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Microbiologie & Sciences Humaines

Equipe n°11: Cheikh SOKHNA IR2 IRD

Paludisme et maladies émergentes en Afrique: détection, épidémiologie et lutte.

1. Team composition

  

1. Oleg MEDIANNIKOV (CR1, IRD, HDR)

2. Cheikh SOKHNA (CR1, IRD, HDR)

 

2. Achievements

Scientific output

 

This program is based on six Health and demographic surveillance systems (HDSS) in Senegal and a regional network in Western Africa. Four of the studied areas are specially adapted for large-scale studies and mortality impact measurement:

- The HDSS in Bambey/Mbour/Fatick, operational since September 2008, includes 602 000 people in 756 different villages and 54 heath dispensaries. It is, at the present time, the biggest HDSS in Africa.

- The HDSS of Niakhar (45 000 inhabitants, continental sudanic-sahelian climate) (70), Mlomp (8 200 inhabitants, guinea savannah climate) and Bandafassi (13 600 inhabitants, Soudan savannah climate) (93) are part of program. They belong to the international INDEPHT network (103,104,105,106,107). Niakhar HDSS was initiated and has been managed by IRD since 1962. It is the oldest demographic surveillance system in Africa. Mlomp and Bandafassi HDSS are jointly managed by IRD and INED. Since 2008, URMITE entirely administer data management, demographic and mortality causes monitoring in Niakhar, Mlomp and Bandafassi (61,137, 141).

- The Dielmo and Ndiop stations are specially designed to study morbidity and biological mechanisms implicated. They comprise 750 people who undergo daily clinical and epidemiological monitoring. Since the beginning of the project, their management is jointed between IRD and Institut Pasteur in Dakar. An agreement has been signed in 2004 with Senegalese Ministry of Health and is regularly renewed. Designed to study malaria, the protocol used for 25 years has demonstrated that is was also suitable for other emerging pathgens as borreliosis, reckettiosis, Q fever, Whipple disease, etc. 

1) Malaria epidemiology in Dielmo and Ndiop: Identifications, measurement and epidemiological analysis of determinants for human resistance to malaria

 

                Malaria remains the most prevalent vector-borne infectious disease and has the highest rates of morbidity and mortality. Over the last decade, important changes have occurred in the management of malaria in Africa. These changes include a shift from chloroquine to artemisinin-based combination therapies (ACTs)in the first line of treatment (170,173) and the mass distribution and use of insecticide-treated bed nets (LLINs). Together, these strategies have led to a dramatic reduction in the prevalence, morbidity, and mortality of malaria in most African countries (18, 19,69,172). In Senegal, the proportion of morbidity due to malaria fell from 33.57% in 2006 to 3.1% in 2009. RDTs for malaria were introduced in recent years, and their use became widespread at health posts. These RDTs allowed for the rational treatment of malaria by limiting it to documented cases (121,171). Prevalence, morbidity, mortality and transmission data collected in our sites and its impact in the management of malaria in West Africa were presented.

 

a) Plasmodium falciparum malaria in Dielmo: epidemiology and impact of the control strategies

                These studies were performed from 1990 to 2016 in Dielmo, a Senegalese malaria endemic village where ACT was introduced in 2006 and LLINs in 2008. LLINs were renewed successively in July 2011, August 2014 and May 2016. The results show the efficacy of the use of ACT and LLINs to decrease dramatically malaria morbidity (1). Indeed, malaria declined significantly during two years after the nets implementation from 0.58 attacks per person per year to 0.05 and 0.04 attacks per person per year the first and the second year of nets use respectively (p1,24) and the second time 3 years after the renewal of LLINs (2013-2014) (128). They were associated with the non use of LLINs, the loss of immunity, the anopheles resistance to pyrethroid insecticides and the fact of watching television outside the house during the night (24,128,132). During the upsurges of malaria, LLINs use remains effective to reduce malaria (P(132). The increase of malaria was more important among adults compared with children. Indeed, about half of clinical attacks occurred among adults from 2010 to 2015 while before LLINs implementation, this figure was only 15% (132).

Another study in this village in order to investigate the risk of malaria among mosquito collectors showed that the method of human landing catches (HLC) did not increase the risk of malaria among the mosquito collectors compared to the non-collector when adjusting for confounders from 1990 to 2011. Indeed, Clinical malaria attacks accounted for 6.3% both in collectors and non-collectors, and asymptomatic malaria infections accounted for 21% and 20% in collectors and non-collectors, respectively (131).

 

b) Risk factors for Plasmodium falciparum gametocyte positivity:

                Cluster analysis of a 19-year long cohort study for gametocyte carriage revealed spatially defined gametocyte hotspots that occurred during the time when chloroquine was the drug used for clinical case treatment. In addition to known risk factors for gametocyte carriage, notably young age (P. falciparum episode up to 30 days previously (5,134,127).

 

c) Association of antibody responses to the conserved Plasmodium falciparum merozoite surface protein 5 with protection against clinical malaria in Dielmo and Ndiop:

                We investigated by ELISA, the association of anti-PfMSP5 IgG levels, in the context of responses to two other conserved Ags PfMSP1p19 and R23, with protection from clinical episodes of malaria in cross-sectional prospective studies in two different transmission settings (Ndiop: mesoendemic and Dielmo: holoendemic). Blood samples were taken before the transmission season (Ndiop) or before peak transmission (Dielmo) and active clinical surveillance was carried out during the ensuing 5.5-month follow-up. Anti-PfMSP5 antibodies predominantly IgG1 were detected in 60-74% of villagers, with generally higher levels in older age groups. Importantly, PfMSP5 antibody levels were significantly associated with reduced incidence of clinical malaria in all three cohorts. Inclusion of IgG to PfMSP1p19 in the poisson regression model did not substantially modify results (101,140).

