Chikungunya Outbreak in Kedougou, Southeastern Senegal in 2009–2010

Chikungunya Outbreak in Kedougou, Southeastern Senegal in 2009–2010 Open Forum Infectious Diseases MAJOR ARTICLE Chikungunya Outbreak in Kedougou, Southeastern Senegal in 2009–2010 1,10,11 1 2 2 3 1 2 Abdourahmane Sow, Oumar Faye, Mawlouth Diallo, Diawo Diallo, Rubing Chen, Ousmane Faye, Cheikh T. Diagne, 3 4 5 6 7 7 Mathilde Guerbois, Manfred Weidmann, Youssoupha Ndiaye, Cheikh Sadibou Senghor, Abdourahmane Faye, Ousmane M. Diop, 1 1 8 9 10 11,a 3,a 1 Bakary Sadio, Oumar Ndiaye, Douglas Watts, Kathryn A. Hanley, Anta T. Dia, Denis Malvy, Scott C. Weaver, and Amadou Alpha Sall 1 2 3 Institut Pasteur Dakar, Arbovirus and Viral Hemorrhagic Fevers Unit, Senegal; Institut Pasteur Dakar, Medical Entomology Unit, Senegal; Institute for Human Infections and Immunity, Center 4 5 for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston; Department of Virology, University Medical Center Göttingen, Germany; District Sanitaire 6 7 8 9 de Saraya, Senegal; District Sanitaire de Kedougou, Senegal; Institut Pasteur Dakar, Medical Virology Unit, Senegal; University of Texas at El Paso; Department of Biology, New Mexico State 10 11 University; Institut Santé et Développement, Université Cheikh Anta Diop, Dakar, Senegal; INSERM 1219, University of Bordeaux, France Background. In Senegal, Chikungunya virus (CHIKV), which is an emerging mosquito-borne alphavirus, circulates in a syl- vatic and urban/domestic cycle and has caused sporadic human cases and epidemics since 1960s. However, the real impact of the CHIKV sylvatic cycle in humans and mechanisms underlying its emergence still remains unknown. Methodology. One thousand four hundred nine suspect cases of CHIKV infection, recruited from 5 health facilities located in Kedougou region, south-eastern Senegal, between May 2009 to March 2010, together with 866 serum samples collected from school- children from 4 elementary schools in May and November 2009 from Kedougou were screened for anti-CHIKV immunoglobulin (Ig)M antibodies and, when appropriate, for viral nucleic acid by real-time polymerase chain reaction (rPCR) and virus isolation. In addition, mosquitoes collected in the same area from May 2009 to January 2010 were tested for CHIKV by rPCR and by virus isola- tion, and 116 monkeys sera collected from March 2010 to May 2010 were tested for anti-CHIKV IgM and neutralizing antibodies. Results. e m Th ain clinical manifestations of the CHIKV suspect cases were headache, myalgia, and arthralgia. Evidence for CHIKV infection was observed in 1.4% (20 of 1409) of patients among suspect cases. No significant difference was observed among age or sex groups. In addition, 25 (2.9%) students had evidence of CHIKV infection in November 2009. Chikungunya virus was detected in 42 pools of mosquitoes, mainly from Aedes furcifer, and 83% of monkeys sampled were seropositive. Conclusions. Our findings further documented that CHIKV is maintained in a sylvatic transmission cycle among monkeys and Aedes mosquitoes in Kedougou, and humans become infected by exposure to the virus in the forest. Keywords. Chikungunya virus; Kedougou; outbreak; Senegal; sylvatic circulation. Chikungunya virus (CHIKV) is a member of the Semliki [5]. Several studies have reported CHIKV circulation in West Forest virus antigenic group of the genus Alphavirus (family Africa, especially in Nigeria between 1963 and 1977 [6, 7]. Togaviridae) and was first isolated in 1953 in Tanzania [1, 2]. Major CHIKV epidemics were recently reported in Africa and This arthtrogenic virus is composed of 2 lineages: African and Asia [8]. A significant outbreak occurred in Italy in 2007, and Asiatic. When symptomatic, cute CHIKV infection in human numerous imported cases have been detected elsewhere in can cause a flu-like syndrome with rash, but joint pains are the Europe and the United States of America [9–13], emphasizing dominant complaint and might evolve to persistent and inca- CHIKV as a re-emerging public health threat worldwide. pacitating manifestations. In Africa, CHIKV is maintained in In Senegal, CHIKV was first isolated in 1962 from bats nature among nonhuman primates and forest-dwelling Aedes [14, 15], and CHIKV outbreaks and sporadic cases were sub- sp. In rural areas, these sylvatic vectors can be responsible for sequently reported in 1966, 1982, 1996, 1997, and 2004–2006 sporadic cases or small outbreaks [3, 4]. In urban areas, CHIKV [4, 16, 17] (Figure 1). In addition, CHIKV has been repeatedly is transmitted to humans by Aedes aegypti and Aedes albopictus isolated from wild caught Aedes furcifer, Aedes luteocephalus, and Aedes taylori in a sylvatic focus near Kedougou in south- eastern Senegal [5, 18–20] as part of an entomological surveil- Received 14 July 2017; editorial decision 18 November 2017; accepted 27 November 2017. lance programme that began in 1972 [21]. Various vertebrates, D. M. and S. C. W. contributed equally to this work. Correspondence: A.  Sow, MD, MPH, Professional Officer/Epidemics Control-Public Health including monkeys, bats, gophers, and galagos, have been Laboratories, West African Health Organisation (WAHO), 175 Avenue Ouezzin Coulibaly, BP proven for implication as hosts of CHIKV by serological evi- 153, Bobo-Dioulasso, Burkina Faso (asow20@gmail.com). dence or viral isolation [5]. Amplifications of CHIKV in the Open Forum Infectious Diseases © The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases Kedougou area have occurred at approximately 5-year intervals, Society of America. This is an Open Access article distributed under the terms of the Creative which is hypothesized to be the time necessary for the turno- Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any ver of susceptible vertebrate hosts [5]. Despite active circulation medium, provided the original work is not altered or transformed in any way, and that the work of CHIKV in the sylvatic cycle in Senegal, limited informa- is properly cited. For commercial re-use, please contact journals.permissions@oup.com tion is available on its transmission on human and wildlife DOI: 10.1093/ofid/ofx259 Chikungunya Outbreak in Kedougou, 2009 • OFID • 1 Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 50 km MAURITANIA 30 mi St-Louis Rao 1963 Louga 2006 Touba 1981 Thies 1982, 2006 Bambey 2006 SENEGAL Rufisque 1966 Diourbel 2006 Dakar Bandia 1966 Niakhar 1997 Kaolack 2005 Karine 1996 MALI Tambacounda GAMBIA Ziguinchor Kedougou 2004 GUINEA-BISSAU GUINEA Figure  1. Map of Senegal showing locations where Chikungunya outbreaks have been reported. Red circle indicates the localities and the years of Chikungunya virus outbreaks. populations. To address this question, we conducted passive of the protocol implementation was regularly conducted by the surveillance for CHIKV infection among patients attending 5 authorities of the National Ethics Committee of Senegal. health clinics with febrile illness, together with active surveil- Study Areas lance of students recruited from 4 schools in Kedougou. In a The study was conducted in the Kedougou region of southeast- parallel effort, we also investigated monkey serosampling in ern Senegal (12°32’N, 12°11’W) (Figure 2). The human popula- the region and surveyed mosquitoes in the area for CHIKV. In tion is 133 487 inhabitants with an average density of 8 persons this study, we report the investigation of the CHIKV zoonotic per km , 55% of whom are under 20  years of age. The climate amplification that occurred among monkeys, mosquitoes, and is Sudano-Guinean with a single rainy season extending from humans in the Kedougou region by 2009–2010. May to November. The landscape consists of wooded grassland MATERIALS AND METHODS or woodland and dense gallery forest. The average annual tem- perature is 28.2°C. The main economic activity in the region is Ethics Statement agriculture and livestock, but hunting and logging are a source of The protocols for human and monkey studies were approved by human contact with the forest. Currently, gold mining is expand- the National Ethics Committee of Senegal and the University of ing as one of the most important economic activities in the area. Texas Medical Branch Institutional Review Board and Animal Care and Use Committees, respectively. The protocol used for Human Surveillance animals adheres to the Senegal national guideline and approved Passive human surveillance focused on patients who attended by the Institutional Review Board of the Interstate School for local health facilities for acute febrile illness, and active sur- science and veterinary medicine. Written informed consent veillance involved the periodic seromonitoring of a prospec- for adults and children was obtained. Concerning children, tive cohort of schoolchildren living in the Kedougou region. sera were collected with the consent of the parents. An audit All patients