Impact of Plasmodium vivax malaria on executive and cognitive functions in elderlies in the Brazilian Amazon

The exact path leading to cognitive impairment that goes beyond malaria is unclear, but it appears to be the result of interactive factors. Time of exposure to disease and recurrences are potentially major determinant variables. Cognitive impairment is described mainly in children, rarely in adults. The disease in high endemic areas usually does not affect elderlies, because of acquired immunity over time. However, this population is relatively more frequently sick in lower endemic areas, such as in the Amazon. This study assessed the effect of Plasmodium vivax malaria on the executive and cognitive functions of elderlies, in the Brazilian Amazon. A cohort study was conducted to evaluate executive and cognitive functions one week (T0), two months (T2) and eight months (T8) after the malaria episode. Mini-Mental State Examination (MMSE), Beck Depression Inventory II (BDI-II), Clock Drawing Test (CDT), Wechsler adult intelligence scale (WAIS-III), and Wisconsin Card Sorting Test (WCST) were used to assess executive and cognitive functions. One hundred-forty elderlies were enrolled (70 with P. vivax malaria and 70 without malaria). P. vivax malaria was associated with impairment of the executive and cognitive functions in elderlies for up to 8 months after acute P. vivax malaria. Prior history of malaria, recurrences and higher parasitemia were independently associated with various surrogates of executive and cognitive impairment. With the increase in life expectancy, elderlies living in malaria endemic areas will deserve more attention from health authorities, to guarantee improvement of their quality of life in the tropics.


Malaria and other diseases diagnosis
Thick blood smears were performed for the malaria diagnosis in all symptomatic patients 19 .All participants (exposed and unexposed) were instructed to seek the care unit if they manifested symptoms.In addition, all participants were followed in online official Malaria Epidemiological Surveillance Information System (Sivep-Malaria).At the time of assessment of executive and cognitive functions, in T2 and T8, peripheral venous blood was collected for assessment of asymptomatic infection by molecular techniques For DNA extraction, the miniblod Qiagen Kit was used, according to the manufacturer's instructions.Qmal Taqman PCR was used to detect Plasmodium spp. in DNA samples, as previously described 20 .Malaria recurrences over the study period have been confirmed in Sivep-Malária.Patients diagnosed with malaria were treated according to the Brazilian Ministry of Health's treatment guidelines.
Hepatitis C, HIV and syphilis were also investigated by serological rapid tests (Abbot) performed according to the manufacturer's recommended instructions, as potential concomitant causes of cognitive impairment at

Sample size calculation
The sample size calculation assumed a 20% prevalence of cognitive impairment due to infection caused by P. falciparum in adults 29 .For the general population, the prevalence was assumed to be 6.1% 30 .Thus, considering a test of differences in proportions between two groups of the same size, 90% of power and 5% alpha error, 244 participants were required (122 per group).Adding 15% of losses, the final sample size was ~ 280 participants.Sample calculation was performed using software and R, version 4.0.5 (R project for Statistical Computing, http:// www.R-proje ct.org) 31 .Interim analysis was scheduled after the inclusion and follow-up of 140 patients.

Statistical analysis
Normal distribution was assessed for variables with the Shapiro-Wilk test.Differences between baseline characteristics were evaluated by ANOVA, for continuous variables with normal distribution, Wilcoxon rank-sum test for continuous variables not normally distributed, and Pearson's Chi Squared test was applied for categorical variables.Multivariate analysis included age, sex, and education variables, as well as depression (expressed by BDI-II indicators).
The main and subgroup analyzes were performed with Poisson regression.The main analyzes compared executive and cognitive fucntions between subjects with malaria and without malaria.Additional subgroup analysis was performed for malaria patients considering three variables: (1) malaria episodes before enrollment; (2) recurrences between T0 and T8; and parasitemia higher than the median number of parasites/mm 3 .Previous univariate analysis was performed for multivariable variable selection.Variables with a p-value lower than 0.2 were included.All analyses were conducted in Stata 13.0 (Statacorp, 2013).

