Risk Factors and Clinical Profile of Sapovirus-associated Acute Gastroenteritis in Early Childhood: A Nicaraguan Birth Cohort Study.

BACKGROUND: Sapovirus is increasingly recognized as an important cause of acute gastroenteritis (AGE) in children. We identified risk factors and characterized the clinical profile of sapovirus AGE in a birth cohort in León, Nicaragua. METHODS: We conducted a case-control study nested within a birth cohort (n = 444). Fieldworkers conducted weekly household AGE surveillance. AGE stools were tested for sapovirus by reverse transcriptase quantitative polymerase chain reaction. For each first sapovirus episode, we selected 2 healthy age-matched controls and estimated independent risk factors of sapovirus AGE using conditional logistic regression. We compared clinical characteristics of sapovirus AGE episodes with episodes associated with other etiologies and identified co-infections with other enteric pathogens. RESULTS: From June 2017 to July 2019, we identified 63 first sapovirus AGE episodes and selected 126 controls. Having contact with an individual with AGE symptoms and vaginal delivery were independent risk factors for sapovirus AGE. All cases experienced diarrhea, lasting a median 6 days; 23% experienced vomiting. Compared with children with AGE due to another etiology, sapovirus AGE was similar in severity, with less reported fever. Most cases experienced co-infections and were more likely than controls to be infected with diarrheagenic Escherichia coli or astrovirus. CONCLUSIONS: Sapovirus was a commonly identified AGE etiology in this Central American setting, and symptoms were similar to AGE associated with other etiologies. The association between vaginal delivery and sapovirus is a novel finding. Gut microbiome composition might mediate this relationship, or vaginal delivery might be a proxy for other risk factors. Further investigation into more specific biological mechanisms is warranted.

Pediatric norovirus GII.4 infections in Nicaragua, 1999-2015.

OBJECTIVES: Investigate clinical and epidemiological factors of pediatric GII.4 norovirus infections in children with acute gastroenteritis (AGE) in Nicaragua between 1999 and 2015. METHODS: We retrospectively analyzed laboratory and epidemiologic data from 1,790 children≤7years with AGE from 6 hospitals in Nicaragua (n=538), and 3 community clinics (n=919) and households (n=333) in León, between 1999 and 2015. Moreover, asymptomatic children from community clinics (n=162) and households (n=105) were enrolled. Norovirus was detected by real-time PCR and genotyped by sequencing the N-terminal and shell region of the capsid gene. RESULTS: Norovirus was found in 19% (n=338) and 12% (n=32) of children with and without AGE, respectively. In total, 20 genotypes including a tentatively new genotype were detected. Among children with AGE, the most common genotypes were GII.4 (53%), GII.14 (7%), GII.3 (6%) and GI.3 (6%). In contrast, only one (1.4%) GII.4 was found in asymptomatic children. The prevalence of GII.4 infections was significantly higher in children between 7 and 12months of age. The prevalence of GII.4 was lowest in households (38%), followed by community clinics (50%) and hospitals (75%). Several different GII.4 variants were detected and their emergence followed the global temporal trend. CONCLUSIONS: Overall our study found the predominance of pediatric GII.4 norovirus infections in Nicaragua mostly occurring in children between 7 and 12months of age, implicating GII.4 as the main norovirus vaccine target.

Etiology of childhood diarrhea after rotavirus vaccine introduction: a prospective, population-based study in Nicaragua.