 

d) The rise and fall of malaria in Dielmo:

                The changes in malaria epidemiology in a Dielmo village, Senegal, over 22 years (From 1990 to 2012) were monitored and analysed. Malaria treatment changed over the years, from quinine (1990-94), to chloroquine (1995-2003), amodiaquine plus sulfadoxine-pyrimethamine (2003-06), and finally artesunate plus amodiaquine (2006-12). Insecticide-treated nets (ITNs) were introduced in 2008. Entomological inoculation rate ranged from 142·5 infected bites per person per year in 1990 to 482·6 in 2000, and 7·6 in 2012 (6). Parasite prevalence in children declined from 87% in 1990 to 0·3 % in 2012. In adults, it declined from 58% to 0·3%. About 23 546 fever episodes were recorded during the study, including 8243 clinical attacks caused by Plasmodium falciparum, 290 by Plasmodium malariae, and 219 by Plasmodium ovale. The incidence of malaria attacks ranged from 1·50 attacks per person-year in 1990 to 2·63 in 2000, and to only 0·046 in 2012. The greatest changes were associated with the replacement of chloroquine and the introduction of ITNs (3,95).

 

e) Dramatic declines in seropositivity as determined with crude extracts of Plasmodium falciparum schizonts:

                In Dielmo and Ndiop, two villages of Senegal with different malaria endemicity, infections have been followed longitudinally for 20 years, during which time there have been several control interventions leading to substantial decreases of transmission. This study aimed to compare malaria antibody responses of the inhabitants of these two villages, between 2000 and 2010, using schizont crude extracts of a local strain of P. falciparum (Pf Sch07/03). Overall seroprevalence to P.fSch07/03 decreased between 2000 and 2010 in both villages: from 94.4% to 44.4% in Dielmo and from 74.4% to 34.6% in Ndiop. The difference between Dielmo and Ndiop was highly significant in 2000 (p0.20). The decrease in seroprevalence was larger in younger (more than 40%) than older (less than 19%) inhabitants. Longitudinal monitoring of the younger group showed that seroprevalence decreased between 2000 and 2010 in Dielmo from 98.7 to 79.3, but not in Ndiop from 67.6 to 66.7. The magnitude of antibody responses in seropositive individuals was significantly higher in 2000 than 2010 for both villages (90).

 

f) 20-year longitudinal study of Plasmodium ovale and P. malariae prevalence and morbidity:

                Little is known about the burden caused by Plasmodium ovale and Plasmodium malariae. The inhabitants of Dielmo village, Senegal, were monitored between June, 1990, and December, 2010 for fever and quarterly measurements of parasitemia were performed. Parasitological and clinical data were analyzed in a random-effect logistic regression model to investigate the relationship between the level of parasitemia and the risk of fever and to establish diagnostic criteria for P. ovale and P. malariae clinical attacks. The prevalence of P. ovale and P. malariae infections in asymptomatic individuals were high during the first years of the project but decreased after 2004 and almost disappeared in 2010 in relation to changes in malaria control policies. The average incidence densities of P. ovale and P. malariae clinical attacks were 0.053 and 0.093 attacks per person per year in children94).

g) Changing malaria epidemiology and diagnostic criteria for Plasmodium falciparum clinical malaria

                The aim of this study is to investigate how changes in malaria control and endemicity modify diagnostic criteria of Plasmodium falciparum attacks. Parasitological and clinical data from the population of Dielmo, Senegal, monitored during 20 years, are analyzed in a random-effect logistic regression model to investigate the relationship between the level of parasitemia and risk of fever. Between 1990 and 2010, P. falciparum prevalence in asymptomatic persons declined from 85% to 1% in children 0-3 years and from 34% to 2% in adults ≥50 years. Thresholds levels of parasitemia for attributing fever episodes to malaria decreased by steps in relation to control policies. Using baseline threshold during following periods underestimated P. falciparum attacks by 9.8-20.2% in children and 18.9-40.2% in adults. Considering all fever episodes associated with malaria parasites as clinical attacks, overestimated P. falciparum attacks by 42.2-68.5% in children and 45.9-211.7% in adults.  Malaria control modifies in all age-groups the threshold levels of parasitemia (27).

2) Malaria entomology in Dielmo and Ndiop: impact of long-lasting insecticidal nets (LLINs) on malaria transmission, Anopheles population dynamics and resistance to insecticides

 

a) Dynamic of malaria transmission and Anopheles populations:

                A longitudinal survey from 2006 to 2016 was conducted in Dielmo to study the impact of LLINs use on Anopheles population dynamics and malaria transmission.This period of study was divided in four periods according to LLIN implementation and their renewal. The P1 period, from January 2006 to June 2008 corresponds to the period before LLIN implementation. The P2 period, from the implementation of LLIN to their first renewal (July 2008 to June 2011), P3 period, from the first to the second LLIN renewal (July 2011 to July 2014) and the P4 period, from the second to third renewal (jAugust 2014 to May 2016). The use of LLIN during the period of study has induced a decrease of An. gambiae aggressiveness from 11,76 bites/person/night in P1 to 12,24, 5,09 and 1,731 b/p/n  in P2, P3 and P4, respectively. The same trend was observed with An. funestus  with a decrease from 17,24 b/p/n at P1 to 1,17, 1,731 et 2 at P2, P3 et P4, respectively. At the same time, a collapse of malaria transmission was observed with the decline of the entomological inoculation rate (EIR) from 207 infected bite/person/night at P1 to 53, 26 and 22 ib/p/n at P2, P3 and P4, respectively. In addition, malaria transmission is now seasonal in Dielmo due to the use of LLIN use.

   The implementation of LLIN has also induced a shift in species composition in the Anopheles gambiae complex. During the P2 period, a dramatic decrease in the proportions of Anopheles coluzzii and An. gambiae was observed while the proportion of Anopheles arabiensis increased becoming the most prevalent species (195). This tendency is maintained until P4 period despite a rebond of An. gambiae s.s.

   During the P3 period, An. funestus showed a behavioural change in biting activity. This species conserved its anthropophilic and endophilic activity but adopted a diurnal feeding, essentially on humans. Six times more An. funestus were captured in broad daylight than at night (6992). Moreover, during the P4 period, An. funestus was the main malaria vector in Dielmo (144).