Results
Out of 470 subjects, 140 were considered eligible (70 exposed and 70 non exposed) (Fig. 1).A total of 110 patients performed tests for HIV, syphilis and CHV (none was positive).Median age was 66 years (63.0-71.5).Women represented 51% from the total population.Exposed and no exposed differed in terms of age, sex, and education (Table 1).
According to official information system, no previous malaria infections were reported in the control group, while 57.1% of cases had previous episodes in the period.In the case group, 75.7% did not present recurrences of malaria until assessment in T8, and 69.1% had higher than 500 parasites/mm 3 parasitaemia at T0 infection (Supplementary Table 2).At T2 and T8, no parasites were identified in the blood smear.

Discussion
This cohort study is one of the first attempts to examine the association of P. vivax malaria between executive and cognitive functions in elderlies.The findings suggest that elderlies with P. vivax malaria experienced deficits in their executive and cognitive functions from 2 months to at least 8 months post-infection.Higher parasitemia, prior and recurrent malaria negatively impacted executive functions in the elderlies.Thus, given the high number of P. vivax infections and an ageing population worldwide, the magnitude of this problem might not be negligible.The recognition of cognitive deficits and variability resulting from ageing, infectious diseases, or metabolic processes, requires early identification since there is a risk of evolution to dementia and a negative impact on the quality of life in the elderlies and their families [32][33][34][35][36] .Poorer cognitive function is associated with an increased risk of depression, social withdrawal, and dependence, and may contribute to decreased quality of life 33,36 .
The impact of malarial infections on cognition has been assessed in severe and non-severe falciparum and vivax malaria, and the studies are almost exclusively performed in children [11][12][13][14][15][16]37 . Thee studies show that malaria affects language development, attention span in children and adolescents and negatively impacts their cognitive development and school performance.Cognitive and executive losses associated with infectious diseases are poorly studied amongst elderlies.This lack of studies may be related to the difficulty in establishing whether the losses are associated with senility or to the infectious process per se.However, some infectious agents have been thought as potential factors contributing to dementia 38,39 .The clinical pattern of malaria, especially severe malaria, differs between children and adults.However, it is uncertain whether these differences reflect the age of affected individuals or other differences between populations in the characteristics of host, parasite, pattern of exposure or provision of health services 40 .These factors may interfere in distinguishing the cognitive effects associated with malaria between children and the elderly, however it is important to note that the impact of cognitive impairment on the child's life may be more significant, as it reflects on delays and important losses in school development, especially, that can be reflected throughout their adult life 7,[11][12][13][14][15][16]41 .
Executive and cognitive assessment is a valuable clinical tool and usually involves the use of two types of psychological instruments or tests (screening tests and diagnostic or confirmatory tests).In this study, we used tools with different sensitivities, which resulted in different results regarding the determination of cognitive and executive functions.However, it is important to emphasize that screening tests MMSE and CDT have high specificity and are used to signal mild cognitive impairment and a maximum number of suspected psychological processes and are suitable to safely exclude false positives.The other tests used WCST and (WAIS/WMI) are high sensitivity instruments and used to confirm any psychological condition and confirm true positives, and are used to signal mild cognitive impairment and a maximum number of suspected psychological processes 42 .
Infections by herpes simplex virus type 1 (HSV-1) 43 and cytomegalovirus (CMV) 39 , HIV 44 , and bacteria, such as Chlamydia pneumoniae 45 and Helicobacter pylori 46 and Lyme neuroborreliosis 47 have been associated with cognitive impairment.In older adults with pneumonia and urinary tract infection, for example, the risk of cognitive loss is about 1.4 times higher and previous episodes are positively associated with cognitive loss 48 .Inflammatory reactions have been highly related to cognitive decline and risk of dementia 49 .Inflammatory cytokines produced www.nature.com/scientificreports/by the nervous system act on neural substrates to produce behavioral symptoms that can be related to cognitive changes 50 .Thus, like the diseases, vivax malaria is capable of significantly altering cognitive and executing functions for up to 8 months, which, in turn, may affect the quality of life of the elderly.In children, parasitemia, the number of previous and recurrent malaria has a negative impact on cognition and executive functions, with a cumulative negative effect on school performance being observed 7,[11][12][13][14][15][16]41 . In he elderlies, we observed similar effects up to 8 months after the malaria episode.Parasitemia at T0, number of previous and recurrent malaria had a negative impact on cognition and executive functions.
