We evaluated the temporal dynamics of reported diarrheal disease and characteristics of patients presenting to select health facilities during outbreak periods in Chobe District, Botswana. The questionnaire survey tool was administered by existing medical staff in attendance and provided important data that would otherwise not have been available in this remote and resource poor setting. To our knowledge, this is the first prospective assessment of diarrheal patient characteristics in this District. Cases of diarrhea were identified among all villages in the District. Death occurred in both outbreaks involving outpatient children under-5 (n = 1 each outbreak). Although medical staffing of health facilities in the District was extremely limited (Table 1), a reasonable proportion of patients were surveyed during outbreak periods despite higher caseloads relative to the outbreak (dry season = 23% and wet season = 19% surveyed, Table 4). We discuss these study results and implications to diarrheal disease control. We then evaluate the effectiveness and limitations of study approaches and their application in resource poor settings.
Seasonality of diarrheal disease
Our examination of passive surveillance data for the region identifies a seasonal pattern of acute diarrhea in under-5 across years that coincides with major hydrological phenomena occurring seasonally in the area (rainfall and flood recession, Figure 2A). Diarrheal case incidence among this same age group during our study in 2011–2012 was similar in temporal pattern (Figure 2B). This pattern of diarrheal disease at the level of Chobe District appears to diverge from the national pattern  where diarrheal case incidence (1974–2003) peaks in March and October. Botswana is an arid country with limited surface water. Villages in the study area with the exception of Pandamatenga rely principally on municipal water obtained from the Chobe River. Potential interactions between rainfall and flooding effects on surface water quality and human diarrheal disease would be an influence limited to those areas where surface water occurs and may be associated with divergence in patterns of diarrheal case incidence. While the temporal pattern of case incidence suggests a strong climatic signature to diarrheal disease in Chobe District, the relative influence of various meteorological variables is unknown. Weather events, however, can strongly influence diarrheal disease as seen before in eastern Botswana in 2006 where unusually heavy rains, flooding, and apparent overflow of pit latrines precipitated a 25-fold increase in mortality with more than 547 under-5 children dying during an associated diarrheal outbreak .
Variation in causality by outbreak
We were interested in understanding whether patient characteristics varied by season of outbreak, as this would provide insight into causality. Responses, however, did not vary significantly by outbreak period (Figure 2A). This is particularly important given the potential for nonrandom patient selection affects, individual variation in the interpretation of questions and patient answers by nursing staff, and limited sample sizes. Furthermore, on cluster analysis, patients did not group by outbreak period (wet or dry season) or village but rather by age, with two distinct clades representing those patients under-5 and those patients 5+ (Figure 3). Lack of variation in patient characteristics by outbreak and seasonal patterns of occurrence support the hypothesis of environmental drivers influencing outbreaks in this region.
Using a decision tree approach, membership in the eight identified clusters was related dominantly to age with patient gender and hospitalization differentiating among patients under-5 and diarrhea in the household the most important varying characteristic among patients 5+ (Figure 4). Variable selection was, by necessity, limited and may not have included key variables important to identifying other important divergent qualities among patients involved in these 2 outbreaks. These results suggest, however, that secondary household transmission may be an important characteristic in adult diarrheal disease and that interventions that dampen these within household transmission pathways may contribute to a reduction in adult diarrheal disease in the study area. For example, improper disposal of children’s feces by the mother or caregiver can contribute importantly to secondary household transmission of diarrhea causing pathogens. A recent national survey found that only 49% of rural households disposed of children’s feces safely . Public health messages and education programs directed at improving hygiene and sanitation practices among mothers and caregivers may strongly contribute to a reduction in secondary household transmission and diarrheal disease.
Clinical presentation, pathogens, and diarrheal disease
A history of bloody diarrhea was reported at low levels in the first outbreak only, but there were no significant differences in the occurrence of this feature between outbreaks when passive surveillance data were evaluated for this same time period for all ages, health facilities, and mobile health visits in the District (overall 3.9% and 5%, dry and wet season respectively, Table 5). As might be expected, characteristics that occur at very low levels may not be accurately detected given potentially limited sample sizes, which should be considered when applying this approach. This type of clinical information, however, can be very valuable as a number of important bacterial pathogens can cause bloody diarrhea. These include Salmonella spp., Campylobacter jujuni, Shigella spp., and enterohemorrhagic and enteropathogenic Escherichia coli, some of which were identified previously among patients presenting to the primary hospital or clinics in the District (Table 3). Concurrent immunosuppressive conditions (e.g., HIV/AIDS) can increase the severity of disease as well as the occurrence of bloody diarrhea . The low frequency of this clinical sign suggests that these pathogens did not play a dominant role in outbreak occurrence and case presentation.
