Extent of asymptomatic Plasmodium spp. infections and associated risk factors at household and individual levels in Korhogo health district, Northern Côte d’Ivoire: implications for malaria control

Background: Plasmodium spp. asymptomatic carriers are potential reservoirs contributing to the persistence of malaria transmission in endemic areas. The study was designed to assess the extent of Plasmodium spp. asymptomatic infection at household and individual levels and associated risk factors in Northern Côte d’Ivoire. Methodology: A cross-sectional survey was conducted in July 2016 at household level in the health district of Korhogo. A questionnaire was administered to household’s head to capture socio-demographic information and practices including malaria treatment and preventive measures. In each household, adults without malaria symptoms or history of fever during the week before recruitment were screened. Capillary blood samples were collected and used for the detection of Plasmodium spp. infections using both conventional microscopy and a loop-mediated isothermal DNA amplification (LAMP) assay. Logistic regression was used to determine variables that influenced Plasmodium spp. asymptomatic infections. Findings: In total, 376 households and 1’011 asymptomatic adults were screened. Asymptomatic Plasmodium spp. infections were identified in 12.5% [47/376] and 38.3% [144/376] of the households and in 5.2% [53/1011] and 18.8% [190/1011] of the individuals screened, according to microscopy and LAMP, respectively. At household level, asymptomatic carriers increased about two times when using mosquito repellent coils compared to those where it is not used (OR: 1.8; p=0.005). At individual level, men’s risk to be infected was about two times that of women (OR: 1.9; p<0.001). The odd to be infected was also two times higher in population living in periurban areas compared to those living in the urban centre (OR: 2.3; p<0.001). Additionally, age appear to be risk factor, with younger individuals being at higher risk of infection than elders (OR: 0.5; p=0.001). Conclusion: Plasmodium spp. asymptomatic carriers are important in Northern Côte d’Ivoire and male, age under 30 and periurban living area appear as significant risk factors. Interventions aiming malaria elimination in the study context should primarily target among other strategies, asymptomatic infections and periurban zones.