 

b) Study of LLINs physical integrity and residual bio-efficacy and Anopheles trophic preferences:

                A survey was conducted to assess the  physical integrity and bio-efficacy of LLINs used in Dielmo. During P3 period, 77% of the LLIN was in good condition of use and 70% conserved their bio-effcacity against a strain of Anopheles susceptible to deltametrhin (article in prep). The study of Anopheles trophic preferences confirmed the efficacy of LLIN with a shift observed in their feeding preferences from human to domestic animal (PhD thesis).

 

c) Susceptibility of Anopheles vectors to insecticides:

                During the P4 period, the susceptibility of Anopheles population pyrethroid, carbamate, organophophate and organochlorine insecticides was evaluated. The population was composed of An. arabiensis (93%), An. coluzzii (2,1%) et An. gambiae s.s. (4,3%). The mosquitoes were totally susceptible to lambda-cyahalothrin (pyrethroid), bendiocarb (carbamate) and fenitrothion (organophosphate) with a mortality of 100%. The Anopheles susceptibility was reduced (90 to 97% of mortality) against other pyrethroïd (permethrin, deltamethrin, alphacypermethrin, etofenprox) and organophosphate (pirimiphos-methyl). However, the resistance to DDT (organochlorine) was confirmed with a mortality of 62%. Anopheles pre-exposure to ethacrynic acid a synergist, did not restore their fully susceptibility to this DDT (mortality ≤90%). The kdr-west (L1014F) mutation was found only in An. arabiensis and at a low frequency (0.1) suggesting therefore that detoxification mechanisms could be involved in mosquito resistance against insecticide (PhD thesis).

 

d) Maldi-Tof applied to entomological monitoring

                Since 2014, we are using the Maldi-Tof for mosquitoes species identification. Currently, we are able to identify the mosquitoes belonging to the genus of AedesCulex, Anopheles and Mansonia (66). Furthermore, we can discriminate 12, 10, 5 and 2 species of the genus of AedesAnopheles, Culex and Mansonia, respectively. In addition the Maldi-Tof is used to determine the origin of Anopheles blood meal. (Master thesis).

 

3) Other malaria studies

a) Malaria intervention: trial of targeted control to eliminate malaria in Central Senegal

                From 2012 to 2015 we implemented a cluster-randomized trial of targeted control to eliminate malaria in Central Senegal. The study was to evaluate the extent to which a targeted malaria control strategy combining vector control with indoor residual spraying (IRS) and chemotherapy, delivered by district health staff to hotspot villages, can substantially reduce the overall incidence of malaria. We also sought to determine if transmission reduction contributes to this impact, by evaluating the effect of targeted control in non-target villages. The trial was also designed to determine whether the chemotherapy should be delivered to all members of targeted communities (MDA = Mass Drug Administration) or only those who have been tested and are known to be infected (MSAT = Mass Screening and Treatment).

40 clusters were randomized. Each cluster was a health post and the population it serves. In 30 clusters, all households in hotspot villages were targeted to receive IRS with Actellic 300CS (the organophosphate insecticide pirimiphos-methyl, formulated in capsules to provide a long-lasting effect) in July each year. In 15 of these 30 clusters, IRS was followed by MDA with dihydroartemisinin-piperaquine (DHA-PQ) administered to all persons in the household, except pregnant women and children under 3 months of age, at the end of August and again in October. In the other 15 clusters, instead of MDA, all persons in the household were screened using a malaria Rapid Diagnostic Test (RDT, First Response) and those who tested positive treated with DHA-PQ. 10 clusters served as controls. In all three arms, health promotion activities encouraged care seeking for fever, and free long-lasting insecticide-treated bednet was provided to each patient treated for malaria at health facilities in order to top-up coverage. The intervention strategy was delivered over two years (2013 and 2014), and the primary outcomes were the incidence of malaria, and the prevalence of parasitaemia just after the main peak period of transmission, in year 2.

In 2013, 228 out of 716 villages (32%) were categorized as hotspots. The villages which were hotspots in 2013, were less likely to be hotspots in 2014 in the IRS+MDA arm than was the case in the control arm (relative odds ratio 0.29, 95%CI 0.14,0.59, P=0.001), and than in the IRS+MSAT arm (relative odds ratio 0.41, 95%CI 0.20,0.83, P=0.013). Households in target villages received IRS with Actellic 300-CS at the start of each rainy season. In 2013, only 23% of households were treated due to a shortage of insecticide. In 2014, 75% households were treated (PhD thesis).

 

b) Ecology of malaria vectors in flooded urban areas: Dakar Region

                The Dakar region is an area where malaria still persist with high levels of transmission in some localities (4, 30). The study of Anopheles ecology in Dakar region marked by recurrent flooding could help to implement effective malaria control strategies. From 2012 to 2015, the population dynamics of Anopheles was studied in 3 of 4 departments of the Dakar region. During this work, only Anarabiensis was found at both larval and adult stages. An. arabiensis larval densities was high, particularly in the peak of the rainy season, with an average of 109 larvae per liter of water. The proliferation of these larvae is reduced by turbid water (IRR = 0.39 ; P = 0.019), the presence of fish (IRR = 0.50 ; P = 0.004) but also in the water bodies located more than 10 m of human dwellings. The abundance of these larval habitats and the human biting rate of Anarabiensis evolved according to the level of the flooding. Indeed, the number of Anarabiensis larval habitats was 2 times higher in a municipality severely affected by flooding compared to localities less flooded. The circumsporozoitique index was negative in the low flooded localities, while in the municipality severely flooded, the infection rate is higher in rainy season (4.6%) compared to the dry season (0.98%) (X² = 2.76; = 0.04). Hence malaria transmission was more important  in flooded localities with an entomological inoculation rate estimated at 0.3 infective bites per person per night for the two seasons combined. This shows the importance of the influence of micro-ecological conditions on the intensity of malaria transmission (194). Furthermore, treatment of Anarabiensis larval habitats with low doses of microbial larvicides (WG Vectobac® and VectoBac®GR and VectoMax® CG) and a growth regulator (MétaLarv® S-PT) has effectively eliminate the immatures stages of malaria vectors in the study area. These biological larvicides and MétaLarv can be used alternately for larviciding, which would be very beneficial in reducing the insecticide pressure (193).

