article: Buruli ulcer is an infectious disease involving the skin, caused by Mycobacterium ulcerans. Its exact transmission mechanism remains unknown. Several arguments indicate a possible role for insects in its transmission. A previous case-control study in the Nyong valley region in central Cameroon showed an unexpected association between bed net use and protection against Buruli ulcer. We investigated whether this association persisted in a newly discovered endemic Buruli ulcer focus in Bankim, northwestern Cameroon. We conducted a case-control study on 77 Buruli ulcer cases and 153 age-, gender- and village-matched controls. Participants were interviewed about their activities and habits. Multivariate conditional logistic regression analysis identified systematic use of a bed net (Odds-Ratio (OR) = 0. 4,95% Confidence Interval [95%CI] = [0. 2–0. 9], p-value (p) = 0. 04), cleansing wounds with soap (OR [95%CI] = 0. 1 [0. 03–0. 3], p<0. 0001) and growing cassava (OR [95%CI] = 0. 3 [0. 2–0. 7], p = 0. 005) as independent protective factors. Independent risk factors were bathing in the Mbam River (OR [95%CI] = 6. 9 [1. 4–35], p = 0. 02) and reporting scratch lesions after insect bites (OR [95%CI] = 2. 7 [1. 4–5. 4], p = 0. 004). The proportion of cases that could be prevented by systematic bed net use was 32%, and by adequate wound care was 34%. Our study confirms that two previously identified factors, adequate wound care and bed net use, significantly decreased the risk of Buruli ulcer. These associations withstand generalization to different geographic, climatic and epidemiologic settings. Involvement of insects in the household environment, and the relationship between wound hygiene and M. ulcerans infection should now be investigated.
  Over the last five years, Buruli ulcer (BU) care has been greatly improved by implementation of antimicrobial therapy and more effective diagnosis methods. However, much about the transmission of this devastating skin disease remains to be learnt. The incidence of BU disease is still on the rise (e. g. in Ghana, Togo and Gabon), and its emergence or increasing incidence have been linked with environmental modifications in rural areas, such as construction of dams or irrigation systems [1]. The main control strategy, based on early detection and early treatment, is hampered in many places by the lack of awareness of the disease and limited access to health services. So far, no primary prevention strategy has been proposed for the population of rural regions where the disease is endemic. Studies aimed at identifying BU risk factors have repeatedly shown an association between the disease and activities such as wading [2], [3] or washing clothes [4] in marshy areas of stagnant or slow-flowing waters. Farming in short clothes was also found to increase the risk of disease [3], [4]. Using a protected water source was associated with a decreased risk in some settings [2], [5]. Proper care of wounds, such as using alcohol to cleanse wounds, and hygienic practices, such as using soap for bathing, were also repeatedly found protective [2], [3], [6]. In 2006, a case-control study performed in Akonolinga, in the endemic region of the Nyong valley in central Cameroon, showed for the first time that the use of a bed net was associated with protection against BU [3]. This association was strong and independent of the socio-economic level of individuals, as it was found when comparing cases to both village and family controls. This finding is consistent with a possible role of insects in the transmission of Mycobacterium ulcerans. Several water bugs (aquatic Hemipterans) species [7], [8], [9], [10], as well as mosquitoes [11], have been shown to carry M. ulcerans in endemic areas. Furthermore, saliva of Hemipterans captured in the environment has been shown to harbor M. ulcerans, and the first M. ulcerans culture from an environmental sample was obtained from a semi-aquatic Hemipteran [12]. Laboratory experimental infection of mice bitten by infected water bugs provides important biological support for their involvement [9], [13], and mosquitoes-related risk factors have been associated with the disease in Australian epidemiologic studies [14]. Protection against insect bites using a bed net could present a simple, easily implementable means of BU prevention. We designed this replication study [15] to investigate the association between bed net use and protection against BU in the health district of Bankim, in north-western Cameroon (Figure S1). BU disease was first identified in this region in 2004 [16]. The Bankim BU focus shows several specificities compared to the endemic region of the Nyong river basin in central Cameroon. First, the disease was unknown in the area until recent years (Boussinesq, M. personnal communication). It was reported 15 years after construction of a dam on the Mape River which caused major modifications in the environment and relocations of populations and activities after submersion of villages and farmland. Secondly, the region' s geographic, climatic and demographic context differs from that of the forested Nyong River basin. Bankim is located in a transitional zone between forest and savanna and at the foot of the Mambila Mountains; its tropical seasonality is more contrasted than in the equatorial south; and the region is populated by a rich diversity of ethnic groups with specific cultural features. Due to its ecological, demographic and ethnic heterogeneity, Bankim district represented an adequate setting to challenge the hypothesis of bed net use as a protective factor against BU. Realizing a study in Bankim provided an opportunity to document the other risk factors for BU in this newly discovered focus. It also contributed to raising awareness of BU among Bankim' s diverse populations, who considered the disease to result from occult origins and who frequently under-report it.