Introduction
Reinforced antimalarial interventions led to a global decline of this disease since 2000 [1]. As a result, Plasmodium spp. infections tend to become gradually restricted to foci of residual transmission in settings with declining malaria transmission. Capitalizing on this success, the World Health Organisation (WHO) has adopted a Global Technical Strategy for the 2015-2030 period with the ambitious goal to reduce malaria mortality by 90% by 2030 [2]. This strategy acknowledges that successful control and elimination strategies need to specifically target transmission reservoirs. Such reservoirs include asymptomatic carries, which typically harbour hard-to-detect low density infections, that can, nevertheless, contribute significantly to malaria transmission in such settings [3].
Asymptomatic infection is defined by the WHO malaria terminology as the presence of asexual parasite in the blood without any fever or malaria-related symptoms [4]. Asymptomatic carriage of P. falciparum may protect against clinical malaria [5]. However, this infection is often undetected and untreated, resulting in a significant source of gametocytes for the transmission of the disease by local mosquito vectors [6]. Asymptomatic carriage of P. falciparum is not detected by routine heath care system and require active case detection strategies using highly-sensitive diagnostic tools [7].
Molecular diagnostics have shown that this asymptomatic parasitic reservoir is more widespread than expected, even in low-endemic areas [8]. Strategies that specifically target this parasite reservoir are therefore needed to accelerate malaria elimination in low-and medium-transmission settings [9]. Benié EMA, et al. However, in some countries, such as Côte d'Ivoire, national policies have not yet specifically targeted asymptomatic carriers of Plasmodium spp. for planning antimalarial elimination interventions [10].
Côte d'Ivoire remains a malaria endemic country, where malaria transmission is stable and occurs throughout the year, while morbidity increases during the rainy season [11,12]. A National cross-sectional survey conducted in 2012, in school age children, reported an average prevalence as high as 70% [13]. The National antimalarial policies have been improved over the last decade to reinforce control measures. Insecticide-treated nets (ITNs) have been widely distributed free-of-charge to the entire population through regular mass campaigns at the country level. In 2006,370'000 ITNs were distributed and total of 30'000'000 were distributed between 2010 and 2014 [14]. Thus, ITNs household coverage increased from 27% in 2006 to 66% in 2012 and 95% in 2015. In parallel, malaria prevalence decreased from 43% to 33% from 2010 to 2014 [13]. Building on this result, the national malaria control program (NMCP) aimed to halve the malaria morbidity by 2020 [14]. In order to reach this target, the NMCP strategy is based on three pillars: (i) an appropriate diagnosis and an effective treatment of clinical cases; (ii) the distribution of ITNs free of charge among population, and (iii) the intermittent preventive treatment (IPT) during pregnancy [10].
This strategic plan is mainly focused on symptomatic cases, identified through a passive detection system within the health system. Intervention targets are youngest children and pregnant women in priority because they are at higher risk of clinical malaria [15]. Asymptomatic infections are not specifically targeted in this strategic plan because of limited available resources which are currently targeted to better control until eliminationspecific measures can be considered.
It has been shown that asymptomatic Plasmodium spp. infections are frequent in the country [16]. In Korhogo, city of Northern Côte d'Ivoire, twenty years ago, a 70% prevalence of asymptomatic Plasmodium spp. Infections has been reported; however, another study carry out in 2015, in the same city showed a prevalence below 20% and 7.7% during the rainy and dry season, respectively [10,12]. Both studies have used routine malaria diagnostic tools for the detection of Plasmodium infections and are likely to have missed a significant number of low-density infections. Sensitive molecular techniques, based on nucleic acid amplification and providing much lower limit of detection (LOD) than routine diagnostic methods, in the order of magnitude of 0.02 parasite per microliter of blood (p/µL) at best, are available to detect Plasmodium infections [17]. Simplified molecular assays, using loop-mediated isothermal amplification (LAMP), have also been developed and enable the sensitive detection of malaria infections, in the range of 1 to 5 p/µL, in remote laboratories with limited equipment and capacity. Commercially available LAMP kits have shown a similar performance than PCR, especially when used in remote settings, making it a useful tool to investigate the prevalence of malaria infections, including asymptomatic low-density infections, in endemic areas [18].
Plasmodium spp. asymptomatic carrier is potential malaria reservoir that not considered in our national malaria policy because of limited resources. Theirs weights should underrate based on routine diagnosis test and LAMP test should be more suitable to detect asymptomatic. This study assesses the extent of asymptomatic Plasmodium spp. infections in Northern Côte d'Ivoire, based on result of a cross-sectional survey using both microscopy and LAMP diagnostic kits. We also investigated the risk factors associated with asymptomatic infections and discuss their relevance for malarial control interventions.

Study area
The study was carried out from 17 to 28 July 2016 in the health district of Korhogo, North Côte d'Ivoire. The district population was estimated at 536 851 inhabitants in 2014 [19]. The climate is characterized by two seasons: a rainy season that occurs from May to October and a dry season from November to April. The yearly rainfall is 1200 mm and the average temperature is 26.6°C [20]. A dam has been built in 1972 to provide water for irrigation of fields and market gardening. Anopheles gambiae is the most prevalent vector species in this district [20]. Among malaria parasites, Plasmodium falciparum is the quasi-exclusive species, with rare cases of P. malariae and P. ovale also being reported [10].

Study design and procedures
The study is a cross-sectional household survey. As described in a recent study carried out in Korhogo, 390 households were randomly selected from 30 demographic zones [10]. Three teams (each including medical doctor or a nurse, a laboratory technician, and questionnaire administrator) have enrolled participants using a households-based approach for 10 days. The study area was divided into three parts: urban centre, intermediate zone and periurban. Households located in town square were considered belong to urban centre. Those were situated in a peripheral of this city were considered belong to periurban. Households between centre and periurban areas were assigned to the intermediate zone ( Figure 1). Participant inclusion criteria were: (i) absence of fever (axillary temperature >37.5°C) or self-reporting history of fever during the last seven days; (ii) self-reporting less than two malaria-suggesting symptoms, such as headache, shiver, sweat, nausea, vomiting, dizziness, tiredness, abdominal pain; (iii) no self-reported use of antimalarial treatments over the four previous weeks; (iv) written informed consent of the participant.
A questionnaire was addressed to the household's head or his/her representative. A finger-prick blood sample was collected for Plasmodium spp. infection detection by microscopy and LAMP. Participants without malaria symptom, positive for malaria diagnosis test were considered as asymptomatic Plasmodium spp. carriers.