 

c) Several studies were conducted to validate human IgG antibody response to salivary peptides as biomarker for evaluating exposure to Aedes aegypti and albopictus bites (56,57,59), dengue transmission (126), to evaluate differential acquisition of human antibody responses against Plasmodium falciparum according to the intensity of exposure to Anopheles bites (58,64) and the Salivary biomarkers in the control of mosquito- borne diseases (174).

 

d) Safety survey of malaria treatments with artesunate/amodiaquine combination in villages of Casamance (Mlomp, Elinkinde, Djembereng) and toucar in the health district of Niakhar

                Safety of malaria treatment using a combination with artemisinin derivatives is crucial to evaluate side effects and risks linked to their use. Artesunate/amodiaquine has been experimentally used in Casamance since 2001 and became in 2006 a national recommendation in all parts of Senegal for treatment of uncomplicated confirmed malaria cases. The collect of data began with a support of Sanofi pharmaceutical company in 2008 and continued by annual amendment until 2016. Up to now, 4 130 surveys of artesunate/amodiaquine treatment are available and no serious side effect has been observed (20). We also managed of fever cases between 2000 and 2014. Data were extracted from registers of the healh centers of Mlomp, Elinkinde, Djembereng, Oussouye in Casamance and Toucar in Niakhar district between 2000 and 2014. This study concerned a large number of patients i.e. 70 892 patients attending the clinic of Mlomp (51.3% attending for fever) and 65 108 in Toucar (54.6% attending for fever). Study of these data afforded advances in fever management according to national recommendations for diagnosis and treatment of fever between 2000 and 2014 (20,55,102).

 

e) The impact of atopy and allergy on the risk of clinical malaria in Dielmo.

                A clinical and immunological allergy cross-sectional survey in a birth cohort of 175 children from 1 month to 14 years of age followed for up to 15 years in a longitudinal open cohort study of malaria in Senegal. Results showed that there was no effect of allergy on the number of non-malaria clinical presentations. Individuals with allergic rhinoconjunctivitis did not have an increased risk of clinical malaria nor any difference in parasite densities (88).

 

4) Emerging pathogens biodiversisty

 

During the period of 2011-2016, the research activities of the emerging pathogens group were determined by three major axes: (1) "Molecular strategy of the identification of the causes of acute fevers in Africa"; (2) "Isolation and culture of fastidious vector-borne bacteria"; and (3) "Medical entomology: search for vector-borne transmitted pathogens".

 

Axe 1. Molecular strategy of the identification of the causes of acute fevers in Africa

                The causes of the acute febrile conditions in Africa were the object of a few studies despite of the fact that the fever is the first cause of the consultations in rural dispensaries in Africa. For a long time the malaria was considered as a major cause of febrile conditions. However, recently the overall morbidity and mortality due to malaria have dropped significantly, mostly because of the use of artemisinin-based combination therapies and insecticide-treated nets. Moreover, the wide availability of rapid diagnostic tests for malaria diagnostics (malarial antigen detection) testify the existence of a high (sometimes, up to 40-50%) proportion of non-malarial fevers that, in the absence of laboratory diagnostics may lead to about 80% of unnecessary treatments. The repertoire of the non-malarial causes o acute febrile conditions in West Africa stays largely unknown and under investigated. Moreover, some preliminary data show that at least in several regions a large proportions of the febrile conditions may be caused by such fastidious emerging bacteria as BorreliaRickettsiaCoxiella burnetii etc., so the diagnostic may not be done in routine bacteriological laboratories. From the other side, these bacteria are often susceptible to a proper antibiotics treatment.

                So, this background brought us to the development of the project of the investigation of the causes of febrile conditions in Africa.

- The concept

             The concept of the project is to perform the molecular screening of acute febrile patient's samples (mostly, capillary blood samples) for the multiple pathogens using the molecular biology methods (qPCR, standard PCR coupled with sequencing). We used the approach of "decreasing mesh size fishing net": all samples are screened with large sensitivity qPCRs (family/genus specific) and then in case of positive results, the identification of the pathogen is made by specific qPCRs and/or by sequencing of appropriates genes. Samples are collected in rural dispensaries of several African countries: Senegal, Guinea, Côte d'Ivoire and Gabon.

- Preparative stages

             We have developed the field-adapted modification of the DNA extraction method.

Many clinical samples (especially fresh blood samples) are very fragile and cannot be stored for a long time and/or in inappropriate conditions, otherwise the results will be biased. It concerns especially molecular techniques for the identification of bacterial pathogens: bacterial DNA in the blood is susceptible to be digested rapidly. Several solutions may be proposed in order to evade this DNA degradation: either to transport the sample (or patient) in the appropriate facility (laboratory or hospital) or to preserve the samples. First variant is too costly and logistically difficult. We proposed and tested the method of field-adapted partial DNA extraction method based on commercially available DNA extraction columns.

The commercially available kits (QIAGEN, MACHERY-NAGEL etc.) are transported to the dispensary where specially trained personnel may perform the partial DNA extraction even in the absence of electricity. The digestion is done in adapted water bath and the centrifugation is replaced by manual pumps.

Obtained DNA is not eluted from the columns but stored and sent regularly to the reference laboratory where the columns are additionally washed and the then DNA is eluted.

The quality of obtained DNA is regularly verified by the qPCR targeting human actin gene. We have demonstrated that the DNA extracted by this method may be stored before elution for several days at the room temperature and to 12 months in +4C without evident degradation.

The DNA is extracted from 3-4 drops of capillary blood took from febrile patient (patients in dispensaries often refuse the vein puncture.

 

a) Results: Senegal

Overall, we have installed the program in 5 sites (including 14 rural dispensaries) in Senegal including almost all climatic zones of Senegal.