  We conducted a matched case-control study in the district of Bankim, Adamaoua region, Cameroon. Study enrollment was voluntary. Information was provided first to the village population then individually to the participants. Written informed consent was obtained from case patients and control subjects or from their parents or guardians if participants were younger than 18 years. All BU case patients had received or were currently receiving free treatment for BU at the district hospital, a local clinic or the reference hospital for BU in Ayos. The study protocol was approved by the National Ethics Committee and the Cameroon Ministry of Public Health. The sample size computed for this matched case-control study required 71 groups of one case and two community controls to document an odds-ratio of 0. 4 or less as statistically significant, with the following assumptions: proportion using bed nets in the control population = 0. 5 (as hypothesized based on previous data available [3]), case/control ratio = 0. 5, power (1−β) = 0. 8, significance level (alpha) = 0. 05, correlation of exposure between groups in the case-control set (phi) 0. 2, calculated using PASS (2008, NCSS, trial version). The requested sample size was the same for the case/family control arm, although we anticipated difficulties for including 2 matched controls in some households. From June 4 to August 16,2009, study personnel administered a standard questionnaire to participants concerning demographic, environmental and behavioral risk factors. The questionnaire was adapted from Pouillot et al. [3] to fit to the Bankim setting and further questions about bed nets were introduced. Questions addressed fishing, bathing, washing clothes and domestic water supply activities, as well as farming activities. For all those activities, frequency, localization, and clothes worn were documented, as well as activity-specific details. Frequency was addressed as “daily”, “weekly”, “monthly”, “less than monthly” or “never”. Less than monthly frequencies were rarely reported, thus weekly, monthly, and less than monthly frequencies were grouped as “rare to often”. “Daily” was coded as “always”. Bed net use was considered systematic when people reported sleeping under a bed net every night, all year long. When bed net use was reported seasonal, or irregular, it was classified as “occasional”. All questions were close-ended and asked in French whenever possible. Translations into common local languages (Pidgin-english, Fulfuldé and Tikar) were provided by the same interpreter when needed. Cases were interviewed about their activities during the year before the onset of disease, while controls were asked to report their activity during the previous year. Data were analyzed using STATA version 10. 0 (STATA Corp. College Station, Texas). Univariate analysis was performed using conditional logistic regression to calculate odds-ratio (OR) and 95% confidence intervals (95%CI). Missing values for a given variable were not included in the univariate analysis of this variable. Variables with p-values lower than 0. 25 in univariate analysis were included in a multivariate model. Multivariate analysis was performed on these variables using multivariable conditional logistic regression after a first step of intra-exposure category selection. Independently associated variables among an exposure category were in a second step introduced in a multi-exposure category model. At each of the two steps, a stepwise backwards procedure was used to remove from the model the variable with the highest p-value until all p-values were less than 0. 05. This procedure (clogit) compared nested conditional logistic regression models by a likelihood ratio test. In the final model, the “interviewer” effect was assessed by creating a variable “interviewer” coded “0” or “1”, and by testing whether odds ratios of the significant associations of the final model were “modified” when adding an interaction term between each variable of the final model and the variable “interviewer”. Interactions between variables of the final model and age or gender variables were tested using the same method. Age was recoded in 4 categories according to quartiles (<10,10–13,14–35, >35). Preventive fraction was defined as the proportion of cases that could be avoided by systematic exposition of the population to a given protective factor. For instance, the preventive fraction for adequate wound hygiene is the proportion of Buruli ulcer cases that would be avoided if every person in the population cleansed their wounds with soap regularly. Preventive fraction was calculated for each protective factor using the odds ratio estimated in the final model according to the formula developed by Miettinen [17] and Bruzzi [18]: Preventive Fraction = [P (having disease/unexposed) −P (having disease) ]/P (having disease/unexposed) = P (Exposed) × (1−OddsRatio).
