Pierre Fongho Suh^1,2, Emmanuel Elanga‐Ndille³, Magellan Tchouakui³, Maurice Marcel Sandeu^3,4, Darus Tagne^1,5, Charles Wondji^1,6 and Cyrille Ndo^1,7*

Impact of insecticide resistance on malaria vector competence: a literature review

Abstract

Since its first report in Anopheles mosquitoes in 1950s, insecticide resistance has spread very fast to most sub‐Saharan African malaria‐endemic countries, where it is predicted to seriously jeopardize the success of vector control efforts, leading to rebound of disease cases. Supported mainly by four mechanisms (metabolic resistance, target site resist‐ ance, cuticular resistance, and behavioural resistance), this phenomenon is associated with intrinsic changes in the resistant insect vectors that could influence development of invading Plasmodium parasites. A literature review was undertaken using Pubmed database to collect articles evaluating directly or indiretly the impact of insecticide resist‐ ance and the associated mechanisms on key determinants of malaria vector competence including sialome composi‐ tion, anti‐Plasmodium immunity, intestinal commensal microbiota, and mosquito longevity. Globally, the evidence gathered is contradictory even though the insecticide resistant vectors seem to be more permissive to Plasmodium infections. The actual body of knowledge on key factors to vectorial competence, such as the immunity and micro‐ biota communities of the insecticide resistant vector is still very insufficient to definitively infer on the epidemiological importance of these vectors against the susceptible counterparts. More studies are needed to fill important knowl‐ edge gaps that could help predicting malaria epidemiology in a context where the selection and spread of insecti‐ cide resistant vectors is ongoing.

Keywords Plasmodium, Anopheles, Insecticide resistance, Vector competence

Impact of insecticide resistance on malaria vector competence: a literature review

Background

Malaria is the biggest killer among vector-borne dis- eases [1] and has claimed the lives of milllions of peo- ple over centuries [2]. In 2020, 241 million cases were reported leading to 627,000 deaths. The African region has paid the highest tributes with 96% of all deaths [3]. Malaria disease is caused by Plasmodium parasites, which are transmitted to humans by the bites of infected female mosquitoes of the genus Anopheles [4]. In Africa, Plasmodium falciparum is the most epidemiologically important of malaria parasites infecting humans [5], and Anopheles gambiae, Anopheles coluzzii, Anopheles funes- tus and Anopheles arabiensis are the dominant vector species [6].

Malaria control includes medical treatment of cases and protective measures against the vectors to prevent

Impact of insecticide resistance on malaria vector competence: a literature review

and/or limit contacts with human hosts during which transmission occurs. The control of mosquito popula- tions on a large scale using insecticide-treated nets (ITNs) and indoor residual spraying, associated with increase case management, has led to a remarkable reduction in malaria burden from 81.1 cases per 1000 population in 2000 to 58.9 in 2015 [3]. After this period, the impact of control efforts on malaria burden have dwindled, coinciding with the spread of insecticide resistant vec- tors across most endemic countries [3, 7]. Resistance of Anopheles mosquitoes to insecticides, reported for the first time in Africa in the 1950s [7], concerns four main classes of insecticides used in public health for vector control purposes, namely pyrethroids, organochlorines, organophosphates and carbamates [7, 8]. There are four mechanisms deployed by mosquitoes to become insensi- tive to the insecticides, including by order of importance (1) degradation of insecticide molecules by detoxification enzymes (metabolic resistance), (2) modification of the target affinity of the insecticide (target site resistance), (3) reduced penetration of the insecticide (cuticular resist- ance) and, (4) avoidance of insecticide-treated surfaces (behavioural resistance). Of these four mechanisms tar- get site and metabolic resistances are most likely to lead to control failure [9].

In target site resistance, a change (leucine changed to a phenylalanine or a serine at position 1014) occurring in the amino acid sequence of the voltage gate sodium channel (vgsc) leads to a reduced sensitivity of mosqui- toes to pyrethroids and organochlorines. This phenotype is known as knock down resistance or kdr [10, 11]. When the amino acid change (glycine replaced by serine at position 119) occurs in the neurotransmitter acetyl-cho- linesterase, it occasions resistance to organophosphates and carbamates, termed ace-1 resistance [12, 13]. About metabolic resistance, insecticide resistant mosquitoes increase the expression of detoxification enzymes, such as the cytochrome P450 monooxygenases, glutathione S-transferases (GSTs) and esterases, that eliminates xeno- biotic compounds (including insecticides) before they reach their target. In another instance, an amino acid substitutions in the sequence of detoxification enzymes could modifiy its affinity with the insecticides in insect vectors [14]. For example, several cytochrome P450 genes (CYP6P9a, CYP6P9b and CYP6M7) are involved in resistance to pyrethroids in the species An. funestus [15, 16]; while a substitution of leucine by phenylalanine at position 119 in the epsilon class of GST (GST2– L119F) confers a cross-resistance to dichloro-diphenyl-trichloro- ethane (DDT) and pyrethroids in the same vector species [17].

Despite the widespread distribution of insecticide resistance, its impact on overall malaria epidemiology remains unclear and is currently a subject of intense debate. The evaluation of the potential impact of insec- ticide resistance on vectorial competence is therefore becoming an important and urgent research theme whose findings will help understanding whether it alters or enhances the permissiveness of malaria vectors to Plasmodium parasites, from its early stage (ookinete) to the infective form (sporozoite). In this review, the evi- dence of insecticide resistance impact on the infectivity of mosquitoes to Plasmodium was explored in the litera- ture, and changes in intrinsic factors that could predict or explain the outcome of an infectious blood meal intake were broached. Finally, the knowledge gaps were pointed out.

Search strategy

A literature search was undertaken in the PubMed data- base to extract articles addressing the following themes: (1) Plasmodium infection in insecticide resistant malaria vectors, (2) sialome of insecticide resistant malaria vec- tors, (3) effect of insecticide resistance on the immunity of malaria vectors, (4) microbiota of insecticide resist- ant malaria vectors and infection, and (5) fitness cost of insecticide resistance in malaria vectors. The first search terms were “Anopheles” and “insecticide resistance” and they were associated with either “Plasmodium infection”, “vector competence”, “salivary gland”, “sialome”, “micro- biota”, “gene expression” or “longevity”. Additional articles were extracted from the references lists of the full pub- lications. The search was done between February and August 2022 and there was no restriction regarding the date of publication of the articles. A total of 560 articles were obtained from the search. Articles that addressed insecticide resistance in Anopheles in a broad manner, and not in relation with either Plamodium infection, vec- tor competence, sialome, or longevity were discarded. Therefore, 28 articles related to the themes mentioned above were selected and used for the review.

Malaria vector competence

Vector competence is the intrinsic ability of anophe- line species or populations to allow the development of Plasmodium parasites from ookinete to infective sporo- zoites. When a mosquito takes an infectious blood meal from human, the gametocytes ingested begin their development in the midgut. The male gametocyte transform into eight microgametes after three rounds of mitosis, meanwhile the female gametocytes matures into macrogametes [4]. These cells fuse to form zygotes that thereafter change into ookinetes in the lumen of the intestine. The ookinetes then strive through the epithelium of the midgut and once in its basal side, transform into oocysts. The oocysts undergo several rounds of asexual multiplication (sporogony) leading to the production of thousands of haploid sporozoites in each oocyst. Mature occysts rupture and release sporo- zoites in the hemocoel, which immediately migrate to the salivary glands. The extrinsic incubation period of the parasite is about 14 days with the transition from ookinetes to mature oocysts having the highest dura- tion (about 10 days) [18, 19].

