Conjugative transfer of multi-drug resistance IncN plasmids from environmental waterborne bacteria to Escherichia coli.

Watersheds contaminated with municipal, hospital, and agricultural residues are recognized as reservoirs for bacteria carrying antibiotic resistance genes (ARGs). The objective of this study was to determine the potential of environmental bacterial communities from the highly contaminated La Paz River basin in Bolivia to transfer ARGs to an Escherichia coli lab strain used as the recipient. Additionally, we tested ZnSO4 and CuSO4 at sub-inhibitory concentrations as stressors and analyzed transfer frequencies (TFs), diversity, richness, and acquired resistance profiles. The bacterial communities were collected from surface water in an urban site close to a hospital and near an agricultural area. High transfer potentials of a large set of resistance factors to E. coli were observed at both sites. Whole-genome sequencing revealed that putative plasmids belonging to the incompatibility group N (IncN, IncN2, and IncN3) were predominant among the transconjugants. All IncN variants were verified to be mobile by a second conjugation step. The plasmid backbones were similar to other IncN plasmids isolated worldwide and carried a wide range of ARGs extensively corroborated by phenotypic resistance patterns. Interestingly, all transconjugants also acquired the class 1 integron intl1, which is commonly known as a proxy for anthropogenic pollution. The addition of ZnSO4 and CuSO4 at sub-inhibitory concentrations did not affect the transfer rate. Metal resistance genes were absent from most transconjugants, suggesting a minor role, if any, of metals in the spread of multidrug-resistant plasmids at the investigated sites.

Pharmaceutical pollution of the world's rivers.

Environmental exposure to active pharmaceutical ingredients (APIs) can have negative effects on the health of ecosystems and humans. While numerous studies have monitored APIs in rivers, these employ different analytical methods, measure different APIs, and have ignored many of the countries of the world. This makes it difficult to quantify the scale of the problem from a global perspective. Furthermore, comparison of the existing data, generated for different studies/regions/continents, is challenging due to the vast differences between the analytical methodologies employed. Here, we present a global-scale study of API pollution in 258 of the world's rivers, representing the environmental influence of 471.4 million people across 137 geographic regions. Samples were obtained from 1,052 locations in 104 countries (representing all continents and 36 countries not previously studied for API contamination) and analyzed for 61 APIs. Highest cumulative API concentrations were observed in sub-Saharan Africa, south Asia, and South America. The most contaminated sites were in low- to middle-income countries and were associated with areas with poor wastewater and waste management infrastructure and pharmaceutical manufacturing. The most frequently detected APIs were carbamazepine, metformin, and caffeine (a compound also arising from lifestyle use), which were detected at over half of the sites monitored. Concentrations of at least one API at 25.7% of the sampling sites were greater than concentrations considered safe for aquatic organisms, or which are of concern in terms of selection for antimicrobial resistance. Therefore, pharmaceutical pollution poses a global threat to environmental and human health, as well as to delivery of the United Nations Sustainable Development Goals.

Development and performance evaluation of an In-House ELISA for the detection of group A rotavirus in diarrheal stool samples from children and domestic South American camelids.

Group A rotavirus (RVA) is a prevalent pathogen causing acute gastroenteritis (AGE) in young children and animals. We developed an in-house ELISA (ROTA-GeFeK) for RVA detection, based on the expression of native recombinant VP6 protein in E. coli. To detect the RVA antigen, rabbit polyclonal IgG antibodies, produced against rVP6,were used as capture and detector antibodies in a sandwich ELISA. To validate the ROTA-GeFeK, 252 stool samples from children with AGE, were evaluated by conventional RT-PCR and commercial ELISA. Compared to RT-PCR, the ROTAGeFeK had a sensitivity of 88.2 % and a specificity of 94.4 %. Total detection rates with the ROTA-GeFeK, commercial ELISA and RT-PCR were 58 %, 58 % and 64 % respectively. The limit of detection was equal to 2.1 × 10 4 CCID 50 of the RVA strain RIX4414. No cross-reactivity with other enteric pathogens was observed. The RVApositive samples detected by the assay belonged to a diversity of G and [P] genotypes.This assay displayed reactivity and was proved to be useful for the detection of RVA in diarrheal samples of domestic South American Camelids. We suggest that the ROTAGeFeK can be used as an epidemiologic tool for rotavirus surveillance and for RVA detection in other animal species.