BACKGROUND: Nicaragua was the first developing nation to implement routine immunization with the pentavalent rotavirus vaccine (RV5). In this RV5-immunized population, understanding infectious etiologies of childhood diarrhea is necessary to direct diarrhea treatment and prevention efforts. METHODS: We followed a population-based sample of children <5 years in León, Nicaragua for diarrhea episodes through household visits. Information was obtained on RV5 history and sociodemographics. Stool samples collected during diarrhea episodes and among healthy children underwent laboratory analysis for viral, bacterial and parasitic enteropathogens. Detection frequency and incidence of each enteropathogen was calculated. RESULTS: The 826 children in the cohort experienced 677 diarrhea episodes during 607.5 child-years of exposure time (1.1 episodes per child-year). At least 1 enteropathogen was detected among 61.1% of the 337 diarrheal stools collected. The most common enteropathogens among diarrheal stools were: norovirus (20.4%), sapovirus (16.6%), enteropathogenic Escherichia coli (11.3%), Entamoeba histolytica/dispar (8.3%), Giardia lamblia (8.0%) and enterotoxigenic E. coli (7.7%), with rotavirus detected among 5.3% of diarrheal stools. Enteropathogenic Escherichia coli and enterotoxigenic E. coli were frequently detected among stools from healthy children. Among children with diarrhea, norovirus was more commonly detected among younger children (< 2 years) and G. lamblia was more commonly detected among older children (2-4 years). The mean age of rotavirus detection was 34.6 months. CONCLUSIONS: In this Central American community after RV5 introduction, rotavirus was not commonly detected among children with diarrhea. Prevention and appropriate management of norovirus and sapovirus should be considered to further reduce the burden of diarrheal disease.

Characterization of enterotoxigenic Escherichia coli strains isolated from Nicaraguan children in hospital, primary care and community settings.

Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhoea among young children in developing countries. ETEC vaccines offer promise in reducing the burden of ETEC disease, but the development of these vaccines relies on the characterization of ETEC isolates from a variety of settings. To best reflect the full spectrum of ETEC disease in León, Nicaragua, the aim of this study was to characterize ETEC strains isolated from children with diarrhoea attending different settings (hospital, primary care clinics and in the community) and children from different age groups. We characterized ETEC isolates in terms of their colonization factors (CFs) and enterotoxins, and determined whether these factors varied with setting and age group. Diarrhoeal stool samples were obtained from children under the age of 60 months from: (1) the regional public hospital, (2) four public primary care clinics, and (3) a population-based cohort. In total, 58 ETEC-positive isolates were analysed by multiplex-PCR assays for the identification of CFs (CS1, CS2, CS3, CS4, CS5, CS6, CS7, CS8, CS12, CS13, CS14, CS15, CS17, CS18, CS19, CS20, CS21, CS22 and CFA/I), and enterotoxins [heat-labile toxin (LT) and heat-stable variants STh and STp]. The frequency of CFs and enterotoxins was compared among the three settings and for different age groups, using Fisher's exact test or a χ(2) test. At least one CF was detected among one-half of samples; CS19 was detected among all strains in which a CF was identified, either alone or in combination with another CF. Among all CFs detected, 91.7 % were identified as members of the class 5 fimbrial family. CFs were detected more commonly among samples from infants captured in the health facility setting compared with the community setting. Overall, LT was detected among 67.2 % of samples, STh was detected among 20.7 % and both enterotoxins were detected among 12.1 %. The enterotoxin STh was detected more commonly among cases in the community, whilst a combination of STh and LT was detected more commonly among cases treated in health facilities. Our results suggest that, to protect against diarrhoeal cases associated with this E. coli pathotype in León, Nicaragua, an ETEC vaccine that effectively targets the archeotype CFA/I of the class 5 fimbrial family would be the most effective in this setting.

Community diarrhea incidence before and after rotavirus vaccine introduction in Nicaragua.

We estimated the incidence of watery diarrhea in the community before and after introduction of the pentavalent rotavirus vaccine in León, Nicaragua. A random sample of households was selected before and after rotavirus vaccine introduction. All children < 5 years of age in selected households were eligible for inclusion. Children were followed every 2 weeks for watery diarrhea episodes. The incidence rate was estimated as numbers of episodes per 100 child-years of exposure time. A mixed effects Poisson regression model was fit to compare incidence rates in the pre-vaccine and vaccine periods. The pre-vaccine cohort (N = 726) experienced 36 episodes per 100 child-years, and the vaccine cohort (N = 826) experienced 25 episodes per 100 child-years. The adjusted incidence rate ratio was 0.60 (95% confidence interval [CI] 0.40, 0.91) during the vaccine period versus the pre-vaccine period, indicating a lower incidence of watery diarrhea in the community during the vaccine period.

Rotavirus prevalence in the primary care setting in Nicaragua after universal infant rotavirus immunization.