In mosquito host, Plasmodium face several immune- related bottlenecks deployed to prevent the success- ful transition from its early stage in the midgut to the sporozoite stage in the salivary glands [18]. The out- come of the parasite infection is reported to depend mainly on the mosquito-Plasmodium genetics adapta- tion [19, 20]. Another very important factor that influ- ences the above outcome is the compatibility of the duration of parasite development with the longevity of the mosquitoes [21, 22]. Only species in which Plas- modium reaches infective form are referred to as com- petent vectors and could ensure malaria transmission. The impact of vector competence on the transmission of malaria can be estimated using Ro (Fig. 1), the basic reproductive number developed by McDonald in 1957. The McDonald model gives the threshold for a disease to persist or spread (Ro greater than 1) or to disappear (Ro less than 1) [23]. The Ro represents the number of individuals in a susceptible human population that are expected to get infected via a mosquito bite when a sin- gle infected individual is present in the population [24, 25]. In the Ro equation, two parameters are related to vector competence: probability of mosquito infection (b)andmosquitolongevity(p)(Fig.1).Modificationsof the values of components of this equation for a given vector population will cause either an augmentation or reduction in the transmission dynamics of the disease, leading probably to a change in the epidemiological profile of the locality concerned. It was established that an increase in b will increase the Ro, whereas a decrease in p will cause the opposite [26].

Insecticide resistance and malaria vector infectivity
to Plasmodium parasite

The rapid spread of insecticide resistance among malaria vectors accross endemic countries in the past dec- ade have raised several questions among which that of knowing what is its impact on mosquito permissiveness to Plasmodium? Only a limited number of studies have tried to elucidate this question [27–35]. These stud- ies compared P. falciparum infection rates in resistant Anopheline vectors with susceptible ones, either caught in the field or experimentally infected (Table 1).

Anopheles gambiae strain bearing kdr resistance allele (Vgsc–L1014S) was found naturally more infected by sporozoites than the susceptible counterpart [27]. Similar findings were experimentally observed in the same spe- cies, as well as in Anopheles coluzzii [30, 32]. Contrary to kdr resistance, An. gambiae with ace-1 resistance allele did not differ from individuals that have the wild type allele (not conferring insecticide resistance) on infec- tion rate despite significantly higher oocyst prevalences were observed in the resistant strain [32]. More studies using field populations are needed to ascertain whether a lower longevity suspected by the author and/or other factors are involved.

Regarding metabolic resistance, recent breakthroughs in designing simple PCR-based assays to detect glu- tathione S-transferase (GST)-based and cytochrome P450-mediated resistance in An. funestus sensu stricto provided a unique opportunity to assess its impact on the mosquito’s ability to develop the parasites. The L119F- GSTe2 resistant genotypes of this species showed, in an experimental infection study, higher permissiveness to oocyst infections than susceptible ones [31]. Similarly, in naturally infected populations of the same species, homozygote L119F-GSTe2 genotypes were found more infected by sporozoites though no significant difference was found at the level of oocyst prevalence [28]. In other hands, Lo and Coetzee [36], infecting experimentally two selected sub-colonies of FUMOZ displaying different degree of pyrethroid resistance by Plasmodium berghei, found that the insecticide resistant colonies were less permissive to infection than the susceptible ones. No investigation has so far explored the relationship between P450s genes implicated in insecticide resistance and P. falciparum infection in An. funestus. Moreover, because of the absence of markers of metabolic resistance in An. gambiae sensu lato such studies are still lacking in these species.

Impact of insecticide resistance on mosquito sialome

Bloodsucking arthropods, like mosquitoes, have evolved saliva containing a mixture of pharmacologically active molecules that help them counteract the hemostatis and inflammatory responses of the vertebrate host during bites, thus facilitating blood meal intake [37]. However, the activity of these molecules goes beyond the scope of ensuring blood meal success, as they possibly influ- ence the completion of Plasmodium development in the salivary gland of malaria vectors. Proteins secreted by

the salivary gland belong to several families (D7, mucin, gSG1, gSG2, gSG6 peptide, gSG7, cE5, 8.2-kDa, 6.2-kDa, etc.) [38] whose function include (1) cytoskeletal and structural activities (2) digestion, (3) circadian rythm and chemosensory, (3) immunity, (4) metabolism and other [39]. The development of insecticide resistance in malaria vectors is accompanied by physiological changes [26] that may affect the sialome composition with consequences on the vector competence. Few studies have investigated changes in the sialome in the insecticide resistant vectors [40, 41].

The secretory protein 100 kDa, which is encoded by Saglin (a cytoskeletal and structural gene present in An. gambiae salivary gland) was considered as the binding target of P. falciparum and P. berghei on salivary gland prior to penetration into the latter [42]. This protein was found down-regulated in ace-1 bearing An. gam- biae strain, suggesting an impact on the vector infectiv- ity to Plasmodium [43]. However, a recent study showed that the 100 kDa Protein is unevenly distributed on the salivary glands lobes. Its absence on the primary site of sporozoites occupancy in the salivary glands, the distal lateral lobes, implies that this protein may instead have a secondary role in the infection of the organ [44–46].

The D7 salivary family has been identified in malaria vectors among the most expressed proteins involved in the antihemostatic activity and probably in digestion of blood meal [47–50]. Elanga et al. [40] showed that two short forms of the D7 family genes (D7r3 and D7r4) are over-expressed in pyrethroid resistant An. funestus (L119F–GSTe2), whereas almost all D7 genes are under- expressed in pyrethroid resistant An. gambiae (kdr, L1014F). A comparable observation was made in insec- ticide resistant Culex quinquefasciatus (ace-1 resistance) [51] as well as in two strains of Aedes aegypti (homozy- gotes resistant C1534 and G1016 kdr) [52]. These find- ings show that insecticide resistance mechanism may affect the sialome composition differently.

Several immune proteins such as the anti-microbial peptides cecropin and defensin were found in the saliva of mosquitoes [39, 53]. These immune proteins under- score the role of the salivary gland in the refractoriness of the Anopheles to infections [39, 53]. The small num- ber of studies that evaluated the impact of insecticide resistance alleles on salivary gland gene expression in mosquito vectors have not reported significant changes related to immune genes as compared with the suscepti- ble counterparts [41, 43, 51, 52], alluding that the resist- ant status to insecticide does not influence noticeably the immune component of the sialome. If these factors are indeed unchanged regardless of the mosquito allelic composition, nothing is known whether under infection the expression profile of these immune proteins will vary or not according to the mosquito genotype. Das et al. [39] and Djegbe et al. [51] demonstrated that salivary gland genes expression is influenced by blood meal intake and varies towards the period coinciding with the matura- tion of Plasmodium parasites in mosquitoes [54]. This evidence was not previously studied and should be taken into account in subsequent research works that aims at identifying differentially expressed genes of the salivary

gland and elucidating their impact on the malaria vector competence.