Enteric viral pathogens can also contribute to diarrheal disease and their contribution to outbreak dynamics is uncertain. Rotavirus is an important seasonal pathogen causing acute diarrheal outbreaks over much of the globe . As with many of the enteric bacterial pathogens, rotavirus can also be transmitted in sewage polluted waters . Pathogen invasion dynamics can be influenced by local climatic drivers including low temperatures and rainfall as well as flooding (reviewed ), features of the early dry season in the Chobe area. A survey of enteric viral pathogens among children presenting with diarrhea in Gaborone in southern Botswana identified low levels of infection with rotavirus (9.2%) peaking in July during that study . The impact of this pathogen on diarrheal incidence is expected to be reduced with the Government’s incorporation of the Rotavirus vaccine to the under five immunization schedule in July of 2012 . Measles in Africa also occurs in the dry season  and can cause diarrhea. Vaccination coverage for measles in Botswana is estimated to be high (94%) and case identification low (n = 8, 2010), although large outbreaks have occurred . In this study, vaccination coverage was reported at a lower level in the dry season (75% dry season and 100% wet season outbreaks).
Protozoal parasites, such as Cryptosporidium, have previously been implicated in diarrheal disease in Botswana in association with a severe outbreak of diarrhea in the eastern part of the country (60%, n = 75 samples tested, ). Surprisingly, of the samples collected from patients presenting with diarrhea in the wet and dry season outbreaks evaluated here, only low levels of Cryptosporidium were identified (wet season outbreak 25%, n = 12, 95% CI 5% - 57%, dry season outbreaks 3%, n = 30, CI 95% 0% - 17% ). Positive cases were seen among children less than two years of age. In Chobe District, as elsewhere, diarrheal causation is complex and likely involves a number of bacterial, parasitic, and viral pathogens, involving a potential diversity of hosts including zoonotic sources and interdependent transmission pathways.
While Botswana has one of the highest prevalence rates of HIV in the world , Chobe District is identified as one of the top five highest affected Districts within the country (13.1% and 30% among men and women respectively, both sexes = 23%, 2008 ). HIV/AIDS can have an important influence on immunological competence and host susceptibility to pathogen invasion  influencing diarrheal disease in particular . It is unclear what role HIV status had on outbreak dynamics in this study given the barriers in obtaining accurate status information from interviewed patients. However, the high prevalence of HIV infection among the community suggests that this is an important component requiring further investigation. Across Chobe District, a significant percent of patients reporting diarrheal disease were in the 5+ age class (44% and 35% in the dry and wet season outbreaks, respectively). Our survey results did not identify any significant difference by age in patients reporting persistent or chronic diarrhea during outbreak periods (Figure 3). While global surveillance focus is directed at the under-5 age group , this may not be appropriate in areas of high HIV prevalence. Distinguishing between infectious and chronic, noninfectious diarrheal disease in HIV infected individuals may be an important challenge to diarrheal disease surveillance in this group.
Interventions directed at reducing HIV transmission may also influence diarrheal disease dynamics. For example, natural maternal immunity, important in fighting water-associated infection, is absent in a great percentage of children in Africa, particularly in Botswana, as HIV-positive mothers are encouraged to use formula rather than breastfeed to reduce mother-to-child transmission of the virus . In Chobe District in 2007, only 52% of children were reported as having been breast-fed . A similar level was reported among participants in this study with only 44% and 63% reported to have been breast-fed in the dry and wet season outbreaks, respectively (Figure 3A). In the large 2006 diarrheal outbreak in Botswana, affected children were less likely to have been breast fed . There is an urgent need to refine our understanding of these interactions and implications to both HIV and diarrheal disease management and public health strategy.
The Botswana Government, since the early 1980s, has monitored and provided free supplementary foods to any child with malnutrition  or those nutritionally vulnerable, such as orphans associated with the HIV/AIDS . Malnutrition is identified as a critically important prognostic indicator of mortality in diarrheal disease . While malnutrition was not noted in the wet season outbreak, 14% of the under -5 patients were identified in the dry season as suffering from malnutrition by nursing staff filling in survey forms. Nutritional status among children can influence disease susceptibility and diarrheal disease . Consequently, an important focus is directed at ensuring adequate nutrition in early and recovery stages of diarrheal disease . While anorexia can occur as a consequence of diarrheal disease , in many places including Botswana, caregivers may also withhold food and milk products, including breast milk, in response to the onset of diarrheal disease in a child . During the dry season outbreak assessed in this study, several mothers with children in the pediatric ward identified withholding food and milk products, although public health educational campaigns actively discourage this practice in diarrheal disease management. Understanding these and other health care behaviors may be critical to public strategy effectiveness and underscore the need to identify locally held beliefs, risk perceptions and behavior, and home-based, health care practices.