  Of the 195 probable Buruli ulcer cases recorded in Bankim district hospital since January 2007,100 cases were contacted, 88 were questioned, and 77 retained for analysis. Only one BU patient refused to participate. Cases were not included or not considered for analysis if they reported having suffered from BU for ten years or longer (n = 11), if one relative (parent, child or sibling) was already included in the study (n = 5) or if their distant location did not allow us to investigate community controls (n = 6). Details of the inclusion process are provided in Figure 1. Community controls were randomly selected in the population census for 74 cases. For 3 cases living in villages for which census data were not available, controls were included from randomly selected houses. The main reasons for controls not participating were long-term or repeated absence (n = 30,45%), refusal (n = 14,21%), unable to be found (n = 8,12%), or related to a case (n = 5,7%). The main analysis was performed on 77 cases (72 probable and 5 laboratory confirmed cases) matched with 153 community controls. Out of 77 cases analyzed, 76 were matched with two controls and one case was matched with one control only. Familial controls were recruited when available, i. e. mainly for children. Case-familial control analysis was performed on 37 cases and 49 controls. These were distributed as follows: 12 groups of one case and two controls and 25 groups of one case and one control. Among controls, 42 were siblings, 5 were other relatives (half-brother or sister, cousin) and two were unrelated children living in the same household. Most patients were healed, except for 16, who presented with lesions still not scarred (Table S1). Five cases could be confirmed, but laboratory confirmation could not be obtained for 11 who were already undergoing treatment. About as many cases were men (n = 40) as women (n = 37). Median age of analyzed cases was 14 years (Inter-quartile Range (IQR) = [10–36. 5]). Lesions classically presented on lower (n = 39,49%), and upper limbs (n = 29,38%). Only 3 patients presented with multiple lesions, and 8 (10%) presented with head or trunk lesions. Median (IQR) time from onset of disease to day of interview was 17 (9–26) months, ranging from 15 days to 7 years. The majority of patients did not associate their disease with any particular circumstance, but 9 linked it to a wound and 17 to insect bites. The overall population of the study, presented in Table 1, showed an important heterogeneity of ethnic groups and origins. Tikari and Kwadja farmers are indigenous and represented 33% of controls, but Yamba and Mambila farmers originating from neighboring regions were also well represented, respectively 29% and 12% of the control population, respectively. Kotoko people, originating from the Extreme-North Cameroon, are also found in Bankim, where they settled on the banks of the Mappé Lake and currently handle the commercial fishing business. Fulani-related Mbororos live usually in settlements in the savanna where they traditionally raise cattle. Some of them practice transhumant pastoralism. Adults principally engaged in farming activities. Most children between 6 and 12 years old reported going to school (94%), and a majority of them participated in farming during weekends and holidays or during seasons when additional workforce was required (85%). Education level was low with few persons over 12 years of age having secondary education. Univariate analysis showed a few associations that did not reach significance level (table S2). Of note among those were: bed net use which was associated with a decreased risk (OR [95%CI] = 0. 4 [0. 1–1. 4], p = 0. 13); watering a garden, associated with an increased risk (OR [95%CI] = 3. 1 [0. 6–15], p = 0. 13); and reporting scratch wounds after insect bites, associated with an increased risk of BU (OR [95%CI] = 2. 3 [0. 8–6. 7], p = 0. 10). No significant association with BU was revealed in multivariate analysis (table S2). If we assume that the relationship between a protective factor and risk decrease is truly causal, we can calculate the proportion of cases in the general population (represented by the controls) prevented by this protective factor, based on the relative risk and the frequency of the factor in the general population (again, represented by controls). The preventive fraction for systematic bed net use was 32% (proportion of controls systematically using a bed net: 53%, OR = 0. 4). Likewise, the fraction of cases prevented by adequate wound hygiene practices (using soap to cleanse wounds) was 34% (proportion of controls using soap: 38%, OR = 0. 1).
