diarrhoea is the second leading cause of death in children aged < 5 years globally with an estimated 25 billion cases and 15 million deaths in this key age group annually . developing countries bear the brunt of diarrhoeal disease burden in both mortality and morbidity [ 2 , 3 ] . the non - typhoidal members of the bacterial genus salmonella ( i.e. excluding salmonella typhi and salmonella paratyphi pathovars ) represent a significant public health problem in industrializing and industrialized countries and have been estimated to account for 15% of all cases of gastroenteritis in the former group [ 3 , 4 ] . however the source and transmission of gastrointestinal non - typhoidal salmonella infections in developing countries are not well documented . infants and young children suffer substantially more from the effects of salmonella infections compared to other age groups and are therefore also at higher risk of secondary complications . obtaining local , specific epidemiological data on enteric pathogens such as salmonella is crucial to understand and combat paediatric diarrhoea . vietnam is a rapidly developing country with a population of more than 88 million people ( over seven million are aged < 5 years ) , a child mortality rate of 24/1000 live births and an average per capita income of about us$1100 per year [ 1 , 8 ] . diarrhoea is the seventh most common cause of death in children aged < 5 years in vietnam and accounts for 4% of the deaths in this age group per year . to assess important epidemiological aspects of diarrhoeal disease in children aged < 5 years resident in ho chi minh city , vietnam , we conducted a large hospital - based case - control study here , we present results regarding the prevalence , clinical presentation , treatment and risk factors associated with symptomatic salmonella diarrhoeal infections in hospitalized children in this large , urban , industrializing setting . the hospital for tropical diseases ( htd ) is the tertiary referral hospital for infectious diseases in ho chi minh city and surrounding provinces in southern vietnam . children 's hospital 1 ( ch1 ) and children 's hospital 2 ( ch2 ) are the two main paediatric hospitals in ho chi minh city . all children aged < 5 years with acute diarrhoeal disease admitted to the gastrointestinal department of ch1 , ch2 and htd from may 2009 to april 2010 in ho chi minh city were eligible for participation in the study . we defined a case of diarrhoea as 3 loose stools or at least one bloody loose stool within a 24-h period . we defined a case of salmonella infection as a diarrhoeal infection stool culture - positive for salmonella spp . due to resource and personnel constraints , the first five patients meeting the inclusion criteria from each of the three study sites on weekdays were included in the study . children were excluded if they did not live within ho chi minh city , had been pre - treated with antimicrobials , had multiple complications unrelated to diarrhoeal disease or did not have informed consent of the parent or guardian . controls were children aged < 5 years from ho chi minh city attending the nutrition wards of ch1 and ch2 from march 2010 to december 2010 . children who had symptoms of diarrhoea or respiratory illness , were currently on an antimicrobial regimen , were aged > 60 months , did not live in a district of ho chi minh city or did not have informed consent of the parent or guardian were excluded . stool specimens for both cases and controls were collected on the day of admission to hospital and prior to any prescribed antimicrobial therapy . all samples were stored in a refrigerator ( 4  c ) and were transported on the same day to the laboratory for analysis . treating clinicians were asked to complete a simple case report form for information on symptoms and duration of disease . in addition , a short questionnaire was administered to the child 's parent or guardian by study nurses to gather information on basic demographics , socioeconomic indicators and potential risk factors for infection . we additionally recorded the location of the residences of the children with salmonella infections using a gps receiver . stool specimens were cultured on macconkey agar ( mc , oxoid ) , xylose - lysine - deoxycholate agar ( xld , oxoid ) , and selenite broth ( oxoid ) media and incubated at 37  c overnight . the overnight selenite broth , which nourished possible salmonella , was subcultured on mc and xld agars for identification of salmonella organisms . identification of potential salmonella was performed using conventional biochemical ( api 20e biochemical strip tests ) according to the standard operating procedure . all isolated pathogens were recorded and kept for future testing by freeze drying and storage at 20  c . data were entered into a database using excel 2007 ( microsoft , usa ) and analysed using stata / ic version 9.2 ( statacorp . and fisher 's exact tests were used to compare proportions