trials [23][24][25].

Study Procedures
Baseline and longitudinal clinical, laboratory, and epidemiological data were entered using standardized case record forms. Venous blood was taken for baseline investigations and then daily for microscopy and hematology during hospital admission and at the follow-up visit 28 days after treatment initiation. Severe malaria was defined using World Health Organization (WHO) 2014 research criteria [26], including for P. knowlesi: hyperparasitemia threshold of 100 000/μL, and jaundice defined as bilirubin >50 μmol/L with parasite count >20 000/ μL and/or creatinine >132 μmol/L [18]. Nonsevere anemia was defined using WHO age-and sex-based hemoglobin criteria [27]. AKI was evaluated using Kidney Disease Outcomes Quality Working Group (KDIGO) criteria [28]. Chronic disease was defined as hypertension; diabetes mellitus; ischemic heart disease; hyperlipidemia; or chronic kidney, liver, or respiratory disease.

Laboratory Procedures
Microscopic asexual parasite and gametocyte counts were calculated by research microscopists using thick blood smears and quantitated leukocyte count. Standard hospital automated hematology, biochemistry, and microbiology laboratory results were used. Final Plasmodium species confirmation was done using PCR [29,30].

Statistical Analysis
We compared between-group differences with analysis of variance or Kruskal-Wallis testing for continuous variables, and Student t test or the Wilcoxon-Mann-Whitney test for 2-group comparisons according to distribution. For categorical variables, χ 2 or Fisher's exact test was used. Logistic regression models were fitted to determine a priori predictors of severe malaria based on standard clinical and laboratory WHO 2014 research criteria [26] evaluable at time of acute patient presentation to district hospital settings, including testing for model interactions and collinearity. Receiver operating characteristic (ROC) analysis was used to assess their sensitivity and specificity. Multivariate analysis controlled for age and log e parasitemia; patients with hyperparasitemia as a sole severity criterion were considered nonsevere.

Ethical Considerations
This study was approved by the medical research ethics committees of the Ministry of Health, Malaysia; London School of Hygiene and Tropical Medicine, United Kingdom; and Menzies School of Health Research, Australia.

Baseline Features: Adults
Duration of fever for P. knowlesi-infected adults was comparable to both children with P. knowlesi and adults with malaria due to   ; P = .033), although this did not remain statistically significant after controlling for age; and was also comparable to that seen in both adults with P. falciparum and children with knowlesi malaria. Liver aminotransferases were higher in adults with knowlesi malaria compared to those with P. vivax including after controlling for age (P = .001). Of the 322 adults with knowlesi malaria who had blood cultures, only 1 grew a noncontaminant isolate, a 14-year-old with Neisseria meningitidis.

DISCUSSION
This study is the largest series of P. knowlesi malaria cases to date, and the first to prospectively compare the clinical spectrum of disease between adults and children. Although 91% of knowlesi malaria cases were adults, morbidity in children was also demonstrated, with an 11-fold higher risk of anemia at presentation and a similar risk of mild to moderate AKI compared to adults [1,7]. The majority of adults with   [31]. Only a minority had high parasitemia, with parasitemia an independent predictor of severe knowlesi malaria overall.
Notably, there was no coma or convulsions seen in any patient with knowlesi malaria, consistent with previous studies  Abbreviations: ICU, intensive care unit; WHO, World Health Organization. a Includes 2 Plasmodium knowlesi patients with serious underlying medical illness: 1 with worsening of known chronic kidney disease (acute kidney injury) and another with endometriosis-associated bleeding (anemia). Table 5 [6,17,18,32]. No child with knowlesi malaria had severe manifestations (although borderline severe anemia was present in one), in contrast to the severe disease found in pediatric falciparum and vivax malaria in this series and elsewhere [26]. A lower proportion of P. knowlesi infections were in children compared to those with P. vivax or P. falciparum. The lower incidence of clinical disease from P. knowlesi infection in infants and also older children has been attributed to epidemiological factors such as lower forest exposure [1,13,18], although contributing age-related innate protective mechanisms are plausible [33], and asymptomatic infection has been reported in children [34]. Most children with knowlesi malaria had anemia at enrollment, consistent with a previous retrospective report [22]. Although adults with knowlesi malaria had higher parasite counts, nonsevere anemia was more common in children, suggesting that children may have a higher rate of uninfected RBC destruction and/or greater dyserythropoiesis, although underlying mechanisms and baseline community anemia prevalence require further investigation [35]. Children with knowlesi malaria had lower parasitemia and platelet counts compared to children with either P. vivax or P. falciparum infection, in addition to a lower neutrophil count compared to P. vivax. However, there was a comparable risk of nonsevere anemia and AKI seen in P. knowlesi-infected children as in those with P. vivax or P. falciparum.