Diarrheal bacterial pathogens and multi-resistant enterobacteria in the Choqueyapu River in La Paz, Bolivia.

Water borne diarrheal pathogens might accumulate in river water and cause contamination of drinking and irrigation water. The La Paz River basin, including the Choqueyapu River, flows through La Paz city in Bolivia where it is receiving sewage, and residues from inhabitants, hospitals, and industry. Using quantitative real-time PCR (qPCR), we determined the quantity and occurrence of diarrheagenic Escherichia coli (DEC), Salmonella enterica, Klebsiella pneumoniae, Shigella spp. and total enterobacteria in river water, downstream agricultural soil, and irrigated crops, during one year of sampling. The most abundant and frequently detected genes were gapA and eltB, indicating presence of enterobacteria and enterotoxigenic E. coli (ETEC) carrying the heat labile toxin, respectively. Pathogen levels in the samples were significantly positively associated with high water conductivity and low water temperature. In addition, a set of bacterial isolates from water, soil and crops were analyzed by PCR for presence of the genes blaCTX-M, blaKPC, blaNDM, blaVIM and blaOXA-48. Four isolates were found to be positive for blaCTX-M genes and whole genome sequencing identified them as E. coli and one Enterobacter cloacae. The E. coli isolates belonged to the emerging, globally disseminated, multi-resistant E. coli lineages ST648, ST410 and ST162. The results indicate not only a high potential risk of transmission of diarrheal diseases by the consumption of contaminated water and vegetables but also the possibility of antibiotic resistance transfer from the environment to the community.

Effect of infant feeding practices on iron status in a cohort study of Bolivian infants.

BACKGROUND: Iron deficiency (ID) is the most common micronutrient deficiency worldwide, with potentially severe consequences on child neurodevelopment. Though exclusive breastfeeding (EBF) is recommended for 6 months, breast milk has low iron content. This study aimed to estimate the effect of the length of EBF on iron status at 6 - 8 months of age among a cohort of Bolivian infants. METHODS: Mother-infant pairs were recruited from 2 hospitals in El Alto, Bolivia, and followed from one through 6 - 8 months of age. Singleton infants > 34 weeks gestational age, iron-sufficient at baseline, and completing blood draws at 2 and 6 - 8 months of age were eligible for inclusion (N = 270). Ferritin was corrected for the effect of inflammation. ID was defined as inflammation-corrected ferritin < 12 µg/L, and anemia was defined as altitude-corrected hemoglobin < 11 g/dL; IDA was defined as ID plus anemia. The effect of length of EBF (infant received only breast milk with no other liquids or solids, categorized as < 4, 4 - 6, and > 6 months) was assessed for ID, IDA, and anemia (logistic regression) and ferritin (Fer) and hemoglobin (Hb, linear regression). RESULTS: Low iron status was common among infants at 6 - 8 months: 56% of infants were ID, 76% were anemic, and 46% had IDA. EBF of 4 months and above was significantly associated with ID as compared with EBF <  4 months (4 - 6 months: OR 2.0 [1.1 - 3.4]; > 6 months: 3.3 [1.0 - 12.3]), but not with IDA (4 - 6 months: OR 1.4 [0.8 - 2.4]; > 6 months: 2.2 [0.7 - 7.4]), or anemia (4 - 6 months: OR 1.4 [0.7 - 2.5]; > 6 months: 1.5 [0.7 - 7.2]). Fer and Hb concentrations were significantly lower with increasing months of EBF. CONCLUSIONS: Results suggest a relationship between prolonged EBF and ID, but are not sufficient to support changes to current breastfeeding recommendations. More research is needed in diverse populations, including exploration of early interventions to address infant IDA.

Using a monitoring and evaluation framework to improve study efficiency and quality during a prospective cohort study in infants receiving rotavirus vaccination in El Alto, Bolivia: the Infant Nutrition, Inflammation, and Diarrheal Illness (NIDI) study.