  The chosen setting, Bankim district in the Adamaoua region, contrasts with the previous study site. Akonolinga, the previous study site, was characterized by a relative homogeneity: 80% of the population belonged to the same ethnic group, the region was largely forested and water-related activities were centered on a major river, the Nyong [3]. By contrast, Bankim was home to numerous ethnic groups with wider variation in environmental, cultural and health practices. Bankim' s environment is a highly varied mosaic of savanna, mountain, and forests. Water-related activities took place in the Mappé reservoir, the Mbam River and many other smaller water bodies (Figure S1). Our rigorous sampling method, which used census data and repeated appointments with controls, allowed us to capture accurately the heterogeneities of populations and activities in Bankim and to minimize control recruitment associated biases [19]. Case recruitment was performed within the entire district and was not restricted to the most accessible areas. Similar to case-control studies on BU, the present study has several limits inherent in its methodology and context. First, even if we are confident that our sampling method prevented most sampling biases, memory bias could have occurred: cases were interviewed on their activities during the period preceding the onset of disease and could therefore remember them differently from controls, who were interviewed on their activities over the previous year. However, we included mainly cases with disease diagnoses during the two previous years. This ensured that cases and controls would recall their activities on similar time scales and minimized differential bias. Furthermore, having suffered Buruli ulcer disease could have influenced cases' answers to questions, such as the occurrence of scratch wound as discussed below (rumination bias, [19]). However, most of our identified risk or protective factors are related to less subjective behavioral parameters, which are less likely to be influenced by the disease status. Finally, this study was performed in a region that is distant from national or regional level healthcare facilities, and where the disease was only recently reported. But in 2007, because of the increasing number of cases, Bankim health personnel were trained at the national Buruli ulcer reference hospital in Ayos on case detection, systematic recording and basic management of cases according to WHO guidelines. These trained personnel implemented these practices in the district. During the period of the present study, a systematic procedure for laboratory case confirmation was set up although only a few cases were confirmed microbiologically. However, clinical diagnosis has been demonstrated to be highly specific when performed by trained health personnel, so that we are confident that almost all clinically-diagnosed Buruli cases were true cases [20]. In a previous study in Akonolinga, Cameroon, analysis of probable and confirmed cases yielded the same risk factors as confirmed cases only [3]. Bathing for hygiene in the Mbam river was associated with an increased risk of disease. These baths involved people spending long amounts of time near the river for their farming activities and eventually dwelling there during the rainy cultivation season where the waters are high and the banks swampy. Risk increase did not concern leisure baths in the same river, which only took place in the shallow waters during the dry season. This finding is in accordance with numerous previous results identifying slow flowing rivers and stagnant waters as an important source of contamination. The identification of the Mbam river as a risk factor rather than the Mappé reservoir seems at first counter-intuitive, but the reservoir is probably not the direct source of every contamination: some patients had no history of contact with the Mappé lake waters and highest prevalences of BU were not recorded in the fishing settlements on the lake shore but in the villages located between the lake and the Mbam river (figure S1). Of note, M. ulcerans DNA positive water bugs were captured in domestic water collections as well as in the Mappé lake in 2008 [16]. The importance of the peri-domestic water collections as a source of M. ulcerans infections remains to be evaluated in terms of M. ulcerans presence throughout the year as well as potential vectors or reservoirs breeding sites. Captures targeting water bugs indicated important seasonal variations of abundance and positivity [8], which warrants regular follow-up of microbe and insect populations. The dam construction may have contributed to BU expansion by providing a broader habitat suitable for proliferation, dissemination and/or survival of M. ulcerans, one of its hosts, or an eventual vector. The dam construction in this region impacted drastically on endemicity of onchocerciasis and