Impact of insecticide resistance on vector immunity

When the infectious blood meal reaches the midgut of the female Anopheles, the immune system is deployed to prevent infections [20]. In the midgut, P. falciparum faces the peritrophic membrane, a physical barrier developed to prevent infections. It also protects against the damag- ing effects of the human blood factors like antibodies and regulates several digestive enzymes [55, 56]. Enzymes such as trypsin 1 and 2, chymotrypsin, carboxypeptidase, aminopeptidase and serine protease are upregulated during digestion to cleave the large content of proteins in the blood meal [57–60]. These proteases are appar- ently involved in the elimination of Plasmodium infec- tions [61]. Three studies attempted to elucidate the effect of insecticide resistance on vectors’ immunity [62–64]. Mitri et al. [62], in a study evaluating genes implicated in the infectivity of An. coluzzii, demonstrated that the kdr– bearing para gene which carries mutations of the voltage- gate sodium channel (confering insecticide resistance) is not associated with infection but rather the ClipC9 gene directing the synthesis of Serine protease. This suggest that the effect of the resistant character on refractoriness to infection may be due to genes other than that involved in resistance to insecticides, and which happen to be linked to it. The Serine protease plays an important role in the activation of the three major immune signaling pathways in mosquitoes: Toll, Imd and JAK/STAT [20], which cause the release of antimicrobial peptides (AMPs) notably defensins, cecropins, attacin, gambicin and AgSTAT-A, effective against malaria parasites infections. Vontas et al. [63], using pyrethroid and organochlorine resistant An. gambiae strains, showed that defensin and cecropin are upregulated after pre-exposure to perme- thrin. This study sugggests that insecticide resistant mos- quitoes may be better equipped than susceptible ones to combat infections, but these two immune effectors alone may not be decisive in rendering the vector completely refractory to malaria infections as many other pathways activated concomitantly during parasitic invasion are altogether implicated in the outcome of a contamination [20].

In Culex pipiens which is vector of many pathogens including arboviruses [65], filarial worms [66], and protozoa [67], immune response was stimulated in an insecticide resistant field strain by injection of Lipopoly- sacharide (LPS) immune elicitor. As result, no difference was found in the expression of defensin and cecropin as compared to the control group; but only an increase in gambicin was recorded [68]. One point can be drawn from these results to infer what might happen in malaria vectors: Plasmodium infections may trigger the overex- presion of some immune factors while the other may have their expression either down regulated or unchanged.

The reactive oxygen species (ROS) produced by cellu- lar metabolism are another class of effectors of the innate immunity that can negatively affect malaria parasites [69, 70]. They kill the parasites through both lytic and melanization pathways [20]. Ingaham et al. [64] showed that An. coluzzii VK7 colony displaying kdr resistance mechanism, CYP6M6 and CYP6P3 metabolisers, had oxidoreductase overexpressed after sub-lethal exposure to deltamethrin, suggesting that this species could be more refractory to Plasmodium infection. At this point, it is necessary to verify whether under natural conditions, insecticide-resistant Anopheles mosquitoes will display an overexpression of ROS or not.

Cellular immune responses are carried out by varous type of hemocytes that eliminate pathogens by phago- cytosis, lysis and melanization [20]. Organochlorines and organophosphate were found to affect differently the hemocytes abundance including granulocytes in the insect Rhynocoris kumarii [71]. In mosquitoes, studies are needed to ascertain the impact of insecticide resist- ance on cellular immunity and the resulting effect on the infectivity of resistant vector to malaria parasite. Regard- ing melanization of pathogens, it is lead by the phe- noloxidase (PO) produced by Oenocytoids [72, 73] and is regulated by serine protease inhibitors. In field-caught C. pipiens resistant to insecticide through an increase in detoxification (esterase) and target site mutation (ace- 1), PO expression was equal to that of susceptible group [74], suggesting that some genes associated with immu- nity might not be affected by insecticide resistance char- acter in mosquitoes. No studies have verified the effect of insecticide resistance on PO in malaria vectors.

Impact of insecticide resistance on commensal intestinal microbiota of malaria vectors
Bacteria, fungi and viruses colonize the gut, salivary glands and reproductive organs of the mosquitoes. These microorganisms are mainly acquired from the environne- ment and its composition is largely influenced by its aquatic breeding sites [75, 76]. In addition, the microbi- ota composition is highly dynamic, varying greatly with localities and seasons [77–79]. These variations of micro- biota composition within field mosquitoes may partly explain the variability in infection levels in the field [80].

Mosquito microbiota has great potential for impeding the transmission of malaria by altering vectorial capac- ity [81]. Also, the microbiota is capable of influencing the biology of the host such as altering its immunity, nutri- tion, digestion, vectorial competence, reproduction, and insecticide resistance [82–87]. With the growing concerns about the rapid spread of insecticide resistance in Anopheles mosquitoes, some studies have explored the functions of the mosquito’s gut microbial communities in the development of resistance. For example, distinct microbita populations were found associated with organ- ochlorine resistance in An. arabiensis [86] and Anoph- eles albimanus [88]. Similarly, an association between specific microbiota and intense pyrethroid resistance was reported in An. gambiae [89] and Anopheles ste- phensi [90], suggesting a microbiota-mediated insecticide resistance mechanism. Dieme et al. [91] suggested that changes in the feeding behaviour of insecticide resistant vectors may lead to higher microbial diversity. This diver- sity could modify the repertoire of protective bacteria against pathogen infections and/or that of their enhanc- ers, with consequences on the vectorial competence [9, 92]. Recently, Bassene et al. [93] showed that, in the spe- cies An. gambiae and An. funestus, the microbiota was signifanctly different between P. falciparum-infected and non-infected samples, although the resistance status of these mosquitoes was not evaluated. More refined stud- ies are needed to characterize the microbial communities harboured by the insecticide resistant malaria vectors. Also the contribution of microbiota against other fac- tors to the vectorial competence of insecticide resistant malaria vectors remains to be investigated.

Impact of insecticide resistance on the longevity of malaria vectors
Mosquito longevity is a determinant factor for parasite maturation and could influences malaria transmission [94, 95]. In fact, the extrinsic incubation of Plasmodium in its hematophageous host is about 11–14 days. There- fore, only mosquitoes whose lifespan is long enough could allow the complete development of the malaria parasite to the sporozoite infective stage and participate in the transmission of the disease. With the emergence and spread of insecticide resistance [7], many investiga- tions were undertaken to gain knowledge of the effect of this phenomenon on the vectors’ longevity and so on its potential epidemiological impact.

So far, studies on the impact of insecticide resistance on malaria vectors longevity have focused on four spe- cies: An. gambiae, An. arabiensis, An. coluzzii and An. funestus. Globally, the findings revealed a pleitropic effect of insecticide resistance on mosquito lifespan [33, 96–108] (Table 2). The majority of studies (10/14) which used laboratory strains showed that pyrethroid resistant An. funestus and An. gambiae live longer than suscepti- ble ones [100, 106]. Of the studies including field strains, a longer life span was reported in organochlorine and pyrethroid resistant An. funestus strains [104, 105]. In contrast, a shorter life span was observed in An. gambiae strain resistant to organochlorine. It was reported that pre-exposure to insecticide in a manner micmicing field exposure to insecticides, affects the longevity of insecti- cide resistant An. gambiae strain [97], and that delayed mortality observed in the vectors may be dependent on resistance intensity [98]. This later observation indi- cates that findings obtained with laboratory colonies are to be taken with caution given that they may not reflect exactly what is oberved on the field [26]. Nevertheless, such studies remain important as they contribute to the understanding of the potential mechanisms affecting the vectors’ longevity [109, 110], notably resource-based trade-off and oxidative stress.

