Prevalence and Force of Plasmodium Vivax and Plasmodium Falciparum Blood Stage Infection and Associated Clinical Malaria Burden in the Brazilian Amazon

Background: Plasmodium vivax and Plamodium falciparum are co-endemic in much of the Brazilian Amazon, with P. vivax comprising greater than 80% of clinical cases, especially in low transmission settings. The molecular force of blood-stage infection of P. vivax (molFOB) can provide a detailed picture of P. vivax transmission in low transmission settings and help improve malaria measures control and elimination efforts. Methodology: Monthly samples were collected in a cohort of 1,274 individuals of all ages between April 2013 and March 2014 in three peri-urban communities in the Brazilian Amazon. Regression analyses were used to test how factors including age and community were associated with P. vivax molFOB, parasite positivity and clinical episodes. Principal Findings: Respectively, 77.8% and 97.2% of the population remained free of P. vivax and P. falciparum infection. Expected heterozygosity for P. vivax was 0.69 for MSP1_F3 and 0.86 for MS2.


Introduction
The global burden imposed by malaria remains high, with an estimated 219 million cases and 435,000 related deaths in 2017 [1]. Although malaria remains endemic in 91 countries, the majority of the burden (90%) is con ned to the African Region where Plasmodium falciparum predominates [1]. In the Americas, Plasmodium vivax is the predominant species causing over two thirds (69%) of all cases in 2017. Despite most endemic countries in Latin America having achieved a reduction of incidence since 2010, there were still more than half a million (562,300) cases in 2017, with Venezuela and Brazil accounting for more than half of these cases (65%) [1]. Between 2005 and 2017 Brazil has achieved a 70% reduction of malaria cases, with 99% of all cases occurring in the Amazon region in 2017 [1,3]. Of special vulnerability are malaria-naïve individuals recently arrived from malaria-free areas and engaged in agricultural and forest-related activities such as logging, shing and mining [4][5][6][7]. In the 1970s, increased industrial development demanded a large labor force, which evoked a large migratory in ux to the peripheries of bigger cities such as Manaus. Such largely uncontrolled settlements have led to a gradual increase of malaria transmission in peri-urban areas [3].
Anopheles darlingi is the major malaria vector in the Amazon region, and has highly anthropophilic and endophagic behavior [8][9][10][11]. Current vector control measures primarily include the use of long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS), but there are little data from Latin America on the impact of vector control. One study showed that the use of LLINs was associated with malaria protection in the Amazon, however, it seemed to have a more protective effect against P. falciparum malaria in comparison with P. vivax malaria [7]. As in other regions, recent progress in malaria control in Brazil has been accompanied by an increasing proportion of cases caused by P. vivax, underscoring the challenges, namely relapses, yet to be addressed in control and management this species [12]. Understanding the epidemiology of relapses after primary infection is challenging, with an estimated 14-40% of individuals having detectable recurrences, even after treatment with primaquine [7,[13][14][15][16]. A second challenge is the early production of P. vivax gametocytes, along with asymptomatic cases, which enables the transmission of parasites even before the infected individual develops clinical symptoms. Thirdly, P. vivax parasite densities are generally lower than those seen in P. falciparum infections, especially in hypoendemic areas such as in Brazil, therefore requiring more sensitive diagnostic tools [17]. Research has shown that a high proportion of submicroscopic and asymptomatic P. vivax infections usually exist in such settings, and that these infections can easily be missed by routine active and passive case detection [17]. It has been shown that asymptomatically infected individuals are able to infect mosquitoes of the Amazon region, therefore these infections might fuel residual malaria transmission, thereby complicating malaria elimination [18][19][20][21].
In view of these challenges to malaria elimination, it is important to gather data of spatial and temporal patterns of asymptomatic Plasmodium infections as well on gametocyte carriage from low-transmission settings. Therefore, in this study, we investigated risk factors and spatial-temporal patterns of incidence of Plasmodium infection and clinical malaria episodes in a peri-urban area of Manaus, Western Brazilian Amazon. We paid special attention to submicroscopic and asymptomatic infections by measuring the prevalence of P. falciparum and P. vivax blood-stage infections by PCR, the incidence of clinical cases, and estimating the molecular force of P. vivax blood-stage infection (molFOB) derived from molecular detection and genotyping of infections. Understanding the malaria epidemiology through P. vivax molFOB may provide a detailed assessment of malaria transmission by measuring individual exposure and its burden. Such information is of paramount importance when measuring intervention e cacy, host susceptibility and transmission patterns in low transmission and pre-elimination settings such as Brazil.