were interviewed by experienced public health 2 • OFID • Sow et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Kedougou Region Localities of catching Monkeys Health facilities Localities of sampling Mosquitoes Figure 2. Map of Kedougou showing the sites sampled for Chikungunya surveillance during this study. Red circles indicates locations of monkey sampling, blue squares indicate health facilities, and green triangles show sites of mosquito sampling. workers. The clinical manifestations and demographic data schools were visited twice a week to identify students reaching were recorded for each patient on a standardized interview criteria for the “suspect case” definition. Students not attending form. In addition, similar data were obtained from both the during the weekly visit at school were visited in their household healthy nonsymptomatic and febrile students. to check whether they even met the “suspect case” definition. Suspect students cases were sent to the health center involved Passive Surveillance of Chikungunya in Health Facilities in the study to perform a blood sample for CHIKV virological Five healthcare facilities located in Kedougou region were diagnostic testing. selected for human surveillance, including the Ninefesha Surveillance of Chikungunya Among Primates Hospital, the Kedougou Health Center, the Bandafassi Clinic, The study focused on 3 species of monkeys in the Kedougou the Military Health Centre, and the Catholic Mission, which area that are considered potential reservoirs of arboviruses: has a mobile team that provides healthcare to indigenous popu- African green monkeys ([AGMs] Chlorocebus sabaeus), Patas lations in remote areas (Figure 2). Suspected CHIKV cases were monkeys (Erythrocebus patas [EP]), and Guinea baboons defined as patients over 1 year of age with a fever over 38.5°C (Papio papio [PP]). Monkey blood sampling was conducted and at least 2 of the following clinical symptoms: headache, during the dry season (January–May 2010) around temporary maculopapular cutaneous exanthema, eye pain, joint pain or ponds around the villages of Silling, Bafoundou, and Ngari- injury, myalgia, fatigue, vomiting, dyspnea, diarrhea, jaundice, Sekoto (Figure  2). The monkeys were trapped in large cages disorientation, and hemorrhagic manifestations. baited with peanuts and anaesthetized with ketamine. Five milliliters of blood was collected and centrifuged, and the Active Surveillance of Chikungunya Among Students’ Cohorts serum fraction was collected and stored at −20°C or −80°C Four primary schools in Kedougou Department were selected until tested for antibody as described below. The capture date, for active surveillance including the Ninefesha, Bandafassi, and location, age, sex, and weight for each captured monkey were Ngari primary schools and the Catholic Mission School with systematically recorded. some boarding residents as well as daily attendees (students from villages around Kedougou city). Serum samples were Laboratory Analysis obtained from students each year before and after the rainy sea- Malaria Test son (May and November) and tested for arboviruses of interest Human blood samples were tested by Giemsa-stained blood in the region including CHIKV antibody testing. In addition, smears and rapid malaria tests kits for plasmodia [22]. Chikungunya Outbreak in Kedougou, 2009 • OFID • 3 Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Statistical Analysis Serology Data were analyzed using R software. The χ test was used to Blood samples collected from patients, students, and primates compare the difference between 2 proportions, with a statistical were centrifuged and stored at −20°C until tested for antibody. significance level set at P < .05. Samples were tested for CHIKV by immunoglobulin (Ig)M antigen-capture enzyme-linked immunosorbant assay (ELISA). RESULTS A  differential diagnosis of CHIKV infection among other arboviruses such as yellow fever virus, dengue virus (DENV), Patient Samples West Nile virus, Rift Valley fever virus, and Crimean-Congo One thousand four hundred nine suspect cases of CHIKV were hemorrhagic fever virus, which are endemic in the study area, evaluated from 5 healthcare facilities in Kedougou between May was also performed by IgM antibody-capture ELISA. Monkey 2009 and March 2010 and tested for evidence of acute CHIKV serum samples were analyzed for specific CHIKV-neutralizing infection using virus isolation, viral genome, or IgM antibody antibodies by plaque reduction neutralization tests (PRNTs) as detection. Table 1 summarizes the distribution of findings relating described by De Madrid and Porterfield [23]. to human sera collected, 50.4% of which were negative for active malaria parasitemia. Overall, evidence for CHIKV infection was observed in 1.4% (20 of 1409)  of patients including 6 patients Molecular Detection who tested positive for CHIKV-specific IgM antibody (Table  1). Real-time polymerase chain reaction (rPCR) was used to test Among the 1409 human sera sampled, 144 were referred to acute human sera and mosquito samples for CHIKV. The mosquito stage injury (<5 days of illness) and subsequently tested for CHIKV sampling and testing protocols were extensively describes by detection or for viral genetic material evidence. Hence, CHIKV rib- Diallo et al 2012 [24]. onucleic acid (RNA) was detected by rPCR in 9.7% (14 of 144) of Sequencing of E1 Coding Region patients. Of note, the majority of CHIKV-confirmed cases based on The whole genome of CHIKV strains was initially amplified the detection of virus or viral RNA from acute sera (9 of 14; 64 %) using the Titan One Tube RT-PCR system (Roche, Mannheim, were recruited from the Kedougou healthcare center (Table 1). Germany), following the strategy reported by Volk et al [25]. Due In terms of the spatial distribution and among the 20 con- to the extremely low level of variation in initially assessed genome firmed humans cases, 90% were reported in the Kedougou sequence (data not shown), only the region that corresponded district, suggesting an incidence rate ranging from 0.55 to to the E1 envelope glycoprotein gene was subjected to further 9.38/1000 inhabitants, and 10% (2 of 20) of cases were reported sequencing and analyses [2]. Primer sequences and specific PCR in the Saraya district (Table  1), with an incidence rate at and sequencing protocols are available from the authors. 1.34/1000 inhabitants. e m Th edian age of infected patients was 24  years (7–55), Phylogenetic Analysis the sex ratio male/female was 1.  Adult individuals, especially Genome sequences representing the spatiotemporal distribu- those between 31 and 45  years old, were significantly more tion of CHIKV were downloaded from GenBank and aligned ae ff cted than others. However, there was no significant differ - using MUSCLE [26] and manually adjusted in Se-Al (available at ence between sexes (P > .05). The most common clinical symp- http://tree.bio.ed.ac.uk/software/seal/) according to amino acid toms among patients were headache (70%), myalgia (70%), and sequence homology. The E1 region was then excised and com- arthralgia (60%) followed by vomiting (30%), cough (25%), bined with the 34 newly generated CHIKV E1 sequences, leading diarrhea (10%), and cutaneous rash (5%) (Figure  3A). Among to a final data set containing 103 sequences and 1317 nucleotides. the CHIKV-infected confirmed cases, 20% (4 of 20)  were A  maximum likelihood tree was then inferred using the PAUP diagnosed with malaria coinfection and presented with all the v4.0b package [27], based on the best-fit nucleotide substitution previously described symptoms except rash. Diarrhea and vom- model determined by MODELTEST [28]. The database and operation script is available upon request from the authors. iting were more common in malaria coinfection (33% and 50%, Table 1. Enrolled Patients and CHIKV-Positive ( ) Cases in Kedougou in Five Health Facilities From July 2009 to March 2010 Health Facilities No. Sera Collected No. CHIKV ELISA IgM+ No. Acute Sera Tested No. CHIKV rPCR+ Total Positive Kedougou 319 0 47 9 9 Military Camp 658 5 40 0 5 Bandafassi 205 0 28 3 3 Ninefesha 199 1 29 2 3 Catholic Mission 28 0 0 0 0 Total 1409 6 144 14 20 Abbreviations: CHIKV, Chikungunya virus; ELISA, enzyme-linked immunosorbant assay; IgM, immunoglobulin M; rPCR, real-time polymearse chain reaction. 