BACKGROUND: Implementing rigorous epidemiologic studies in low-resource settings involves challenges in participant recruitment and follow-up (e.g., mobile populations, distrust), biological sample collection (e.g., cold-chain, laboratory equipment scarcity) and data collection (e.g., literacy, staff training, and infrastructure). This article describes the use of a monitoring and evaluation (M&E) framework to improve study efficiency and quality during participant engagement, and biological sample and data collection in a longitudinal cohort study of Bolivian infants. METHODS: The study occurred between 2013 and 2015 in El Alto, Bolivia, a high-altitude, urban, low-resource community. The study's M&E framework included indicators for participant engagement (e.g., recruitment, retention, safety), biological sample (e.g., stool and blood), and data (e.g., anthropometry, questionnaires) collection and quality. Monitoring indicators were measured regularly throughout the study and used for course correction, communication, and staff retraining. RESULTS: Participant engagement indicators suggested that enrollment objectives were met (461 infants), but 15% loss-to-follow-up resulted in only 364 infants completing the study. Over the course of the study, there were four study-related adverse events (minor swelling and bruising related to a blood draw) and five severe adverse events (infant deaths) not related to study participation. Biological sample indicators demonstrated two blood samples collected from 95% (333 of 350 required) infants and stool collected for 61% of reported infant diarrhea episodes. Anthropometry data quality indicators were extremely high (median SDs for weight-for-length, length-for-age and weight-for-age z-scores 1.01, 0.98, and 1.03, respectively), likely due to extensive training, standardization, and monitoring efforts. CONCLUSIONS: Conducting human subjects research studies in low-resource settings often presents unique logistical difficulties, and collecting high-quality data is often a challenge. Investing in comprehensive M&E is important to improve participant recruitment, retention and safety, and sample and data quality. The M&E framework from this study can be applied to other longitudinal studies.

Impact of rotavirus vaccination on child mortality, morbidity, and rotavirus-related hospitalizations in Bolivia.

BACKGROUND: The public health impact of rotavirus vaccination in countries with high child mortality rates remains to be established. The RV1 rotavirus vaccine was introduced in Bolivia in August 2008. This study describes the trends in deaths, hospitalizations, and healthcare visits due to acute gastroenteritis (AGE) and in rotavirus-related hospitalizations, among children <5 years of age, during the pre- and post-vaccination periods. METHODS: Data were obtained from the National Health Information System to calculate vaccine coverage and AGE-related health indicators. Trend reductions in the main health indicators were examined using the pre-vaccine period as baseline. The effect of vaccination on the epidemiology of rotavirus-related AGE was assessed using data from the active surveillance hospitals. RESULTS: Compared with the 2001-2008 pre-vaccine baseline, the mean number of rotavirus-related hospitalizations was reduced by 40.8% (95% confidence interval (CI) 21.7-66.4%) among children <5years of age in the post-vaccine period (2009-2013). Reductions were most pronounced in children <1year of age, eligible for vaccination. The mean proportions of AGE-related deaths, AGE-related hospitalizations, and AGE-related healthcare visits during 2009-2014 were reduced by 52.5% (95% CI 47.4-56.3), 30.2% (95% CI 23.5-36.1), and 12.9% (95% CI 12.0-13.2), respectively. The greatest effect in reduction of AGE-related deaths was found during the months with seasonal peaks of rotavirus disease. Over the post-vaccine period, changes in rotavirus epidemiology were observed, manifested by variations in seasonality and by a shift in the mean age of those with rotavirus infection. CONCLUSIONS: The significant decrease in main AGE-related health indicators in children <5years of age after the introduction of rotavirus vaccine provides evidence of a substantial public health impact of rotavirus vaccination in Bolivia, as a measure for protecting children against AGE.

Early deterioration of iron status among a cohort of Bolivian infants.