between groups and mann  whitney u tests were used for non - parametric data . univariate analyses were performed to assess factors associated with symptomatic non - typhoidal salmonella infection . factors found to be significantly associated with infection in the univariate analysis were then included in a multivariate logistic regression model to simultaneously control for any effects of confounding . evidence of effect modification was also investigated to determine if the effect of one exposure on the outcome of salmonella infection was influenced by other exposures of interest . arcmap software version 9.2 ( esri , usa ) was used for geographical mapping of patient addresses . informed consent was required from all parents or guardians for the child to participate in the study . a total of 1419 children with acute diarrhoea and 571 asymptomatic individuals ( without diarrhoea and with stool samples microbiologically culture negative for salmonella ) were enrolled between may 2009 and december 2010 from the three defined healthcare facilities in central ho chi minh city , vietnam . for 77 ( 54% ) diarrhoea cases , patient stool culture yielded salmonella on the day of hospital admission and therefore met the criteria for a salmonella case . of these , 45 ( 58% ) were serogroup b , ten ( 13% ) were serogroup c , five ( 6% ) were serogroup d , two ( 3% ) were s. arizonae and 15 ( 20% ) were ungroupable . sixty - four per cent of the salmonella diarrhoeal cases and 53% of asymptomatic individuals ( referred hereon as controls ) were male (  test , p  =  0086 ) , as shown in table 1 . patients with salmonella infection were , on average , marginally younger ( median 10 months ) than controls ( median 12 months ) ( mann  whitney u test , p  =  0015 ) , although an equal proportion ( 78% ) of salmonella patients ( 60/77 ) and controls ( 443/571 ) were being actively breastfed , or were breastfed as infants . conversely , a greater proportion of controls ( 70/571 , 12% ) were malnourished ( weight for age z score 2 ) , compared to salmonella patients ( 5/77 , 7% ) ( p  <  0001 ) . table 1.baseline , socioeconomic and behavioural characteristics of salmonella diarrhoea cases and asymptomatic controls aged < 5 years from three hospitals in ho chi minh city , vietnamexposuresalmonella patientscontrolsp 
 n  =  77 ( % ) n  =  571 ( % ) male sex6365320087breastfed7797760947age ( months)0379  06247168  712377345  1318195193  19245298  25367886  37482658  49602653weight for age z score < 265123<0001*contact with symptomatic8118<0001*previous illness143700026*residence0852  rural117124  urban883876household monthly income0690 <  us$145169229  us$146242377347  us$243483312273  us$48472578100  >  us$7256551water source0251  government pipe636587  well286366  other||7847  bottled drinking water2082010953  boil water8969350205toilet0018 *  inside818744  outside117249hand washing after using toilet0015 *  always338398  occasionally14995  no / don't know14962  not applicable365447>4 adults in household2992640524>2 children in household2081020006*daycare / nursery1431540773history of probiotic usage137727<0001*market food7535790004*pet in household2472720646 or fisher 's exact test as appropriate.considered malnourished .responses for symptomatic contact from 74 cases , 565 controls ; for toilet use from 72 cases , 567 controls ; for hand washing from 74 cases , 571 controls ; for daycare / nursery from 77 cases , 566 controls ; for probiotics from 31 cases , 394 controls ; and for market food from 73 cases , 570 controls.||rain water , from a government truck during road construction or other source.child in diapers and therefore would not require hand washing.*p value 005 . baseline , socioeconomic and behavioural characteristics of salmonella diarrhoea cases and asymptomatic controls aged < 5 years from three hospitals in ho chi minh city , vietnam  or fisher 's exact test as appropriate . responses for symptomatic contact from 74 cases , 565 controls ; for toilet use from 72 cases , 567 controls ; for hand washing from 74 cases , 571 controls ; for daycare / nursery from 77 cases , 566 controls ; for probiotics from 31 cases , 394 controls ; and for market food from 73 cases , 570 controls . rain water , from a government truck during road construction or other source . profuse watery diarrhoea was most the commonly reported form of diarrhoea , found in 45/77 ( 59% ) patients with salmonella infection followed by 26 ( 34% ) with diarrhoea containing mucus , and six ( 8% ) with bloody diarrhoea ( table 2 ) . mild ( 37239  c ) and severe ( > 39  c ) fever were common , and the majority of children experienced vomiting . illness prior to hospitalization occurred for a median of 2 days [ interquartile range ( iqr ) 23 ] and children remained in hospital for a median of 5 days ( iqr 38 ) , with discharge permitted only by a treating clinician on the observation of the cessation of symptoms . attending clinicians prescribed treatment