Resource based trade-off is an evolutionary ecology concept that states that when environmental constraints lead to the augmentation of resources to one biological trait, other traits will have their energy budget reduced [111]. Accordingly, when mosquitoes adopt the detoxi- fication mechanism to prevent the effect of insecticide, an increased production of detoxifying enzymes follows and is maintained by the additional resources deployed for the function. Otali et al. [100] have demonstrated

that metabolism and longevity of insecticide resist- ant An. gambiae are lower than that of the susceptible strain. Moreover, they showed that the resistant strain has higher Reactive Oxygen Species (ROS), which are factors determining oxydative stress. In fact, the ROS are multifunctional molecules produced by cells of all organisms during normal metabolism [112, 113]. They have been pointed out as key aging factor in other organ- isms including Anopheles [114]. Therefore, mosquitoes that develop the capacity to cope with oxydative stress are likely to live longer. Oliver and Brooke [103] in an experiment evaluating the effect of oxidative stress on the longevity of both An. arabiensis and An. funestus bear- ing respectively kdr and Cytochrome P450 mechanisms demonstrated that these species live longer, and that Cytochrome P450 activity seems more protective against oxydative stress.

Rivero et al. [26] proposed the potential effect of dif- ferent detoxifying enzymes on vector longevity. For example, Glutathion S-Transferase is considered to pro- tect against oxydative stress. Confirming this point, a longer lifespan implicating Glutathion S-Tranferase in An. funestus was revealed with and without exposure to insecticide [104, 105]. In contrast, monoxygenase, known to be associated with an increase in oxydative stress has not led, as expected, to reduced longevity in An. funes- tus [108]. More studies using field populations and mic- micing field conditions are necessary to ascertain the full impact of insecticide resistance on longevity of the malaria vector.

Conclusion

The need for a comprehensive understanding of the impact of insecticide resistance on malaria vector com- petence is unquestionable. The current state of knowl- edge is not only insufficient but also contradictory to draw a definitive conclusion. A tendency nevertheless emerges from findings that insecticide resistance may increases the infectivity of malaria vectors to Plasmo- dium, thus their vector competence. This is possibly due to changes in the expression of some genes notably those involved in blood-feeding and the immunity. Addition- ally, microbiota communities vary in the resistant mos- quitoes as compared to the susceptible counterparts. The actual effect of these changes in the course of infection and their impact on the infectivity of malaria vectors to P. falciparum is still to be investigated. Finally, the longevity of the vectors is not always affected by insecticide resist- ance mechanisms. It is worth noting that, studies using vectors displaying metabolic resistance were under- represented because molecular markers to diagnose this character were developped only recently, especially in An. funestus. Malaria vectors that bear metabolic resistant mechanism are, on an ecological immunology point of view, expected to have a number of biological functions impaired, including immunity. If established, this situa- tion may cause them to become less refractory to Plas- modium infection. Taking advantage of recent advances in the genomics, transcriptomics and molecular charac- terization of insecticide resistance, more refined studies can now be undertaken to fill knowledge gaps regarding the effect of insectide resistance on key determinants of vectorial competence and subsequently predict changes in the epidemiology of malaria in a context of insecticide resistance escalation.

DDT Dichloro‐diphenyl‐trichloroethane GST Glutathione S‐transferases
kdr Knock down resistance
LPS Lipopolysacharide

PO Phenoloxidase
Vgsc Voltage gate sodium channel

Author contributions

PFS, EEN, MT, MMS, CN wrote the manuscript. TD and PFS prepared the figures and tables. CN and CW acquiered the funding. All the authors revised the manuscript. All authors read and approved the final manuscript.

Funding

The authors acknowledge the financial support from the Wellcome Trust, UK, through the International Intermediate Fellowship (220720/Z/20/Z) granted to Cyrille NDO.

Availability of data and materials

Not applicable.

Declarations
Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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Accepted: 4 January 2023

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“Neuropsychology of migration and impact on family composition”

Dr. Rostand Idriss Tchana Tchamba ( Pharmacist – Neuropsychologist)

Living beings, due to the initial and essential movement of the minimal units that form them (cells), seek various basic functions: respiration, nutrition, development and procreation for the survival of the species.
Therefore, due to the concepts of globalization and the need for security and fulfillment, many individuals are driven to migrate to horizons that they find more suitable to their life models and aspirations: In 2021, Europe will have a total of 1.92 million migrants and currently a total population of 37.5 million people who were born outside the European Union (European Union Commission, Statistics on migration to Europe, EUROSTAT DATAS 2021).
This arrival in a new society requires an awareness of oneself and one’s background, but also a great deal of adaptation in order to find one’s place in society and to make sense of it. In order to do so, integration through the learning of a new language, a way of life with a different history and geography is necessary, education through the learning of new skills and value creation, but also living together through the awareness of religious differences, values and priorities between the different members constituting this cosmopolitan society are to be taken into account.

The current circumstances of the migratory processes in Europe and the complexity of its diversity make the adaptation of children in society depend greatly on family structures and the dialogue that is present in them, the educational potential of them being also determined by the family structure, social, material means and the level of education of the parents (J. Borubin et al., 2015) .
Numerous studies have demonstrated a predisposition to psychological illnesses in families with a migration background due to the difference between the stable social structure of these families before the migration and after it. This is due to the complexity of the society, the psychological influences accompanied by stress and social burden, especially during the migration process, which fatally impact the psychological health of the family and its structure.

It is therefore important to see to what extent the therapeutic support of these families can be integrated, taking into account the different structural elements, parental models, but also the material elements made available for a perfect social integration and a common benefit.

Dr. Tchana Tchamba Rostand Idriss’ presentation at the next CARES conference will focus on the neurocognitive factors involved in family structure for populations with a migration background and family recomposition models.

REFERENCES:

1. G. Burorin, N. Kalinina, Pathological Structural Changes at Migrants’ Families, European Psychiatry, Volume 30, Supplement 1, 2015, Page 860, ISSN 0924-9338,
2. European Comission, EUROSTATS 2022, Overall figure of migrants in European Society: https://ec.europa.eu/info/strategy/priorities-2019-2024/promoting-our-european-way-life/statistics-migration-europe_en#:~:text=Refugees%20in%20Europe,-Based%20on%20data&text=26.6%20million%20refugees%20in%20mid,at%20the%20end%20of%2020.
3. American Psychological Association Task Force on the Psychosocial Effects of War on Children and Families Who Are Refugees From Armed Conflict Residing in the United States (2009). Working with refugee children and families: Update for mental health professionals. Retrieved from http://www.apa.org/pubs/info/reports/refugees-health-professionals.pdf.
4. Abi-Hashem, N. (2011) Working with Middle Eastern immigrant families. In: Zagelbaum, A., Carlson, J. (eds) Working with immigrant families: A practical guide for counselors, New York, NY: Taylor & Francis Group, pp. 151-180.
5. Black, N. (2001) Evidence based policy: Proceed with care. British Medical Journal 323: 275-279.
6. Joop T. V. M. de Jong, Ortal Slobodin, Family interventions in traumatized immigrants and refugees: A systemic review : Trascultural psychiatry

Lalaina V.Rahajamanana^1,2*, Paulin Andrianjakasolo², Dera S.Andriatahiana², Zakasoa Ravaoarisaina³, Liliane J. Raboba¹, Andry Rasamindrakotroka²

¹Department of Pediatrics, Mother and child Teaching Hospital Tsaralalàna, Antananarivo, Madagascar

² Department of Biology, Faculty of Medicine, Antananarivo, Madagascar

³Department of Pediatrics, Mother and child Teaching Hospital,Ambohimiandra Antananarivo, Madagascar

*Corresponding author contact information:

Lalaina Vonintsoa RAHAJAMANANA

CHU Mère Enfant Tsaralalàna

Antananarivo-101- Madagascar

Tel: +26134 19 321 79

E-mail address : v_lalaina@yahoo.fr

Co-authors email address

Paulin Andrianjakasolo : docteurrazanakolona@gmail.com

Dera S.Andriatahiana : deraandriatahiana@gmail.com

Zakasoa Ravaoarisaina : zakasoa.ravaoarisaina@yahoo.com

Liliane J. Raboba : liliane_raboba@yahoo.fr

Andry Rasamindrakotroka : andryrasamindrakotroka@idriss-tchana

ABSTRACT

Background: Diarrheal diseases are a major public health problem in developing countries with high mortality and morbidity rates, especially among children. Shigella sp is the leading cause of pediatric bacterial diarrhea and shigellosis data are very scarce in Madagascar.

