BACKGROUND:Prenatal Zika virus (ZIKV) infection leads to microcephaly and adverse neurodevelopment. The effects of postnatal ZIKV infection on the developing brain are unknown. We assessed the neurodevelopmental outcomes of children exposed postnatally during the ZIKV epidemic. METHODS:A prospective study enrolled infants 0-3 months of age and their mothers, and children 1.5-3.5 years of age in rural Guatemala from 2017 and were followed for 12 months until 2019. Neurodevelopment was evaluated using the Mullen Scales of Early Learning (MSEL). ZIKV and dengue virus (DENV) infections were identified by polymerase chain reaction (PCR) using active surveillance. Serological analyses, stratified by age group flavivirus serostatus at enrollment, were conducted using a focus reduction neutralization test. RESULTS:Of 1371 enrolled participants, 1187 (86.6%) completed the study. No PCR-confirmed ZIKV infections were identified during the study period. One-third of 1.5-3.5-year-old children were ZIKV-seropositive at enrollment (likely postnatal infection). Twenty participants (5.8%) tested positive for DENV by PCR (11 infants, 5 children and 4 mothers); 15 (75%) were DENV-3 infections and 5 were DENV-2. The incidence of DENV infection in infants was 2.6%. No significant differences in MSEL scores were found between infants born seropositive versus seronegative for ZIKV or DENV. DENV seropositivity at enrollment in 1.5-5-year-old children was associated with lower MSEL scores for fine motor, visual reception and language, and microcephaly at 12 months versus seronegative children (all P < 0.05). CONCLUSIONS:Postnatal ZIKV infection in children from rural Guatemala was not associated with worse neurodevelopmental outcomes. DENV seropositivity was associated with a higher risk of microcephaly in infants and worse neurodevelopmental outcomes in children.

Objective We aimed to evaluate the association between cumulative illness with neurodevelopment and growth outcomes in a birth cohort of Guatemalan infants. Study Design From June 2017 to July 2018, infants 0-3 months of age living in a resource-limited region of rural southwest Guatemala were enrolled and completed weekly at-home surveillance for caregiver-reported cough, fever and vomiting/diarrhea. They also underwent anthropometric assessments and neurodevelopmental testing with the Mullen Scales of Early Learning (MSEL) at enrollment, six months, and one year. Results Out of 499 enrolled infants, 430 (86.2%) completed all study procedures and were included in the analysis. At 12-15 months of age, 140 (32.6%) infants had stunting (length-for-age Z [LAZ] score <-2 SD) and 72 (16.7%) had microcephaly (occipital-frontal circumference [OFC] <-2 SD of the mean). In multivariable analysis, greater cumulative weeks of reported cough illness (beta=-0.08/illness-week, p=0.06) and febrile illness (beta=-0.36/illness-week, p<0.001) were marginally or significantly associated with lower MSEL Early Learning Composite (ELC) Score at 12-15 months, respectively; there was no association with any illness (cough, fever, and/or vomiting/diarrhea; p=0.27) or with cumulative weeks of diarrheal/vomiting illness alone (p=0.66). No association was shown between cumulative weeks of illness and stunting or microcephaly at 12-15 months. Conclusions These findings highlight the negative cumulative consequences of frequent febrile and respiratory illness on neurodevelopment during infancy. Future studies should explore the inflammatory profile associated with these syndromic illnesses and their impact on neurodevelopment in the first years of life.

During the course of the 2015-2017 outbreak of Zika virus (ZIKV) in the Americas, the emerging virus was recognized as a congenital infection that could damage the developing brain. As the Latin American ZIKV outbreak advanced, the scientific and public health community questioned if this newly recognized neurotropic flavivirus could affect the developing brain of infants and young children infected after birth. We report here the study design, methods and the challenges and lessons learned from the rapid operationalization of a prospective natural history cohort study aimed at evaluating the potential neurological and neurodevelopmental effects of postnatal ZIKV infection in infants and young children, which had become epidemic in Central America. This study enrolled a cohort of 500 mothers and their infants, along with nearly 400 children 1.5-3.5 years of age who were born during the initial phase of the ZIKV epidemic in a rural area of Guatemala. Our solutions and lessons learned while tackling real-life challenges may serve as a guide to other researchers carrying out studies of emerging infectious diseases of public health priority in resource-constrained settings.