to salmonella patients prior to a microbiological stool investigation or an available antimicrobial susceptibility profile . in total , 675% ( 52/77 ) of salmonella infections were prescribed an antimicrobial by the attending physician during their hospital stay . over half ( 38/52 , 73% ) of the prescribed antimicrobials were fluoroquinolones , of which the most commonly used were ciprofloxacin ( 23/52 , 44% ) and norfloxacin ( 10/52 , 19% ) . additionally , rehydration therapy and probioitic supplements were administered to the majority of patients ( lyophilized probiotics reconstituted in a beverage ) . sixty - four per cent ( 33/52 ) of those prescribed antimicrobials were prescribed probiotic therapy concurrently with the antimicrobials . table 2.clinical features of salmonella infections in vietnamese children hospitalized with diarrhoea ( n  =  77)diarrhoea typen%  bloody678  mucoid26338  watery45585patient characteristicsmedianiqr  average no . episodes / day538  length of illness * , days223  length of stay in hospital , days538symptomsn%  fever58754  abdominal pain13169  anorexia35455  cough22286  dehydration565  headache113  vomiting50649iqr , interquartile range.*prior to hospitalization . clinical features of salmonella infections in vietnamese children hospitalized with diarrhoea ( n  =  77 ) iqr , interquartile range . prior to hospitalization . the south of vietnam has two main seasons : a wet season from may to november and a humid dry season from december to april . the proportion of diarrhoea cases presenting to hospital that had a culture - confirmed diagnosis for salmonella was the highest in august ( 12/193 , 129% ) and the lowest in february ( 2/80 , 23% ) . we did not identify a strong association between salmonella infections and average monthly temperature ( spearman 's correlation coefficient r  =  0389 , p  =  0080 ) and there appeared to be no association between salmonella infection and average monthly rainfall ( spearman 's r  =  014 , p  =  0665 ) . the gps coordinates of each case were plotted and case counts per district revealed the highest proportion of salmonella patients ( 12/77 , 16% ) lived in district 8 in ho chi minh city ( fig . however , when comparing the ratio of salmonella cases to cases of diarrhoea caused by other bacterial or viral agents collected for 20092010 ( fig . 1b ) , less centrally located districts such as district 11 , tan binh and binh thanh seemed to have proportionally heavier burdens of salmonella infection with ratios of salmonella culture - positive diarrhoeal cases to culture - negative cases of 018 ( 7/39 ) , 015 ( 8/52 ) and 013 ( 9/69 ) , respectively . 
 fig . the geographical distribution of salmonella cases in children aged < 5 years admitted to the three study hospitals in central ho chi minh city . the hospital locations are as follows :  children 's hospital 1 ;   children 's hospital 2 ;  hospital for tropical diseases . ( a ) proportion of total salmonella cases from each district ; ( b ) ratio of cases of salmonella to cases of another bacterial or viral aetiology . the geographical distribution of salmonella cases in children aged < 5 years admitted to the three study hospitals in central ho chi minh city . the hospital locations are as follows :  children 's hospital 1 ;   children 's hospital 2 ;  hospital for tropical diseases . ( a ) proportion of total salmonella cases from each district ; ( b ) ratio of cases of salmonella to cases of another bacterial or viral aetiology . responses from the questionnaire administered to the parents / guardians of both cases and controls were evaluated to determine trends in socioeconomic or behavioural factors in the overall population as well as any important differences between the two groups . from the resulting questionnaire data , a low proportion of children reported having been in recent contact with an individual symptomatic for diarrhoea or a recent previous diarrhoeal episode , although children with salmonella infection were more likely to have reported both ( table 1 ) . in regard to sanitation and water supply , over half of salmonella patients ( 49/77 , 64% ) and controls ( 335/571 , 59% ) used a municipal government pipeline , which was followed less commonly by wells , 29% ( 22/77 ) and 37% ( 209/571 ) , respectively . roughly 20% of children with salmonella infection ( 16/77 ) and controls ( 117/571 ) reported drinking only bottled water , while almost all salmonella patients ( 69/77 , 90% ) and controls ( 534/571 , 94% ) reported boiling their water prior to drinking . hand washing behaviour differed between the groups , with those positive for salmonella more commonly reporting not washing hands or not knowing if hands were washed ( 36/71 , 15% ) than controls ( 35/571 , 6% ) . yet surprisingly , salmonella patients were more likely to have an indoor toilet ( 63/72 , 82% ) compared to controls ( 142/567 , 74% ) . in order to assess general living conditions , we evaluated