Mogoung-Wafo et al. BMC Infectious Diseases (2019) 19:146 https://doi.org/10.1186/s12879-019-3776-y

RESEARCHARTICLE Open Access Prevalence and intensity of over twenty-three years in three communities of the Mbalmayo health district (Central ) Aude E. Mogoung-Wafo1†, Hugues C. Nana-Djeunga1,2†, André Domche1, Floribert Fossuo-Thotchum1, Jean Bopda1, Steve Mbickmen-Tchana1, Honoré Djomo-Kamga1 and Joseph Kamgno1,3*

Abstract Background: Loiasis is a -borne due to Loa loa and transmitted to humans by tabanids of the Chrysops. Loiasis has been historically considered as the second or third most common reason for medical consultation after , and a recent study has reported an excess mortality associated with the infection. However, the clinical impact of this filarial disease is yet to be elucidated, and it is still considered a benign disease eliciting very little attention. As a consequence of post-treatment severe adverse events occurring in individuals harboring very high Loa microfilarial loads, is not recommended in hypo-endemic areas that are co-endemic for loiasis. Without treatment, it is likely that the transmission of the disease and the morbidity associated with the infection will increase over time. This study aimed at investigating the long-term trends in prevalence and intensity of Loa loa infection in an area where no mass anti-filarial treatment has ever been distributed. Methods: A cross-sectional survey was conducted in three communities of the Mbalmayo health district (Central Cameroon). All volunteers, males and females aged five years and above, underwent daytime calibrated thick blood smears (CTBS) to search for L. loa microfilariae (mf). A structured questionnaire was administered to assess the history of both loiasis related clinical signs and migration of enrollees. Results: The prevalence of loiasis was 27.3% (95% CI: 22.3–32.9) in the three surveyed communities, with a mean mf density of 1922.7 (sd: 6623.2) mf/mL. Loa loa infection rate was higher amongst females than in males (p = 0.0001) and was positively associated with age of (OR = 1.018; p = 0.007). The intensity of infection was higher among males than in females (p < 0.0001), and displayed a convex in form trends with age groups. The follow up over 23 years revealed that both the rate and intensity of infection were similar to baseline. Conclusions: The prevalence and intensity of Loa loa infection 23 years on is stable over time, indicating that this filarial disease might be noncumulative as regarded till now. Keywords: Loiasis, Prevalence, Intensity of infection, Mbalmayo health district, Cameroon

* Correspondence: [email protected] †Aude E Mogoung-Wafo and Hugues C Nana-Djeunga contributed equally to this work. 1Centre for Research on and other Tropical Diseases, PO Box 5797, Yaounde, Cameroon 3Faculty of Medicine and Biomedical Sciences, University of Yaoundé 1, PO Box 1364, Yaounde, Cameroon Full list of author information is available at the end of the article

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mogoung-Wafo et al. BMC Infectious Diseases (2019) 19:146 Page 2 of 7

