Risk factors for transmission of Salmonella Typhi in Mahama refugee camp, Rwanda: a matched case-control study

Introduction In early October 2015, the health facility in Mahama, a refugee camp for Burundians, began to record an increase in the incidence of a disease characterized by fever, chills and abdominal pain. The investigation of the outbreak confirmed Salmonella Typhi as the cause. A case-control study was conducted to identify risk factors for the disease. Methods A retrospective matched case-control study was conducted between January and February 2016. Data were obtained through a survey of matched cases and controls, based on an epidemiological case definition and environmental assessment. Odd ratios were calculated to determine the risk factors associated with typhoid fever. Results Overall, 260 cases and 770 controls were enrolled in the study. Findings from the multivariable logistic regression identified that having a family member who had been infected with S. Typhi in the last 3 months (OR 2.7; p < 0.001), poor awareness of typhoid fever (OR 1.6; p = 0.011), inconsistent hand washing after use of the latrine (OR 1.8; p = 0.003), eating food prepared at home (OR 2.8; p < 0.001) or at community market (OR 11.4; p = 0.005) were risk factors for typhoid fever transmission. Environmental assessments established the local sorghum beer and yoghurt were contaminated with yeast, aerobic flora, coliforms or Staphylococcus. Conclusion These findings highlight the need of reinforcement of hygiene promotion, food safety regulations, hygiene education for beverage and food handlers in community market and intensification of environmental interventions to break the transmission of S.Typhi in Mahama.


Introduction
Typhoid fever is an infectious disease caused by Salmonella Typhi [1]. It is transmitted through ingestion of food and water contaminated by the feces and urine of patients, or symptomatic carriers of the bacteria [2]. It is characterized by severe systemic illness with insidious onset, fever, severe headache, malaise, anorexia and relative bradycardia [3]. The clinical scope varies from mild illness with a low-grade fever to severe clinical disease with abdominal discomfort and multiple complications, including intestinal hemorrhages and perforations of the ileum [4]. Recent estimates showed that there are approximately 20.6 million cases and 223,000 typhoid-related deaths annually worldwide [5].
Outbreaks of the disease have been documented in many countries and are associated with poor sanitation, inadequate hygiene practices and unsafe food and drinking water [6]. The political crisis in Burundi, a country located in Eastern Africa, which started in late April 2015, forced thousands of Burundians to flee their homes. This resulted in an influx of refugees into neighboring countries, including Rwanda. In April 2015, a refugee camp was established in the Mahama sector of Kirehe District in the Eastern Province, to accommodate some of these refugees. As of February 2016, the number of refugees living in the camp was estimated to be 48,000 [7]. Commencing in early October 2015, the camp health facility began to record an increase in the incidence of a disease  [8]. Following confirmation of the outbreak, prevention and control measures such as active surveillance, case management, community mobilization and hygiene education, improvement in sanitation and provision of clean water were scaled up in the camp.
Despite these interventions, which resulted in availability of adequate clean water and sanitation facilities, the number of cases continued to increase, demonstrating the need for a better understanding of the risk factors and pathways for transmission of the disease. The objectives of the study were to characterize further the epidemiology of the outbreak, identify risk factors for transmission and to use these data to propose additional recommendations for controlling S. Typhi outbreaks in Mahama and other refugee camps. The study tested the null hypothesis that "the risk factor for transmission of the outbreak is not consumption of contaminated water"

Study design:
We conducted a retrospective case-control study from January to February 2016, in the Mahama refugee camp, to identify the risk factors associated with the transmission of S. Typhi.
Epidemiological data were collected through a survey of matched cases and controls. We also conducted an environmental assessment through collection of environmental samples from community water sources and markets and observation of hygiene practices.

Study area: Mahama camp is located in a remote area in Eastern
Province, Rwanda, next to the Akagera River, which separates Rwanda from the United Republic of Tanzania. Half of the population is women and 47% of refugees are under 18 years of age [7]. The population lives in 18,481 households, in 15 villages, numbered from 1 to 11 on the old site of the camp (Mahama I) and 17 to 20 on the new site (Mahama II) [7]. Refugees in Mahama I reside in tents, while those in Mahama II live in semi-permanent houses. Water supply to both sites is via a mini-water treatment plant, which draws water supplies from the Akagera River. As of January 2016, water supply to the camp had exceeded the Sphere standard recommendation of 15 liters per person per day [9].
Human waste disposal is through pit latrines, which are strategically located in the camp. Two health centers offer both therapeutic and preventive services, and are sufficiently staffed by qualified doctors, nurses and biotechnologists, however only one was fully functional at the time of this study. Public health challenges in the camp Page number not for citation purposes 3 include poor sanitation and hygiene, overcrowding and childhood malnutrition.