4 • OFID • Sow et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 AB fever myalgia headache arthralgia vomiting cough CHIKV GE+ CHIKV GE– diarrhea rash 0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100% Percentage of symptoms Percentage of symptoms − + Figure 3. Clinical features of all Chikungunya (CHIKV) cases (A) and cases with and without malaria coinfection (3B). CHIKV GE were negative for malaria, whereas GE were positive for malaria. respectively) than in the sole CHIKV infection with no evidence at weeks 2 and 3, and finally dropped to 1 case at week 4 of of active malaria (6% and 25%) (Figure 3B). However, no signifi- November 2009. No case was detected between December 2009 cant difference was observed in the frequency of signs and symp- and February 2010. In March 2010, 1 more confirmed CHIKV toms between the malaria and CHIKV-only groups (P > .50). case was detected during the relating second week (Figure 4). Analysis of temporal distribution of CHIKV-positive mos- quito pools and CHIKV-infected cases showed an overlap Entomological Findings between the 2, with a lag of approximately 1 month between the From June 2009 to January 2010, a total of 39 799 mosquitoes, first detection of CHIKV-infected mosquitoes and first detec- grouped in 4211 pools, were collected and analyzed. The most tion of a human case (Figure  4). The epidemic curve showed abundant species among the potential CHIKV vectors were intermittent peaks that began in October 2009 with the first Aedes vittatus (23.0% of the host-seeking females), A furcifer peak in week 2 (6 cases). In November 2009, the number of (18.7%), Aedes dalzieli (15.6%), and A luteocephalus (13.1%). CHIKV-infected confirmed cases peaked in the first week 1 (6 A  total of 42 CHIKV-infected pools were obtained by rPCR cases), and then declined gradually by half (3 cases per week) from September to December 2009 mainly from A furcifer (16 Infected humans Infected Mosquitoes 7 12 0 0 Weeks Figure 4. Temporal distribution of Chikungunya cases and detection of Chikungunya-infected mosquito pools from May 2009 to March 2010 in Kedougou. W, week; W1, the first week of the month. Chikungunya Outbreak in Kedougou, 2009 • OFID • 5 Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 W1 September 09 W3 September 09 W1 October 09 W3 October 09 W1 November 09 W3 November 09 W1 December 09 W3 December 09 W1 January 10 W1 February 10 W1 March 10 W3 March 10 Symtoms Number of human cases Number of mosquitoes pools pools), A taylori (5 pools), and A luteocephalus (5 pools), which immunosorbent assay (ELISA) IgM was performed on all represented 66.9% of the CHIKV-positive pools. sera collected. Table  3 shows that no evidence of recent CHIKV infection was detected in May. However, ELISA Analysis of the E1 Sequences IgM performed on sera collected from the same children in As shown in Figure  5, all of our samples from 2009, including November 2009 showed that 25 were positive for CHIKV in those from both mosquitoes and patients, were closely related Bandafassi, Ninefesha, and Catholic Mission schools with to each other. All were within the West-Africa lineage, with a infection rates of 4% (7 of 171), 8% (15 of 180), and 0.6% closest neighbor from Senegal in 2005. All of the CHIKV strains (3 of 464), respectively. The infection rates were significantly had an alanine residue at E1 position 226, consistent with enzo- different among the schools (P < .000003; Fisher’s exact test). otic strains rather than the recent strains responsible of Asian or However, in Bandafassi and Ninefesha schools, the infec- South American epidemics. tion rate was statistically similar (P = .12; Fisher’s exact test). Active Surveillance Among School Children Between November 2009 and May 2010, a total of 65 sus- A total of 866 students were investigated for CHIKV circula- pected students were followed. However, none was positive tion in May and November 2009 in 4 schools. Enzyme-linked for CHIKV test. ECSA lineage Asian lineage CHIK/Senegal/PM201813/2009 CHIK/Senegal/PM201651/2009 CHIK/Senegal/K006709/2009 CHIK/Senegal/PM202059/2009 CHIK/Senegal/PM203816/2009 CHIK/Senegal/PM201819/2009 CHIK/Senegal/PM200900/2009 CHIK/Senegal/PM201841/2009 CHIK/Senegal/PM201991/2009 CHIK/Senegal/PM201811/2009 CHIK/Senegal/PM201822/2009 CHIK/Senegal/PM203827/2009 CHIK/Senegal/PM200904/2009 CHIK/Senegal/PM202037/2009 CHIK/Senegal/PM202067/2009 CHIK/Senegal/PM203817/2009 CHIK/Senegal/PM201701/2009 CHIK/Senegal/PM201727/2009 CHIK/Senegal/PM200828/2009 CHIK/Senegal/PM200905/2009 CHIK/Senegal/PM200765/2009 CHIK/Senegal/PM200844/2009 CHIK/Senegal/PM200862/2009 West Africa lineage CHIK/Senegal/PM200916/2009 CHIK/Senegal/K006809/2009 CHIK/Senegal/Ke005609/2009 CHIK/Senegal/K007909/2009 CHIK/Senegal/B014309/2009 CHIK/Senegal/PM201881/2009 CHIK/Senegal/ArD196168/2008 CHIK/Senegal/PM201800/2009 CHIK/Senegal/K003809/2009 CHIK/Senegal/N009309/2009 CHIK/Senegal/B013909/2009 CHIK/Senegal/HD180760/2005 CHIK/Ivory Coast/ArA2657/1981 CHIK/Ivory Coast/ArA30548/1993 CHIK/Senegal/ArD93229/1993 CHIK/Senegal/37997/1983 CHIK/Nigeria/IbAn4824/1965 0.03 CHIK/Nigeria/IbH35/1964 100 CHIK/Senegal/SH3013/1966 CHIK/Senegal/ArD30237/1979 CHIK/Senegal/PM2951/1966 CHIK/Senegal/SH2830/1966 Figure 5. Maximun likelihood tree of the E1 gene of Chikungunya virus. Isolates from humans and mosquitoes obtained between May 2009 and March 2010 in Kedougou are highlighted in blue, and the most recent, previous Senegal strain is highlighted in yellow. Bootstrap value higher than 70 are labeled along the major branches. 6 • OFID • Sow et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Table  2. Spatial Distribution, Gender, and Age Distribution of CHIKV- DISCUSSION Positive ( ) Cases in Kedougou From July 2009 to March 2010 Chikungunya virus is a re-emerging mosquito-borne viral infection. In the Kedougou region, Southeastern Senegal, sev- Recruited Localities Patients CHIKV+ eral epizootics of CHIKV have been reported at intervals of approximately 5 years since 1972, but little information is avail- District Number % Population Number IR (1/1000) able on the impact of CHIKV among human populations and Bandafassi 94 6.67 972 1 1.028 Boundoucondi 16 1.13 177 1 5.64 mechanisms involved in its emergence. In the present study, we Ibel 17 1.20 1000 1 1 reported a sylvatic outbreak of CHIKV in the Kedougou region Thioketian 17 1.20 512 1 1.953 that was evidenced from patients recruited in 5 health facilities Niemeneke 1 0.07 800 1 1.25 and among a prospective cohort of living locally students. Ninefesha 34 2.41 213 2 9.38 Overall, a combination of serological IgM, rPCR testing, and Kedougou 834 59.19 19 731 11 0.55 virus isolation performed on 1409 patients enrolled revealed 20 Others 394 27.96 0 0 Subtotal 1407 99.85 23 405 18 0.769 that were infected by CHIKV in the survey area. Seventy per- Sabadola 2 0.15 1454 2 1.375 cent of CHIKV-positive patients had symptoms matching the Total 1409 100 25 859 20 0.804 case CHIKV disease definition. This result is similar to that Sex reported during CHIKV epidemics that occurred in Thailand Male 747 53 11933 10 0.838 [29] and in the Indian Ocean [30]. Overall, the clinical manifes- Female 661 47 12926 10 0.773 tations observed (algo-febrile eruptive illness, with vomiting and Age Group 0–4 years 165 11.71 4227 0 0 diarrhea) were similar to those described previously for acute 5–14 years 494 35.06 6960 6 0.862 CHIKV infection [11]. Our study also revealed coinfections of 15–29 years 427 30.30 6960 5 0.718 CHIKV with malaria in 20% of CHIKV-confirmed cases and 30–44 years 194 13.76 3729 6 1.609 supporting previous observations on CHIKV-malaria coinfec- >45 years 106 7.54 2983 3 1.005 tions reported in the Democratic Republic of the Congo and Missing 23 1.63 — — — in Senegal [31, 32]. Except for the absence of cutaneous rash, Total 1409 100.00 24859 20 0.804 the coinfected patients presented the same symptoms and signs Abbreviations: CHIKV, Chikungunya virus; IR, incidence rate. described above for CHIKV-only infection. Diarrhea and vom- iting were more frequently reported among confirmed malaria Primates Samples Analysis cases coinfected with CHIKV, but no significant difference was One hundred seventeen monkeys including 77 PP, 33 observed when comparing with patients infected with CHIKV Chlorocebus sabaeus (AGM), and 7 EP were sampled from only. These results (1) underscore the difficulties and challenges March to May 2010 in Silly, Ngari, and Bafoundou (Figure 1). in clinically differentiating these conditions in areas where No IgM antibody-positive animals were detected, but malaria is endemic [3] and (2) reinforce malaria as an “umbrella” CHIKV-neutralizing antibodies were found by PRNT in 87% infection potentially masking arboviral disorders in the context (66 of 76)  of PP, 75% (25 of 33)  of AGM, and 71% (5 of 7)  of sub-Saharan Africa. Therefore, in suspected cases of malaria, of EP; 34% of infant and juvenile primates had neutraliz- CHIKV infection should be included as an alternative diagno- ing antibodies. Chikungunya virus seroprevalences did not sis, as well as other arboviral infection options. As previously differ significantly among the 3 primates species (P  >  .22; described, fever and joint involvement were the most frequent Fisher’s exact test). manifestations, ae ff cting most of Chikungunya patients [11, 30]. Also our study showed that 60% of infected patients presented arthralgia for which 10% experienced persistent arthralgia. Table  3. Number of Student Samples Tested and Number of Students Our finding of equal prevalence of CHIKV infection between Positive ( ) by ELISA IgM Against CHIKV in May and November 2009 the genders and the age groups is also consistent with a rural outbreak of CHIKV that occurred in Cameroon [33]. In con- CHIKV+ trast to studies carried out in Nigeria in the 1960s–1970s, School Number Students May 2009 November 2009 where children from 1 to 4 years of age were significantly more Bandafassi 171 0 7 infected [34, 35], all age groups were found to be to be suscep- Ninefesha 180 0 15 tible for infection with CHIKV. Distinctly, an exception for Ngari 51 0 0 children under 4  years was noteworthy, suggesting that little Catholic Mission 464 0 3 or no domestic transmission of CHIKV occurred during this Total 866 0 25 outbreak. Indeed, children younger than 4  years of age tend Abbreviations: CHIKV, Chikungunya virus; ELISA, enzyme-linked immunosorbant assay; to remain in the household, unlike adults and schoolchildren IgM, immunoglobulin M.  