Water shortages and quality
Human health is directly related to the quality and quantity of readily available water, which, in turn, influences exposure to waterborne pathogens, hygiene and sanitation practices, and the occurrence of diarrheal illness . This is particularly true for much of the African continent, where poverty, water quality, and sanitation deficiencies have strongly influenced declining human health levels and, in particular, diarrheal illness . Water shortages appear to be an important common element in both outbreaks across seasons. In Botswana, in general, water shortages occur commonly. This is particularly so in more remote regions as a result of equipment breakdowns, fuel shortages, and HIV/AIDS absenteeism from work related to water services . Many communities suffer chronic disruptions in water delivery that can result in the use of poor water quality sources such as river water, ephemeral pans, and open wells . In a municipal system, water shortages or cessation in delivery may also lead to declines in water quality. In this study, many respondents identified dirt or discoloration in municipal water. In Chobe District, as elsewhere, large centralized water tanks provide water for sections of the municipal system. During shortages, these tanks are quickly drained through local consumption, but retain small quantities of standing water and sediment from previous fillings. Re-suspension of both bacteria and sediment on resumption of delivery may be possible. Shortages, which occur year round, might be more important when cyclic changes occur in water quality related to seasonal hydrological phenomena (rainfall, flooding) identifying potential linkages where humans health and environmental conditions are coupled.
In Chobe District, like many African rural areas, alternate sources of clean water are unavailable during water shortages or periods of poor quality, particularly for the poor where purchasing of bottled water is economically infeasible. Use of unsafe water resources can lead to exposure to waterborne pathogens and diarrheal disease. In our study site, the Chobe River is accessible to most community members living in the District with the exception of Lesoma and Pandamatenga villages (Figure 1). Direct exposure to surface water through swimming, cleaning vegetables, and/or washing laundry was infrequently reported (6.4% and 0% by outbreak period, respectively) among patients and no one reported using the Chobe River directly as a drinking water source. This is an important finding, as the potential use of the Chobe River for drinking and other household uses during water shortages may have been considered an important hypothesized contributor to diarrheal outbreaks.
Recurrent water shortages lead to an increase in water storage practices , elevating the potential for post source contamination to occur, an increasing concern in Africa and elsewhere . Water is also stored in containers in order to increase water access where water is not piped into the home (Figure 3A). Storage of water was reported among most patients surveyed. Contamination of stored household water can occur at the source or post collection during utilization by the family . However, while there is great concern regarding the potential for post-collection contamination of water, other comparative studies suggest that source water quality is still more significant than water storage practices in determining water quality and diarrheal disease incidence .
The majority of respondents reported a lack of waterborne sanitation with most reporting the use of pit latrines (Figure 3A) reflective of the community in general (unpublished data). Only one individual reported lack of access and the use of a bush latrine (open air defecation) although lack of sanitation is not uncommon in the study area (unpublished data). It may be that a higher number of individuals did not have access to sanitation facilities but were reluctant to disclose this information during an interview. Previous studies have identified the benefits of sanitation on diarrhea and other diseases with more significant reductions achieved with flush toilets than pit latrines . However, access to flush toilets may not be possible even with heavy government investment in sanitation infrastructure as poorer people are often still unable to identify funds necessary for connection fees and development of associated infrastructure. Simple pit latrines, commonly used in the area may also, however, provide increased excreta access for flies, increasing fly population density and potential fly borne transmission of disease causing diarrheal organisms (reviewed ). Heavy rainfall events and overflowing of pit latrines has also been linked to the occurrence of major diarrheal outbreaks where high levels of morbidity and mortality occurred .
Pit latrines have also been associated with declines in water quality through bacterial contamination and nitrate leaching of water resources (reviewed in ). Saturated soils can facilitate this process with microorganism movement occurring up to several hundred meters through subsurface water flow [61–63]. The complicated manner in which sanitation may influence health in the region is a critical area of future research.
Appraisal of approach, limitations, and lessons learned
Our study data were available prior to collation of summary patient attendance data and provided insight into the relative magnitude of the outbreak and unique data on the characteristics of patients involved in the respective outbreaks.
The most important elements influencing the effectiveness of this method appeared to be motivation of individual and particularly, senior staff, appropriate variable selection crafted to the area and questions at hand, and the need to keep the instrument short in order to increase its use during outbreak periods where higher patient - staff ratios are experienced. Improved delivery and return of questionnaires and medical staff participation may be enhanced if implemented through existing institutional and hierarchical structures and not externally driven with essentially volunteer contributions, as was the case here. Factors influencing participation were not systematically assessed, however, but such an evaluation will be important to improving the approach and replicating it successfully in other medical facilities. Finally, feedback of study results by the lead author to the involved facility medical staff (doctors, nurses, and laboratory staff) and District health officials contributed to a greater understanding of diarrheal disease dynamics and more positive views of the exercise and resultant investment of time. These latter activities are important components of the study approach, increasing the use of study results and identifying local value in such an exercise.
The methods and data acquired here cannot be used to make any characterizations or inferences regarding the larger Chobe population experiencing diarrheal disease and does not take the place of a case–control study. It does, however, provide a mechanism to evaluate patient characteristics during outbreaks, inform future study hypotheses and approaches, and potentially identify immediate public health interventions. This information is acquired without relying on any additional funding or staffing resources. These latter considerations dominate resource poor settings and contribute to a continued lack of information regarding characteristics of disease outbreaks in these areas.