Ethics statement
This study was approved by the Brazilian National Committee of Ethics (CONEP) (349.211/2013) and the Committee of Ethics for Clinical Investigation of the Barcelona Hospital Clinic (7306/2012). All participants were informed about the objectives of the study as well as the potential risks and bene ts of their participation in the study. An informed consent form was signed by all study participants or by a parent or legal guardian in case of participants that were under 18 years of age. Children between 12 and 17 years of age signed an additional assent form. As routinely done, malaria patients received treatment with 25 mg/kg of chloroquine over a 3-day period (10 mg/kg on day 0 and 7.5 mg/kg on days 1 and 2.
Primaquine was prescribed at the dosage of 0.5 mg/kg per day, during 7 days.

Study design and subjects
This cohort study was conducted in the Brasileirinho, Ipiranga and Puraquequara communities, located in Manaus peri-urban area, between April 2013 and March 2014 ( Figure 1A). A detailed description of the study area has been presented elsewhere [17]. According to a census performed by a Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD) eld team, the population of the study area was estimated to be approximately 2,400 inhabitants before the start of the study, in 2012. Each community has access to a malaria clinic for microscopy-based malaria diagnosis and treatment. A total of 1,200 participants of any age were enrolled into the study in April 2013.

Data and sample collection
For each study participant, a questionnaire was completed containing personal information such as age, gender, occupation, pregnancy, history of travel, as well as information on malaria preventive measures, previous malaria episodes and current health status. Upon enrolment and monthly during follow-up, nger-prick blood 151 samples (~300 μL) were collected using Microtainer® tubes containing EDTA and sodium uoride (Becton Dickinson, USA). In infants, blood was obtained by either heel or toe puncture. Within one hour of collection, 50 μL of blood were transferred into a reaction tube containing 250 μL of RNAprotect (QIAGEN, Germany) in order to preserve RNA for downstream analyses [22] and 200 μL of whole blood was transferred to another reaction tube. Samples were stored in cooling boxes until arrival in the laboratory, where the 200 μL sample was separated into a red blood cell (RBC) pellet and plasma.
All samples were frozen at -80ºC until further processing. If the collected blood volume was <250 μL, the actual volume was recorded.

Clinical symptoms
In the case of symptoms related to malaria or in 161 the case of increased body temperature (>37.5ºC), a thick blood smear (TBS) was prepared according to World Health Organization guidelines [23]. When positive for malaria, appropriate treatment was provided in accordance with the national guidelines of the Brazilian Ministry of Health [24]. An asymptomatic infection was de ned as presence of a malarial infection by TBS, but absence of fever and any other malaria related symptoms (chills, sweating, headache, vomit, abdominal pain) at the moment of sample collection, or anytime in the preceding 48 hours.
Plasmodium spp. infection, clones and gametocyte carriage Pelleted RBCs, obtained from 200 μL of whole blood, were re-suspended in PBS and genomic DNA was extracted using FavorPrep 96-well Genomic DNA Kit (Favorgen, Taiwan) according to the manufacturer's instructions. DNA was eluted with 2x 100 μL of elution buffer and stored at -20ºC until assayed by PCR. If the amount of whole blood available for DNA extraction was ≤100 μL, the DNA volume was vacuum concentrated until reaching the original blood volume recorded. RNA from 50 μL whole blood, stored in RNAprotect, was extracted using RNeasy Plus 96-well kit (QIAGEN, Germany) and eluted in 50 μL RNasefree dH2O as described previously [22]. All DNA samples were subject to a generic Plasmodium species (QMAL) qPCR targeting a conserved region of the 18S rRNA gene [22]. QMAL-positive samples were further analysed by species-speci c qPCR assays targeting the18S rRNA genes of P. falciparum and P. vivax, as previously described [22,25]. For detection of P. falciparum, a modi ed reverse primer was used [26]. For quanti cation of 18S rRNA gene copy numbers, in each experiment three dilutions of control plasmids containing the respective amplicons were included in triplicates (102, 104 and 106 copies/μL). For genotyping individual P. vivax clones, the molecular markers msp1F3 and MS16 were typed using capillary electrophoresis for highly precise fragment sizing allowing for longitudinal follow up of individual parasite clones. Details of the genotyping methods have been described previously [27]. RT-qPCR assays were performed on RNAs from all P. vivax and/or P. falciparum positive samples to detect gametocyte-speci c transcripts of the pvs25 (P. vivax) and pfs25 (P. falciparum) genes. For quanti cation of pvs25 and pfs25 transcript numbers, control plasmids containing the ampli ed region were included as standards in each run. All qPCR and RT-qPCR assays were performed on a 7500 Fast Real-Time PCR System (Applied Biosystems).