Iron deficiency (ID) and iron deficiency anemia (IDA) are major contributors to infant and maternal morbidity worldwide. There is limited longitudinal data on iron status in young infants and on methods to adjust iron biomarkers for inflammation. We aimed to quantify the prevalence of inflammation-adjusted ID, anemia, and IDA over the first year in a cohort of Bolivian infants and their mothers. Healthy mother-infant dyads were recruited from two peri-urban hospitals. Infants provided three blood draws (2, 6-8, and 12-18 months; N = 160); mothers provided two blood draws (1 and 6-8 months postpartum [plus third anemia measurement at 12-18 months]; N = 250). Blood was analyzed for hemoglobin, ferritin, soluble transferrin receptor, C-reactive protein (CRP), and alpha(1)-acid glycoprotein (AGP). Iron biomarkers were adjusted for inflammation using CRP and AGP; hemoglobin cutoffs were adjusted for altitude. Inflammation (elevated CRP or AGP) was 17% among toddlers 12-18 months of age. ID (inflammation-adjusted ferritin) increased with age (<1%, 56%, and 79% at each blood draw), as did anemia and IDA (anemia: 70%, 76%, and 81%; IDA: <1%, 46%, and 68%). Maternal ID declined from the first to second assessment (39% vs. 27%). Inflammation-adjusted ID prevalence was up to 15 percentage points higher than unadjusted estimates. The high prevalence of ID, anemia, and IDA in this cohort of Bolivian infants beginning at 6-8 months of age suggests that early interventions may be necessary in vulnerable populations.

Predictors of Inflammation in a Cohort of Bolivian Infants and Toddlers.

Inflammation has been associated with cardiovascular disease and other health outcomes in children and adults, yet few longitudinal data are available on prevalence and predictors of inflammation in infants. We aimed to identify the prevalence of inflammation in a cohort of Bolivian infants and estimate its association with acute (recent illnesses) and chronic (overweight, stunting) morbidities and potential pathogen exposure (represented by water, sanitation, and hygiene [WASH] resources). We measured plasma concentrations of two acute phase proteins (C-reactive protein [CRP], marking acute inflammation, and alpha(1)-acid-glycoprotein [AGP], marking chronic inflammation) at three time points (target 2, 6-8, and 12-18 months). Of 451 singleton infants enrolled in the parent study, 272 had the first blood draw and complete data. Anthropometry and sociodemographic and recent illness data (2-week recall of cough, diarrhea, and fever) were collected at each visit. Inflammation was defined as CRP > 5 mg/L or AGP > 1 g/L. The prevalence of inflammation increased from early infancy (3% at first blood draw) to later infancy (15-22% at later blood draws). Recent cough, recent fever, and age in months were significantly associated with relative increases in CRP (7-44%) and AGP (5-23%), whereas recent diarrhea was only significantly associated with an increase in CRP (48%). Neither anthropometry nor WASH was significantly associated with inflammation. Results confirm the role of recent acute illness in inflammation in infants, and indicate that adiposity and WASH are not as important to inflammation in this age category.

Impact of urban contamination of the La Paz River basin on thermotolerant coliform density and occurrence of multiple antibiotic resistant enteric pathogens in river water, irrigated soil and fresh vegetables.

La Paz River in Andean highlands is heavily polluted with urban run-off and further contaminates agricultural lowlands and downstream waters at the Amazon watershed. Agricultural produce at this region is the main source of vegetables for the major Andean cities of La Paz and El Alto. We conducted a 1 year study, to evaluate microbial quality parameters and occurrence of multiple enteropathogenic bacteria (Enterohemorrhagic E. coli-EHEC, Enteroinvasive E. coli or Shigella-EIEC/Shigella, Enteroaggregative E. coli-EAEC, Enteropathogenic E. coli-EPEC Enterotoxigenic E. coli-ETEC and Salmonella) and its resistance to 11 antibiotics. Four sampling locations were selected: a fresh mountain water reservoir (un-impacted, site 1) and downstream sites receiving wastewater discharges (impacted, sites 2-4). River water (sites 1-4, N = 48), and soil and vegetable samples (site 3, N = 24) were collected during dry (April-September) and rainy seasons (October-March). Throughout the study, thermotolerant coliform density values at impacted sites greatly exceeded the guidelines for recreational and agricultural water uses. Seasonal differences were found for thermotolerant coliform density during dry season in water samples nearby a populated and hospital compound area. In contrast to the un-impacted site, where none of the tested enteropathogens were found, 100 % of surface water, 83 % of soil and 67 % of vegetable samples at impacted sites, were contaminated with at least one enteropathogen, being ETEC and Salmonella the most frequently found. ETEC isolates displayed different patterns of toxin genes among sites. The occurrence of enteropathogens was associated with the thermotolerant coliform density. At impacted sites, multiple enteropathogens were frequently found during rainy season. Among isolated enteropathogens, 50 % were resistant to at least two antibiotics, with resistance to ampicillin, nalidixic acid, trimethoprim-sulfamethoxazole and tetracycline commonly present. Moreover, some Salmonella isolates were distinguished by their multi-resistance to ≥8 antibiotics, within soil and vegetable samples. Overall, this study demonstrates that La Paz River-an affluent of the Amazon macrobasin-is heavily polluted along the year with a high density of thermotolerant coliforms and is a reservoir of multiple antibiotic resistant enteropathogens, present in river water, soil and vegetables. These data highlight health risk associated with food and waterborne diseases at the region.