Material and methods: A 4-year retrospective study, from January 2018 to December 2020, at the University Hospital Mother and Child Tsaralalàna laboratory was performed to assess the bacteriological and epidemiological characteristics of laboratory confirmed shigellosis cases

Results: During the study period, 223 stool samples were examined, of which 45 (20.7%) were positive for Shigella sp. The mean age of infected children was 29.8 months, predominantly in the 24-59 month age group. The infection was found mainly in male children (54.3%). Most isolates of Shigella sp showed resistance to co-trimoxazole and amoxicillin. All the strains were susceptible to third-generation cephalosporins. Of the isolated Shigella sp, 14 strains were tested for species identification and serotyping, twelve of which were Shigella flexneri and two were Shigella sonnei. The most frequent serotypes were Shigella flexneri 1b and 2a.

Conclusion: This study found a Shigella sp positivity rate of 20.7%. This pathogen frequently infects infants age group. Bacteriology laboratory surveillance and a multicenter survey are essential to control the spread of drug-resistant Shigella and to monitor circulating strains and the burden of this disease. Awareness of water, hygiene, and sanitation (WASH) and community water supply is also necessary to reduce this infection.

Keywords: antibiotic, laboratory, Paediatry, serotype, Shigella,

INTRODUCTION

Shigellosis or bacillary dysentery is a bacterial diarrhea due to bacteria of the genus Shigella sp¹. The relatively high morbidity affects mostly children under 5 years of age. ² The clinical symptoms are related to the invasive nature of Shigella sp on the intestinal epithelium associated with a strong inflammatory reaction in the lamina propria. Additionally to this invasive mechanism, toxin secretion leads to intestinal hypersecretion. These physiopathological mechanisms result clinically in a dysenteric syndrom with bloody stools, abdominal pain, tenesma and fever. ⁴ In older children and young people, the disease can be mildly symptomatic. ^5,6 The diagnosis of shigellosis is based on the laboratory identification of Shigella by bacteriological examination. Coproculture remains the gold standard in the laboratory. Rarely, a blood culture is requested to diagnose shigellosis. Other biological tests, like molecular biology, are currently available for the detection of Shigella sp but they do not allow antibiotic susceptibility testing. The Shigella genus contains 4 species: Shigella flexneri, Shigella boydii, Shigella dysenteriae and Shigella sonnei, which are classified into several serotypes according to the biochemical and antigenic characteristics of the bacteria.⁷ Shigella sp is the second leading cause of death from infectious diarrhea in children after rotavirus and is the leading cause of bacterial diarrhea.⁸ Furthermore, shigellosis in the past was very different from the current situation. In the past, Shigella dysenteriae was the commonest species responsible for severe disease and it has been replaced by Shigella flexneri in many countries.^9,10 The treatment is based on antibiotic therapy.¹¹

Also the emergence of multidrug-resistant bacteria is threatening worldwide due to the irrational use of antibiotics both in developed and developing countries¹², so that the WHO established a global antimicrobial resistance surveillance to tackle these new problems and Shigella sp is one of its target pathogens. Data on shigellosis in Madagascar are very scarce. This study aims to describe the epidemiological and bacteriological profile of Shigella sp strains circulating in Antananarivo Madagascar.

MATERIALS AND METHOD

It was a 3-year descriptive retrospective study, from January 2018 to December 2020, performed at the Mother and Child University Hospital of Tsaralalana (CHUMET) bacteriology laboratory. This 82-bed public pediatric referral hospital in the capital of Madagascar offers care primarily to local patients under 15 years of age, although there are some patients from elsewhere in the country.

All hospitalized and outpatient children who performed a stool culture at the CHUMET laboratory with a positive result for Shigella sp with all available results were included. Coprocultures positive for other pathogens were excluded. The laboratory logbook and patient records were used to collect data and the analysis was performed with EPI info v7.0. The studied variables were: socio-demographic characteristics (age, gender, origin of the sample), bacteriological results (species and serotypes of Shigella sp isolated, susceptibility profile to antibiotics routinely used in practice). Stool samples were plated on a selective culture medium Hektoen agar (Biokar Diagnostics, Allonne, France). A phenotypic method by the appearance of colonies was used for identification. The appearance of suspect colonies (lactose negative) on this medium was followed by the isolation of three isolated colonies on another Hektoen agar and simultaneously on a chromogenic agar Uriselect (BioRad, Californie, Etats – Unis). Lactose-negative colonies on Hektoen agar with a small white appearance on chromogenic agar were further tested by biochemical identification using the API 20E gallery (BioMérieux, Marcy l’Etoile, France). Antibiotics susceptibility testing of Shigella sp. were performed by Kirby Bauer disc diffusion method according to the current CASFM/EUCAST standard. The antibiotics tested were amoxicillin, amoxicillin/ clavulanic acid, ceftriaxon, Imipenem, gentamicin, amikacin, ciprofloxacin, and sulfamethoxazole/trimethoprim. All the Shigella sp isolated strains were stored in a 20% glycerol – Brain Infusion (BHI) storage medium at -80°C. A total of 14 Shigella sp strains were tested for serological identification or serotype by slide agglutination according to the manufacturer’s instructions (Shigella anti-sera, Eurobio scientific®, France) (Figure 1) which enabled identification of Shigella dysenteriae, flexneri, sonnei or bodyii species and serotypes of each. The test kit includes ready-to-use polyvalent and monovalent sera for the determination of serotypes belonging to each group.