. Selected Clinical and Laboratory Predictors of Severe Knowlesi Malaria at Presentation
The proportion of adults with severe disease from P. knowlesi infection was comparable to that seen in P. falciparum. The risk of severe knowlesi malaria in this primary referral setting in Sabah, 6.2% in adults, was similar to district hospital presentations in Sarawak (9.3%) [17], and lower than that demonstrated in a tertiary hospital setting of 29% [18]. Severe AKI was the most frequent severity criterion, and has commonly been reported in other adult studies [17,18,32]. Severe anemia was present in a larger proportion of adults with severe knowlesi malaria than in a previous tertiary-referral study, which reported anemia as a severe criterion in only 5% of adults, 1 of whom was splenectomized [18]. Plasmodium knowlesi parasitemia [18,32] and age [33] independently predicted severe disease in this study, in addition to abdominal pain and dyspnea, which have not been previously demonstrated. Parasite counts were higher in severe knowlesi malaria than in uncomplicated disease despite no difference in the number of preceding days of fever, which suggests differences in efficacy and tropism of normocyte invasion and parasite multiplication [31]. With age an independent risk factor for both parasitemia and severity, the immunosenescence that occurs with aging [33,36] may also result in impaired control of parasite multiplication.
The pathophysiological mechanisms in severe knowlesi malaria are not well understood but likely differ from P. falciparum, with coma remaining unreported and a lack of the retinal microcirculatory changes found in severe falciparum malaria [37]. With endothelial activation and systemic inflammation at least as high in response to P. knowlesi as in P. falciparum infection [33,36], these processes also likely contribute to pathogenesis, particularly with the comparatively low parasite biomass able to produce severe disease observed in this study. The nature and role of microvascular accumulation of parasitized RBCs, a key mechanism of severe knowlesi malaria in rhesus macaques and also observed in a single human autopsy report [21], requires investigation. RBC deformability is reduced in proportion to disease severity in knowlesi malaria [38], however the role of hemolysis and endothelial dysfunction, other key pathogenic mechanisms also present in severe falciparum malaria [39], require further investigation. Phenotypic glucose-6-phosphate dehydrogenase deficiency has been shown to protect against knowlesi malaria [13]. Other host genetic factors related to selection pressure from historical human-only Plasmodium transmission may also modulate disease severity.
Current knowlesi malaria management guidelines in Sabah recommend referral for tertiary care and initial treatment with intravenous artesunate for any patients >50 years of age or with a parasitemia >20 000/μL [18,40]. Along with appropriate intravenous artesunate administration for severe malaria due to any Plasmodium species, these management guidelines have contributed to a decline in reported malaria case-fatality rate [6,18,20]. In the current study, predictors were limited to severe disease given the low case-fatality rate, with a parasite threshold of 15 000 parasites/μL giving a high negative predictive value of 98.5%. A conservative approach would be to recommend early administration of intravenous artesunate initially for any knowlesi malaria case with a parasite count above this threshold, given the potential delay or inability to evaluate other laboratory markers of severe disease in most primary care settings. In conclusion, although the majority of cases are uncomplicated, P. knowlesi infection causes morbidity at comparatively low parasitemia in both adults and children. Adults are at risk of severe and fatal disease, in contrast to children, among whom this was not demonstrated. A conservative treatment approach utilizing parasite counts to predict severe disease is warranted.

Supplementary Data
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