The economic burden of pediatric gastroenteritis to Bolivian families: a cross-sectional study of correlates of catastrophic cost and overall cost burden.

BACKGROUND: Worldwide, acute gastroenteritis causes substantial morbidity and mortality in children less than five years of age. In Bolivia, which has one of the lower GDPs in South America, 16% of child deaths can be attributed to diarrhea, and the costs associated with diarrhea can weigh heavily on patient families. To address this need, the study goal was to identify predictors of cost burden (diarrhea-related costs incurred as a percentage of annual income) and catastrophic cost (cost burden ≥ 1% of annual household income). METHODS: From 2007 to 2009, researchers interviewed caregivers (n = 1,107) of pediatric patients (<5 years old) seeking treatment for diarrhea in six Bolivian hospitals. Caregivers were surveyed on demographics, clinical symptoms, direct (e.g. medication, consult fees), and indirect (e.g. lost wages) costs. Multivariate regression models (n = 551) were used to assess relationships of covariates to the outcomes of cost burden (linear model) and catastrophic cost (logistic model). RESULTS: We determined that cost burden and catastrophic cost shared the same significant (p < 0.05) predictors. In the logistic model that also controlled for child sex, child age, household size, rural residence, transportations taken to the current visit, whether the child presented with complications, and whether this was the child's first episode of diarrhea, significant predictors of catastrophic cost included outpatient status (OR 0.16, 95% CI [0.07, 0.37]); seeking care at a private hospital (OR 4.12, 95% CI [2.30, 7.41]); having previously sought treatment for this diarrheal episode (OR 3.92, 95% CI [1.64, 9.35]); and the number of days the child had diarrhea prior to the current visit (OR 1.14, 95% CI [1.05, 1.24]). CONCLUSIONS: Our analysis highlights the economic impact of pediatric diarrhea from the familial perspective and provides insight into potential areas of intervention to reduce associated economic burden.

The burden of pediatric diarrhea: a cross-sectional study of incurred costs and perceptions of cost among Bolivian families.

BACKGROUND: Worldwide, acute gastroenteritis represents an enormous public health threat to children under five years of age, causing one billion episodes and 1.9 to 3.2 million deaths per year. In Bolivia, which has one of the lower GDPs in South America, an estimated 15% of under-five deaths are caused by diarrhea. Bolivian caregiver expenses related to diarrhea are believed to be minimal, as citizens benefit from universal health insurance for children under five. The goals of this report were to describe total incurred costs and cost burden associated with caregivers seeking treatment for pediatric gastroenteritis, and to quantify relationships among costs, cost burden, treatment setting, and perceptions of costs. METHODS: From 2007 to 2009, researchers interviewed caregivers (n=1,107) of pediatric patients (<5 years of age) seeking treatment for diarrhea in sentinel hospitals participating in Bolivia's diarrheal surveillance program across three main geographic regions. Data collected included demographics, clinical symptoms, direct costs (e.g. medication, consult fees) and indirect costs (e.g. lost wages). RESULTS: Patient populations were similar across cities in terms of gender, duration of illness, and age, but familial income varied significantly (p<0.05) when stratified on appointment type. Direct, indirect, and total costs to families were significantly higher for inpatients as compared to outpatients of urban (p<0.001) and rural (p<0.05) residence. Consult fees and indirect costs made up a large proportion of total costs. Forty-five percent of patients' families paid ≥1% of their annual household income for this single diarrheal episode. The perception that cost was affecting family finances was more frequent among those with higher actual cost burden. CONCLUSIONS: This study demonstrated that indirect costs due to acute pediatric diarrhea were a large component of total incurred familial costs. Additionally, familial costs associated with a single diarrheal episode affected the actual and perceived financial situation of a large number of caregivers. These data serve as a baseline for societal diarrheal costs before and immediately following the implementation of the rotavirus vaccine and highlight the serious economic importance of a diarrheal episode to Bolivian caregivers.