RESULTS

During the study period, 298 stool specimens from patients under the age of 15 years were studied, of which 48 (16.1%) were positive for Shigella sp (Figure 1). The ages of the Shigella patients range from 8 to 96 months with a mean age of 29.7 months. The peak frequency was in the age range 24 – 60 months (Table 1). The sex ratio was 1.1. Twenty-eight (n=28, 58.3%) stool cultures were from hospitalized patients. Shigellosis cases in this study were all community acquired infections. The Kirby-Bauer disc diffusion method showed marked drug resistance of the Shigella sp strains to cotrimoxazole (93.7%), aminopenicillin (87.5%), amoxicillin-clavulanic acid (54.1%) (Table 2). No extended-spectrum beta-lactamase (ESBL)-producing strains were found. Fourteen Shigella sp strains were identified by species in this study, with a predominance of Shigella flexneri (n=12, 85.7%) and Shigella sonnei (n=2, 14.3%). (Figure 1)

DISCUSSION

Shigellosis or bacillary dysentery is a fecal peril disease caused by Enterobacteriaceae of the genus Shigella sp. This disease has been responsible for important epidemics in wartime. They currently persist in endemic form in tropical countries, where they occur frequently, particularly during the hot, wet season of the year.¹³ During this study, two hundred and ninety-eight coprocultures (n=298) were performed in the bacteriology laboratory of CHUMET, which represented 10.1% of all samples received in the laboratory. Forty-five (n=45) were positive for Shigella sp, giving a positivity rate of 16.1%. This finding was lower compared with other studies in resource-limited countries like Nepal and Togo where the positivity rate was 52.2%, 47% respectively. ^14,15,16

This rate may be certainly underestimated, since coproculture is not routinely performed in all dysenteric syndromes, due mainly to the accessibility of this test in public hospitals, as well as to the high cost of the assay and to the lack of good laboratory capacity. The conventional bacterial culture remains the gold standard for the biological diagnosis of shigellosis. It also allows for antibiotic susceptibility testing, which is particularly important in this era of antimicrobial resistance. The mean age of the children in our study was 29.7 months, with range of 8 and 96 months. Most of the children were less than 60 months of age (95.8%), with high frequency of positivity in the 24-59 month age range. Our results are consistent with other studies from 2017 and 2018 that reported a high frequency of shigellosis in this age group. ^16,17 The vulnerability of children under 5 years of age to shigellosis might be due to their dietary behavior as well as a problem of sanitation and accessibility to safe water. According to the literature, children under 5 years of age are the principal targets of shigellosis and they are rarely infected before the age of 6 months if they are breast-fed.^12,18

We found the predominance of males. This result was similar to other published studies reporting a male predominance. ^{15, 16, 19} Genetics factors could explain this male over female infectious predominance.

Shigellosis is a highly infectious disease. ^{20, 21} The most common symptom of shigellosis is the dysenteric syndrome, which is manifested by afecal, frequent, glairy, bloody, and sometimes mucopurulent stools, abdominal pain, epithelial discharge, tenesmus with false needs. The majority of the patients with shigellosis were hospitalized which was also found by other authors.^14,22

The clinical manifestations of shigellosis may vary in different degree, they can be well tolerated by patients but they can lead to hospitalization because of complications which can be immediate or delayed, like dehydration with hydroelectric losses, neurological damage (convulsions, consciousness disorders), hemolytic uremic syndrome (HUS) and severe malnutrition. ^12,18

In contrast to other diarrhoeal diseases, the therapy for shigellosis cannot be treated by rehydration alone. The first-line treatment is based on antibiotics, which generally allow a rapid recovery without sequelae. ¹¹

The classical therapy for Shigellosis consisted of the use of aminopenicillin- amoxicillin or cotrimoxazole, but since several years, a spread of drug-resistant strains has been reported. ^{12, 23} Monitoring of disease incidence and the antimicrobial susceptibility of the strains are important for appropriate curative treatment and patient management. Acquired resistance was noted in this study with a high proportion of resistant strains to sulfamethoxazole/trimethoprim, amoxicillin and amoxicillin and clavulanic acid. On the other hand, all strains were susceptible to third-generation cephalosporin and imipenem, 95.7% and 91.3% to gentamicin and ciprofloxacin, respectively. Multiple drug resistance was observed in more than two-thirds of Shigella isolates. Lango-Yaya et al in 2017 from the Central African Republic reported a high level of amino penicillin and co-trimoxazole resistance of about 100%. ²⁴ Shigella sp is group 0 in the beta-lactam susceptibility phenotypic classification. They are naturally susceptible to all beta-lactams and antibiotics used in practice. During the last half century, an alarming increase in antimicrobial resistance has been reported, especially in developing countries, where use of these medications is relatively limited. In fact, the extraordinary ability of Shigella to acquire plasmid-encoded resistance to antimicrobial drugs previously considered as first-line treatments has been demonstrated. ²⁵ The spread of bacterial resistance is due to the irrational overuse of antibiotics in veterinary medicine. The use of azithromycin is currently recommended for the treatment of shigellosis ²⁸ but this antibiotic was not tested in our study due to the non-availability of the disc from local suppliers. The frequency of the different Shigella species varies in different parts of the world. ^27,28 People living in resource limited countries have natural immunity to Shigella sonnei from exposure to feces contaminated water containing Pleisomonas shigelloid O17 ^29,30 which has a

similar O type antigen as this strain. Also, Shigella sonnei is phagocytized by the Acanthomoebeoe casthalanni, ubiquitous amoeba that phagocytizes S sonnei in nature and provides an intracellular environment immune to the use of chlorination and other forms of sanitation processes. In contrast, S flexneri is lethal to the amoeba A. castellanii and cannot have this protection. ^31,32 Despite the numbers studied strains, Shigella serotypes isolated were Shigella flexneri 1b and Shigella flexneri 2a. Shigella sonnei serotype g. Knowledge of the serotype has no immediately impact on the management of the patient. However, it is critical for the monitoring of Shigella virulence, for epidemiologic surveillance and for vaccine development. 

CONCLUSION Shigellosis or bacillary dysentery is a public health problem with a high morbidity in the developing countries. Despite the limits of the number of sites and the number of tested strains in this study, it showed a picture of Shigella species and serotypes circulating and the antibiotic susceptibility for treatment in Madagascar. Bacteriology laboratory has a crucial role in diagnosis and treatment of shigellosis cases as well as in epidemiological surveillance which needs to be ongoing and extend to other parts of the country.

ACKNOWLEDGMENT

We thank the following people :

1. Medical Staff, medical trainee, staff laboratory from CHUMET, Antananarivo
2. Doctor Collard Jean Marc, microbiologist

DECLARATIONS

Funding : None

Conflict of interest : None declared

Ethical approval : Not required

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Tropical disease caused by plasmodium faciparum study made in 2017 by Dr Rostand Idriss Tchana

OVERVIEW OF MALARIA

In this chapter, we will talk about malaria, the world situation regarding this pathology, the case of the Democratic Republic of Congo, the agent responsible for it, the medical diagnosis of the disease and finally the current methods taken in terms of treatment.

I. REMINDERS ON MALARIA

Malaria is a parasitic disease caused by a blood protozoan of the type Plasmodium (Lariviere, 1987; AECP, 2007; Hunt, 2007). This acute or chronic parasitaemia is caused by a protozoan of the type Plasmodium which is transmitted to humans by a vector agent, the anopheles. Transmission to humans is through the bite of a musquito, the female Anopheles (Fattorusso et al. 2004; Yuda et al., 2004; Quevauvilliers et al., 2007; Quevauvilliers et al., 2009; Aubry et al., 2015 ).

This pathology is characterized by several symptoms, including: cyclic febrile states, shivers, nausea, emesis, sudation, headaches, diarrhea, stiffness, splenomegaly, anemia, jaundice and several other symptoms that may be individual. The disease is complicated by the passage from a simple state to a severe state, often accompanied by severe anemia, renal failure, coma, high fevers, central nervous system involvement, respiratory problems often leading to death. Those most affected are children under 5 years old, non-immune adults and pregnant women (WHO, 2015).

II. MALARIA AROUND THE WORLD

In 2016, 91 countries were listed as malaria-endemic territories. The number of malaria cases was estimated at 214 million and the number of associated deaths at 437,000. In Africa, access to tools to prevent and treat the disease is still very difficult (WHO, World Malaria Report 2016).