Background been reduced in some areas under community directed Loiasis, better known as the African eye worm, is a treatment with ivermectin (CDTI) [12]. However, iver- parasitic disease caused by the filarial Loa mectin is not recommended in hypo-endemic areas for loa and transmitted to humans by tabanids belong- onchocerciasis (< 40% mf or < 20% nodules prevalence) ing to the genus Chrysops. Chrysops dimidiata and that are co-endemic for loiasis [11]. One can anticipate C. silacea, commonly known as deer and mango an increase in transmission and morbidity as well as life or mangrove flies, are the two main anthropophilic expectancy reduction of infected individuals [7]. vector species, only found in [1]. The disease The objective of the present study was to follow up is restricted to western and central Africa where a the prevalence and intensity of Loa loa infection over 23 geostatistical analysis of history of eye worm has years in three neighboring communities of the Mbal- mapped high risk levels of loiasis in 10 countries mayo health district (Central Cameroon) in order to where an estimated 14.4 million people live [2]. Al- investigate the long-term trends in infection parameters though most cases of loiasis are asymptomatic [3], in an area where no mass anti-filarial treatment was ever the most striking clinical manifestations of this filar- distributed. ial infection are the migration of the adult worm under the bulbar conjunctiva, and the migratory transient called Calabar swellings. In addition, Methods loiasis has been historically ranked as the second or Study area and population third most common reason for medical consultation This study was conducted in the Mbalmayo health dis- after malaria and pulmonary diseases in some com- trict (Nyong-et-So’o Division, Centre Region) located at munities of Congo and [4, 5]. Data reporting about 50 km south of Yaoundé, the political capital city implication of loiasis on human health are very of Cameroon. In this forested environment, the Nyong scanty, and its clinical impact is yet to be clarified River is the main watercourse of the study area. [6]. Loiasis is still considered a benign disease elicit- Together with its numerous tributaries, this low flowing ing very little attention, although a recent survey has river generates swamps where raffia grows, thus pro- reported an excess mortality associated with the in- viding suitable breeding sites for chrysops. The mean fection [7]. annual rainfall is 1600 mm and the mean annual Due to the benign nature attributed to this tropical temperature is 24.1 °C. disease, no large-scale control program is dedicated to The population of this area is mainly made of Beti. loiasis though four chemotherapeutic agents (diethylcar- The major occupations of the populations are subsist- bamazine, ivermectin, and mebendazole) ence agriculture and cocoa cultivation. The urbanization have been proven effective against the parasite [6]. How- of the Mbalmayo town, head of the Nyong-et-So’o div- ever, it is now accepted that the most effective drugs on ision, has generated many activities attracting popula- L. loa infection are not really safe when individuals are tions of the neighboring villages, especially youth. heavily infected, and the safer ones are not completely efficient [6, 8]. Indeed, although benzimidazoles are safer for the treatment of loiasis, their effects on microfilarial Study design loads are quite modest or transitory [8, 9]. In contrast, The study area, known to be endemic to loiasis [2, 13, and ivermectin are very efficient 14], has sheltered a clinical trial in 1993, aiming at deter- against L. loa, but therapeutic accidents with sometimes mining whether large-scale ivermectin treatment would fatal outcomes have been reported in some individuals have an impact on the transmission of loiasis [15]. Base- harboring high microfilarial loads (>8000 microfilariae line data on loiasis were thus collected, and the present per milliliter, mf/mL) after treatment with each of these study aimed to investigate the prevalence and intensity two drugs [10]. of Loa loa infection over 23 years. To do this, a In areas endemic for loiasis and where onchocerciasis cross-sectional survey was conducted in three communi- is meso- (> 40% mf or > 20% nodule prevalence) or ties of the Mbalmayo health district (Central Cameroon) hyper-endemic (> 60% mf or > 40% nodule prevalence), to assess the prevalence and intensity of Loa loa infec- ivermectin treatments have been recommended since tion. Eligible individuals were both males and females, the clinical impact and consequences associated to on- aged 5 years and above, who had already lived in the chocerciasis outweighed the risk of post-treatment se- selected community for at least 5 years. All volunteers vere adverse events (SAEs), under specific surveillance underwent blood sample collection to search for Loa system for early detection and management of SAE cases loa microfilariae, and a structured questionnaire was [11]. As a consequence of the collateral impact of iver- administered to assess the history of both loiasis mectin on Loa loa, the transmission of infection has related clinical signs and migration of populations. Mogoung-Wafo et al. BMC Infectious Diseases (2019) 19:146 Page 3 of 7