Sample size, methods and exclusion criteria:
For the purpose of this study we used the epidemiological definition, which defined a typhoid fever case as any person who presented with fever for 3 or more days with or without: malaise; headache; abdominal pain; constipation or diarrhea; joint pains; chills or cough, from 1 October 2015 to 28 January 2016 and residing in the camp since its establishment in May 2015. Controls were defined as any person who did not fall into the epidemiological case definition but resided in the camp. The exclusion criteria for cases were any person who was not a permanent resident of Mahama camp. Exclusion criteria for the controls was anyone who had been permanent resident for less than 1 month and anybody who had developed symptoms similar to those of typhoid fever in the past 6 months. We calculated the sample size using a method described by Lemeshow et al [10].
This method established a sample size of 259 cases; based on a ratio of one case to three controls, the number of controls was calculated to be 777, giving a total sample size of 1,036. Cases were selected using a systematic random sampling technique from a database of all cases. The data collection team worked with community leaders to identify and visit the households of the cases.
For every typhoid fever case (suspected or confirmed), participants enrolled on the study and three neighborhood controls, matched for age and gender, were identified and enrolled. Each control was selected using a systematic selection method in which the interviewer, after exiting the household of the case, chose the first matched control from the second household on the right side of the case house then skipped two households before choosing the second matched control; the procedure was repeated on the left side of the case house to identify the third control. In the event that the selected household had no eligible candidate, the surveyor moved to the next household without skipping any houses.  Table 2).

Bivariate analysis:
The bivariate analysis identified that for people who had spent more than 6 months in the camp, the risk of contracting typhoid fever was nearly three times that of individuals who had spent less than 6 months in the camp (OR 2.89; p < 0.001). Those who completed pre-school education were two times more likely to be infected (OR 2.07; p = 0.021), while participants who had a family member affected by typhoid fever in the past 3 months were more likely to be infected by S. typhi than those whose family members had not been affected by the disease (OR 2.81; p < 0.001). Poor awareness about typhoid fever was also significantly associated with the illness (OR 2.06; p < 0.001); while subjects who reported only sometimes washing their hands after using the latrine were twice as likely to develop typhoid fever compared with those who do it regularly (OR 2.52; p < 0.001). The risk of infection with S. typhi was also higher in respondents who reported eating food prepared at home (OR 4.06; p < 0.001) and those who used the community market as source of food on a daily basis (OR 23.88; p < 0.001), compared with a community kitchen.
Respondents who reported only washing their jerry-can sometimes were also more likely to contract typhoid fever (OR 2.07; p = 0.002), compared with those who always washed it (Table 3).

Multivariable logistic regression:
The multivariable regression analysis identified having a family member who had typhoid fever in the last 3 months (OR 2.65; p < 0.001), low awareness about typhoid fever (OR 1.63; p = 0.011) and inconsistent hand washing after use of the latrine (OR 1.78, p = 0.003), were risk factors significantly associated with typhoid fever. Additionally, the risk of developing the disease was higher for respondents who reported eating food prepared at home (OR 2.75; p < 0.001) or food prepared at community market (OR 11.39; p = 0.005) compared with a community kitchen (Table 3). Table 3 shows results from the clustered logistic regression. The goodness-of-fit test showed no significant difference between the clustered and non-clustered multivariate analysis.