Tropical or sub-tropical Africa accounts for more than 80% of cases and 90% of mortality (Greenwood et al.,2005, Steketee et al., 1996; Fidock et al.,2004). Moreover, if we consider the indirect effects of malaria and its manifestations correlated with other diseases, the number of deaths would be even greater than that which is reported each year (Rogers et al., 2002; Hay et al., 2004; Christopher et al., 2012). On the highly exposed African continent (Figure 1), malaria is the leading cause of morbidity and mortality, contributes greatly to underdevelopment and poverty, and thus establishes a great barrier to socioeconomic development. It has been estimated that malaria costs Africa more than US$12 billion each year (Dorsey et al., 2000; Bloland et al., 2002).

Since the early 1990s, with a global awareness of the management of malaria and other neglected diseases, and with the aim of reducing malaria mortality and morbidity, the founding organizations of Roll Back Malaria (RBM) and the Abuja Declaration Action Plan in Nigeria have developed new approaches to reduce malaria, tuberculosis, and HIV/AIDS worldwide (Ridley, 2001; 2002).

In the last ten years, Africa is threatened by the decline of its health system, lack of infrastructures, climate change, instability, permanent atmosphere of insecurity, and manifestations of other serious pathologies (HIV/AIDS). All of these factors have a negative impact on immunity and lead to the development of malaria in areas with temperatures above 18°C and high humidity (where P. falciparum is still involved in more than 90% of cases) (Greenwood et al., 2005).

III. EPIDEMIOLOGY :

Malaria is a major public health problem in endemic countries, particularly sub-Saharan African countries with tropical and sub-tropical climates. Temperature, humidity, wind, and rainfall are factors that influence the complete cycle of Plasmodium and its transmission from one person to another (Jonathan, 2006; Pascual, 2006). Transmission can also occur in the absence of mosquitoes, for example in the case of pregnant women with congenital transmission via the placenta (Menendez, 1994).

IV. CARRIERS

The vector of the disease is a female insect of the Diptera order, of the Culicidae family, subfamily Anophelinea and Anopheles gender (Figure 2). There are about 3500 different species of mosquitoes grouped in 41 genii. Among these, the genus Anopheles has at least 430 species, of which 30-40 are known to transmit disease to humans (CDC) (Figure 3).

The life expectancy of an Anopheles is 2-3 weeks. The Anopheles that survive for a long time allow the accomplishment of the Plasmodium cycle (e.g. 10 days at 25°C for P. falciparum).

Figure 2. Anopheles Arabiensis during its blood meal (Source: www.vectorbase.org, 2017)  

V. PLASMODIUM 

The Plasmodium responsible for malaria is an intracellular, amoeboid protozoan parasite that colonizes red blood cells and produces a pigment. During the life cycle, the parasite alternates between asexual reproduction (schizogony) in the vertebrate host and sexual reproduction (sporogony) in the invertebrate host, the Anopheles (Molez, 1993).

Plasmodium is an amoeboid intracellular parasite (Fig.4), of the phylum Sporozoa or Apicomplexa. It belongs to the order Heamosporidea, the latter being composed of only one family: Plasmodiidae; various genus are described, the genus Plasmodium having particular characteristics, such as the male and female gametocyte stage with different morphology.

VI. Taxonomy :

Plasmodium falciparum belongs to the domain, Eukaryota of the kingdom Chromalveolata, of the division Alveolata, it is included in the phylum Apicomplexa, of the class Aconoidasida, in the Order Haemosporida, of the family Plasmodiidae.

There are over 136 species of plasmodium (Marchiafava and Celli, 1895), the most important of which are the parasites of humans and rodents.

Five species are pathogenic to humans: P. vivax, P. ovale, P. malariae and P. falciparum; it has recently been confirmed that P. knowlesi (a parasite of primates in Southeast Asia, of the subgenus Plasmodium) is also capable of infecting humans (Singh et al., 2004). (Singh et al., 2004; Cox- singh et al., 2008, Chitnis and Miller, 1994).

For our research, we have selected Plasmodium yoelii, a rodent pathogen that is the model of choice for the study and simulation for human malaria. Plasmodium yoelii is one of many types of rodent parasites. Studies have shown that this parasite has many similarities with human Plasmodium: similarities in structure, physiology and life cycle (Diggs et al., 1977). In spite of these similarities, small differences remain, such as a variation in certain surface proteins that allow the invasion of red blood cells. The cycle of Plasmodium yoelii involves two hosts: the rodent, the intermediate host, hosts the asexual or schizogonic multiplication of the parasite; the mosquito of the genus Anopheles, Anopheles stephensi, which is the definitive host in which the sexual or sporogonic multiplication takes place.

VII. Growth cycle :

There are two phases: an asexual phase that occurs in the vertebrate host and is subdivided into two sub-phases (exo-erythrocytic phase and intra-erythrocytic phase) and a sporogonic phase that occurs in the invertebrate host (anopheles mosquito) (Figure 5).

The asexual cycle in the vertebrate host is as follows: During her blood meal, the female anopheles inoculates the human with sporozoites present in her salivary glands. These sporozoites invade the blood and rapidly reach the liver by invagination, where they begin the exo-erythrocytic phase, forming hepatic schizonts. These multiply by schizogony. This phase goes unnoticed and is asymptomatic. At the end of this phase, thousands of merozoites are generated by bursting of the hepatic schizonts. This phase may last one to two weeks depending on the species. In P. vivax and P. ovale, there is a delayed schizogony (hypnozoites) and the release of merozoites into the bloodstream can take place up to 18 months later. The erythrocytic phase begins with the invasion of red blood cells by merozoites via a ligand-receptor mechanism involving parasite proteins and red blood cell receptors (Gaur et al., 2004), thus there is a difference in host cell preference by the parasite (reticulocytes, aged red blood cells). Maturation into schizonts (2 to 3 days depending on the species), via young trophozoites (ring) and mature trophozoites, leading to the destruction of red blood cells and the release of new merozoites that infect red blood cells again. The breakdown of the red blood cells leads to fever attacks and an increase in parasitaemia characteristic of malaria attacks. For its growth, the parasite imports nutrients either from the globular cytoplasm (hemoglobin, amino acids, fatty acids, p-aminobenzoic acid, glucose) or from the globular membrane surface. It also exports proteins; some particular proteins located at the level of the red blood cell membrane promote the adhesion of parasitized and non-parasitized red blood cells in order to escape the splenic cleaning action.

When the red blood cells burst, pyrogenic substances are released as well as TNF cytokines which have a necrotizing action on the vessels and further aggravate the disease (Grau et al., 1989).

The infected erythrocyte experiences changes in structure and size, from a biconcave to a globular crenellated sphere, and its deformability is reduced. Some merozoites, after a number of cycles, are differentiated into male and female gametocytes which differ in size of the nucleus and cytoplasm and ensure the sexual cycle in the invertebrate host. Maturation of the gametocytes takes place in the human and the fertilization in the stomach of the female mosquito.

The sexual cycle in the invertebrate host is as follows: The anopheles during its blood meal inoculates the sporozoites while recovering the gametocytes from the infected vertebrate host. After 10 minutes, in its stomach, the male gametocytes transform by exflagellation and fertilize the female gametocytes to form a zygote (ookinetes). The ookinetes adhere to the stomach wall after 24 hours and pass through it, becoming oocysts. The growth of the oocysts takes 4 to 21 days, the duration of the process depending on the ambient temperature. From an initial oocyst several hundred sporozoites are formed, migrate to the salivary glands and are ready to be injected during a new puncture (Grau et al., 1989).