Nicaragua was the first developing nation to implement universal infant rotavirus immunization with the pentavalent rotavirus vaccine (RV5). Initial studies of vaccine effectiveness in Nicaragua and other developing nations have focused on the prevention of hospitalizations and severe rotavirus diarrhea. However, rotavirus diarrhea is more commonly treated in the primary care setting, with only 1-3% of rotavirus cases receiving hospital care. We measured the prevalence of rotavirus infection in primary care clinics in León, Nicaragua, after introduction of the immunization program. In the post-vaccine period, 3.5% (95% confidence interval = 1.9-5.8) of children seeking care for diarrhea tested positive for rotavirus. A high diversity of rotavirus genotypes was encountered among the few positive samples. In conclusion, rotavirus was an uncommon cause of childhood diarrhea in this primary care setting after implementation of a rotavirus immunization program.

Case-control study of the effectiveness of vaccination with pentavalent rotavirus vaccine in Nicaragua.

BACKGROUND: In 2006, Merck & Co., Inc., partnered with the Nicaraguan Ministry of Health to demonstrate the public health impact of routine universal vaccination by delivering more than 1.3 million doses of the oral, pentavalent rotavirus vaccine (RV5) in a 3-year period. METHODS: A matched case-control study evaluated the effectiveness of RV5 in reducing the risk for severe wild-type rotavirus gastroenteritis (RGE) resulting in hospitalizations and emergency department visits among children who completed the recommended 3-dose regimen as part of the routine national vaccine program. Cases were identified from 6 hospitals from February 2007 to October 2009 and were age-matched with hospital controls and community controls. Vaccine effectiveness was calculated using conditional logistic regression. RESULTS: Three hundred RGE cases eligible for analysis were matched to 792 hospital and 851 community controls. Vaccine coverage of RV5 in the community reached 92%. Vaccine effectiveness during 2 years of follow-up against severe disease in children receiving 3 doses of RV5 was 87% (95% confidence interval [CI], 74-93) for community controls, 64% (95% CI, 44-78) for hospital controls, and 76% (95% CI, 63-84) when the groups were combined. For the combined groups, vaccine effectiveness was 85% (95% CI, 66-93) among children <12 months old at the time of RGE onset. CONCLUSIONS: The Merck-Nicaragua Rotavirus Vaccine Partnership promoted rapid and widespread uptake of a novel vaccine in a developing country. Vaccine effectiveness was greatest for children younger than 12 months of age who were at the highest risk for severe rotavirus disease.

Changes in childhood diarrhea incidence in nicaragua following 3 years of universal infant rotavirus immunization.

BACKGROUND: Although the pentavalent rotavirus vaccine was highly efficacious against rotavirus diarrhea in clinical trials, the effectiveness of vaccine under field conditions in the developing world is unclear. In October 2006, Nicaragua became the first developing nation to implement universal infant immunization with the pentavalent rotavirus vaccine. To assess the effect of the immunization program, we examined the incidence of diarrhea episodes between 2003 and 2009 among children in the state of León, Nicaragua. METHODS: We extracted data on diarrhea episodes from health ministry records. We used scaled Poisson regression models to estimate diarrhea incidence rate ratios for the period following the program's implementation to the period before implementation. RESULTS: Following implementation of the immunization program, diarrhea episodes among infants were reduced (incidence rate ratios: 0.85, 95% confidence interval: 0.71-1.02) during the rotavirus season, but appear to have increased during other months. CONCLUSIONS: Although the immunization program appears effective in reducing diarrhea episodes during the rotavirus season, a large burden of diarrhea still persists during the remainder of the year.

Antibiotic resistance patterns of intestinal Escherichia coli isolates from Nicaraguan children.