AbstractMicrocephaly, an anthropometric marker of reduced brain volume and predictor of developmental disability, is rare in high-income countries. Recent reports show the prevalence of microcephaly to be much higher in lower resource settings. We calculated the prevalence of microcephaly in infants and young children (n = 642; age range = 0.1–35.9 months), examined trends in occipitofrontal circumference (OFC) growth in the year after birth and evaluated the relationship between OFC and performance on the Mullen Scales of Early Learning (MSEL) in rural Guatemala. Multivariable regression analyses adjusted for age were performed: (1) a model comparing concurrent MSEL performance and OFC at all visits per child, (2) concurrent OFC and MSEL performance by age group, and (3) OFC at enrollment and MSEL at final visit by age group. Prevalence of microcephaly ranged from 10.1% to 25.0%. OFC z-score decreased for most infants throughout the first year after birth. A significant positive association between continuous OFC measurement and MSEL score suggested that children with smaller OFC may do worse on ND tests conducted both concurrently and ∼1 year later. Results were variable when analyzed by OFC cutoff scores and stratified by 6-month age groups. OFC should be considered for inclusion in developmental screening assessments at the individual and population level, especially when performance-based testing is not feasible.

Background: Although performance-based assessment of early childhood development is preferred, there are a number of limitations to this methodology in low resource settings (LRSs). Hence, clinicians and researchers often rely on caregiver report screening tools. The Ages and Stages Questionnaire 3 (ASQ) is one of the most widely used caregiver report measures globally. Adequate psychometric properties have been demonstrated in high income settings, especially when used in older children, high-risk children, or those with severe neuro-developmental delays. However, its utility is more variable within very young children and for use in LRSs. Methods: The reliability and validity of the ASQ was determined for children ages 0-5 years living in rural Guatemala. Internal consistency and test-retest reliability were assessed, as well as concurrent and predictive validity. Sensitivity, specificity, positive and negative predictive values related to performance-based developmental assessment (Mullen Scales of Early Learning; MSEL) and growth status (i.e. stunting) were also calculated. Results: Internal consistency reliability for the ASQ was adequate, except when results were limited by small sample size. Test-retest reliability ranged from low to moderate (r = 0.08-0.43; p < 0.05-0.01). However, there was significant variability in mean scores over time across ASQ domain scores. In terms of validity, the ASQ did not discriminate adequately between children who performed within or below age-expectations on performance-based developmental testing or those with and without stunting. Conclusions: The ASQ did not demonstrate adequate psychometric properties in rural Guatemala, consistent with concerns documented in other LRSs. These results indicate that existing caregiver report screening measures of early childhood development should be utilized with caution in LRSs, and alternative methods for assessment or in the development and utilization of caregiver report measures should be considered.

In this study, we review the implementation, reliability, and validity of the Pediatric Quality of Life Inventory (PedsQL), a measure of health-related quality of life, in young children in rural Guatemala. Mothers of 842 children (age range = 1-60 months) completed the PedsQL Generic Core Scales 4.0 serially for 1 year. Low (Pearson's r = 0.28, P < .0001) to moderate (Pearson's r = 0.65, P < .0001) consistency in responding over time was shown. The PedsQL did not discriminate reliably between healthy children and those with stunting or wasting. PedsQL scores were not lower during the time of an acute illness. While we found low to moderate evidence for the reliability of the PedsQL in healthy children, it did not discriminate between healthy children and those with stunting, wasting or other acute illness.

BACKGROUND:A growing literature base supports the use of tests developed in high-income countries to assess children in low resource settings when carefully translated, adapted, and applied. Evaluation of psychometric properties of adapted and translated measures within populations is necessary. The current project sought to evaluate the reliability and validity of an adapted and translated version of the Mullen Scales of Early Learning (AT-MSEL) in rural Guatelama.METHODS:The reliability and validity of the AT-MSEL in rural Guatemala were analyzed for children ages 0-5 years.RESULTS:Interrater reliability coefficients (ICC = 0.99-1.0) and internal consistency (Cronbach's alpha = 0.91-0.93) were excellent for all subscales. General linear models utilizing paired data showed consistency between standard scores (p < 0.0001). Mean raw scores increased with chronological age, as expected. Across age groups, subscales were significantly, positively correlated with one another (p < 0.05 - < 0.001) with one exception, visual reception and expressive language at the 0-10 month age range (p = 0.43).CONCLUSIONS:The AT- MSEL showed strong psychometric properties in a sample of young children in rural Guatemala. Findings demonstrate that the AT-MSEL can be used validly and reliably within this specific population of children. This work supports the concept that tests developed in high-income countries can be used to assess children in low resource settings when carefully translated, adapted and applied.