VIII. DIAGNOSIS 

Malaria is diagnosed by two types of methods: direct and indirect. The direct ones are parasitological, while the indirect ones are based on immunology or molecular biology (Mouchet, 2004). Diagnosis is based on the detection of erythrocytic forms of plasmodium in a peripheral blood sample (Gentilini, 1993). Classically, the thick drop and the blood smear are used (Courte-joie, 2000). However, their performance in terms of sensitivity and reliability depends directly on the experience of the microscopist and the parasitemia of the infected subject (Gentilini, 1993). It is noted that some complementary examinations are to be done; we have the hemoglobin (Hb) that should be measured systematically in case of clinical anemia and severe malaria, the glycemia that should be measured systematically to detect hypoglycemia (< 3 mmol/l or < 55 mg/dl) in case of severe malaria or associated malnutrition (WHO, 2010; WHO, 2013; WHO, 2014).

IX. ENVIRONMENTAL AND PERIDOMESTIC SANITATION PRACTICES

            These are preventive methods that are done either by mechanical or chemical control. Mechanical control consists of: destroying and emptying regularly objects likely to retain water such as vehicle wrecks, old tires, cans (sardines, tomatoes, etc.. Use insecticide-treated mosquito nets. Improve housing (use mosquito netting and mesh to cover windows and ventilation holes, fill in holes and cracks that are hiding places for mosquitoes); building of septic tanks; regular disposal and storage of solid waste (garbage); vegetation weeding and tree pruning; disposal of household waste in individual containers (garbage cans) or in bins placed along the streets by the administration; de-cluttering of living quarters (Fattorusso et al. , 2004). Chemical control consists of: spreading waste oil, mineral oil or diesel on the surface of stagnant water, using pesticides (insecticides with a spray machine, a fumigator…).                               

X. CARE PLANS

          Curative treatment has two aspects: biomedicine and traditional medicine. In biomedicine, treatment involves several molecules that are classified according to their mechanisms of action as schizonticides, nucleic acid or anti-metabolite inhibitors, inhibitors of mitochondrial functions, inhibitors of protein synthesis and gametocytocides (Katzung, 2006; Njomnang, 2008). Blood schizonticides includes quinoline derivatives and artemisinin derivative. The quinoline derivatives include the four amino quinolines (Chloroquine, Amodiaquine) and the amino alcohols (Mefloquine, Halofantrine). These molecules interfere with the digestion of hemoglobin by inhibiting the formation of hemozoin. Being weak bases, these molecules concentrate in the digestive vacuoles of the parasite and act by binding to free heme and thus inhibiting heme polymerization. The detoxification process by haemozoin is blocked, resulting in the accumulation of the toxic haem for the plasmodium (Moulin and Coqueret, 2008). This mechanism is used in the present study to evaluate the antimalarial activity. Artemisinin derivatives (Artesunate, Artemether, Artemether) have a gametocytocidal action that reduces transmission and limits the chances of resistance emergence. They interfere also in the digestion of hemoglobin by releasing free radicals that are toxic for the parasites (Moulin and Coquerel, 2008). Anti-metabolics block the division of the parasite nucleus. They incorporate the anti-folics (sulfonamides) and anti-folinics (proguanil, pyrimethamine). They act by inhibiting respectively dihydrofolate synthetase and dihydrofolate reductase necessary for the biosynthesis of the parasite’s nucleic acids (Moullin and Coquerel, 2008). Mitochondrial function inhibitors are naphthoquinones (Atovaquone). The latter is a powerful inhibitor of mitochondrial functions by blocking dihydropteroate dehydrogenase. It is always used in combination with an anti-metabolite (Proguanil) to avoid the appearance of resistance (Musset, 2006). Protein synthesis inhibitors are essentially antibiotics (tetracyclines, macrolides and lyncosamides), they can inhibit the protein synthesis of the parasite; they are used in combination with other products in case of multiple resistance (Njomnang, 2008). There are also combinations, including Artemether and Lumefantrine, Artesunate and Sulfadoxine-Pyrimethamine, and Artesunate with Amodiaquine. They are recommended to limit the emergence of resistance cases.  The above groups of drugs are used in the therapeutic treatment of severe and uncomplicated malaria (WHO, 2013).

In traditional medicine, antimalarial treatment uses mainly plants (N’guessan et al., 2009). In this health care system, diagnosis is based only on the symptoms of the disease, which is a source of ambiguity. The disappearance of signs, however, is evidence of the effectiveness of traditional medicines. In many countries, studies on the use of plants have confirmed their value in the treatment of malaria (Boubacar, 2005).

                        Several plants are currently used for that purpose. These include: Cinchona sp (Rubiaceae) bark used as a decoction, infusion or maceration; Azadirachta indica (Meliaceae) leaves as a decoction; and Artemisia annua (asteraceae) leaves as an infusion (Benoit, 1996; Njomnang, 2008). Figure 6 shows the targets of antimalarials.

XI. FOCUS ON THE DEMOCRATIC REPUBLIC CONGO (DRC) AND MALARIA

The Democratic Republic of Congo is a highly endemic country and the vast majority of the population is exposed to malaria (Figure 1). The disease is a major public health and development issue in the country. WHO statistics for 2016 show approximately 19 million cases of malaria and 42,000 related deaths. In 2007, 68% of outpatient visits recorded and 30% of hospitalizations were due to malaria, while adding that only 20% of the population attends medical centers (NMCP, 2007). The DRC is ranked after Nigeria among the most affected countries in sub-Saharan Africa (WHO, 2010). On the average, a Congolese child suffers 7-10 episodes of malaria per year. More than 27,000,000 cases of malaria are recorded each year with at least 180,000 deaths, the most affected being children under five and pregnant women (WHO, 2011).

In this large country, three species of Plasmodium are commonly encountered in malaria: P. vivax, P. malariae, and P. falciparum. Of the three, P. falciparum is the most common, accounting for 95% of cases, and it alone is responsible for the majority of morbidity and mortality. In addition, it very often presents resistance to the most commonly used and commercially available antimalarial drugs (Delacollette et al. 1983; Wilson, 1989; Mesia, 2009).

Prior to antimalarial drug resistance, chloroquine was used as a first-line drug. This resistance led to the use of the combination of sulfadoxine and pyrimetamine. A few years later, this combination encountered the same problem of resistance leading to a change in malaria management policy (Kazadi, 2003). Today, in most endemic areas, ACTs (Artemisinin Combination Therapy) are prescribed as first-line treatment for uncomplicated malaria. Quinine, which has been used for years, continues to demonstrate its efficacy and is used as monotherapy in the treatment of simple or complicated malaria in children as well as in pregnant women, both in DRC and in other endemic countries (Achan et al., 2011).

In terms of therapy in DRC, several ACTs are available: Artemisinin-derived combinations with Amodiaquine, Sulfadoxine/Pyrimethamine, Lumefantrine, Mefloquine or Piperaquine. All of these products are circulating as generics with over 60 different names. The cost of treatment does not make it accessible to everyone despite the different strategies put in place by the PNLP (National Malaria Control Program), which leads to the use of on-board resources or the return to plants in the form of raw or improved products (MTA, Improved Traditional Medicines).