the level of household crowding by measuring the proportion of salmonella patients and controls who reported having more than the median number of adults ( n  =  4 ) and children ( n  =  2 ) in the household , as estimated from our larger population of 1419 diarrhoeal cases and 571 controls . the households of ~30% of salmonella patients ( 23/77 ) and controls ( 151/571 ) had more than four adults yet the patients ' households had greater than the median of two children more frequently ( 16/77 , 21% ) than controls ' households ( 57/571 , 10% ) . additionally , monthly income distributions were comparable between salmonella patients and controls , with the majority of households ( cases and controls ) having an income of between us$145 and us$480 per month . more children with salmonella reported living in households regularly purchasing meat and vegetables from outdoor markets ( 55/73 , 75% ) compared to only 58% ( 330/570 ) of controls . pet ownership ( mainly cats and dogs ) , was ~25% in children with salmonella infection ( 19/77 ) and controls ( 155/571 ) . finally , salmonella patients reported regularly consuming probiotics prior to diarrhoea much less frequently ( 7/51 , 14% ) than controls ( 280/440 , 64% ) . symptomatic salmonella gastrointestinal infection was associated with having contact with a recently symptomatic individual [ odds ratio ( or ) 490 , 95% confidence interval ( ci ) 17139 ] , living in a household where meat and vegetables were primarily purchased at an outdoor market ( or 222 , 95% ci 1339 ) , recent previous diarrhoeal illness ( or 221 95% ci 1145 ) , not washing hands or not knowing if hands were washed after using the toilet ( or 267 , 95% ci 1355 ) , age ( or 098 , 95% ci 096100 ) and having > 2 children living in the household ( or 232 , 95% ci 1248 ) . the type of toilet ( indoor / outdoor ) present in the household was also found to be important . living in an urban or rural district modified the effect of toilet type on the risk of contracting a salmonella infection ( test for homogeneity of ors , p  =  0038 ) such that living in a household with an outdoor toilet was not a risk factor in rural districts ( or 241 , 95% ci 0691 ) , whereas it appeared to be strongly protective for those living in urban districts ( or 024 , 95% ci 0106 ) . after controlling for the factors that were found to be statistically significant in the univariate analysis as listed above , several risk factors remained independently associated with salmonella infections in the multivariate model ( table 3 ) . these risk factors included having had a symptomatic contact [ adjusted or ( aor ) 598 , 95% ci 18204 ] , age ( aor 097 , 95% ci 094099 ) , living in a household where meat and vegetables were primarily purchased at an outdoor market ( aor 227 , 95% ci 1242 ) , having > 2 children in the household ( aor 232 , 95% ci 12447 ) individuals with an outdoor toilet living in predominantly urban districts ( aor 025 , 95% ci 009072 ) . table 3.selected univariate and multivariate analysis of risk factors for symptomatic salmonella gastroenteritisrisk factoror95% ciaor95% ciprevious illness221108452177073426symptomatic contact3141128835981762036age ( months)098096100097094099absence of hand washing267129553199085463purchasing market food222127388227122424>2 children in household232126429232115467outside toilet  urban027011070025009072  rural167041681153030786or , odds ratio ; c , : confidence interval ; aor , adjusted odds ratio . selected univariate and multivariate analysis of risk factors for symptomatic salmonella gastroenteritis or , odds ratio ; c , : confidence interval ; aor , adjusted odds ratio . the epidemiology of gastrointestinal infections caused by salmonella has been extensively studied in developed countries but there is a paucity of data regarding the prevalence and potential transmission routes of salmonella in developing countries . this is the first study to exclusively evaluate the epidemiology and risk factors of non - typhoidal salmonella gastrointestinal infections in children in vietnam . we found that fever , anorexia , vomiting and either watery or mucoid diarrhoea were all common features of patients with salmonella infections , which are typical globally of salmonella infection presentation . cases of salmonella were not found to be associated with average monthly temperature in ho chi minh city , although there was a proportional increase during the warmer months of the year . previous studies conducted in the uk and australia have shown that a higher mean ambient temperature leads to an increase in the number of salmonellosis notifications , possibly through an increase in bacterial reproduction at various points along the food chain [ 11 , 12 ] . although , whether their findings are an important transmission factor in a developing - country setting remains unclear currently . additionally , although it is important to consider that our geographical data is biased as