Resurgence risk for malaria, characterization of a recent outbreak in a Amazonian border area French Guiana/Brazil-CURRENT

Background In 2017, inhabitants along the border between French Guiana and Brazil were affected by a malaria outbreak primarily due to Plasmodium vivax ( Pv ). While malaria cases have steadily declined between 2005-2016 in this Amazonian region, a resurgence was observed in 2017. Methods Two investigations were performed according to two different spatial scales and type of information details. Firstly, a local study on the French Guiana border, which enabled a thorough investigation of the malaria cases treated at the local village health center and of the entomological situation in the most affected neighborhood and secondly a regional and cross-border study, which enabled exploration of the regional spatial-temporal epidemic dynamic. Number and location of malaria cases were estimated using French and Brazilian surveillance systems. Results On the French Guiana side of the border in Saint Georges de l’Oyapock, the attack rate was 5.5% (n=219/4000), reaching 51.4% (n=90/175) in one Amerindian neighborhood. Entomological findings suggest a peak of Anopheles darlingi density in August and September. Two female An. darlingi (n=2/1104, 0.18%) were found Pv positive during this peak. During the same period, aggregated data from passive surveillance conducted by Brazilian and French Guianese border health centers identified 1,566 cases of Pv infection. Temporal distribution during the 2007-2018 period displayed seasonal patterns with a peak in November 2017. Four clusters were identified among the epidemic profiles of the localities of the cross-border area. All localities of the first two clusters were Brazilian. The localization of the first cluster suggests an onset of the outbreak in the Amerindian reservation, subsequently expanding to French Amerindian neighborhoods and to non-Native communities. Conclusions The current findings demonstrate a potential increase in malaria cases in an

area with otherwise declining numbers. This is a transborder area where human mobility and remote populations challenge malaria control programs. This investigation illustrates the importance of international border surveillance and collaboration for malaria control, particularly in Amerindian villages and mobile populations.

Background
After two decades of global decreases in malaria incidence, rates are increasing for the first time since 2016 [1]. In the Americas, the largest increase was recorded in Brazil and Venezuela [1]. Thus, malaria remains a public health challenge in South America. In Brazil, transmission is mainly entrenched in the Amazon Basin, which accounts for 99.5% of Brazil's malaria burden [2].
French Guiana is a European overseas, malaria-endemic territory. The Oyapock river forms the border between French Guiana and Brazil. This area has engaged in a regional malaria control program [3], which faces challenges in controlling malaria among autochthonous and illegal gold miner populations living generally in remote areas and along the borders with Surinam and Brazil [2][3][4][5]. Along the Oyapock river, Plasmodium falciparum has declined over the past several years and P. vivax is now responsible for most malaria cases [6,7]. Although transmission decline is observed on a global scale, local heterogeneities persist and must be addressed through targeted control initiatives in order to achieve malaria elimination [8,9]. This is particularly important in border areas, which complicate effective implementation of malaria control interventions [2,10].
Anopheles darlingi is the predominant species and a major malaria vector in this territory [11,12].
The aim of this article is to describe the malaria outbreak in 2017 through an entomoepidemiological investigation in the Saint Georges de l'Oyapock (STG) area along the Oyapock river. The investigation is associated with a retrospective study using a spatio-temporal analysis of malaria cases data from surveillance health systems of French Guiana and Brazil.

Outbreak detection
The malaria surveillance system in French Guiana is based on three data sources: remote health centers (CDPS) reports and laboratory and military health services notification.
Since the end of May 2017, data from the CDPS of Saint Georges de l'Oyapock showed a slow and persistent increase of P. vivax cases in the Native neighborhood of Trois Palétuviers. These clusters and the subsequent local outbreak have no apparent link to any other transmission sites in French Guiana, nor with the epidemiological situation in the municipality of Oiapoque, an immediate neighbor on the Brazilian side of the border.
The transmission continued, and between September and October 2017, increases in the number of malaria cases were observed in downtown STG, French Guiana. This number largely exceeded the expected cases over the past three years for this endemic area [7].
Retrospectively, the data provided by the Brazilian malaria epidemiological surveillance information system (SIVEP-Malária) revealed an increase in the number of cases of malaria infections in the Oiapoque municipality, including among the Uaçá, Galibi and Juminã indigenous territories (Amapá, Brazil).

Study area and population
On the French side, the cross-border Oyapock region includes three municipalities: Camopi, STG and Ouanary, with approximately 1,700, 4,000 and 100 residents respectively in 2017 according to the STG health center census. The region marks the north-eastern border between French Guiana and Brazil (Figure 1). On the Brazilian side, the cross-border area includes the municipality of Oiapoque, which holds 25,514 inhabitants according to the 2015 estimation by the Brazilian National Institute of Statistics, IBGE), and lies along the Oyapock river (Figure 1). To the north and east of the Oiapoque municipality, there are a large number of small villages. Amerindian territories (Uaçá, Galibi, Juminã) are predominant. This cross-border area consists of a vast remote territory covered by the Amazonian rainforest associated with highly variable population densities. There is also a great diversity of populations, including Amerindians (mainly Wayãpi, Teko, Palikur, Karipuna, Galibi-Marworno and Galibi tribes), Creoles, Saramaka and migrants from other Brazilian states who migrate to the area to work in the gold mining sector (mainly illegal gold mining or border supply zones). Therefore, daily transborder exchanges exist among these populations who share a unique living, fishing and hunting area. The climate is equatorial, with four alternating seasons: a long rainy season from April to June, a dry season from July to December, a short, rainy season from January to February and a short dry season in March. The cities of STG and Oiapoque are persistent, low malaria endemicity areas [7].