In general, we have found different fastidious and emerging pathogens playing important roles in local pathology. One of the most unusual results was the striking incidence of Rickettsia felis-associated fevers in different regions of Senegal: it was found in the blood of up to 15.6% of all febrile patients in Senegal (28,63, 68,162,187). Tropheryma whipplei, the agent of Whipple's disease, was found to be responsible for occasional bacteremia (2,62). Also we have detected the outbreak probably related with the food-borne clonal transmission of T. whipplei in Senegal (198). Tick-borne relapsing fever caused by Borrelia crocidurae continues to be an important endemic infection in the West Africa: overall it is responsible for 7,6% of acute fevers, competing in several regions with malaria for the first place (11,25,60,99,165). Bartonella quintana, the agent of the trench fever is also prevalent in Senegal, especially in the region of Sine-Saloum (36,63,96,119). Q fever, as it was shown serologically in many African countries, is endemic also in Senegal, it may cause up to 2,3% of fevers in certain (mostly desertic) regions (29,63,89).

Malarial plasmodia were also identified in the samples. We found only P. falciparum in about 23% of all samples (63,68).

- Point-of-Care (POC) laboratory

                MIE team implemented POC laboratory in Sine-Saloum region of Senegal (Dielmo village) (63). During the first year of the study (February 2011 to January 2012), 440 blood specimens from febrile patients were collected in Dielmo and Ndiop villages. We identified DNA from at least one pathogenic bacterium in 80/440 (18.2%) of the samples from febrile patients. B. crocidurae was identified in 35 cases (9.5%), and R. felis DNA was found in 30 cases (6.8%). The DNA of Bartonella spp. was identified in 23/440 cases (4.3%), and DNA of C. burnetii was identified in 2 cases (0.5%). T. whipplei (0.2%) was diagnosed in one patient. Malaria was diagnosed in 54 cases. In total, at least one pathogen (bacterium or protozoa) was identified in 127/440 (28.9%) of studied samples. The laboratory continues to work, for the moment of the present report, around 2750 blood samples of acute febrile patients were analyzed. The second POC laboratory was constructed and inaugurated in another region of Senegal: Niakhar since November 2015(data not published).

Due to the options of rapid diagnostic available in POC laboratory, we have diagnosed and reported the case of primary R. felis-associated eruptive fever (A30), isolated and characterized several strains of B. crocidurae from acute relapsing fever patients (112,113,161) as well as several strains of B. quintana from acute trench fever patients (data not published).

Another interesting result was the report about unexpectedly high frequency of Mansonella perstans filariosis in Senegal (152).

 

b) Results: Guinea

                We have successfully installed the similar project in the Republic of Guinea. Two sites participated in the study: Centre National de Formation et de Recherche en Santé Rurale (CNFRSR) in Maferinyah village and ex-Pasteur Institut of Guinea (IPG) in Kindia. Preliminary data obtained showed the presence of multiple tick-borne rickettsiae in the region (34). We have obtained 440 DNA samples extracted from acute febrile pateints and 127 from healthy donors. The samples are under analysis now, preliminary results showed the presence of such pathogens as C. burnetiiRickettsia spp., T. whippleiBartonella spp., Salmonella spp. (including S. typhi and S. paratyphi), Staphylococcus aureusStreptococcus pyogenesStreptococcus pneumoniaeMansonella perstans, and Plasmodium spp.

We also studied the role of different bacteria in the chronic skin ulcers. Samples from patients were collected by CNFRSR in Maferinyah village (Ehounoud C. et al. PLOS Negl. Trop. Dis, submitted). Eighty-four skin samples from the wounds of 20 patients (42 edge swabs and 42 center swabs) and twenty-two skin samples from 11 healthy people were analyzed by real-time quantitative PCR and standard PCR assays combined with sequencing. Pseudomonas aeruginosa was the most frequently detected bacterium, which was significantly more prevalent in patients (80%, 16/20) than in healthy people (9%, 1/11; pStaphylococcus aureus which was only detected in patients (60%, 12/20; pStreptococcus pyogenes was also more frequently detected in patients (30%, 6/20) than in healthy people (9%, 1/11) but the difference was not statistical significant. Rickettsia felis was also detected for the first time in Guinea, in one patient. Finally, species of the genus Acinetobacter were also frequently and exclusively detected in patients (80%, 16/20). Acinetobacter baumannii (2/20, 10%), Acinetobacter nosocomialis (10%), Acinetobacter junii (1/20, 5%), Acinetobacter lwofii (5%), and Acinetobacter guangdongensis (5%), which was detected for the first time in skin, were identified. A. junii and A. lwofii were observed in different samples from the same patient.

 

c) Results: Côte d'Ivoire

                As in Guinea, we have began the similar project in Côte d'Ivoire.

We have performed the scrupulous search of pathogens in ticks from Côte d'Ivoire and found multiple tick-associated pathogens, including Q fever agent C. burnetii, several pathogenic rickettsiae and potentially new species of borrelia, anaplasmae and ehrilichiae(175). In collaboration with Centre médico-social Walé, in Yamoussoukro, we have collected 325 DNA samples from acute febrile patients and 364 DNA samples from healthy controls.  These materials are still in teh process of study, preliminary results showed the presence of R. felisT. whippleiBartonella spp., Mansonella perstansS. aureusS. pneumoniaeS. pyogenesSalmonella spp. and P. falciparum in the samples. Interesting, that no Borrelia spp. or Bartonella spp. were identified.

d) Results: Gabon

                The similar project in Gabon was initiated in 2014 in collaboration with Parasitological laboratory (Prof. J.-B. Lekana-Douki) of the Centre International de Recherches Médicales de Franceville (CIRMF, www.cirmf.org).

Preliminary data showed the presence of R. felis in the blood of acute febrile patients from Gabon in 10,2% (155). Another interesting finding is the detection of a novel genetic variant of Mansonella sp. in the blood of febrile and healthy children (157). This genotype may present either new human pathogenic species of Mansonella, or, probably, one of the previously reported ape-associated species that were never characterized molecularly.