Discussion
This study describes the epidemiology of Salmonella transmission in the Mahama refugee camp. These data were useful for evaluating and implementing the outbreak response strategy and ultimately contributed to the control of the outbreak. This study identified having a family member affected by typhoid fever in the last 3 months, poor awareness about how to prevent and control the disease, inconsistent hand washing after going to the latrine, consumption of food prepared at home and from the community market as risk factors for the disease. This proves our study hypothesis that transmission was not associated with consumption of contaminated water. Furthermore, the laboratory analysis of the water samples taken from the camp showed that the water was biologically safe for consumption, which further supports this theory.
In addition, the epidemic curve of this outbreak illustrates a propagated outbreak, which is highly suggestive of person-toperson transmission, and confirms our finding of having a family member affected as one of the main risk factors for transmission.
The association of increased risk with poor awareness of typhoid fever is not surprising, as lack of awareness is known to be associated with poor compliance with typhoid prevention and control practices [12]. The association between eating food prepared at home and in the community market with higher risk of disease transmission could be because of a number of factors. The laboratory analysis showed that the food and beverages sampled at the community market were contaminated. Furthermore, observations during the environmental assessment highlighted poor enforcement of food safety and hygiene in the community markets.
Utensils such as cups were not properly cleaned and were being shared between multiple clients of the alcoholic beverage sellers, while house fly control was also a major challenge. Additional sampling and epidemiological analysis showed clustering of typhoid fever cases around the pit latrines. There was lack of adequate space in the camps, therefore many families were observed to be preparing and consuming their food next to open waste water trenches and pit latrines, which could increase the risk of food contamination. This is corroborated by previous studies in Zimbabwe and Indonesia, which showed that crowded living conditions and poor sanitation were associated with outbreaks of typhoid fever [13,14].
Additionally, inconsistent washing of hands after using the latrine, which is another risk factor identified in this study, may have  [20]. This is consistent with the finding that typhoid fever cases were clustered around latrines, where houseflies are usually prevalent. The signs and symptoms such as fever, abdominal pain and headache that were reported in this study are consistent with the usual symptoms of typhoid fever. The long duration of time (on average 5.7 days) before seeking treatment, which was observed in this study, could be because of many factors. Lack of information about how the disease is prevented and controlled could have been responsible for Page number not for citation purposes 6 this. The considerable proportion of people self-medicating (23.5%) could have delayed seeking treatment. Additionally, the long waiting time before receiving treatment at the only fully operational health center at the time of the study may have discouraged early attendance at the center. Teke et al [21] reported lack of good services as being the major motivator for self-medication in refugees in Durban, which further supports this hypothesis.
Furthermore, delay in seeking health care was also reported by Srikantiah et al [22] during an investigation of typhoid fever in Uzbekistan.

Study limitations:
The study was conducted 3 months after the typhoid fever outbreak and prevention and control interventions had already been introduced, therefore knowledge and attitudes regarding sanitation and hygiene practices may have changed. As a result, common risk factors associated with the disease may have been masked during our investigation. Information was retrospectively self-reported and provided by mothers or care givers for participants aged under 15 years, which could have introduced some recall bias into the study. These limitations were addressed through rigorous training of data collectors, pre-testing of the questionnaire and reformulation of questions, where necessary.
There is a high proportion of asymptomatic illness associated with S. Typhi, therefore it is possible that some asymptomatic carriers may have been misclassified as controls. These would markedly reduce the observed association between cases and potential exposure because of the similarity between cases and controls. In addition, given the broad symptomatology associated with typhoid fever, which is similar to other viral enteritis illnesses, there could have been an overestimation of the true typhoid fever cases. To address these limitations, the ratio of controls to cases was increased from two to three.

Conclusion
This study identified having a family member affected by typhoid fever, inconsistent hand washing, poor awareness about preventive measures and consumption of food prepared at home and from the community market as the main risk factors for typhoid fever in Mahama camp. In view of these findings, we proposed four main recommendations to facilitate timely containment of this and future outbreaks. First, hygiene promotion activities should be reinforced in the camp, particularly among those who are at highest risk of being infected such as those households accommodated near the latrines and those with previously infected people in the household.
Furthermore, we recommend continued respect of the sanitation corridor between latrines and households during shelter planning in future. In addition, when planning relocation of refugees to semipermanent houses, priority should be given to refugees accommodated in the crowded hangars in the old site, to reduce the overcrowded conditions. Second, food safety and hygiene regulations should be enforced in the community markets. This should be complemented with engagement of the market association members and market users to improve hygiene education. Furthermore, provision of more hand washing facilities, particularly in the community markets should be prioritized. Third, hygiene education for alcohol beverage sellers and food handlers in the market on potential diseases outbreaks associated with poor water, sanitation and hygiene should be increased. Finally, environmental interventions, particularly those related to sanitation, should be reinforced during critical times, such as the rainy season and periods of increased temperature, where an increase in the house fly population is possible [23].
What is known about this topic  The risk factors associated with typhoid fever are commonly known as contaminated water and food;  The population in situations of mass displacement such as refugee camps is exposed to communicable diseases such as typhoid fever;  Improved quantity and quality of drinking water in refugee camp, enhanced knowledge, attitude and practice towards prevention and control of poor hygiene related outbreaks among displaced people reduce the risk of water and foodborne disease outbreaks such as typhoid fever.

Competing interests
The authors declare no competing interests.

Acknowledgments
The authors would like to thank the WHO Country Office Rwanda for providing the funds for conducting this study. We also thank MIDIMAR and UNHCR Rwanda for providing the administrative approval of the study and Mr. Waltaji Kutane for supporting the environmental assessment. The authors alone are responsible for the views expressed in this article, which do not necessarily represent the views, decisions, or policies of the institutions with which they are affiliated.

Case n=260; Controls n=770
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