In developing countries, diarrhoeal diseases are one of the major causes of death in children under 5 years of age. It is known that diarrhoeagenic Escherichia coli (DEC) is an important aetiological agent of infantile diarrhoea in Nicaragua. However, there are no recent studies on antimicrobial resistance among intestinal E. coli isolates in Nicaraguan children. The aim of the present study was to determine the antimicrobial resistance pattern in a collection of 727 intestinal E. coli isolates from the faeces of children in León, Nicaragua, between March 2005 and September 2006. All samples had been screened previously for the presence of DEC by multiplex PCR. Three hundred and ninety-five non-DEC isolates (270 from children with diarrhoea and 125 from children without diarrhoea) and 332 DEC isolates (241 from children with diarrhoea and 91 from children without diarrhoea) were analysed in this study. In general, antimicrobial resistance among the 727 intestinal E. coli isolates was high for ampicillin (60 %), trimethoprim-sulfamethoxazole (64 %) and chloramphenicol (11 %). Among individual E. coli categories, enteroaggregative E. coli isolates from children with and without diarrhoea exhibited significantly higher levels of resistance (P<0.05) to ampicillin and trimethoprim-sulfamethoxazole compared to the other E. coli categories. Resistance to ceftazidime and/or ceftriaxone and a pattern of multi-resistance was related to CTX-M-5- or CTX-M-15-producing E. coli isolates. The results suggest that E. coli isolates from Nicaraguan children have not reached the high levels of resistance to the most common antibiotics used for diarrhoea treatment as in other countries.

Asymptomatic norovirus infections in Nicaraguan children and its association with viral properties and histo-blood group antigens.

BACKGROUND: It has been previously reported that histo-blood group antigens (HBGAs) and particularly secretor status provides protection against symptomatic norovirus infection, but it remains unclear to what extent this includes asymptomatic infections in children. METHODS: To explore whether HBGAs or certain viral genotypes are associated with asymptomatic norovirus infections in a pediatric population in Nicaragua, we investigated 163 children ≤5 years of age, without a recent history of diarrhea (≤10 days). RESULTS: Asymptomatic norovirus infections were observed in 11.7% (19/163), with children ≤6 months of age being most frequently infected (16%). Of the 19 norovirus-positive children, 4 (21%) and 10 (53%) were infected with genogroups GI and GII, respectively, and 4 children (21%) were infected with viruses of both genogroups. Most children had ≥10 viral genomes per gram of feces. Nucleotide sequence analysis (15/19) revealed uncommon genotypes, such as, GII.7 (n = 5) and GII.2 (n = 3). An interesting observation was the low frequency of norovirus GII.4 strains among the asymptomatic children. AB blood type, Lewis a (Le) phenotype and nonsecretor genotype (sese) were not found among the asymptomatic children, but they occurred in population controls. CONCLUSIONS: Frequency of asymptomatic norovirus infections was similar to that observed in symptomatic children from Nicaragua. Norovirus GII.2 and GII.7 were frequently detected but the globally dominating GII.4 was infrequent. Host genetic factors previously observed to be associated with protection against symptomatic norovirus infection were not found in this study.

Diarrheagenic Escherichia coli markers and phenotypes among fecal E. coli isolates collected from Nicaraguan infants.

We analyzed the prevalence of diarrheagenic Escherichia coli (DEC) markers and common phenotypes in 2,164 E. coli isolates from 282 DEC-positive samples. Enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) were very diverse and were not correlated with diarrhea. Enterotoxigenic E. coli (ETEC) estA and enterohemorrhagic E. coli (EHEC) belonged to a few phenotypes and were significantly correlated with diarrhea.

Diversity of intestinal Escherichia coli populations in Nicaraguan children with and without diarrhoea.