Ethical consideration
The study was approved in 2016 by the National Ethic and Research Committee of Côte d'Ivoire (NCER) (N/Réf: 120/MSHP/CNER-dk), NMCP and the local health district authorities. In agreement with recommendations of NMCP pertaining to the national policy on the management of malaria cases, asymptomatic participants positive to Plasmodium spp. were not treated. All of them received ITNs. They were also advised, if any malaria symptom would appear, to rapidly present to a health centre for malaria diagnosis and, if needed, treatment.

Data collection Questionnaire
A questionnaire relative to promiscuity (household size, household crowd), use of preventive measures (ITNs, mosquito's repellents coils and insecticide sprayed) and treatment seeking behaviour was addressed to the household's head or his/her representative. Socio-demographic data (sex and age) were recorded for all members of the selected household.

Microscopy
Microscopy is gold standard. It was used as routine malaria diagnosis test. A thick and thin blood film was made on a slide and air dried on site. This diagnostic was performed as described elsewhere. A slide was classified as negative if no Plasmodium asexual forms or gametocytes were found after counting 500 white blood cells [10].

LAMP
LAMP is a novel malaria diagnosis. According its similar performance as PCR, it was used in this study [21]. A finger-prick blood sample was collected for Plasmodium spp. infection detection by LAMP (LoopampTM MALARIA Pan/Pf Detection Kit, Eiken Chemical Company). Sixty µL of capillary blood were collected from finger prick, immediately mixed with an equal volume of extraction buffer, stored at ambient temperature, until processed the same day for LAMP as described elsewhere and using a "boil and spin" sample  preparation [22]. The LAMP reactions were conducted according to the manufacturer's recommendations using a standard heat block and results were read under a UV lamp. Extracted DNA samples were first tested using a Pan LAMP assay detecting all human-infecting Plasmodium spp. All panpositive samples were further tested using a P. falciparum specific LAMP assay (Pf LAMP). Samples were classified as negative (Pan LAMP negative), P. falciparum positive (Pan and Pf LAMP positive) or non-falciparum positive (Pan LAMP positive and Pf LAMP negative).

Data analysis
Double data entry was done using the Epidata 3.1 software. Labelling, and data transcription was carried out from software SPSS 18.0.0. Statistical analysis was conducted using STATA 12.1. Data were analysed both at household and at individual levels. For both levels, the dwelling setting was also considered. A household with at least one member diagnosed as positive for Plasmodium infection was considered as positive.
Prevalence of asymptomatic carrier was determined with 95% confidence interval. Pearson chi-square or Fisher's exact tests was used to determine association between asymptomatic Plasmodium spp. infection prevalence and promiscuity, prevention, treatment seeking behaviour, sex, ages groups and dwelling setting. The age group defined were <30 years, 30-40 years and >40 years [23]. Bivariate logistic regression was used to identify crude odds ratios (cOR) of variables associated with Plasmodium spp. asymptomatic infection and multiple logistic regression model to identify adjusted odds ratios (aOR).Variables were considered significant when the p value was < 0.05.

Study population characteristics
Out of 390 households visited, 376 were included in this study. Fourteen households without any eligible participant were excluded. A total of 1'149 individuals were screened for eligibility and 1'011 eligible participants were enrolled in the study. The 138 non-eligible participants were spread across the 14 excluded households and 61 included ones. All enrolled participants underwent malaria testing as described above.

Household characteristics
The mean number of people per household's was 7.7 persons.    Table 3).

Individual characteristics
Household Plasmodium spp. infection risk is summarized in Table 4. The risk to be infected in household using mosquito repellent coil compared to those not doing so increased slightly according to microscopy (OR 1.4, p=0.308) and about two-fold (OR 1.8, p=0.005) according to LAMP. Household risk to be infected was not associated to promiscuity, malaria morbidity and treatment seeking behaviour (Table 4).