Abstract Background Little is known about the epidemiology of Cytomegalovirus (CMV) infection in low resource countries. We evaluated the frequency and effects of post-natal CMV infection in infants from a prospective cohort study designed to assess the effects of post-natal Zika on neurodevelopment (ND) in rural Guatemala. Infants with CMV infection (blue bars) were older compared CMV-negative (red bars) infants. Methods Infants were evaluated for CMV infection by PCR using urine samples collected at 0-3 months of age. ND testing was conducted by local psychologists using a culturally adapted Mullen Scales of Early Learning (MSEL). We explored associations between CMV infection and microcephaly, neurological, visual and hearing deficits, malnutrition and ND outcomes at 1 year of age. Results The infant cohort (N = 469) had a mean age at enrollment of 1.5 (SD 0.75) months; 47% were female and 71% were breastfeeding at 1 year. A total of 103 (22%) were CMV positive and the majority of these (97%) were > 4 weeks of age at testing. Infants > 4 weeks of age were more likely to be CMV positive (P < 0.0001) (Figure). Gender was not correlated with CMV positivity. Among children with head circumference (HC) measurements, microcephaly (HC < 2 SD) was present in 9/87 (10.3%) CMV positive and 35/338 (10.4%) CMV negative infants at 0-3 months of age (p =0.99). Among 438 infants who underwent screening for hearing deficits and a complete ophthalmologic evaluation, none of the CMV positive children had abnormal vision or hearing. Abnormal neurological exams in the first year of life occurred in 50/100 (50%) CMV positive and 166/365 (45.5%) CMV negative infants (p =0.56). There was no association between CMV infection at 0-3 months and MSEL overall or subdomain scores at 1 year (overall Relative risk (RR) 1.02, 95% CI 0.99-1.05, p=0.16). Malnutrition at 0-3 months (RR: 1.53, 95% CI 0.89-2.66, p = 0.13) and 1 year (RR: 1.10, 95% CI 0.77-1.58, p=0.59) was not associated with CMV infection at 0-3 months. Conclusion In a cohort of Guatemalan infants, postnatal CMV infection was common (22%) and more likely to occur after the neonatal period. There was no correlation between CMV infection and microcephaly at 0-3 months or at 1 year of age, nor with abnormal nutritional, neurologic, ophthalmologic, hearing or ND deficits at 1 year of age. This is the first epidemiologic report on CMV infection in early life in rural Guatemala. Disclosures Molly Lamb, PhD, BioFire (Grant/Research Support)

Antimicrobial resistance (AMR) is an increasing global threat to public health, particularly in Latin America. Most published data are based on adults with limited pediatric reports regarding resistance trends. Our study evaluated AMR rates in a large tertiary pediatric hospital in Guatemala City and the association with clinical outcomes. We analyzed AMR rates for six bacterial species (Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus) identified from blood cultures from the WHONET database between 2005-2019. Resistance was determined using CLSI cut-offs on the VITEK and Sensititre systems. Student’s t tests and simple linear regression models were performed. A retrospective review was performed on 99 pediatric patient charts with positive blood cultures (June 2018-May 2019) to assess clinical outcomes. Klebsiella and Acinetobacter were the most prevalent organisms throughout the 15 years of surveillance, with 2019 sensitivities demonstrating carbapenem-resistance in 99 (57%) and 57 (91%) of isolates, respectively. Increased resistance rates were noted for all Gram-negative organisms evaluated, with particular clinical and statistical significance noted for K. pneumoniae with imipenem (4.3% average resistance increase per year (PARPY), p-value < 0.0001), ciprofloxacin (4.5 PARPY, < 0.0001), and piperacillin-tazobactam (3.4 PARPY, < 0.0001), as well as A. baumannii with imipenem (2.9 PARPY, p-value < 0.0001), cefepime (1.7 PARPY, < 0.0001), and ciprofloxacin (2.5 PARPY, 0.0002). In contrast, resistance rates decreased for S. aureus with oxacillin (-2.7 PARPY, 0.0015). A mortality rate of 20% among our 99-patient cohort was detected. Of the 37% who received optimal therapy, the median time to optimal therapy was 90 hours. Acinetobacter baumannii resistance to imipenem, 2005-2019 Klebsiella pneumoniae resistance to imipenem, 2005-2019 Staphylococcus aureus resistance to oxacillin, 2005-2019 Significant rises in AMR among pediatric patients in a large tertiary hospital in Guatemala City have occurred over 15 years. This likely contributed to delays in optimal antimicrobial therapy, increased exposure to broad spectrum antibiotics, and potentially increased mortality. Improved antimicrobial stewardship, infection prevention, and rapid diagnostic testing are needed in order to combat this growing problem. Kelly E. Graff, MD, BioFire Diagnostics, LLC (Grant/Research Support) Samuel Dominguez, MD, PhD, BioFire (Consultant, Research Grant or Support)