Escherichia coli remains an important aetiological agent of infantile diarrhoea in Nicaragua. However, little is known about whether there is a high prevalence of endemic strains or whether infection is due to the epidemic spread of virulent clones. This study was undertaken to determine the diversity and distribution of clonal groups in a population of intestinal E. coli isolated from the faeces of children from León, Nicaragua, with (n=381) and without (n=145) diarrhoea, between March 2005 and September 2006. All samples had been screened previously for the presence of diarrhoeagenic E. coli (DEC) markers by multiplex PCR. From each sample, 8 E. coli colonies (where available) were analysed by biochemical fingerprinting (PhP-RE system), yielding a total of 4009 tested isolates. On average, three different biochemical phenotypes (BPTs) were found among the eight colonies analysed from each sample. The total diversity, measured as Simpson's diversity index (Di), was 0.97 among all 4009 isolates studied. Cluster analysis of data from all 4009 isolates revealed 24 common BPTs (identified in at least 1 % of the isolates) and 234 less common BPTs. Similar Di values were obtained among isolates from infants with and without diarrhoea, indicating that no widespread outbreak of DEC had occurred. Moreover, among samples that were positive for the DEC types enteroaggregative E. coli, enteropathogenic E. coli and enterotoxigenic E. coli (ETEC) carrying the eltB gene, the diversities were almost as high as among non-DEC samples, whereas samples positive for ETEC carrying estA, enteroinvasive E. coli and enterohaemorrhagic E. coli showed lower diversities, indicating the prevalence of virulent clonal groups among these samples. The PhenePlate patterns of the 24 common BPTs identified here were compared with those obtained from E. coli isolated in a cohort infant study performed in 1991-1992 in the same area. Only 4 % of the isolates from the 1990s were similar to any of the common BPTs found in the present study.

Prevalence of diarrhoeagenic Escherichia coli in children from León, Nicaragua.

Diarrhoeal disease is a public health problem worldwide, mostly affecting children in developing countries. In Nicaragua, diarrhoea is the second greatest cause of infant mortality. During the period March 2005 to September 2006, a total of 526 faecal samples from children aged 0-60 months (381 with and 145 without diarrhoea) from León, Nicaragua, were studied. In order to detect five different diarrhoeagenic Escherichia coli pathotypes simultaneously [enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC) and enteroinvasive E. coli (EIEC)], a mixture of eight primer pairs was used in a single PCR. At least one diarrhoeagenic E. coli pathotype was detected in 205 samples (53.8%) of the diarrhoea group and in 77 samples (53.1%) in the non-diarrhoea group. ETEC was detected significantly more often in children with diarrhoea (20.5%) than in children without diarrhoea (8.3%) (P=0.001). Atypical EPEC, EIEC and EAEC were detected with slightly lower frequencies in children with (16.0, 0.8 and 27.8%, respectively) than in children without (20.7, 1.4 and 33.1%, respectively) diarrhoea. EHEC was only detected in children with diarrhoea (2.1%). In conclusion, ETEC continues to be an important agent associated with diarrhoea in children from León, Nicaragua. Although not very frequent, the only findings that were 100% associated with diarrhoea were ETEC estA (4.7%) and EHEC (2.1%). Nevertheless, EAEC and EPEC were also frequent pathotypes in the population under study. In children with severe diarrhoea, more than half had EAEC, ETEC or EPEC, and EAEC was the most prevalent pathotype.

Genetic susceptibility to symptomatic norovirus infection in Nicaragua.

Host genetic resistance to Norovirus (NoV) has been observed in challenge and outbreak studies in populations from Europe, Asia, and USA. In this study, we have investigated if histo-blood group antigens can predict susceptibility to diarrhea caused by NoV in Nicaragua, Central America, and if this can be reflected in antibody-prevalence and titer to NoV among individuals with different histo-blood group antigen phenotypes. Investigation of 28 individuals infected with NoV and 131 population controls revealed 6% of non-secretors in the population and nil non-secretors among patients infected with NoV, suggesting that non-secretors may be protected against NoV disease in Nicaragua. Surprisingly, 25% of the population was Lewis negative (Le(a-b-)). NoV infections with genogroup I (GI) and GII occurred irrespective of Lewis genotype, but none of the Lewis a positive (Le(a + b-)) were infected. The globally dominating GII.4 virus infected individuals of all blood groups except AB (n = 5), while the GI viruses (n = 4) infected only blood type O individuals. Furthermore, O blood types were susceptible to infections with GI.4, GII.4, GII.7, GII.17, and GII.18-Nica viruses, suggesting that secretors with blood type O are susceptible (OR = 1.52) and non-secretors resistant. The overall antibody-prevalence to NoV GII.3 VLP was 62% with the highest prevalence among blood type B carriers (70%) followed by A (68%) and O (62%). All four investigated individuals carrying blood type AB were antibody-negative. Among secretors, 63% were antibody-positive compared to 33% among non-secretors (P = 0.151). This study extends previous knowledge about the histo-blood group antigens role in NoV disease in a population with different genetic background than North American and European.