Prevalence, seasonality and severity of disease caused by pathogenic Escherichia coli in children with diarrhoea in Bolivia.

The prevalence of infection caused by different categories of diarrhoeagenic E. coli (DEC) strains, including enteroaggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC) and enterohaemorrhagic (EHEC) E. coli, in children who suffered from diarrhoea (n = 3943) or did not have diarrhoea (n = 1026) were analysed in two areas in Bolivia over a period of 4 years. We also analysed the seasonality of DEC infections and severity of diarrhoea in children with DEC infection and compared antibiotic resistance in DEC strains isolated from children with and without diarrhoea. Stool samples were analysed for the presence of DEC by culturing followed by PCR. The most prevalent DEC categories in samples from the children were: EAEC (11.2 %); ETEC (6.6 %); EPEC (5.8 %); and EIEC and EHEC (<1 %). DEC strains were isolated significantly more often from diarrhoea cases (21.6 %) than from controls (17.6 %; P = 0.002). The number of children with diarrhoea associated with EAEC, EPEC and ETEC infections peaked in the Bolivian winter (April-September), although the proportion of DEC-positive stool samples was higher during the warm rainy season (October-March). High levels of antibiotic resistance were detected among the DEC strains. In particular, resistance to tetracycline and sulfamethoxazole-trimethoprim was significantly higher in strains isolated from individuals with diarrhoea than in samples from controls. The severity of disease in children infected with EAEC, EPEC and ETEC varied from mild to severe diarrhoea, although disease severity did not differ significantly between the different DEC categories. ETEC, EPEC and EAEC are commonly found in Bolivia and may cause severe disease in children.

Effectiveness of monovalent rotavirus vaccine in Bolivia: case-control study.

OBJECTIVE: To evaluate the effectiveness of two doses of a monovalent rotavirus vaccine (RV1) against hospital admission for rotavirus in Bolivia. DESIGN: Case-control study. SETTING: Six hospitals in Bolivia, between March 2010 and June 2011. PARTICIPANTS: 400 hospital admissions for rotavirus, 1200 non-diarrhea hospital controls, and 718 rotavirus negative hospital controls. MAIN OUTCOME MEASURES: Odds of antecedent vaccination between case patients and controls; effectiveness of vaccination ((1-adjusted odds ratio)×100), adjusted for age and other confounders; and stratified effectiveness by dose, disease severity, age group, and serotype. RESULTS: In comparison with non-diarrhea controls, case patients were more likely to be male and attend day care but less likely to have chronic underlying illness, higher level maternal education, and telephones and computers in their home. Rotavirus negative controls were somewhat more similar to case patients but also were more likely to be male and attend day care and less likely to have higher level maternal education and computers in their homes. The adjusted effectiveness of RV1 against hospital admission for rotavirus was 69% (95% confidence interval 54% to 79%) with rotavirus negative controls and 77% (65% to 84%) with non-diarrhea controls. The effectiveness of one dose of RV1 was 36% and 56%, respectively. With both control groups, protection was sustained through two years of life, with similar efficacy against hospital admission among children under 1 year (64% and 77%) and over 1 year of age (72% and 76%). RV1 provided significant protection against diverse serotypes, partially and fully heterotypic to the G1P[8] vaccine. Effectiveness using the two control groups was 80% and 85% against G9P[8], 74% and 93%% against G3P[8], 59% and 69% against G2P[4], and 80% and 87% against G9P[6] strains. CONCLUSION: The monovalent rotavirus vaccine conferred high protection against hospital admission for diarrhea due to rotavirus in Bolivian children. Protection was sustained through two years of life against diverse serotypes different from the vaccine strain.

Rotavirus genotype distribution during the pre-vaccine period in Bolivia: 2007-2008.