 

Axe 2. Isolation and culture of fastidious vector-borne bacteria

 

The isolation of bacteria in pure culture is the essential step for the description of a new species, as well as for most studies of pathogenicity, diagnostic methods and genetic structure of pathogenic bacteria.

We have implied the most advanced techniques of cell culture, animal models and culturomics in order to isolate in pure culture and to describe the following bacteria:

  1. Bartonella chomeli (13)
  2. Diplorickettsia massiliensis gen. nov. sp. nov. (37)
  3. Acinetobacter baumannii (38, 39)
  4. Ricketsia helvetica (42)
  5. Arsenophonus nasoniae (50)
  6. Coxiella burnetii (197),
  7. Bartonella senegalensis sp. nov. (77)
  8. Bartonella florenciae sp. nov. (80)
  9. Bartonella massiliensis sp. nov. (100)
  10. Occidentia massiliensis gen. nov. sp. nov. (111).
  11. Borrelia crocidurae (112)
  12. Rickettsia senegalensis (116)
  13. Bacillus dielmoensis sp. nov. (153)
  14. Necropsobacter massiliensis sp. nov. (160)
  15. Weeksella massiliensis sp. nov. (163)
  16. Chryseobacterium senegalense sp. nov. (179)
  17. Haemophilus massiliensis sp. nov. (183)
  18. Dermabacter indicis sp. nov. (184)
  19. Streptococcus varani sp. nov. (188)
  20. Bacillus ndiopicus sp. nov. (153)
  21. Bartonella lascolai sp. nov. (197)
  22. Bartonella rolaini sp. nov. (197)
  23. Lascolabacter vaginalis gen. nov. sp. nov. (190)
  24. Bartonella raoultii sp. nov.
  25. Bartonella mastomydis sp. nov.
  26. Bartonella saheliensis sp. nov.

 

Many isolates, especially those constituting human skin microbiota are still under the isolation/description.

 

Axe 3. Medical entomology: search for vector-borne transmitted pathogens

 

                Vectors are organisms that transmit pathogens and parasites from one infected person (or animal) to another, causing serious diseases in human populations. Vector-borne diseases account for 17% of the estimated global burden of all infectious diseases. The most deadly vector-borne disease, malaria, caused an estimated 627 000 deaths in 2012. Studies of vectors and associated pathogens are one of the priorities of URMITE scientific activities.

 

a) Ticks (Ixodidae and Argasidae) and tick-transmitted diseases

                Ticks are second only to mosquitoes in the transmission of diseases to man and animals. Every year tick-borne diseases affect hundreds of thousands people in the world and cause billions of global economic losses.

Detection of tick-associated pathogens is an important issue that elucidate the circulation of pathogens in nature and directly lead to preventive measures. Among the works published by MIE team are the manuscripts reporting the emergence of R. africae in Oceania (9), R. aeschlimannii in Germany (10), relapsing fever borreliae in soft ticks in Bolivia (12) and, Ethiopia (75), spotted fever group rickettsiae and bartonellae in Egypt (32), Guinea and Liberia (34), Slovakia (37,42,49, 50), France (46) and French Giana (197), Democratic Republic of Congo (48), Algeria (51), Senegal (77,78, 80,109,110,111,113) and Côte d'Ivoire (175)

b) Fleas

                Fleas play n important role in the transmission of such pathogens as Yersinia pestisRickettsia typhiRickettsia felisBartonella spp. etc. The detection of different pathogens in fleas is one of the important activities of URMITE. Our works identified the presence of different flea-associated pathogens in New Caledonia (8), Senegal (31, 116), Slovakia (35), Ethiopia, (41) and Democratic Republic of Congo (48).

 

c) Lice

                Human lice Pediculus humanus with two biotypes (head and body lice) attract the attention of infectious diseases specialists as the vectors of such deadly diseases as an epidemic typhus, trench fever and louse-borne relapsing fever. Since the discovery of their roles in the transmission of these diseases, only human body lice were thought to be implied. However, our works show that, probably mitochondrial clade A (that include both head and body lice), probably, is capable of the pathogen transmission. Many examples of the presence of B. quintana and B. recurrentis in the clade A human lice suggest that it may be a genotype (clade A) and not phenotype (head or body) that determine the ability to transmit B. quintana and B. recurrentis (15,36,72, 96). We have also identify new genotypes of an important nosocomial pathogen, Acinetobacter baumannii, and carbapenem resistance genes in headlice from Ethiopia and Senegal (38,39).

 

d) Tsetse flies

                We have identified the presence of new genotype of rickettsia (that may represent a new species) in 100% of Glossina morsitans submorsitans collected in Senegal (A11). It may be another endosymbiotic bacterium associated with tsetse flies: the biological role of this rickettsia is to be udentified.

 

e) Biting midges

             Blood-sucking Ceratopogonidae of the genus Culicoides are important pest transmitting not only veterinary pathogens, but also human pathogens, Mansonella spp. and some viruses (Oropuche etc.). A vast diversity of species of this genus and very few studies of African Culicoides make the identification of biting midges a real challenge.

MIE team developped a tool of detection of West African biting midges based on revolutinary MALDI-TOF mass spectrometer (147).

             The role of Culicoides in the transmission of M. perstans in Senegal was never confirmed. We tried to identify the species, but, surprisingly, did not find any culicoides infected by Mansonella filariae in the endemic region of South-Eastern Senegal (152).

Collaborating with metagenomic team, we have identified in Senegalese Culicoides spp. important veterinary pathogens such African horse sickness, bluetongue and epizootic haemorrhagic disease viruses. We also identified the novel Thogotovirus species and isolated Faustovirus-like Asfarvirus from these biting midges (164,181).