Malaria case definition
Malaria diagnosis was performed in health centers using the Rapid Diagnosis Test (RDT) -SD Bioline ® Malaria Ag Pf/Pan in French Guiana, and thick and thin smears or RDTs were used in Brazil. A malaria case was defined as a patient with RDT or microscopy-positive results. Data included passive monitoring of cases in border health centers in French Guiana and Brazil, but also active case detection around positive cases in the city of Oiapoque.
Before treatment with primaquine, a glucose-6-phosphate dehydrogenase (G6PD) deficiency test was conducted at least two weeks after the malaria infection in French Guiana [13].
In French Guiana, P. vivax relapse was defined as having a medical history of malaria within a period of 7 to 90 days since the last malaria diagnosis. This interval was considered adequate in length to distinguish follow-up (0-7 days), relapse (7-90 days) and new infection (>90 days) [7,14].
In Brazil, the absence of a unique patient identification code did not permit the use of the same relapse identification method. However, malaria attacks related to patient follow-up, treatment failures and potential relapses were identified during the medical consultation and denoted as treatment verification slide (Lâmina de Verificação de Cura, LVC) in the database. A malaria attack was considered as an LVC for P. vivax if the patient was positive for P. vivax and received a treatment against P. vivax malaria during the last 60 days. It is worth noting that P. vivax relapses are expected to be less likely to occur in Brazil due to a systematic primaquine administration (except for specific cases such as pregnancy), which did not exist on the French Guiana side.
As listed below, two investigations were performed according to two different spatial scales and type of information details: 1.
a local study on the French Guiana border, which enabled a thorough investigation of the malaria cases treated at the STG health center and of the entomological situation in the most affected neighborhood; 2. a regional and cross-border study, which enabled exploration of the regional spatialtemporal epidemic dynamic.

Epidemiological description
Data from medical records of the STG health center allowed a retrospective analysis of epidemics. Malaria cases diagnosed between January 1, 2017 and January 31, 2018 were included, and the following variables analyzed: age, gender, outcome and location of acute P. vivax malaria cases and relapses treated in the only STG health center of the study area. Census data were retrieved from the health center in order to calculate the incidence and the attack ratio by neighborhood. Factors associated with risk of attack/relapse were identified by univariate analysis.

Entomological investigations
The entomological investigation focused on Trois-Palétuviers, a neighborhood of STG experiencing large numbers of malaria cases during this period. Mosquitoes were collected monthly from August to November 2017 over 2 to 3 consecutive nights per month, then identified morphologically. Two octenol-baited Mosquito Magnet® traps were used to collect anopheline species and were supplemented with BG-Sentinel and CDC light traps [15]. The collections were performed periodically from 18:00 to 07:00 am. Intradomiciliary aspirations were done inside four houses between 19:00 and 20:00 in August.
The infectious status of the Anopheles spp. captured from August to October was investigated. The head and thorax of 1,218 females were dissected and placed in an agitator with grinding beads. The DNA of each sample was extracted using a Magjet Genomic DNA kit (Thermo Scientific, K2722), then 10 female DNA samples were pooled for polymerase chain reaction (PCR) detection. The presence of P. falciparum, P. vivax and P. malariae parasites was investigated by nested PCR using the Snounou et al. method [16].
Individual confirmation was performed for each positive pool.

Registries
French Guiana data came from border area health centers (Delocalized Centers for Prevention and Care CDPS) in French Guiana.
Brazilian data came from the Brazilian information system dedicated to epidemiological surveillance (Sistema de Informações de Vigilância Epidemiológica da Malária, SIVEP-Malária).
The harmonization of the epidemiological information provided by the French and Brazilian malaria surveillance systems was performed by the cross-border malaria information system described in Saldanha et al., submitted.

Epidemic profile clustering
In order to describe the spatial-temporal epidemic dynamics in the study area, the epidemic profiles of the cross-border area localities were defined and clustered, and the resulting clusters were represented and interpreted both temporally and spatially. To achieve this, first we selected localities presenting a significant number of cases, followed by each country individually. The method included:

Epidemiological description of malaria cases within the French Guiana border area (STG health center):
During the study period from January 1, 2017 to January 31, 2018, 219 people were infected with P. vivax (primary attack The density of An. darlingi at the village outskirts was higher in August and September than in October and November 2017 (p= 0.045), with no significant peak in August ( Figure   3). Anopheles darlingi was captured at all hours of the collection period. It was also the only Anopheline species found biting inside households in the evening in August.