OBJECTIVES: Rotavirus is the most important etiology of severe diarrhea in Bolivia. The monovalent attenuated human oral rotavirus vaccine Rotarix(®) was introduced in Bolivia in 2008. We describe the molecular epidemiology of circulating rotavirus strains before vaccine introduction. METHODS: Two thousand one hundred thirty-five diarrheal samples were collected from hospitals in four Bolivian cities during 2007-2008. Forty-three percent (445 of 1030 rotavirus-positive samples) were analyzed for G and P genotypes. Among those, 331 were electropherotyped by polyacrylamide gel electrophoresis. Disease severity was quantified using a modified Vesikari scale. RESULTS: Among the 445 samples, five genotypes were found to be prevalent: G9P[8] (33%), G1P[6] (17%), G2P[4] (13%), G9P[6] (12%), and G1P[8] (4%). Co-infections with two or more strains accounted for 14% of samples. The most prevalent strain, G9, showed greater electropherotype diversity compared to other serogroups. Strain G1P[6] generally infected younger children and peaked later in the year than other strains. No particular genotype was associated with a higher severity score, though there was a significant difference in the duration of diarrhea between genotypes. CONCLUSIONS: During the 2-year pre-vaccine period, substantial diversity of rotavirus co-circulating strains was observed. These data constitute a baseline against which changes in circulating strains post-vaccine introduction can be monitored.

Molecular characterization of enterotoxigenic Escherichia coli isolates recovered from children with diarrhea during a 4-year period (2007 to 2010) in Bolivia.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of childhood diarrhea. This study aimed to characterize ETEC strains isolated from Bolivian children aged <5 years according to enterotoxin profile, colonization factors (CFs), suggested virulence genes, and severity of disease. A total of 299 ETEC isolates recovered from children with diarrhea and 55 ETEC isolates from children without diarrhea (controls) were isolated over a period of 4 years. Strains expressing heat-labile toxin (LT) or heat-stable toxin (ST) alone were about equally common and twice as common as ETEC producing both toxins (20%). ETEC strains expressing human ST (STh) were more common in children aged <2 years, while ETEC strains expressing LT plus STh (LT/STh) were more frequent in 2- to 5-year-old children. Severity of disease was not related to the toxin profile of the strains. CF-positive isolates were more frequently identified in diarrheal samples than in control samples (P = 0.02). The most common CFs were CFA/I and CS14. CFA/I ETEC strains were more frequent in children aged <2 years than CS1+CS3 isolates and CS14 isolates, which were more prevalent in 2- to 5-year-old children. The presence of suggested ETEC virulence genes (clyA, eatA, tia, tibC, leoA, and east-1) was not associated with disease. However, east-1 was associated with LT/STh strains (P < 0.001), eatA with STh strains (P < 0.001), and tia with LT/STh strains (P < 0.001). A minor seasonal peak of ETEC infections was identified in May during the cold-dry season and coincided with the peak of rotavirus infections; this pattern is unusual for ETEC and may be important for vaccination strategies in Bolivia.

Clonal relatedness of enterotoxigenic Escherichia coli (ETEC) strains expressing LT and CS17 isolated from children with diarrhoea in La Paz, Bolivia.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller's and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNP(bol) in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNP(bol)) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns. CONCLUSION/SIGNIFICANCE: The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors.

Cost-effectiveness of rotavirus vaccination in Bolivia from the state perspective.

BACKGROUND: In Bolivia, in 2008, the under-five mortality rate is 54 per 1000 live births. Diarrhea causes 15% of these deaths, and 40% of pediatric diarrhea-related hospitalizations are caused by rotavirus illness (RI). Rotavirus vaccination (RV), subsidized by international donors, is expected to reduce morbidity, mortality, and economic burden to the Bolivian state. Estimates of illness and economic burden of RI and their reduction by RV are essential to the Bolivian state's policies on RV program financing. The goal of this report is to estimate the economic burden of RI and the cost-effectiveness of the RV program. METHODS: To assess treatment costs incurred by the healthcare system, we abstracted medical records from 287 inpatients and 6751 outpatients with acute diarrhea between 2005 and 2006 at 5 sentinel hospitals in 4 geographic regions. RI prevalence rates were estimated from 4 years of national hospital surveillance. We used a decision-analytic model to assess the potential cost-effectiveness of universal RV in Bolivia. RESULTS: Our model estimates that, in a 5-year birth cohort, Bolivia will incur over US$3 million in direct medical costs due to RI. RV reduces, by at least 60%, outpatient visits, hospitalizations, deaths, and total direct medical costs associated with rotavirus diarrhea. Further, RV was cost-savings below a price of US$3.81 per dose and cost-effective below a price of US$194.10 per dose. Diarrheal mortality and hospitalization inputs were the most important drivers of rotavirus vaccine cost-effectiveness. DISCUSSION: Our data will guide Bolivia's funding allocation for RV as international subsidies change.