4) Strategies to control emerging pathogens

 

a) An Alternative Strategy of Preventive Control of Tick-borne Relapsing Fever in Rural areas of Senegal

                In Senegal, tick-borne relapsing fever (TBRF) is a major cause of morbidity and a neglected public-health problem (81). Borreliosis cases commonly detected in two villages, led us to implement a borreliosis preventive control including cementing of floors in bedrooms and outbuildings attended by inhabitants to avoid human contacts with tick vectors. Epidemiological and medical monitoring of the TBRF incidence was carried out at Dielmo and Ndiop by testing the blood of febrile patients since 1990 and 1993 respectively. Intra-domiciliary habitat conditions were improved by cementing, coupled with accompanying measures, from March 2013 to September 2015. Application of this strategy was associated with a significant reduction of borreliosis incidence. This was more evident in Dielmo, dropping from 10.55 to 2.63 cases per 100 person-years (p < 0.001), than in Ndiop where it changed from 3.79 to 1.39 cases per 100 person-years (p < 0.001). Thirty-six cases of TBRF were estimated to be prevented at a cost of 526 Euros per infection. The preventive control strategy was successful in Dielmo and Ndiop, being associated with decreased incidence by 89.8% and 81.5% respectively, suggesting that TBRF may be widely decreased when the population is involved. Public health authorities or any development stakeholders should adopt this effective tool for promoting rural health through national prevention programs (paper in press).

 

b) Head lice probably resistant to ivermectin recovered from two rural girls in Dielmo

                Recently, investigations were carried out to determine the prevalence of head lice carriage in females from Dielmo and Ndiop, two villages located in the Sine-Saloum region of Senegal for which daily epidemiological and medical monitoring has been performed for more than 25 years. The prevalence of head lice carriage was found to be 34.8% in Dielmo and 60.6% in Ndiop. Given the possibly huge impact of head lice carriage on the health of these populations, we decided to take action to eradicate them from the Dielmo and Ndiop human populations. In the past, action to eradicate head lice consisted of using anti-lice lotions based on insecticides such as pyrethroids and organophosphates. However, as head lice resistant to these insecticides have been increasingly observed, resulting in treatment failure, new therapeutic alternatives are needed. Oral treatment based on ivermectin was identified to be the most promising option for treating head lice. Therefore, from 29 May 2014 to 23 June 2014, an ivermectin oral treatment trial was initiated in Dielmo to eradicate head lice in 52 females aged >2 years and weighing >15 kg. The ultimate goal of this trial was to propose to the health authorities a low cost, easy-to-use and safe treatment to fight head lice carriage. Ivermectin (400 µg/kg of body weight) was orally administered in two intakes every 7 days to each of the 52 infested persons. Unexpectedly, suspected ivermectin resistance in head lice populations from four females (8%) (Females 5, 6 and 8 plus one other female) and possible head lice re-infestation in six other females (12%) (Females 2, 4, 7 and three other females) were identified 15 days later. In November 2015, we decided to begin another ivermectin-based head lice treatment trial in Dielmo to confirm or invalidate our previous observations, particularly given the suspicion of ivermectin resistance previously observed.

                The treatment protocol was similar to the previous one; other than that the treatment efficacy time was reduced to 24 h, the dose was given in one intake, and we decided to treat only the ten females with suspected resistance or re-infestation observed previously. Unfortunately, only six of the ten females were ultimately available for the second trial (Females 2, 4, 5, 6, 7 and 8). We therefore decided to include two other females who had already been treated during the first trial and in whom no resistance or re-infestation phenomena were observed (Females 1 and 3). After 24 h of ivermectin treatment, two cases (25%) of probable ivermectin resistance were observed (Females 2 and 5). Head lice from these two girls were collected to determine the resistance genes possibly involved in ivermectin resistance (199).

 

5) Schistosomasis studies in Senegal

 

                Urinary schistosomiasis is the most common form of schistosomiasis in Senegal and remains a public health problem. Transmission is continuous in foci along the Senegal River Basin (21) and occurs seasonally in most parts of the country. The purpose of these following studies was to understand the epidemiology of urinary schistosomiasis in the district of Niakhar, an area of seasonal transmission in Senegal. The main objective of these works was to show if with repeated mass treatment, it is possible to eliminate urinary schistosomiasis in seasonal transmission foci Senegal. To achieve this goal, we first measured in 2010 the endemicity of the disease by determining the prevalence and intensity of infection among children aged 7 - 15 years. We then evaluated in 2011 the effectiveness and impact of a single treatment on re-infection among 329 children aged 5 – 15 years. We also studied from 2011 to 2013 the dynamics and the role of the snail intermediate hosts. Finally, we evaluated the impact of an annual treatment administered in 2011, 2012 and 2013 on the transmission of S. haematobium in a cohort of 624 children and adults aged 5 – 60 years.

 

a) Prevalence and intensity of urinary schistosomiasis among school children in the district of Niakhar, Senegal

                This study was carried out to determine for the first time, the prevalence and intensity of urinary schistosomiasis in 12 villages of Niakhar (Fatick, Senegal). A total of 210 schoolchildren, aged 7 to 15 years, were enrolled, and urine samples were examined for S. haematobium eggs using a standard urine filtration technique. Of these children, 121 (57.6%) were found to be infected with a mean geometric count of 185 eggs per 10 ml of urine. This study show that the district of Niakhar is endemic for urinary schistosomiasis and that a control program to decrease the prevalence and intensity should be implemented in this area to improve community health. These results were shared with the national program against schistosomiasis and have allowed the Niakhar district to be enrolled for three years mass treatment with PZQ (91).

 

b) Efficacy of praziquantel against urinary schistosomiasis and reinfection in Senegalese school children where there is a single well-defined transmission period

                This study was carried out in 2011 in a cohort of 329 children aged five to 15 years enrolled from six villages in Niakhar to determine the efficacy of one dose of PZQ. A parasitological test was performed in June to determine the baseline prevalence of S. haematobium, and then a single dose of PZQ was administered to all selected subjects in August. The efficacy of PZQ treatment and reinfection were monitored respectively five weeks after in September and from February March 2012. At baseline, the overall prevalence and the heavy intensity of infection were 73.2 % and 356.1eggs/10 ml of urine. A single dose of PZQ significantly reduced the prevalence of S. haematobium infection from 73.2 % to 4.6 % and the geometric mean intensity of infection from 356.1 to 43.3 eggs/10 ml of urine. The cure rates ranged from 89.4 % to 100 %. The egg reduction rates also ranged from 77.6 % to 100. The overall rate of reinfection was 12.6 %. The prevalence at this period was 13.8 %, which was significantly lower than the prevalence at baseline (73.2 %). This study showed that the Niakhar area remains a hot spot of urinary schistosomiasis in Senegal. This study suggests that when transmission is strictly seasonal, Praziquantel shows the expected efficacy in reducing the prevalence and intensity of infection (135).