Chikungunya Outbreak in Kedougou, 2009 • OFID • 7 Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 (7 years old and older) whose activities (agriculture, traditional while outside of their homes but within their villages. Such an gold extraction) and daily school attendance from remote vil- hypothesis is reinforced by the fact that no domestic transmis- lages expose them to forests and forest-living mosquitoes. sion of CHIKV occurred given the absence of infection among Our data also showed low CHIKV incidence rates (0.55– children under 4  years, who are more likely to spend time 9.38/1000), in comparison with those reported from Kaffrine within the household. Our results also suggest that the CHIKV (with an incidence rate of 35.3%), Senegal in 1996–1997 [3] and epizootic began in September 2009. Hence, sylvatic circulation on the Maldive islands (65.2/1000) [36]. This observation may must precede epidemic amplification in the emergence area be explained by the low density of the human population in [38, 39]. The CHIKV outbreak episode that emerged in March Kedougou, which is 8 people/km and probably not sufficient for 2010 raises a question about the transmission of the virus human-mosquito-human CHIKV transmission. Previous stud- during the dry season in southeastern Senegal. Based in our ies showed that human population densities of approximately data, the Anopheles genus found infected with CHIKV could 3000–7000/km are needed to produce CHIKV outbreaks [37]. be partly involved in transmission, consistent with a previous It is also likely that only severe cases attend healthcare centers study that demonstrated experimental CHIKV transmission by and clinics, and the asymptomatic forms of the disease—which Anopheles spp [40]. is believed to be a vast majority of cases—were missed by the e sy Th lvatic CHIKV cycle in Senegal is believed to be main- recruitment process used in this investigation. tained through the nonhuman primates. We found evidence for Among the cohort of schoolchildren, 25 seroconverted for past CHIKV infection in 3 monkey species collected between CHIKV IgM antibody in Bandafassi, Ninefesha, and Catholic March and May 2010. Although sampling of primates occurred Mission schools between May and November 2009, suggesting aer o ft ur studies of humans and mosquitoes (May 2009 to that CHIKV circulated during the rainy season. The infection March 2010), the detection of CHIKV-neutralizing antibodies rate was observed in students of Ninefecha and Bandafassi among infant primates strongly suggest that they were proba- schools, which are located in the forest area. In addition, the con- bly infected recently during the sylvatic amplification between firmed CHIKV cases belonging to the Catholic Mission School May and November 2009. Previous studies have shown that the (located in Kedougou city) were students who returned to their turnover of monkey populations is necessary for the emergence villages during week ends and holidays. Overall, the exposure of arboviruses [41]. of these students to CHIKV could be explained by their prox- As shown in our phylogenetic tree (Figure  5), the CHIKV imity to the forest environment, which could facilitate con- strains isolated from patients in Kedougou in 2009 clustered tact with CHIKV-infected mosquito vectors. This explanation in the West African genotype and were closely related to pre- is further strengthened by the level of difference for infection vious isolates from Senegal, suggesting continuous circulation rates between the student cohort (2.88%) and the framework of of CHIKV in rural or sporadic local transmission of the virus. the local healthcare facilities (1.42%). Furthermore, up to 36% e i Th solates sequenced exhibited an alanine at amino acid resi- of the CHIKV patients found in the Kedougou area were evi- due 226 of the E1 envelope glycoprotein. Previous studies have denced with a history of recent travel to forested areas. demonstrated that a substitution of valine (A226V) provides Chikungunya virus isolates obtained from humans and mos- a selective advantage for the replication and transmission of quitoes [24] were found between October and December 2009, CHIKV by A albopictus [42, 43]. However, no A albopictus was confirming previous studies showing that in the Kedougou area detected so far in the study area, in Senegal and in turn in West arboviruses are mostly isolated from October to December, Africa. Our phylogenetic data supported by the sylvatic ampli- which corresponds to the end of the rainy season [18, 19]. fication of CHIKV described by Diallo et al [24], are similar to Chikungunya virus was mostly isolated from A furcifer, which previously studies based on DENV, which suggest that sylvatic is probably the primary vector of this outbreak. In a companion cycle is a natural source of emergence [20, 44]. study [24], we showed that A furcifer occurred in all 5 major CONCLUSIONS land cover classes in the Kedougou region (forest, village, agri- culture, savannah, and barren). On this behalf, there were no In conclusion, the present study highlights the importance of significant differences in the abundance of this species among surveillance including clinical observations, laboratory diag- these land cover classes, and the A furcifer species was rarely nosis, and cohort serosurveillance to detect outbreaks caused found inside households in villages and was distinctly more by CHIKV and other arboviruses in the Kedougou region. The abundant on the periphery than inside the villages. In addition, combination of serological and molecular tests identified an CHIKV was also detected at equal rates in all 5 lands cover epidemic of CHIKV in Kedougou after an amplification from classes. Given that, humans are rarely staying in forests during the sylvatic cycle that would otherwise have been overlooked. the moment when sylvatic CHIKV vectors are the more active. Our results support the hypothesis that sylvatic enzootic circu- Because A furcifer shows high biting rates in villages [24], we lation can be a source of emergence of CHIKV into domestic or suggest that humans are likely exposed to sylvatic CHIKV urban transmission cycles and underscore the value of effective, 8 • OFID • Sow et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 21. CRORA. Centre Collaborateur OMS pour la Recherche sur les Arbovirus (CRORA), real-time surveillance to improve the detection and prevention Base de données. 07/04/2015]; Available at: http://www.pasteur.fr/recherche/ of arboviral diseases. banques/CRORA 22. Stauffer WM, Cartwright CP, Olson DA, et al. Diagnostic performance of rapid Acknowledgments diagnostic tests versus blood smears for malaria in US clinical practice. Clin Infect Dis 2009; 49:908–13. We thank the population, healthcare workers, and professional of health 23. De Madrid AT, Porterfield JS. A simple micro-culture method for the study of facilities in Kedougou for their support and cooperation in conducting group B arboviruses. Bull World Health Organ 1969; 40:113–21. this study. 24. Diallo D, Sall AA, Buenemann M, et  al. Landscape ecology of sylvatic Disclaimer. e f Th unders had no role in study design, data collection Chikungunya virus and mosquito vectors in southeastern Senegal. PLoS Negl and analysis, decision to publish, or preparation of the manuscript. Trop Dis 2012; 6:e1649. Financial support. e r Th esearch was funded by the National Institutes 25. Volk SM, Chen R, Tsetsarkin KA, et  al. Genome-scale phylogenetic analyses of of Health (Grant Number 5R01A 1069145).  Chikungunya virus reveal independent emergences of recent epidemics and vari- Potential conifl cts of interest. All authors: No reported conflicts of ous evolutionary rates. J Virol 2010; 84:6497–504. 26. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high interest. All authors have submitted the ICMJE Form for Disclosure of throughput. Nucleic Acids Res 2004; 32:1792–7. Potential Conflicts of Interest. Conflicts that the editors consider relevant to 27. Swofford DL. PAUP: phylogenetic analysis using parsimony (and other meth- the content of the manuscript have been disclosed. ods), version 4. Sunderland (Massachusetts): Sinauer; J Virol. 2010; 84(13): 6497–504. Available at: https://www.researchgate.net/publication/271205405_ References PAUP_Phy logenet ic_Ana lysis_Using_Parsimony_and_O t her_Met ho ds_ 1. Ross RW. The Newala epidemic. III. The virus: isolation, pathogenic properties Version_40b10. Accessed 12 December 2017. and relationship to the epidemic. J Hyg (Lond) 1956; 54:177–91. 28. Posada D, Crandall KA. MODELTEST: testing the model of DNA substitution. 2. Powers AM, Brault AC, Shirako Y, et al. 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Open Forum Infectious Diseases MAJOR ARTICLE Chikungunya Outbreak in Kedougou, Southeastern Senegal in 2009–2010 1,10,11 1 2 2 3 1 2 Abdourahmane Sow, Oumar Faye, Mawlouth Diallo, Diawo Diallo, Rubing Chen, Ousmane Faye, Cheikh T. Diagne, 3 4 5 6 7 7 Mathilde Guerbois, Manfred Weidmann, Youssoupha Ndiaye, Cheikh Sadibou Senghor, Abdourahmane Faye, Ousmane M. Diop, 1 1 8 9 10 11,a 3,a 1 Bakary Sadio, Oumar Ndiaye, Douglas Watts, Kathryn A. Hanley, Anta T. Dia, Denis Malvy, Scott C. Weaver, and Amadou Alpha Sall 1 2 3 Institut Pasteur Dakar, Arbovirus and Viral Hemorrhagic Fevers Unit, Senegal; Institut Pasteur Dakar, Medical Entomology Unit, Senegal; Institute for Human Infections and Immunity, Center 4 5 for Tropical Diseases and Department of Pathology, University of Texas Medical Branch, Galveston; Department of Virology, University Medical Center Göttingen, Germany; District Sanitaire 6 7 8 9 de Saraya, Senegal; District Sanitaire de Kedougou, Senegal; Institut Pasteur Dakar, Medical Virology Unit, Senegal; University of Texas at El Paso; Department of Biology, New Mexico State 10 11 University; Institut Santé et Développement, Université Cheikh Anta Diop, Dakar, Senegal; INSERM 1219, University of Bordeaux, France Background. In Senegal, Chikungunya virus (CHIKV), which is an emerging mosquito-borne alphavirus, circulates in a syl- vatic and urban/domestic cycle and has caused sporadic human cases and epidemics since 1960s. However, the real impact of the CHIKV sylvatic cycle in humans and mechanisms underlying its emergence still remains unknown. Methodology. One thousand four hundred nine suspect cases of CHIKV infection, recruited from 5 health facilities located in Kedougou region, south-eastern Senegal, between May 2009 to March 2010, together with 866 serum samples collected from school- children from 4 elementary schools in May and November 2009 from Kedougou were screened for anti-CHIKV immunoglobulin (Ig)M antibodies and, when appropriate, for viral nucleic acid by real-time polymerase chain reaction (rPCR) and virus isolation. In addition, mosquitoes collected in the same area from May 2009 to January 2010 were tested for CHIKV by rPCR and by virus isola- tion, and 116 monkeys sera collected from March 2010 to May 2010 were tested for anti-CHIKV IgM and neutralizing antibodies. Results. e m Th ain clinical manifestations of the CHIKV suspect cases were headache, myalgia, and arthralgia. Evidence for CHIKV infection was observed in 1.4% (20 of 1409) of patients among suspect cases. No significant difference was observed among age or sex groups. In addition, 25 (2.9%) students had evidence of CHIKV infection in November 2009. Chikungunya virus was detected in 42 pools of mosquitoes, mainly from Aedes furcifer, and 83% of monkeys sampled were seropositive. Conclusions. Our findings further documented that CHIKV is maintained in a sylvatic transmission cycle among monkeys and Aedes mosquitoes in Kedougou, and humans become infected by exposure to the virus in the forest. Keywords. Chikungunya virus; Kedougou; outbreak; Senegal; sylvatic circulation. Chikungunya virus (CHIKV) is a member of the Semliki [5]. Several studies have reported CHIKV circulation in West Forest virus antigenic group of the genus Alphavirus (family Africa, especially in Nigeria between 1963 and 1977 [6, 7]. Togaviridae) and was first isolated in 1953 in Tanzania [1, 2]. Major CHIKV epidemics were recently reported in Africa and This arthtrogenic virus is composed of 2 lineages: African and Asia [8]. A significant outbreak occurred in Italy in 2007, and Asiatic. When symptomatic, cute CHIKV infection in human numerous imported cases have been detected elsewhere in can cause a flu-like syndrome with rash, but joint pains are the Europe and the United States of America [9–13], emphasizing dominant complaint and might evolve to persistent and inca- CHIKV as a re-emerging public health threat worldwide. pacitating manifestations. In Africa, CHIKV is maintained in In Senegal, CHIKV was first isolated in 1962 from bats nature among nonhuman primates and forest-dwelling Aedes [14, 15], and CHIKV outbreaks and sporadic cases were sub- sp. In rural areas, these sylvatic vectors can be responsible for sequently reported in 1966, 1982, 1996, 1997, and 2004–2006 sporadic cases or small outbreaks [3, 4]. In urban areas, CHIKV [4, 16, 17] (Figure 1). In addition, CHIKV has been repeatedly is transmitted to humans by Aedes aegypti and Aedes albopictus isolated from wild caught Aedes furcifer, Aedes luteocephalus, and Aedes taylori in a sylvatic focus near Kedougou in south- eastern Senegal [5, 18–20] as part of an entomological surveil- Received 14 July 2017; editorial decision 18 November 2017; accepted 27 November 2017. lance programme that began in 1972 [21]. Various vertebrates, D. M. and S. C. W. contributed equally to this work. Correspondence: A.  Sow, MD, MPH, Professional Officer/Epidemics Control-Public Health including monkeys, bats, gophers, and galagos, have been Laboratories, West African Health Organisation (WAHO), 175 Avenue Ouezzin Coulibaly, BP proven for implication as hosts of CHIKV by serological evi- 153, Bobo-Dioulasso, Burkina Faso (asow20@gmail.com). dence or viral isolation [5]. Amplifications of CHIKV in the Open Forum Infectious Diseases © The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases Kedougou area have occurred at approximately 5-year intervals, Society of America. This is an Open Access article distributed under the terms of the Creative which is hypothesized to be the time necessary for the turno- Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any ver of susceptible vertebrate hosts [5]. Despite active circulation medium, provided the original work is not altered or transformed in any way, and that the work of CHIKV in the sylvatic cycle in Senegal, limited informa- is properly cited. For commercial re-use, please contact journals.permissions@oup.com tion is available on its transmission on human and wildlife DOI: 10.1093/ofid/ofx259 Chikungunya Outbreak in Kedougou, 2009 • OFID • 1 Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 50 km MAURITANIA 30 mi St-Louis Rao 1963 Louga 2006 Touba 1981 Thies 1982, 2006 Bambey 2006 SENEGAL Rufisque 1966 Diourbel 2006 Dakar Bandia 1966 Niakhar 1997 Kaolack 2005 Karine 1996 MALI Tambacounda GAMBIA Ziguinchor Kedougou 2004 GUINEA-BISSAU GUINEA Figure  1. Map of Senegal showing locations where Chikungunya outbreaks have been reported. Red circle indicates the localities and the years of Chikungunya virus outbreaks. populations. To address this question, we conducted passive of the protocol implementation was regularly conducted by the surveillance for CHIKV infection among patients attending 5 authorities of the National Ethics Committee of Senegal. health clinics with febrile illness, together with active surveil- Study Areas lance of students recruited from 4 schools in Kedougou. In a The study was conducted in the Kedougou region of southeast- parallel effort, we also investigated monkey serosampling in ern Senegal (12°32’N, 12°11’W) (Figure 2). The human popula- the region and surveyed mosquitoes in the area for CHIKV. In tion is 133 487 inhabitants with an average density of 8 persons this study, we report the investigation of the CHIKV zoonotic per km , 55% of whom are under 20  years of age. The climate amplification that occurred among monkeys, mosquitoes, and is Sudano-Guinean with a single rainy season extending from humans in the Kedougou region by 2009–2010. May to November. The landscape consists of wooded grassland MATERIALS AND METHODS or woodland and dense gallery forest. The average annual tem- perature is 28.2°C. The main economic activity in the region is Ethics Statement agriculture and livestock, but hunting and logging are a source of The protocols for human and monkey studies were approved by human contact with the