Epidemic profile analysis
The total number of cases reported in registries during this period (from January 1, 2017 to January 31, 2018) was 1,664 (1,434 in Brazil, 182 in French Guiana). Forty eight (48) cases were associated with unspecified localities of residence and were ignored (nine were notified in Brazil, with Brazil as the country of residence; the 39 remaining cases were notified in French Guiana without any mention of country of residence). Figure 5 illustrates the results of the epidemic curve clustering. A dendrogram was dissected to define four clusters that corresponded to both low intra-cluster and high inter-cluster variances. Clusters were ranged chronologically by considering the time interval during which the weekly case numbers reached their maximum, corresponding to the inflection points of the cumulated curves. The inflection point corresponded approximately to the moment when the number of cases reached 50% of the total number of the cases during the whole considered period ( Figure 5). All localities of the first two clusters are Brazilian ( Figure 6 and Supplement S2). Figure 6 is a map of the localities and clusters. All localities are situated in the northern part of the study area. Amerindian areas were reached first, demonstrating high incidence rates compared to the French STG or Brazilian Oiapoque city centers ( Figure 6). Cluster 1 included two Amerindian localities, with a much earlier start of the epidemic than elsewhere (between weeks 21-25) ( Figure 6 and Supplement S2). The Firstly, there has been a shift from P. falciparum to P. vivax infections in this area over the last decade with greater difficulty (contraindication, availability of G6PD blood test and treatment) in treating with primaquine and thus in avoiding relapses [6,17].
Secondly, hikes in infections may be due to environmental causes such as the exceptional rainy season in 2017 -which can raise vector density -and the impacts of increased deforestation in Amazonia [18,19]. Thirdly, in 2017, a new municipal government was inaugurated in Oiapoque. With that, leadership roles shifted in the Special Indigenous Health District (DSEI), an entity responsible for providing health care to indigenous people in Oiapoque. At the onset of this type of change in power dynamics, there is often an administrative discontinuity of local governments, which may have consequences on public services [20]. Finally, political and economic regional crises, particularly in Venezuela but also in Brazil, have led to large-scale human migration into Brazil and is associated with an increase of illegal gold mining [4,21]. Unexpectedly, our results on incidence and spatial distribution revealed an additional human factor within the specific population of Amerindians. The study of the epidemic dynamics tends to show that the number of cases began increasing in Brazilian Amerindian villages before increasing on the French side of the border. These communities appeared to be particularly at risk for malaria infection and participated in factors driving this epidemic. Remote Amerindian communities have specific behaviors, precarious living conditions, poor access to health care, higher mobility, as well as commercial, cultural or family links among populations from both sides of the border. This may explain the cross-border epidemic dynamic, and these communities represent a key population for malaria control. However, further investigations should be conducted to: i) clearly identify these hypothetical mechanisms; ii) confirm that such a scenario may have occurred in previous years; iii) show that observed events did not occur independently. Indeed, studies of the transmission dynamics for each Plasmodium spp. genotype could help provide an overview of the spread of infection in this cross-border population.
Present and past studies show the influence of vector dynamics on malaria transmission [12,22]. In 2017, the heaviest rainfall hit the area since 2000 and may have had an impact on mosquito density [18]. Our collections of An. darlingi confirm the well-known exo/endophagic behavior of this vector, and the need to prevent host-vector contact inside households (with mosquito nets) and outside (use of repellants and loose clothing).
Several recent publications refer to insecticide resistance within this species in Brazil [23], and also increased daytime activity [22]. Outbreak control measures in French Guiana enabled the creation of a specific malaria team with community health mediators from November to February. This team administered radical treatment to a large majority of cases. A previous retrospective study in the same area did not deploy the same strategy and reported less than 5% complete treatments [17].

Conclusion
The current findings demonstrate a potential increase in malaria cases in an area with otherwise declining numbers. This is a transborder area where human mobility and remote populations challenge malaria control programs. P. vivax elimination requires greater, well-coordinated efforts and border-transcending malaria control. New strategies may be vital to achieving elimination, particularly in Amerindians communities.

Ethics approval and consent to participate
The French Guianese database was anonymized and declared to the Commission Nationale Informatique et Libertés (CNIL) (authorization N°1939018). Brazil's surveillance registries database was anonymized prior to being sent. The cross-border malaria information system (Saldanha et al., submitted) was also approved by the CNIL (N°2135463).

Consent for publication
Not applicable

Availability of data and materials
The datasets generated and analyzed during the current study are not publicly available due to the requirement of special authorization to transfer databases provided by the CNIL. Upon prior CNIL authorization, the datasets can be made available from the corresponding author upon reasonable request.

Competing interests
The authors declare that there is no conflict of interest.

Funding
This study was funded by the French Guianese Regional Health Agency and by the European Funds for Regional Development, "ELIMALAR" N°Synergie: GY0012082 and     cluster (same color code as in Figure 1).The size of the triangles is proportional to the incidence rate. The colors of the triangles correspond to the color code used in Figure 1. Bottom right: normalized cumulated curves for the averaged number of cases per cluster (same color code as in Figure 1).