loasis [26] as well as schistosomiasis (Mouchet F, personal communication). Increase in BU disease may also result from increased exposure to the environment surrounding the Mbam River where relocated villagers exploit new fields after the dam construction. The second risk factor identified in this study is more difficult to interpret. Reporting occurrence of scratch wounds after being bitten by an insect was found associated with Buruli ulcer disease. This could be seen as an indication that insect bites are involved in the transmission, either through an insect vector or through unspecific skin lesions from scratching representing a port of entry for M. ulcerans. We would however recommend considering this factor with great caution, as it was only reported and actual sensitivity to insect bites was not assessed. Furthermore, BU cases often reported having suffered intense itching before the onset of the lesion, which they often attributed to an insect bite. This event may have led cases to report being more sensitive to insect bites than controls. Finally, several risk factors commonly evoked for BU did not present statistically significant associations with the disease in this study. Statistical power was probably insufficient to investigate properly several associations, such as education level (very low) and short clothing related associations (infrequently used for farm work or fishing). Bankim environment' s heterogeneity probably prevented us from identifying major water sources of exposure: there is a large number of water bodies, and people change both water-related activities and locations throughout the year because these water sources dry up during the dry season. This may explain why in this study, four behavioral factors and only one location-dependent factor had statistically significant associations with BU in the final model. Through this study, we have demonstrated that the association between bed net use and protection against BU, found in a previous case-control study in Akonolinga, can be generalized to another climatic, geographical, environmental and ethnic context. We also identified good wound hygiene as a key protective factor, as reported in several previous studies. These findings support the hypothesis of a domestic or peri-domestic transmission of M. ulcerans, by direct (skin lesions) or indirect (insect bites) contamination involving peri-domestic water collections. We believe that it is now of critical interest to confirm or to rule out the domestic vector hypothesis by investigating insects in the domestic and peri-domestic environment of BU endemic foci. Such study would also help to determine if implementation of bed net protection would help prevent BU transmission. Furthermore, good hygiene appears again as a key protective factor: using soap to cleanse wounds was found protective, while hygiene baths in the Mbam River increased the risk of disease. Hygienic water sources and behaviors appear to be connected. The link between hygiene and M. ulcerans transmission merits investigation in greater detail, so as to promote specific hygienic practices through prevention strategies. Finally, we believe that while clinical research is making progress in developing oral treatment of Buruli ulcer [27], the research on ecology and transmission of M. ulcerans infection should be encouraged so that meaningful and effective prevention strategies can be identified and proposed.
summary: Mycobacterium ulcerans is the causative agent of Buruli ulcer disease, which causes skin wounds and often results in disabilities. The transmission of M. ulcerans remains unknown. Environmental and biological studies have gathered evidence that insects could play a role in M. ulcerans circulation. A case-control study performed in central Cameroon in 2007 unexpectedly illuminated an association between bed net use and a decreased risk of Buruli ulcer. As this result suggested a potential domestic transmission involving insects, we set up a new study to investigate whether this association existed in Bankim, a newly discovered Buruli ulcer endemic site in northwestern Cameroon. Our results confirm the protective effect of bed nets in this region, despite very different population, environment, and climate factors. They also confirm the role of good hygienic practices, a protective factor repeatedly identified in previous studies. These repeated associations now warrant further research on a possible domestic or peri-domestic transmission of the disease, involving local water collections and possibly insects.
keywords: medicine
  bacterial diseases
  infectious diseases
  buruli ulcer
  public health and epidemiology
  behavioral and social aspects of health
  neglected tropical diseases
  public health
title: Adequate Wound Care and Use of Bed Nets as Protective Factors against Buruli Ulcer: Results from a Case Control Study in Cameroon