forest. Currently, gold mining is expand- the National Ethics Committee of Senegal and the University of ing as one of the most important economic activities in the area. Texas Medical Branch Institutional Review Board and Animal Care and Use Committees, respectively. The protocol used for Human Surveillance animals adheres to the Senegal national guideline and approved Passive human surveillance focused on patients who attended by the Institutional Review Board of the Interstate School for local health facilities for acute febrile illness, and active sur- science and veterinary medicine. Written informed consent veillance involved the periodic seromonitoring of a prospec- for adults and children was obtained. Concerning children, tive cohort of schoolchildren living in the Kedougou region. sera were collected with the consent of the parents. An audit All patients were interviewed by experienced public health 2 • OFID • Sow et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Kedougou Region Localities of catching Monkeys Health facilities Localities of sampling Mosquitoes Figure 2. Map of Kedougou showing the sites sampled for Chikungunya surveillance during this study. Red circles indicates locations of monkey sampling, blue squares indicate health facilities, and green triangles show sites of mosquito sampling. workers. The clinical manifestations and demographic data schools were visited twice a week to identify students reaching were recorded for each patient on a standardized interview criteria for the “suspect case” definition. Students not attending form. In addition, similar data were obtained from both the during the weekly visit at school were visited in their household healthy nonsymptomatic and febrile students. to check whether they even met the “suspect case” definition. Suspect students cases were sent to the health center involved Passive Surveillance of Chikungunya in Health Facilities in the study to perform a blood sample for CHIKV virological Five healthcare facilities located in Kedougou region were diagnostic testing. selected for human surveillance, including the Ninefesha Surveillance of Chikungunya Among Primates Hospital, the Kedougou Health Center, the Bandafassi Clinic, The study focused on 3 species of monkeys in the Kedougou the Military Health Centre, and the Catholic Mission, which area that are considered potential reservoirs of arboviruses: has a mobile team that provides healthcare to indigenous popu- African green monkeys ([AGMs] Chlorocebus sabaeus), Patas lations in remote areas (Figure 2). Suspected CHIKV cases were monkeys (Erythrocebus patas [EP]), and Guinea baboons defined as patients over 1 year of age with a fever over 38.5°C (Papio papio [PP]). Monkey blood sampling was conducted and at least 2 of the following clinical symptoms: headache, during the dry season (January–May 2010) around temporary maculopapular cutaneous exanthema, eye pain, joint pain or ponds around the villages of Silling, Bafoundou, and Ngari- injury, myalgia, fatigue, vomiting, dyspnea, diarrhea, jaundice, Sekoto (Figure  2). The monkeys were trapped in large cages disorientation, and hemorrhagic manifestations. baited with peanuts and anaesthetized with ketamine. Five milliliters of blood was collected and centrifuged, and the Active Surveillance of Chikungunya Among Students’ Cohorts serum fraction was collected and stored at −20°C or −80°C Four primary schools in Kedougou Department were selected until tested for antibody as described below. The capture date, for active surveillance including the Ninefesha, Bandafassi, and location, age, sex, and weight for each captured monkey were Ngari primary schools and the Catholic Mission School with systematically recorded. some boarding residents as well as daily attendees (students from villages around Kedougou city). Serum samples were Laboratory Analysis obtained from students each year before and after the rainy sea- Malaria Test son (May and November) and tested for arboviruses of interest Human blood samples were tested by Giemsa-stained blood in the region including CHIKV antibody testing. In addition, smears and rapid malaria tests kits for plasmodia [22]. Chikungunya Outbreak in Kedougou, 2009 • OFID • 3 Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Statistical Analysis Serology Data were analyzed using R software. The χ test was used to Blood samples collected from patients, students, and primates compare the difference between 2 proportions, with a statistical were centrifuged and stored at −20°C until tested for antibody. significance level set at P < .05. Samples were tested for CHIKV by immunoglobulin (Ig)M antigen-capture enzyme-linked immunosorbant assay (ELISA). RESULTS A  differential diagnosis of CHIKV infection among other arboviruses such as yellow fever virus, dengue virus (DENV), Patient Samples West Nile virus, Rift Valley fever virus, and Crimean-Congo One thousand four hundred nine suspect cases of CHIKV were hemorrhagic fever virus, which are endemic in the study area, evaluated from 5 healthcare facilities in Kedougou between May was also performed by IgM antibody-capture ELISA. Monkey 2009 and March 2010 and tested for evidence of acute CHIKV serum samples were analyzed for specific CHIKV-neutralizing infection using virus isolation, viral genome, or IgM antibody antibodies by plaque reduction neutralization tests (PRNTs) as detection. Table 1 summarizes the distribution of findings relating described by De Madrid and Porterfield [23]. to human sera collected, 50.4% of which were negative for active malaria parasitemia. Overall, evidence for CHIKV infection was observed in 1.4% (20 of 1409)  of patients including 6 patients Molecular Detection who tested positive for CHIKV-specific IgM antibody (Table  1). Real-time polymerase chain reaction (rPCR) was used to test Among the 1409 human sera sampled, 144 were referred to acute human sera and mosquito samples for CHIKV. The mosquito stage injury (<5 days of illness) and subsequently tested for CHIKV sampling and testing protocols were extensively describes by detection or for viral genetic material evidence. Hence, CHIKV rib- Diallo et al 2012 [24]. onucleic acid (RNA) was detected by rPCR in 9.7% (14 of 144) of Sequencing of E1 Coding Region patients. Of note, the majority of CHIKV-confirmed cases based on The whole genome of CHIKV strains was initially amplified the detection of virus or viral RNA from acute sera (9 of 14; 64 %) using the Titan One Tube RT-PCR system (Roche, Mannheim, were recruited from the Kedougou healthcare center (Table 1). Germany), following the strategy reported by Volk et al [25]. Due In terms of the spatial distribution and among the 20 con- to the extremely low level of variation in initially assessed genome firmed humans cases, 90% were reported in the Kedougou sequence (data not shown), only the region that corresponded district, suggesting an incidence rate ranging from 0.55 to to the E1 envelope glycoprotein gene was subjected to further 9.38/1000 inhabitants, and 10% (2 of 20) of cases were reported sequencing and analyses [2]. Primer sequences and specific PCR in the Saraya district (Table  1), with an incidence rate at and sequencing protocols are available from the authors. 1.34/1000 inhabitants. e m Th edian age of infected patients was 24  years (7–55), Phylogenetic Analysis the sex ratio male/female was 1.  Adult individuals, especially Genome sequences representing the spatiotemporal distribu- those between 31 and 45  years old, were significantly more tion of CHIKV were downloaded from GenBank and aligned ae ff cted than others. However, there was no significant differ - using MUSCLE [26] and manually adjusted in Se-Al (available at ence between sexes (P > .05). The most common clinical symp- http://tree.bio.ed.ac.uk/software/seal/) according to amino acid toms among patients were headache (70%), myalgia (70%), and sequence homology. The E1 region was then excised and com- arthralgia (60%) followed by vomiting (30%), cough (25%), bined with the 34 newly generated CHIKV E1 sequences, leading diarrhea (10%), and cutaneous rash (5%) (Figure  3A). Among to a final data set containing 103 sequences and 1317 nucleotides. the CHIKV-infected confirmed cases, 20% (4 of 20)  were A  maximum likelihood tree was then inferred using the PAUP diagnosed with malaria coinfection and presented with all the v4.0b package [27], based on the best-fit nucleotide substitution previously described symptoms except rash. Diarrhea and vom- model determined by MODELTEST [28]. The database and operation script is available upon request from the authors. iting were more common in malaria coinfection (33% and 50%, Table 1. Enrolled Patients and CHIKV-Positive ( ) Cases in Kedougou in Five Health Facilities From July 2009 to March 2010 Health Facilities No. Sera Collected No. CHIKV ELISA IgM+ No. Acute Sera Tested No. CHIKV rPCR+ Total Positive Kedougou 319 0 47 9 9 Military Camp 658 5 40 0 5 Bandafassi 205 0 28 3 3 Ninefesha 199 1 29 2 3 Catholic Mission 28 0 0 0 0 Total 1409 6 144 14 20 Abbreviations: CHIKV, Chikungunya virus; ELISA, enzyme-linked immunosorbant assay; IgM, immunoglobulin M; rPCR, real-time polymearse chain reaction. 