 

c) Study of intermediate snail hosts of S. haematobium in Niakhar, Fatick, Senegal

                Since no malacological investigation has been conducted in the Niakhar area, we realized a third study to identify the snail intermediate hosts but also to study their ecology and epidemiological role. This survey was conducted from 2011 to 2013 in the ponds and backwater of 11 villages. B. senegalensis and B. umbilicatus were the only snails intermediate hosts collected in the Niakhar study area. For B. umbilicatus, 58 snails were collected and tested in 2012. In 2013, 290 were collected and 281 tested. The overall rates of schistosome cercarial shedding were 0 % in 2012 and 0.12 % in 2013 for B. senegalensis and 13.79 and 4.98 % in 2012 and 2013 respectively for B. umbilicatus. This study reported for the first the presence of B. umbilicatus in the region of Fatick and its ability to maintain Schistosoma spp. larvae during 7 months of drought and may transmit the disease in early July, increasing the period and the risk of transmission. This study recommends that the malacological control strategies must take into account the phenomena of drought resistance and the capacity of the snails to maintain parasite during aestivation. Therefore, treatment of ponds with Bayluscide at the end of the rainy season in November and upon onset of rains in July would be more profitable to the control of snails thereby reducing transmission of urogenital schistosomiasis in the Niakhar area (136).

 

d) Impact of annual praziquantel treatment on urogenital schistosomiasis in a seasonal transmission focus

                After showing the efficacy of one dose of PZQ in the previous study, we realized a fourth study wich aim was to determine the effect of annual repeated treatment over 3 years on the seasonal transmission dynamics of S. haematobium in 9 villages in the Niakhar district. A total of 624 adults and children aged between 5 and 60 years were surveyed from 2011 to 2014. Urine samples were collected door-to-door and examined for S. haematobium eggs at baseline in June 2011, and all participants were treated in August 2011 with PZQ (40 mg/kg). After this first assessment, evaluations were conducted at 3 successive time points from September 2011 to March 2014, to assess the efficacy of the annual treatments and the rates of reinfection. Each year, from July to November-December, corresponding to the transmission period, malacological surveys were carried out in the fresh water bodies of each village to evaluate the infestation of the snail intermediate hosts in correlation with the human reinfection.

                At baseline, the overall prevalence of S. haematobium infection was 57.7%, and the proportion of heavy infection was 45.3%, but one month after the first treatment high cure rates (92.9%) were obtained. The overall infection prevalence and proportion of heavy infection intensities were drastically reduced to 4.2% and 2.3%, respectively. At follow-up time points, prevalence levels varied slightly between reinfection and treatment from 9.5% in June 2012 to 0.3% in March 2013, 11.2 in June 2013, and 10.1% in April 2014. At the end of the study, overall prevalence was significantly reduced from 57.7% to 10.1%. The overall rate of infested Bulinid snails was reduced after repeated treatment from 0.8% in 2012 to 0.5% in 2013. Repeated annual treatments are suggested to have a considerable impact on the transmission dynamics of S. haematobium in Niakhar, due to the nature of the epidemiological system with seasonal transmission. Thus, to maintain this benefit on the fight against urogenital schistosomiasis, other approaches should be integrated into the strategy plans of the National program to achieve the goal of urogenital schistosomiasis elimination in seasonal foci in Senegal (143).

 

6) Influenza studies in Senegal

 

Flu effectiveness vaccine project on children in Senegal (TIV), MF59-adjuvanted TIV and Live Attenuated Influenza Vaccine (LAIV) (2009-2014)

                The Project (Path, CDC, IPD, IRD) was conducted in order to generate data on the occurrence and burden of influenza in tropical developing Africa, the effectiveness of the vaccination of children with current trivalent influenza vaccine (TIV) to reduce disease in those vaccinated and unvaccinated, and to evaluate new vaccines that might be appropriate for controlling influenza in such settings. The Project includes 6 primary studies conducted from 2009 through 2014. These are a) three annual evaluations of the effectiveness of TIV in children, b) an evaluation of the duration of protection of inactivated TIV greater than 12 months post-vaccination, c) an evaluation of the safety and immunogenicity of MF59-adjuvanted TIV and full-dose TIV and d) an efficacy trial of LAIV in children, included in the first two annual evaluation of TIV are substudies of the immunogenicity and safety of TIV in children, and included in the efficacy trial of LAIV is vaccines take and extended safety substudy.

The overall effectiveness of TIV compared to IPV for laboratory-confirmed AFRI with infection with any influenza A and B was 34.7% (95% CI: 8.2, 53.5). The overall effectiveness of TIV compared to IPV for laboratory-confirmed AFRI with infection with influenza A (H3N2) was 55.4% (95% CI: 29.4, 71.8). The indirect effectiveness in the non-age eligible (

                LAIV was well-tolerated in this population of children in Senegal. No wheezing signal in this population with low prevalence of wheezing illness at baselineH1N1 efficacy similar to effectiveness findings from US studies. Unable to assess efficacy against matched H3N2 or B lineage due to no circulation and low attack rate, respectively. Efficacy point estimates against all strain (primary) and vaccine-matched strain (secondary) objectives driven by H1N1

 

7) Meningitis studies

 

a) Evaluation of the antibody persistence in African subjects up to five years after immunization with MenA    

a) Evaluation of the antibody persistence in African subjects up to five years after immunization with MenAfriVacTM, a meningococcal A conjugate vaccine (2007-2013)


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