Enterotoxins, colonization factors, serotypes and antimicrobial resistance of enterotoxigenic Escherichia coli (ETEC) strains isolated from hospitalized children with diarrhea in Bolivia.

UNLABELLED: Enterotoxigenic Escherichia coli (ETEC) is recognized as the main cause of bacterial diarrhoea among children in Asia, Africa and Latin America but less investigated in Bolivia. OBJECTIVE: To determine the relation between enterotoxins, CFs and serotypes as well as the antimicrobial resistance patterns in a set of ETEC isolates collected from hospitalized children with acute diarrhea. In the present study we characterized 43 ETEC strains isolated from 2002 to 2006 from hospitalized children (0-5 years) with acute diarrhea in Bolivia. The strains were analyzed for heat-labile (LT) and heat-stable (ST) enterotoxins and colonization factor (CF) profiles, as well as for serogroups and antimicrobial resistance using phenotypic (ELISA, dot blot, slide agglutination and disc diffusion) and genotypic (Multiplex PCR) methods. Among the ETEC isolates tested, 30 were positive for LT, 3 for STh and 10 for LT/STh. Sixty-five percent (28/43) of the strains expressed one or more CF. The most common CFs were CS17 (n = 8) and CFA/I (n = 8). The phenotypical and genotypical results for toxins and CFs were congruent except for CS21 that was amplified in 10 of the strains by multiplex PCR, but CS21 pili was only detected phenotypically in four of these strains. The ETEC strains had diverse O and H antigens and the most common types were O8:H9 LT CS17 (n = 6; 14%) and O78:HNM LT-ST CFA/I (n = 4; 9%). The analysis of antibiotic resistance showed that 67% (n = 29/43) of the strains were resistant to one or several of the antimicrobial agents tested. Presence of CFs was associated with antibiotic resistance. CONCLUSION: The most common toxin profile was LT 70%, LT/STh 23% and STh 7%. High antimicrobial resistance to ampicillin among serogroups O6, O8 and O78 were the most common.

Enterotoxins, colonization factors, serotypes and antimicrobial resistance of enterotoxigenic Escherichia coli (ETEC) strains isolated from hospitalized children with diarrhea in Bolivia

Enterotoxigenic Escherichia coli (ETEC) is recognized as the main cause of bacterial diarrhoea among children in Asia, Africa and Latin America but less investigated in Bolivia. OBJECTIVE: To determine the relation between enterotoxins, CFs and serotypes as well as the antimicrobial resistance patterns in a set of ETEC isolates collected from hospitalized children with acute diarrhea. In the present study we characterized 43 ETEC strains isolated from 2002 to 2006 from hospitalized children (0-5 years) with acute diarrhea in Bolivia. The strains were analyzed for heat-labile (LT) and heat-stable (ST) enterotoxins and colonization factor (CF) profiles, as well as for serogroups and antimicrobial resistance using phenotypic (ELISA, dot blot, slide agglutination and disc diffusion) and genotypic (Multiplex PCR) methods. Among the ETEC isolates tested, 30 were positive for LT, 3 for STh and 10 for LT/STh. Sixty-five percent (28/43) of the strains expressed one or more CF. The most common CFs were CS17 (n = 8) and CFA/I (n = 8). The phenotypical and genotypical results for toxins and CFs were congruent except for CS21 that was amplified in 10 of the strains by multiplex PCR, but CS21 pili was only detected phenotypically in four of these strains. The ETEC strains had diverse O and H antigens and the most common types were O8:H9 LT CS17 (n = 6; 14 percent) and O78:HNM LT-ST CFA/I (n = 4; 9 percent). The analysis of antibiotic resistance showed that 67 percent (n = 29/43) of the strains were resistant to one or several of the antimicrobial agents tested. Presence of CFs was associated with antibiotic resistance. CONCLUSION: The most common toxin profile was LT 70 percent, LT/STh 23 percent and STh 7 percent. High antimicrobial resistance to ampicillin among serogroups O6, O8 and O78 were the most common.