Statistical analysis
Data from questionnaires were imported into databases using Cardiff TeleForm version 10.4.1 (Cardiff Software). Individual databases were combined in Microsoft Access 2010. For incidence calculations (molFOB and clinical malaria incidence), subject data were censored on the last visit before two consecutively missed scheduled follow-up visits in order to reduce bias [28]. Differences in proportions were tested for statistical signi cance using the McNemar X2 test with continuity correction. To achieve normal distribution, qPCR densities were expressed as log10-transformed 18S rRNA genomic copies/μL blood for asexual parasites, and log10-transformed pfs25 or pvs25 transcripts/μL blood for gametocytes. Geometric means of densities were calculated. Differences in densities of asexual or sexual-stage parasites were tested for statistical signi cance using Welch's two-sample t-test.
The molecular force of new P. vivax blood-stage infections (molFOB) was calculated by counting the number of genotypes observed at each visit, that had not been present in the preceding two visits (0-0-1 patterns) and adjusting these counts by the respective times-at-risk. molFOB for P. vivax was determined for both genetic markers combined. Negative binomial regression models were used to assess the in uence of different risk-factors on the incidence of P. vivax and P. falciparum gametocyte positivity as previously described, using positivity counts and times-at-risk over the entire period of observation [28].
Since molFOB is a count variable measured per individual over a speci c exposure time (time at risk), and is overdispersed, a negative binomial regression model was chosen in which the exposure time at risk is used as offset. If we de ne μj as the log of the number of genotypes at visit j, then for each infection pattern j (0-0-0 or 0-0-1) we have μj = exp(βxj + offsetj + νj), where β is a vector of regression coe cients, offsetj=log(exposure time) and νj follows a gamma distribution (to give a negative binomial distribution). Incidence rate ratios (IRR) and adjusted IRR (aIRR) were calculated with their respective 95% con dence intervals. Because using the collapsed data to model molFOB for each individual does not allow for the analysis of time-changing covariates, factors in uencing frequency of parasite positivity and frequency of clinical episodes within the study period were explored using multiple failure time models allowing for time-changing covariates [29]. For multiple failure time models, hazard rate ratios (HRR) are calculated with 95% con dence intervals. In these models, parasite positivity and clinical episodes were equivalent to a 'failed' outcome, respectively. In addition to the adjusted statistical models presented in the main manuscript, univariate analyses and multivariate analyses with backward selection are provided as  Table 1.

Prevalence by active case detection
Monthly P. vivax prevalence by qPCR ranged from 2.5% in June 2013 to 6.5% in November 2013 ( Figure  2A). P. falciparum was not detected in August and September 2013, with the highest prevalence (~1.0%) occurring in March 2014 ( Figure 2B). Both species presented a similar seasonality pro le, with a higher prevalence of P. vivax from October to February, and of P. falciparum from November to March, coinciding with the rainy season ( Figure 2D). P. vivax asymptomatic infections predominated in Puraquequara community, although clinical cases were mostly seen in Ipiranga. For P. falciparum, asymptomatic infections and clinical cases were both predominant in the Ipiranga community.

Risk factors associated with P. vivax positivity
In the multivariate analysis shown in Table 2

Genetic Diversity and Multiplicity of Infections
The heterogeneity in the incidence of malaria infections is shown in Figure 3A. Both P. vivax and P. falciparum infections were restricted to a small proportion of the study population. Overall, 77.8% of the population remained free of P. vivax infection and 97.2% of the population remained free of P. falciparum infection over the course of the study period. Based on the two markers (MSP1 F3 and MS2), expected heterozygosity for P. vivax was 0.69 (MSP1_F3) and 0.86 (MS2), respectively. Overall, the multiplicity of infection was close to the value of 1 as determined with both markers (1.06 for MSP1_F3 and 1.04 for MS2, respectively), indicating that infections were mostly observed as monoclonal. Figure 3B shows the P. vivax molFOB over the entire year of follow-up. Similarly, new P. vivax infections were restricted to approximately 20% of the study population, whereas 80% did not receive new infections. Of individuals who had any P. vivax infection, the majority had molFOB = 1 to 2. The maximum number of genetically distinct infections/individual/year was molFOB = 5.
Factors associated with P. vivax molFOB