4 • OFID • Sow et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 AB fever myalgia headache arthralgia vomiting cough CHIKV GE+ CHIKV GE– diarrhea rash 0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100% Percentage of symptoms Percentage of symptoms − + Figure 3. Clinical features of all Chikungunya (CHIKV) cases (A) and cases with and without malaria coinfection (3B). CHIKV GE were negative for malaria, whereas GE were positive for malaria. respectively) than in the sole CHIKV infection with no evidence at weeks 2 and 3, and finally dropped to 1 case at week 4 of of active malaria (6% and 25%) (Figure 3B). However, no signifi- November 2009. No case was detected between December 2009 cant difference was observed in the frequency of signs and symp- and February 2010. In March 2010, 1 more confirmed CHIKV toms between the malaria and CHIKV-only groups (P > .50). case was detected during the relating second week (Figure 4). Analysis of temporal distribution of CHIKV-positive mos- quito pools and CHIKV-infected cases showed an overlap Entomological Findings between the 2, with a lag of approximately 1 month between the From June 2009 to January 2010, a total of 39 799 mosquitoes, first detection of CHIKV-infected mosquitoes and first detec- grouped in 4211 pools, were collected and analyzed. The most tion of a human case (Figure  4). The epidemic curve showed abundant species among the potential CHIKV vectors were intermittent peaks that began in October 2009 with the first Aedes vittatus (23.0% of the host-seeking females), A furcifer peak in week 2 (6 cases). In November 2009, the number of (18.7%), Aedes dalzieli (15.6%), and A luteocephalus (13.1%). CHIKV-infected confirmed cases peaked in the first week 1 (6 A  total of 42 CHIKV-infected pools were obtained by rPCR cases), and then declined gradually by half (3 cases per week) from September to December 2009 mainly from A furcifer (16 Infected humans Infected Mosquitoes 7 12 0 0 Weeks Figure 4. Temporal distribution of Chikungunya cases and detection of Chikungunya-infected mosquito pools from May 2009 to March 2010 in Kedougou. W, week; W1, the first week of the month. Chikungunya Outbreak in Kedougou, 2009 • OFID • 5 Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 W1 September 09 W3 September 09 W1 October 09 W3 October 09 W1 November 09 W3 November 09 W1 December 09 W3 December 09 W1 January 10 W1 February 10 W1 March 10 W3 March 10 Symtoms Number of human cases Number of mosquitoes pools pools), A taylori (5 pools), and A luteocephalus (5 pools), which immunosorbent assay (ELISA) IgM was performed on all represented 66.9% of the CHIKV-positive pools. sera collected. Table  3 shows that no evidence of recent CHIKV infection was detected in May. However, ELISA Analysis of the E1 Sequences IgM performed on sera collected from the same children in As shown in Figure  5, all of our samples from 2009, including November 2009 showed that 25 were positive for CHIKV in those from both mosquitoes and patients, were closely related Bandafassi, Ninefesha, and Catholic Mission schools with to each other. All were within the West-Africa lineage, with a infection rates of 4% (7 of 171), 8% (15 of 180), and 0.6% closest neighbor from Senegal in 2005. All of the CHIKV strains (3 of 464), respectively. The infection rates were significantly had an alanine residue at E1 position 226, consistent with enzo- different among the schools (P < .000003; Fisher’s exact test). otic strains rather than the recent strains responsible of Asian or However, in Bandafassi and Ninefesha schools, the infec- South American epidemics. tion rate was statistically similar (P = .12; Fisher’s exact test). Active Surveillance Among School Children Between November 2009 and May 2010, a total of 65 sus- A total of 866 students were investigated for CHIKV circula- pected students were followed. However, none was positive tion in May and November 2009 in 4 schools. Enzyme-linked for CHIKV test. ECSA lineage Asian lineage CHIK/Senegal/PM201813/2009 CHIK/Senegal/PM201651/2009 CHIK/Senegal/K006709/2009 CHIK/Senegal/PM202059/2009 CHIK/Senegal/PM203816/2009 CHIK/Senegal/PM201819/2009 CHIK/Senegal/PM200900/2009 CHIK/Senegal/PM201841/2009 CHIK/Senegal/PM201991/2009 CHIK/Senegal/PM201811/2009 CHIK/Senegal/PM201822/2009 CHIK/Senegal/PM203827/2009 CHIK/Senegal/PM200904/2009 CHIK/Senegal/PM202037/2009 CHIK/Senegal/PM202067/2009 CHIK/Senegal/PM203817/2009 CHIK/Senegal/PM201701/2009 CHIK/Senegal/PM201727/2009 CHIK/Senegal/PM200828/2009 CHIK/Senegal/PM200905/2009 CHIK/Senegal/PM200765/2009 CHIK/Senegal/PM200844/2009 CHIK/Senegal/PM200862/2009 West Africa lineage CHIK/Senegal/PM200916/2009 CHIK/Senegal/K006809/2009 CHIK/Senegal/Ke005609/2009 CHIK/Senegal/K007909/2009 CHIK/Senegal/B014309/2009 CHIK/Senegal/PM201881/2009 CHIK/Senegal/ArD196168/2008 CHIK/Senegal/PM201800/2009 CHIK/Senegal/K003809/2009 CHIK/Senegal/N009309/2009 CHIK/Senegal/B013909/2009 CHIK/Senegal/HD180760/2005 CHIK/Ivory Coast/ArA2657/1981 CHIK/Ivory Coast/ArA30548/1993 CHIK/Senegal/ArD93229/1993 CHIK/Senegal/37997/1983 CHIK/Nigeria/IbAn4824/1965 0.03 CHIK/Nigeria/IbH35/1964 100 CHIK/Senegal/SH3013/1966 CHIK/Senegal/ArD30237/1979 CHIK/Senegal/PM2951/1966 CHIK/Senegal/SH2830/1966 Figure 5. Maximun likelihood tree of the E1 gene of Chikungunya virus. Isolates from humans and mosquitoes obtained between May 2009 and March 2010 in Kedougou are highlighted in blue, and the most recent, previous Senegal strain is highlighted in yellow. Bootstrap value higher than 70 are labeled along the major branches. 6 • OFID • Sow et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Table  2. Spatial Distribution, Gender, and Age Distribution of CHIKV- DISCUSSION Positive ( ) Cases in Kedougou From July 2009 to March 2010 Chikungunya virus is a re-emerging mosquito-borne viral infection. In the Kedougou region, Southeastern Senegal, sev- Recruited Localities Patients CHIKV+ eral epizootics of CHIKV have been reported at intervals of approximately 5 years since 1972, but little information is avail- District Number % Population Number IR (1/1000) able on the impact of CHIKV among human populations and Bandafassi 94 6.67 972 1 1.028 Boundoucondi 16 1.13 177 1 5.64 mechanisms involved in its emergence. In the present study, we Ibel 17 1.20 1000 1 1 reported a sylvatic outbreak of CHIKV in the Kedougou region Thioketian 17 1.20 512 1 1.953 that was evidenced from patients recruited in 5 health facilities Niemeneke 1 0.07 800 1 1.25 and among a prospective cohort of living locally students. Ninefesha 34 2.41 213 2 9.38 Overall, a combination of serological IgM, rPCR testing, and Kedougou 834 59.19 19 731 11 0.55 virus isolation performed on 1409 patients enrolled revealed 20 Others 394 27.96 0 0 Subtotal 1407 99.85 23 405 18 0.769 that were infected by CHIKV in the survey area. Seventy per- Sabadola 2 0.15 1454 2 1.375 cent of CHIKV-positive patients had symptoms matching the Total 1409 100 25 859 20 0.804 case CHIKV disease definition. This result is similar to that Sex reported during CHIKV epidemics that occurred in Thailand Male 747 53 11933 10 0.838 [29] and in the Indian Ocean [30]. Overall, the clinical manifes- Female 661 47 12926 10 0.773 tations observed (algo-febrile eruptive illness, with vomiting and Age Group 0–4 years 165 11.71 4227 0 0 diarrhea) were similar to those described previously for acute 5–14 years 494 35.06 6960 6 0.862 CHIKV infection [11]. Our study also revealed coinfections of 15–29 years 427 30.30 6960 5 0.718 CHIKV with malaria in 20% of CHIKV-confirmed cases and 30–44 years 194 13.76 3729 6 1.609 supporting previous observations on CHIKV-malaria coinfec- >45 years 106 7.54 2983 3 1.005 tions reported in the Democratic Republic of the Congo and Missing 23 1.63 — — — in Senegal [31, 32]. Except for the absence of cutaneous rash, Total 1409 100.00 24859 20 0.804 the coinfected patients presented the same symptoms and signs Abbreviations: CHIKV, Chikungunya virus; IR, incidence rate. described above for CHIKV-only infection. Diarrhea and vom- iting were more frequently reported among confirmed malaria Primates Samples Analysis cases coinfected with CHIKV, but no significant difference was One hundred seventeen monkeys including 77 PP, 33 observed when comparing with patients infected with CHIKV Chlorocebus sabaeus (AGM), and 7 EP were sampled from only. These results (1) underscore the difficulties and challenges March to May 2010 in Silly, Ngari, and Bafoundou (Figure 1). in clinically differentiating these conditions in areas