Sample collection and processing arithmetic means, and the sampling fluctuations esti- From each participant, a calibrated thick blood film was mated using standard deviation (sd). Chi-square, collected daytime (from 10 am to 4 pm) to take into Mann-Whitney and Kruskal-Wallis tests were used to account the diurnal periodicity of Loa loa [16]. Trained compare loiasis prevalence and mean intensity of infec- and qualified lab technicians collected a 50 μL sample of tion between communities, genders and age groups, finger-prick blood from each study participant using a respectively. non-heparinized capillary tube. Blood samples were col- lected in aseptic conditions using sterile and disposable Results equipment. Standard procedures were used for blood The survey was carried out in three small neighboring samples processing and analysis [17]. Slides were exam- communities (Abang, Akometam and Ngat-Bane) of the ined independently using bright field microscopy (mag- Mbalmayo health district. A total of 271 individuals were nification × 10 and × 40 for identification) by two examined, among whom 140 (51.6%) were females. The experienced laboratory technicians. All microfilariae median age of the population was 50 years old (inter- present in the blood sample were identified and counted, quartile range: 28–63). and the results expressed as microfilariae per mL of blood (mf/mL) [17]. Prevalence Among the 271 individuals examined, 74 (27.3%; 95% Identification of Loiasis-related clinical signs CI: 22.3–32.9) were harboring L. loa microfilariae in The most striking clinical manifestations attributable to their blood. Although the prevalence of loiasis was simi- loiasis were assessed using a standardized questionnaire lar among communities visited (Chi-square: 1.604; p = adapted to the proven Rapid Assessment Procedure for 0.448), a high heterogeneity was observed in Loa distri- Loiasis (RAPLOA) based on the history of eye worm or bution between genders and age groups (Table 1). Calabar swellings [18]. Indeed, the questions asked to Loiasis was more prevalent in females as compared to the participants were “Have you ever experienced or no- males (Chi-square: 13,027; p < 0.0001), and was ticed worms moving along the white of the lower part of gradually increasing in prevalence between age classes your eye?” to assess the experience of eye worm, and (Table 1). Indeed, a positive correlation was observed “Have you ever experienced swellings under the skin between infection with Loa loa and the age of enrollees which changed position and disappeared?” to investigate (OR: 1.018; 95% CI: 1.005–1.032; p = 0.007). the experience of Calabar swellings. Investigators were assisted by local guides to ease communication with vil- Intensity of infection lagers, especially illiterates. Regarding the intensity of infection, the mf density arith- metic mean of was 1922.7 (sd: 6623.2; maximum: Migration history 59,540) mf/mL. Like prevalence, intensity of infection A brief questionnaire was administered to all survey par- was similar between communities (p = 0.528), whereas a ticipants to check whether they were foreigners or na- high heterogeneity was reported between genders and tives of the targeted community. The number of years of age groups (Table 1). The intensity of Loa infection was residence in the selected community was recorded and higher among males (p < 0.0001) and a convex in form foreigners or visitors were asked where they were origin- trends was observed among age groups (p= 0.032). The ating from in order to identify the origin of the disease maximum microfilarial load was reached in individ- in case of infection. uals aged 35 to 49 years old, both in males and females (Table 1;Fig.1). Besides these observations, Data analysis 7.4% (7.7% amongst females) and 1.5% (1.9% All relevant data for loiasis were recorded into a amongst females) of enrollees harbored microfilarial purpose-built Microsoft Access database and exported loads exceeding 8000 mf/mL and 30,000 mf/mL, into PASW Statistics version 18 (SPSS Inc., Chicago, IL, respectively, with similar trends between males and USA) for statistical analysis. Microfilariaemia and clinical females (p >0.816). sign prevalence were expressed as the percentage of in- fected or affected individuals (presenting with microfilar- Morbidity associated with loiasis iae in their blood or having experienced eye worm Among the 177 individuals to whom the standardized migration or Calabar swelling) among the total number questionnaire was administered (the interview was not of individuals examined; the 95% confidence interval performed in the Akometam community for logistic (CI) was calculated using the Wilson method not cor- constrains) 22.5% (95% CI: 15.0–32.2) and 26.0% (95% rected for continuity [19]. The intensity of infection was IC: 20.1–32.9) declared having already experienced computed when the microfilarial count was available as Calabar swellings and sub-conjunctival migration of Mogoung-Wafo et al. BMC Infectious Diseases (2019) 19:146 Page 4 of 7

Table 1 Prevalence and intensity of loiasis in three neighboring communities of the Mbalmayo health district Variables N examined % prevalence (95% CI) Intensity of infection (sd) By community Abang 83 28.9 (20.3–39.4) 22.4 (6435.4) Akometam 89 22.5 (15.0–32.2) 481.8 (8841.9) Ngat-Bane 99 30.3 (22.1–30.3) 446.5 (3945.4) By sex Males 131 37.4 (29.6–45.9) 2594.7 (6035.6) Females 140 17.9 (12.4–25.0) 1294.0 (7160.9) By age group 5–19 24 4.2 (0.7–20.2) 289.2 (1416.6) 20–34 69 23.2 (14.8–34.4) 1127.5 (3559.5) 35–49 40 30.0 (18.1–45.4) 3622.5 (10,562.8) 50-Over 138 32.6 (27.3–43.0) 2111.7 (6811.8) Overall 271 27.3 (22.3–32.9) 1922.7 (6623.2) CI confidence interval, sd standard deviation adult Loa, respectively. The prevalence of these clinical 23-years change in Loa loa endemicity signs was similar among communities, genders, and age Among the 271 individuals tested in 2016, 39 (of whom groups (p > 0.085) except for eye worm whose history 27 males) were already present in 1993. A positive asso- was mostly recorded among individuals aged 21 years ciation was observed between the prevalence of Loa and over (p = 0.012). A positive association was found microfilariaemia and the duration of residence in the between the prevalence of loiasis (evaluated by microfi- community (OR: 1.016; 95% CI: 1.005–1.027; p = 0,003). lariaemia) and history of eye worm (OR: 5.250; 95% IC: The infection rate, when considering only those 39 indi- 2.545–10.829; p < 0.0001) and Calabar swelling (OR: viduals, was relatively higher in 2016 (41.0% (95% CI: 5.039; 95% IC: 2.382–10.656; p < 0.0001). 27.1–56.6) than in 1993 (35.9%; 95% CI: 22.7–51.6), but