a consequence of hospital referral patterns , the highest proportion of salmonella patients came from district 8 of ho chi minh city . this district is the area of the city with the greatest density of canals , waterways and is one of the districts with the largest number of temporary urban settlements . proximity to potentially contaminated water and related poor sanitary conditions may explain the high proportion of salmonella infections from persons living in this area . with respect to our controls , young children with salmonella infections were more likely to have had a symptomatic contact , to be younger , to live in a household where food is purchased primarily at an outdoor market , to live with at least three children in the household and were more likely to use an outdoor rather than an indoor toilet in urban areas . our findings regarding the young age of infection are consistent with other studies , which have shown that younger children are at greater risk , and have more frequent salmonella infections than children in older age groups . the majority of salmonella infections in this study were from infants aged < 1 year , with the peak of infections in those aged between 7 and 12 months . potential reasons for increased susceptibility to salmonella infections include numerous host factors , such as decreased gastric acidity , immaturity of the gut - associated lymphoid tissue or a lack of serogroup - specific maternal antibodies . additionally , research conducted in malawi has demonstrated that children aged between 4 months and 2 years are at increased risk of invasive non - typhoidal salmonella infection due in part to a lack of salmonella - specific antibody , impaired bactericidal activity and waning maternal igg [ 15 , 16 ] , although mechanisms may differ for disease limited to the gastrointestinal tract . previous studies have identified salmonella in the environment of patients with recently identified infections and that multiple cases of the same salmonella strain may occur in the same household , suggesting the potential for intra - household transmission [ 1719 ] . in a large case - control study using hospital laboratory reports in the usa from 2002 to 2004 , however , having had contact with a symptomatic individual is difficult to interpret as it is often unclear if the contact had the same infection , if the period of infectiousness of the contact coincides with the timing of the patient 's illness and whether a shared exposure is responsible for the association . we observed that cases were more likely to have several children living in the same household , which has also been shown to be a risk factor in studies of other enteric pathogens in the congo and bangladesh [ 20 , 21 ] . it is reasonable to suggest that multiple children living in a single household may lead to an increase in the frequency of salmonella transmission , as children aged < 5 years are more likely to be infected and also tend to shed asymptomatically for protracted time periods which would broaden the potential transmission window . the possibility of different primary patterns of salmonella transmission by age warrants more attention . transmission occurs through the food chain , direct person - to - person salmonella transmission has been implicated as a more important transmission route in some non - industrialized countries . we found that living in a household where food was purchased at outdoor markets was a risk factor , a consequence of substandard food handling , storage and preparation practices at such settings . very young children are less likely to eat solid food , so it is likely that a primary risk factor for symptomatic salmonella is by a direct exposure from either the contaminated environment or family members , not specific food products . therefore , ensuring a clean local environment and adequate personal hygiene of caregivers are critical prevention measures to limit symptomatic salmonella infections in very young children in this population . antimicrobials are not universally recommended for the treatment of gastrointestinal non - typhoidal salmonella infections specifically or for treating non - bloody diarrhoea in children in general . treatment with antimicrobials is only advised for children with suspected or confirmed septicaemia or additional secondary complications . almost 70% of the salmonella - infected individuals in this study received at least one antimicrobial , most commonly a fluoroquinolone , which contradicts internationally recognized guidelines . in fact , some data suggest that antimicrobials may actually prolong shedding of the pathogen in the stool , and will presumably increase the potential for the development of antimicrobial resistance . resistance has implications for treatment failure , increasing treatment costs and protracted therapy for infections that do require an antimicrobial , as second - line drugs are often more expensive and typically require