Pediatric norovirus diarrhea in Nicaragua.

Information about norovirus (NoV) infections in Central America is limited. Through a passive community and hospital pediatric diarrhea surveillance program, a total of 542 stool samples were collected between March 2005 and February 2006 in León, Nicaragua. NoV was detected in 12% (65/542) of the children; of these, 11% (45/409) were in the community and 15% (20/133) were in the hospital, with most strains (88%) belonging to genogroup II. NoV infections were age and gender associated, with children of <2 years of age (P < 0.05) and girls (P < 0.05) being most affected. Breast-feeding did not reduce the number of NoV infections. An important proportion (57%) of NoV-infected children were coinfected with diarrheagenic Escherichia coli. A significant proportion (18/31) of NoV-positive children with dehydration required intravenous rehydration. Nucleotide sequence analysis (38/65) of the N-terminal and shell region in the capsid gene revealed that at least six genotypes (GI.4, GII.2, GII.4, GII.7, GII.17, and a potentially novel cluster termed "GII.18-Nica") circulated during the study period, with GII.4 virus being predominant (26/38). The majority (20/26) of those GII.4 strains shared high nucleotide homology (99%) with the globally emerging Hunter strain. The mean viral load was approximately 15-fold higher in children infected with GII.4 virus than in those infected with other G.II viruses, with the highest viral load observed for the group of children infected with GII.4 and requiring intravenous rehydration. This study, the first of its type from a Central American country, suggests that NoV is an important etiological agent of acute diarrhea among children of <2 years of age in Nicaragua.

Mutated G4P[8] rotavirus associated with a nationwide outbreak of gastroenteritis in Nicaragua in 2005.

During February and March 2005, one of the largest national recorded outbreaks of severe acute gastroenteritis occurred in Nicaragua, affecting >or=64,000 individuals and causing >or=56 deaths, predominantly in children under 5 years of age. Through a nationwide laboratory-based study, stool samples were collected and investigated for rotavirus. Of 108 stool samples examined, 72 (67%) were positive for rotavirus. While 69% (50/72) of the positive samples were found in children less than 2 years of age, 50% (6/12) of the adult samples were positive. A mutated G4P[8] strain was the most commonly recognized strain (85%), followed by mixed G strains (8%) and G9P[8] (7%) strains. Phylogenetic analysis of the VP7 gene revealed that the G4 strains belonged to the emerging lineage Ic and was distantly related to the ST3 and VA70 G4 strains. Secondary structure predictions of the VP7 G4 protein revealed an insert of an asparagine residue in position 76, which, combined with additional mutations, surprisingly modified two downstream beta-sheets at amino acid positions 80 to 85 and 115 to 119. The 2005 G4P[8] strain compared to a G4P[8] strain from 2002 had a substitution of an asparagine residue for threonine (Asn-->Thr) at position 96 within antigenic region A, thus eliminating a potential glycosylation site. The mutated G4 virus was introduced in Nicaragua after 2002 and probably emerged from Brazil, Argentina, or Uruguay.

Shifts of rotavirus g and p types in Nicaragua--2001-2003.

The present study reports the diversity of rotavirus strains circulating in León, Nicaragua during three years. There was a shift of G and P genotypes with increment of one specific genotype during the second most important peak of diarrhea occurring in the beginning of every year.

Antibodies in mother's milk protect children against giardiasis.

The protective effect of anti-Giardia antibodies in mother's milk on the acquisition of Giardia infection in their children during the first 2 y of life was analysed as part of a prospective study on infant diarrhoea in a group of 307 mothers and children in Leòn, Nicaragua. Among 24 children acquiring infection within the first 6 months, 23 were born to mothers lacking antibodies. These children also developed more severe diarrhoea. A significant difference between children born to mothers with and without antibodies with respect to the age at which the first Giardia infection was acquired was demonstrated by survival analysis and log rank test (p = 0.036). In conclusion, children born to non-immune mothers are at significantly higher risk of acquiring Giardia infection and developing giardiasis with more severe symptoms compared with children of immune mothers.