Fig. 1 Trends in Loa microfilariaemia according to age groups in the Mbalmayo health district. Grey lines and bars represent males and dark lines and bars represent females Mogoung-Wafo et al. BMC Infectious Diseases (2019) 19:146 Page 5 of 7

the difference was not significant (Chi-square: 0.22; p = clinical signs (migration of adult worm under the bul- 0.639). Microfilarial densities were also similar between bar conjunctiva and the Calabar swelling) rarely har- 1993 (mean: 2280.5; sd: 6074.0) and 2016 (mean: 3080.0; bor L. loa mf [22, 23]; (iii) finally the sample size of sd: 5872.3), though a slight increase was observed in our study was small (only 177 individuals were inter- 2016 (Wilcoxon signed ranks tests, p = 0.351) (Fig. 2). viewed for logistical constraints), thus reducing the Importantly, 50% of heavily infected (microfilarial loads power of statistical tests, and the likelihood to accur- exceeding 8000 mf/mL) enrollees already exhibited high ately capture this relationship. However, a positive as- microfilarial densities 23 years ago. sociation was found between Loa microfilarial prevalence and histories of both migration of eye Discussion worm and Calabar swelling. This repeated cross-sectional study was carried out to The mean intensity of infection was 1922.7 (sd: investigate the long-term trends in prevalence and in- 6623.2; maximum: 59,540) mf/mL, with 7.4 and 1.5% tensity of Loa loa infection in an area that has never of enrollees harboring microfilarial loads exceeding benefitted from community preventive chemotherapy. 8000 mf/mL and 30,000 mf/mL, respectively. The re- The overall prevalence of loiasis was 27.3% in the lationship between prevalence and intensity of infec- three targeted communities; a figure similar to what tion observed in the present study fits with previous was previously observed in the same communities observations [20]. Indeed, it was demonstrated that [13–15] and indicating that this area is highly en- prevalence of Loa microfilariaemia between 20 and demic for African eye worm [20]. The prevalence of 30%, can predict approximately 5–9% of adults har- the African eye worm, estimated by history of either boring microfilarial loads exceeding 8000 mf/mL, and migration of adult worm under the bulbar conjunctiva 1–3% of adults with > 30,000 mf/mL [20]. Since se- or the Calabar swelling, was not accurately predicted vere adverse events have been correlated with high as previously demonstrated [18, 21]. Several reasons Loa microfilarial loads [10], these results (i) indicate can explain this discrepancy: (i) in the present study, that the implementation of ivermectin treatments in the interview was quicker and lighter (no image was the Mbalmayo health district needs to be done with shown) than in previous study; (ii) it was demon- caution, and (ii) confirm why CDTI is yet to be im- strated that people presenting with loiasis associated plemented in this health district [11].

Fig. 2 Individual microfilarial load trends between 1993 and 2016 in the Mbalmayo health district Mogoung-Wafo et al. BMC Infectious Diseases (2019) 19:146 Page 6 of 7