Discussion
The present study highlights well-known differences in the epidemiology of P. vivax and P. falciparum, evaluating the incidence of Plasmodium spp. and clinical cases of malaria in peri-urban communities in the Brazilian Amazon and estimate the P. vivax molFOB in low transmission settings in the Americas. Previous studies have measured molFOB in Papua New Guinean children in observational and randomized clinical trial cohorts, and have related incidence of clinical infection and other factors to this measure [30,31]. The main factors related to P. falciparum molFOB in these earlier studies were seasonality, village of residence and age [30][31][32][33]. As for P. vivax, molFOB was strongly associated with incidence of clinical episodes and a high molFOB likely resulted in rapid acquisition of immunity against P. vivax in children [31].
Recent studies in the Amazon region and other malaria endemic areas in the world have shown a high proportion of submicroscopic P. vivax infections [7,17,34]. In the present study, factors associated with P. vivax positivity were age (20-60 years), seasonality, use of mosquito nets and IRS. In the Brasileirinho, Ipiranga and Puraquequara communities, the distribution of mosquito nets and IRS is mainly focused on areas where there are many cases of malaria (ascertained by active and passive case detection, registered by the SIVEP-Malaria platform). However, this measure may not have been effective in preventing infection in these areas, or it may not be su cient to decrease P. vivax positivity, since many infections may derive from hypnozoites rather than new infections, as suggested by a study in Papua New Guinea [32].
A higher prevalence of malaria infection in the rainy season was also shown in previous studies, corroborating what has already been described for other regions of the Amazon [34,35]. The predictors of P. vivax clinical disease found in our study were seasonality, and marginally, frequency of travel and use of mosquito nets. Koep i [31] also found the seasonality to be a predisposing factor in clinical disease by P. vivax. Protective factors of clinical disease such as being over 60 years old and working in agriculture may be associated to prolonged exposure to P. vivax infection during lifetime. Due to age-and exposuredependent acquired immunity, clinical presentation of malaria becomes rarer in relation to age, thus increasing the number of asymptomatic carriers [31,36].
We also observed that P. vivax prevalence and molFOB in the study area were higher and less affected by seasonality as compared to P. falciparum. Whereas P. vivax prevalence and molFOB peaked in November in Ipiranga community, transmission indicators remained more stable throughout the entire observational period in the other two communities, with almost no seasonality observed in Brasileirinho. In contrast, the annual P. falciparum prevalence pro le was characterized by a very sharp peak in January in Ipiranga community during which the observed prevalence increased by almost 10-fold alongside a sharp rise of clinical malaria cases. In fact, nearly all clinical cases observed in the present study occurred during this period and in Ipiranga community. The outbreak caused by P. falciparum subsided by April and the remainder of the observational period was characterized by very low P. falciparum prevalence in all three communities. These observed differences in the seasonality pro les of the two species indicate a more stable transmission of P. vivax in contrast to in contrast to a more unstable transmission of P. falciparum.
The overall lower P. vivax parasite densities are close to the threshold of detection of even molecular diagnostic tests, thus hampering the characterization of the true incidence of new infections detected by genotyping, as reported elsewhere [37,38]. Therefore, estimation of molFOB can be dominated by clones with higher parasite densities, while low density, sub-dominant clones may not be detected, especially if they have arisen from relapses. As such, the molFOB reported in this study is likely an underestimation of the true force of blood-stage infection.
In conclusion, P. vivax infection prevailed in the area and infections were mostly observed as monoclonal.
High proportions of symptomatic and submicroscopic infections were also found. Previous malaria episodes were associated with signi cantly higher P. vivax molFOB, likely indicating that effective radical cure is an important strategy to be addressed in these endemic communities. Asymptomatic and submicroscopic infections pose substantial challenges for P. vivax malaria control, hampering accurate surveillance efforts needed to pursue elimination, especially in low transmission settings.   Spatial representation of clinical P. vivax and P. falciparum cases (Panel A) and qPCR detected P. vivax and P. falciparum infections (Panel B). Data are shown as incidence (cases/detections per person per year, aggregated to the household level). Increased diameter of the circles represents increased incidence.

Declarations
Maps were created using QGIS 2.18, with geodata collected for this study.  Supplementary Files