The cognitive mechanisms underlying malaria remain poorly understood and experimental models of severe malaria, particularly the murine model using the Plasmodium berghei ANKA (PBA) strain, are a valuable tool for understanding the cognitive and neurological outcomes associated with this condition 51,52 .Studies with an animal model are focused on seeking to understand mainly the cognitive and neurological mechanisms, based on the implementation of CM.Neuroinflammation after PBA infection of mice influences neurotrophin expression, which impairs hippocampal neurogenesis and increases hippocampal cell death.This is associated with impaired memory, but specifically short-term memory after the CM course 51 .Inflammation of the central nervous system, because of CM, determines the release of inflammatory cytokines, which affect neurogenesis and reverberate in cognitive defects 53     activities of a key leukocyte integrin in the immunoinflammatory responses that mediate brain involvement 54 .
In non-severe malaria, the cognitive impairment mechanisms are not known, but they may be associated with cytokine storms during acute infection 55 , the production of neurotoxins by infected red blood cells 56 , impaired coagulation that can impact the correct oxygen supplementation to the tissues of the central nervous system 57 , anemia 58 , multiple infections 59 .
Plasmodium vivax can be associated with neurological and vascular impairment 60 .The predilection for reticulocytes, marked proinflammatory responses, and the reversible microvascular dysfunction typically associated with P. vivax infection may explain the pathogeny of CM in malaria vivax [61][62][63] .Vascular congestion, hypoperfusion, and localized hypoxia in CM occurs mainly in the occipital and parietal lobes, this in turn has important reflexes in visuospatial deficit [64][65][66][67] .In the present work, visuospatial deficits were evaluated with the MMSE and CDT tools and the exposed group's performance was worse than non-exposed group.Executive functions and visuospatial skills are two interrelated cognitive processes and these deficits, which are associated with a decline in the performance of daily activities 68,69 .
It must be considered that chloroquine (CQ) used for the treatment of vivax malaria may have as an adverse event psychiatric and nervous system disorders, which may reflect on changes in cognitive and executive functions during the treatment of vivax malaria 70,71 .Since only the patients exposed in this study had previous malaria, they are in an endemic area, where CQ has been the treatment of first choice for many years and low CQ resistance is defined, we consider that CQ may potentially play an important role in altering neurocognitive functions.CQ is reported as an inhibitor of autophagy in a variety of diseases, including Alzheimer's disease and cerebral ischemia.After inducing brain trauma, treatment with CQ significantly suppressed neuronal autophagy and reduced levels of expression of inflammatory cytokines, interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α), in the hippocampus of the mouse 72 and this can reduce cognitive impairment.In contrast, patients treated with CQ (cases) showed worsening cognition and executing functions.However, the half-life of the drug is highly unlikely to explain the long-term cognitive impairment seen in our patients at T8.
In this study, we did not assess other potential co-infections associated with malaria or underlying diseases that could potentially interfere with cognition.A systematic approach to addressing these confounders would be important, however, the clinical and pre-clinical follow-up of these patients to define potential confounders was hampered in this study population, especially due to home care and to the fact that they present at the hospital unit exclusively for the malaria diagnosis.The use of secondary information from SIVEP is also an important limiting factor.Although the groups exposed and not exposed to malaria were selected from the same transmission area, other variables were not properly paired.The adjustment of variables not properly paired were included in multivariate analyzes and had no impact on cognition alone.Attention and language were evaluated by instruments of low sensitivity.

Concluding remarks
Our results suggest that malaria promotes cognitive, executive, and functional decline in the elderly population for up to 8 months after P. vivax infection, and that factors such as number of previous malaria cases, recurrence, and parasitemia are important predictors of executive impairment in the second and eighth months after infection.In the context of a rapidly ageing population and the high rates of transmission of vivax malaria in subtropical and tropical areas, where vivax malaria is endemic, malaria can contribute with a substantial negative impact on cognition and executing functions that reflect on the life quality.This, in turn, adds to the deleterious events of the normal ageing process (the lack of health guarantee and the risks of developing diseases common to this stage of life, such as dementia).The revelations obtained in this study should serve as a notice to the policy makers and professionals involved in the healthcare and improvement of the elderlies' quality of life, considering that this population is vulnerable to cognitive damage.

Figure 1 .
Figure 1.Flowchart of the study.
. The study organized by Azevedo-Quintanilha et al. revealed that αDβ2 integrin deletion alters the natural course of experimental severe malaria, demonstrating previously unrecognized

Table 1 .
Demographic and clinical characteristics in the baseline.Significant values are in bold.