where No IgM antibody-positive animals were detected, but malaria is endemic [3] and (2) reinforce malaria as an “umbrella” CHIKV-neutralizing antibodies were found by PRNT in 87% infection potentially masking arboviral disorders in the context (66 of 76)  of PP, 75% (25 of 33)  of AGM, and 71% (5 of 7)  of sub-Saharan Africa. Therefore, in suspected cases of malaria, of EP; 34% of infant and juvenile primates had neutraliz- CHIKV infection should be included as an alternative diagno- ing antibodies. Chikungunya virus seroprevalences did not sis, as well as other arboviral infection options. As previously differ significantly among the 3 primates species (P  >  .22; described, fever and joint involvement were the most frequent Fisher’s exact test). manifestations, ae ff cting most of Chikungunya patients [11, 30]. Also our study showed that 60% of infected patients presented arthralgia for which 10% experienced persistent arthralgia. Table  3. Number of Student Samples Tested and Number of Students Our finding of equal prevalence of CHIKV infection between Positive ( ) by ELISA IgM Against CHIKV in May and November 2009 the genders and the age groups is also consistent with a rural outbreak of CHIKV that occurred in Cameroon [33]. In con- CHIKV+ trast to studies carried out in Nigeria in the 1960s–1970s, School Number Students May 2009 November 2009 where children from 1 to 4 years of age were significantly more Bandafassi 171 0 7 infected [34, 35], all age groups were found to be to be suscep- Ninefesha 180 0 15 tible for infection with CHIKV. Distinctly, an exception for Ngari 51 0 0 children under 4  years was noteworthy, suggesting that little Catholic Mission 464 0 3 or no domestic transmission of CHIKV occurred during this Total 866 0 25 outbreak. Indeed, children younger than 4  years of age tend Abbreviations: CHIKV, Chikungunya virus; ELISA, enzyme-linked immunosorbant assay; to remain in the household, unlike adults and schoolchildren IgM, immunoglobulin M.  Chikungunya Outbreak in Kedougou, 2009 • OFID • 7 Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 (7 years old and older) whose activities (agriculture, traditional while outside of their homes but within their villages. Such an gold extraction) and daily school attendance from remote vil- hypothesis is reinforced by the fact that no domestic transmis- lages expose them to forests and forest-living mosquitoes. sion of CHIKV occurred given the absence of infection among Our data also showed low CHIKV incidence rates (0.55– children under 4  years, who are more likely to spend time 9.38/1000), in comparison with those reported from Kaffrine within the household. Our results also suggest that the CHIKV (with an incidence rate of 35.3%), Senegal in 1996–1997 [3] and epizootic began in September 2009. Hence, sylvatic circulation on the Maldive islands (65.2/1000) [36]. This observation may must precede epidemic amplification in the emergence area be explained by the low density of the human population in [38, 39]. The CHIKV outbreak episode that emerged in March Kedougou, which is 8 people/km and probably not sufficient for 2010 raises a question about the transmission of the virus human-mosquito-human CHIKV transmission. Previous stud- during the dry season in southeastern Senegal. Based in our ies showed that human population densities of approximately data, the Anopheles genus found infected with CHIKV could 3000–7000/km are needed to produce CHIKV outbreaks [37]. be partly involved in transmission, consistent with a previous It is also likely that only severe cases attend healthcare centers study that demonstrated experimental CHIKV transmission by and clinics, and the asymptomatic forms of the disease—which Anopheles spp [40]. is believed to be a vast majority of cases—were missed by the e sy Th lvatic CHIKV cycle in Senegal is believed to be main- recruitment process used in this investigation. tained through the nonhuman primates. We found evidence for Among the cohort of schoolchildren, 25 seroconverted for past CHIKV infection in 3 monkey species collected between CHIKV IgM antibody in Bandafassi, Ninefesha, and Catholic March and May 2010. Although sampling of primates occurred Mission schools between May and November 2009, suggesting aer o ft ur studies of humans and mosquitoes (May 2009 to that CHIKV circulated during the rainy season. The infection March 2010), the detection of CHIKV-neutralizing antibodies rate was observed in students of Ninefecha and Bandafassi among infant primates strongly suggest that they were proba- schools, which are located in the forest area. In addition, the con- bly infected recently during the sylvatic amplification between firmed CHIKV cases belonging to the Catholic Mission School May and November 2009. Previous studies have shown that the (located in Kedougou city) were students who returned to their turnover of monkey populations is necessary for the emergence villages during week ends and holidays. Overall, the exposure of arboviruses [41]. of these students to CHIKV could be explained by their prox- As shown in our phylogenetic tree (Figure  5), the CHIKV imity to the forest environment, which could facilitate con- strains isolated from patients in Kedougou in 2009 clustered tact with CHIKV-infected mosquito vectors. This explanation in the West African genotype and were closely related to pre- is further strengthened by the level of difference for infection vious isolates from Senegal, suggesting continuous circulation rates between the student cohort (2.88%) and the framework of of CHIKV in rural or sporadic local transmission of the virus. the local healthcare facilities (1.42%). Furthermore, up to 36% e i Th solates sequenced exhibited an alanine at amino acid resi- of the CHIKV patients found in the Kedougou area were evi- due 226 of the E1 envelope glycoprotein. Previous studies have denced with a history of recent travel to forested areas. demonstrated that a substitution of valine (A226V) provides Chikungunya virus isolates obtained from humans and mos- a selective advantage for the replication and transmission of quitoes [24] were found between October and December 2009, CHIKV by A albopictus [42, 43]. However, no A albopictus was confirming previous studies showing that in the Kedougou area detected so far in the study area, in Senegal and in turn in West arboviruses are mostly isolated from October to December, Africa. Our phylogenetic data supported by the sylvatic ampli- which corresponds to the end of the rainy season [18, 19]. fication of CHIKV described by Diallo et al [24], are similar to Chikungunya virus was mostly isolated from A furcifer, which previously studies based on DENV, which suggest that sylvatic is probably the primary vector of this outbreak. In a companion cycle is a natural source of emergence [20, 44]. study [24], we showed that A furcifer occurred in all 5 major CONCLUSIONS land cover classes in the Kedougou region (forest, village, agri- culture, savannah, and barren). On this behalf, there were no In conclusion, the present study highlights the importance of significant differences in the abundance of this species among surveillance including clinical observations, laboratory diag- these land cover classes, and the A furcifer species was rarely nosis, and cohort serosurveillance to detect outbreaks caused found inside households in villages and was distinctly more by CHIKV and other arboviruses in the Kedougou region. The abundant on the periphery than inside the villages. In addition, combination of serological and molecular tests identified an CHIKV was also detected at equal rates in all 5 lands cover epidemic of CHIKV in Kedougou after an amplification from classes. Given that, humans are rarely staying in forests during the sylvatic cycle that would otherwise have been overlooked. the moment when sylvatic CHIKV vectors are the more active. Our results support the hypothesis that sylvatic enzootic circu- Because A furcifer shows high biting rates in villages [24], we lation can be a source of emergence of CHIKV into domestic or suggest that humans are likely exposed to sylvatic CHIKV urban transmission cycles and underscore the value of effective, 8 • OFID • Sow et al Downloaded from https://academic.oup.com/ofid/article-abstract/5/1/ofx259/4683371 by Ed 'DeepDyve' Gillespie user on 16 March 2018 21. CRORA. Centre Collaborateur OMS pour la Recherche sur les Arbovirus (CRORA), real-time surveillance to improve the detection and prevention Base de données. 07/04/2015]; Available at: http://www.pasteur.fr/recherche/ of arboviral diseases. banques/CRORA 22. Stauffer WM, Cartwright CP, Olson DA, et al. Diagnostic performance of rapid Acknowledgments diagnostic tests versus blood smears for malaria in US clinical practice. Clin Infect Dis 2009; 49:908–13. 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