Loa loa prevalence and intensity of infection were previously reported elsewhere, even though within a found to increase while age was increasing (OR: 1.018; shorter time frame, suggesting that these findings and 95% CI: 1.005–1.032; p = 0.007). A convex in form trend associated interpretations are not unique to our study. was observed among age groups, the maximum microfi- larial load being reached in individuals aged 35 to 49 Conclusions years old (Table 1; Fig. 1). These results might indicate This 23-year repeated cross-sectional study confirmed that the exposure to loiasis is increasing with age, the that the prevalence and intensity of Loa loa infection is disease appearing to accumulate in infected individuals. stable over time, indicating that this filarial disease might In case of accumulation of the parasite consecutive to be noncumulative [28]. It has been argued that infection new infective bites by the vector, one would have ex- with Loa loa can hinder the success of onchocerciasis pected an increase in prevalence and intensity of infec- and control and/or elimination pro- tion over time, particularly in the absence of any grams as a consequence of post-ivermectin SAEs. Given intervention targeting either the parasite or the vector. that this study has demonstrated that loiasis might be However, the convex in form profile displayed in the re- noncumulative and microfilarial densities are somehow lationship between age and intensity of infection might stable over time, an increased risk of post-ivermectin suggest that this accumulation is somehow relative and SAEs in these untreated communities is unlikely or not might reverse in advanced age group. Similar results expected, and the caution with which mass drug admin- have been reported in previous studies, suggesting a istration has to be implemented in loiasis endemic areas noncumulative nature of this filarial disease [14]. Such will not fundamentally change. observations have also been described in [24], another filarial nematode closely related to Abbreviations L. loa CDTI: Community directed treatment with ivermectin; CI: Confidence interval; , and was explained by the fact that in younger in- mf/mL: Microfilariae per milliliter of blood; OR: Odds ratio; SAE: Severe dividuals the parasite population has not reached its adverse event; sd: Standard deviation equilibrium, the number of maturing adult worms in these individual hosts exceeding that of dying worms. Acknowledgements The authors are grateful to the population who willingly accept to On the contrary, in older individuals the number of participate in this study, and to the local authorities for their assistance dying adult worms exceeds that of maturing worms as a during the implementation of this study. consequence to their reduced exposure to chrysops’ bites because of their indoor habit related to their age. Funding This survey was funded by the Centre for Research on Filariasis and other This latter hypothesis supports the decreasing trend in Tropical Diseases (CRFilMT) as part of support received from the Mectizan intensity of infection observed in advanced age. Donation Program. A follow up of individuals already examined 23 years Availability of data and materials ago have shown that both the rate and intensity of infec- The datasets used and/or analyzed during the current study are available tion remained almost unchanged (Fig. 2), indicating that from the corresponding author on reasonable request. the transmission of loiasis is stable over time. This sta- ’ bility in L. loa transmission was already observed in Authors contributions AEMW collected field data, analyzed the data and drafted the manuscript. Gabon [25], Congo [26], and Cameroon (same site like HCND conceived the study and designed the experiments, collected field in our study) [14] over a shorter interval (1–3 years). data, analyzed the data and drafted the manuscript. AD collected field data The hypotheses underpinning this stability were sug- and contributed to the preparation of the manuscript. FFT collected field data and contributed to the preparation of the manuscript. JB collected field gested to be inherent both to the parasite (mf produc- data and contributed to the preparation of the manuscript. SMT collected tion by adult worms) and to the (immune defense field data and contributed to the preparation of the manuscript. HDK mechanism). This long-term stability observed in L. loa participated in the design of experiments, and contributed to the preparation of the manuscript. JK conceived the study and designed the infection is consistent with a noncumulative status of experiments, collected field data, analyzed the data and drafted the the disease observed in the present study. A 13-year fol- manuscript. All authors read and approved the final manuscript. HCND and low up of loiasis transmission in the Lékié division (Cen- JK are guarantors of the paper. tral Cameroon), has also revealed a stability in Competing interest entomological indices, including (but not limited to) The authors declare that they have no competing interests. chrysops infection and potential infective rates [27]. Des- pite these interesting findings, the sample size of this Ethics approval and consent to participate This study received ethical clearance from the National Ethics Committee for follow-up study was small (only 39 individuals examined Human Health Research (N°2016/09/810/CE/CNERSH/SP). After approval of in 1993 were found in 2016), and one can question rep- the local administrative and traditional authorities, the objectives and resentativeness and the generalizability of this small schedules of the study were first explained to community leaders and to all eligible individuals. Written agreements were obtained from those who sample with regards to the general population. It is agree to participate, under the discretion of community leaders. The worth mentioning that similar observations have been approval of parents or legal guardians of minors was necessary before any Mogoung-Wafo et al. BMC Infectious Diseases (2019) 19:146 Page 7 of 7

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