Discussion
Asymptomatic carriers of Plasmodium spp. can act as parasite reservoirs that contribute to malaria transmission in some endemic areas. These infections are characterized by low parasitemia which frequently was missed by RDTs and microscopy. In this study, conducted in the city of Korhogo, Northern Côte d'Ivoire, we used microscopy and LAMP to assess the extent of asymptomatic Plasmodium spp. infections. We also investigated the risk factors associated with asymptomatic infections at household and individual level. This study included 1'011 individuals of 376 households.
In this population, the prevalence of asymptomatic Plasmodium spp. infection was found to be high, with slightly more than one third (38.3%) of all included household having at least one infected individual and approximately one out of five individual (18.8%) being infected overall when using LAMP for asymptomatic infection detection.  ITNs, Insecticide-treated mosquito net   indicates that any active case detection intervention would miss most of all infections if based on microscopy or other methods with similar LOD, such as rapid diagnostic tests. It is likely that a more sensitive method, such as LAMP, which could be successfully implemented for the screening activities described here, would allow to capture a vast majority of all these infections [24]. Importantly, LAMP allowed here to screen a relatively high number of individuals, more than a thousand, over a short period of time, with more than 100 samples tested per day in average over 10 days, showing that this method is compatible with large-scale screening campaigns. It cannot be excluded that additional asymptomatic infections, below the LOD of LAMP, remained undetected, yet the transmissibility of such very low-density infections is unlikely to be significant and, by extension, detecting such infections is likely to be of limited relevance from a programmatic point-ofview. Whether the use of more sensitive methods than microscopy or RDT but also more ones, such as LAMP, is ultimately cost-effective for active case detection screening campaign remains to be determined. Although some studies show high sensibility of LAMP, its performance should be demonstrated in our context [21,25].
Our results show that both at household and individual level, asymptomatic carriers risk increased when moving from the urban centre to the intermediate and periurban zones. It is well reported that malaria infections are more prevalent in the periurban setting compared to urban [26]. The high risk of asymptomatic Plasmodium spp. infection in periurban setting was highlighted by another study [27]. In our case, the high risk observed in periurban setting can be related to the presence of shallows suitable for agricultural activities including market gardening and irrigation rice field. These typically lead to several malaria vectors breeding sites [28]. Previous studies have reported that, the low socioeconomic status observed in periurban areas [26], and living close to a permanent stagnant water (at a distance <1 km from the house) were more likely to increase the risk to be infected with malaria parasite [29].
Malaria prevention measures, particularly mosquito repellent coil, were used in more than half of household. Yet, household reporting the use mosquito repellent coil were found to be at greater risk of asymptomatic Plasmodium spp. infection, an apparently counter-intuitive observation. It can be Benié EMA, et al. hypothesized that coil use is prompted by the actual presence of mosquitos within or near the households in question but that these coils might be only partially effective at preventing bites. Indeed, mosquito mortality due to application of various mosquito coils averagely ranged between 24 and 64% [30]. On the other hand, coil might simply not be used in households free of mosquitos, where the risk of infections would be lower to null.
Age and gender are characteristics often significantly associated with specific risk of asymptomatic Plasmodium spp. infections [23,31]. Our study also found that, older age was associated with a reduction of asymptomatic Plasmodium spp. infection. Asymptomatic prevalence in age <30 years (24%) declined significantly in age >40 years (14.3%, by LAMP). In stable malaria transmission areas, repeated contact with parasites, contribute to increase immunity with age [32].
It is possible that men may spend longer time outdoor during evening and thus are more exposed to risk of infection [33]. A study performed in Eastern Myanmar associated men infection to professional activities. Individuals who worked as farmers or wood and bamboo cutters were found to be at increased risk of asymptomatic Plasmodium infection [23]. In our case, it is possible that national control measures mainly focused women, as ITNs and IPT have been distributed free-of-charge during pregnancy, could partially explain this observation.

Conclusion
Plasmodium spp. asymptomatic infection is highly prevalent in Korhogo. Infection risks appear linked to dwelling setting, gender and age. Better controlling and ultimately eliminating malaria in Korhogo will require taking into consideration these asymptomatic infections. We report here that LAMP molecular assay could represent a valuable and potential cost-effective approach to tackle these infections. Our results also provide guidance as to which populations should be prioritized when investigating such infections and could serve to define specific elimination policies and more effective programmatic interventions in the future.