a longer treatment . furthermore , the treating clinicians prescribed probiotics to over 60% of salmonella - infected patients , with almost half of the patients concurrently receiving probiotics and antimicrobials . diagnostics are seldom performed for diarrhoea in settings like vietnam and patients are prescribed therapy based on clinical presentation and prior to microbiological culture result . more stringent treatment guidelines and the restriction of access to antimicrobials in the community would help to ensure more appropriate antimicrobial practices . our analysis suggests that probiotics have a significant prophylactic effect against symptomatic salmonella in this study . in vietnam , probiotics vary substantially but generally consist of a lyophilized lactobacillus spp . in a single - dose sachet , which is normally reconstituted in water or milk prior to consumption . the use of probiotics was not included in the multivariate model due to possible biases introduced by limitations in study design . as controls were collected from the nutritional ward , parents of these children may have been likely to give their child a product supplemented with probiotics ( very popular and inexpensive in vietnam ) in a specific effort to care for the nourishment of their child . parents of diarrhoeal patients may have been less concerned with nutritional issues and therefore may have been less likely to give their child probiotics . however , the strong effect found in the univariate model warrants further scrutiny . a recent large community - based , randomized , double - blind , placebo - controlled trial was conducted in an urban settlement in kolkata , india , demonstrating that a daily intake of probiotics resulted in significant protection against acute diarrhoea in children aged < 5 years with an overall protective efficacy of 14% ( 95% ci 423% , p these results are positive , as this was the first large , randomized controlled trial for a prophylactic effect of probiotics conducted in a developing country . a recent cochrane review regarding the use of probiotics for treating acute diarrhoea found probiotics to be associated with a reduction in risk and duration of diarrhoeal diseases in children , adding additional plausibility to our observation . there were several limitations to our study , the most important of which is that this data was entirely from passive detection and was dependent on the healthcare - seeking behaviour of the patients . as such , much of the burden of infection due to salmonella could remain undocumented , including those less severe cases that did not require medical care . uncollected differences could have been present between the cases and controls as well , as controls were only collected from two hospitals due to logistical reasons . we found that these individuals were more likely to have a lower weight - for - age z score than the cases which could potentially introduce biases as nutritional state tends to play a strong role in susceptibility to diarrhoeal infections and could skew noted epidemiological associations through influences of unknown confounders . or it is possible that controls were likely to have already acquired salmonella infection which could have contributed to their poor nutritional status . we surmise that while our findings should be generalized with caution , our study provides a reasonable estimate of the proportion of salmonella - associated diarrhoea in hospitalized children and highlights some related risk factors for children resident in ho chi minh city . our findings imply that salmonella is a common cause of paediatric gastroenteritis in this setting and that transmission may occur through direct human contact within the household , offering some palpable and tractable prospective routes for more focused epidemiological investigations in locations in other rapidly developing cities in asia .
summarynon - typhoidal salmonella are an important but poorly characterized cause of paediatric diarrhoea in developing countries . 
 we conducted a hospital - based case - control study in children aged < 5 years in ho chi minh city to define the epidemiology and examine risk factors associated with salmonella diarrhoeal infections . from 1419 diarrhoea cases and 571 controls enrolled between 2009 and 2010 , 77 ( 54% ) diarrhoea cases were stool culture - positive for non - typhoidal salmonella . 
 salmonella patients were more likely to be younger than controls ( median age 10 and 12 months , respectively ) [ odds ratio ( or ) 097 ; 95% confidence interval ( ci ) 094099 ] , to report a recent diarrhoeal contact ( 81% cases , 18% controls ; or 598 , 95% ci 18204 ) and to live in a household with > 2 children ( cases 208% , controls 102% ; or 232 , 95% ci 1247 ) . 
 our findings indicate that salmonella are an important cause of paediatric gastroenteritis in this setting and we suggest that transmission may occur through direct human contact in the home .