Abstract Background Recurrent infections are associated with neurodevelopmental (ND) delay in infants, but the primary drivers are poorly understood. Leveraging an infant cohort from rural Guatemala designed to evaluate the effects of post-natal Zika virus on ND (DMID 16-0057), we evaluated the association between cumulative illness and ND delay and stunting. Methods Infants enrolled at 0-3 months of age underwent weekly at-home surveillance for caregiver-reported syndromic illness, including cough, fever and vomiting/diarrhea for a 12-month period. Anthropometric assessments and ND testing by Guatemalan psychologists using the Mullen Scales of Early Learning (MSEL) were perforrmed at 12-15 months of age. Multivariable generalized linear regression models were used to test associations between syndromic illness in infancy, 12-15-month MSEL Early Learning Composite (ELC) Score, and stunting (height-for-age < -2 SD) at 12-15 months. Results The cohort (n=425) had a mean enrollment age of 1.3 months; 202 (48%) were female, 387 (91%) self-reported a literate mother, and 301 (71%) were breastfeeding at study completion. Infants had reported illness for a median of 16 weeks during the surveillance period; cough was reported most frequently (median=11 weeks, range=0-37 weeks). Lower maternal education (p=0.007) and literacy (p=0.002) as well as infant age (p=0.007) and male gender (p=0.004) were associated with MSEL ELC Score <85 (-1 SD). After adjusting for gender, breastfeeding, age, and maternal literacy, the cumulative number of weeks with reported cough (p=0.0009), fever (p=0.0001), or any syndromic illness (p=0.0007) were associated with decreased 12-month MSEL ECL Score; there was no association with diarrhea/vomiting (p=0.36). There was no association between caregiver-reported syndromic illnesses (any type) and stunting at final study visit. Conclusion In a cohort of Guatemalan infants, cumulative fever and cough episodes were significantly associated with lower MSEL ELC Score, whereas there was no association with diarrhea/vomiting. In this low-resource community, these findings highlight the potential negative ND consequences of febrile illness and persistent cough in the first year of life. NIAID Contract HHSN272201300015I Task Order HHSN27200013 (Co-PIs: FMM and EJA). Disclosures Molly Lamb, PhD, BioFire (Grant/Research Support) Evan J. Anderson, MD, Sanofi Pasteur (Scientific Research Study Investigator)

BACKGROUND:Children in low- and lower middle income countries (LMICs) often have poorer language skills compared with children from high-income countries. Limited availability of culturally and linguistically appropriate assessment measures in LMICs, especially for young children, can hinder early identification and prevention efforts. Here, we describe receptive language (RL) skills among young children in rural Guatemala and report on the validity of a translated and culturally adapted developmental measure of RL.METHODS:Children (n = 157; m = 53.6 months, range = 42-68 months) enrolled in a prospective cohort study of postnatally acquired Zika virus infection were administered the Test de Vocabulario en Imagenes Peabody (TVIP) and the RL scale from a translated and adapted version of the Mullen Scales of Early Learning (MSEL). Performance on the TVIP was compared with the Latin American normative sample. Correlational analysis examined the relationship between performance on the TVIP and the MSEL-RL.RESULTS:Mean scores were significantly below the normative sample mean on the TVIP, t(126) = -11.04, p < .001; d = 1.00. Performance on the TVIP among children who passed the practice items (n = 127) was significantly positively associated with performance on the MSEL-RL (r = .50, p < .001), but not significantly associated with age or gender. Older age (p < .0001) and female gender (p = .018) were associated with passing the TVIP practice items.CONCLUSIONS:Delays in RL vocabulary were identified among young children in rural Guatemala on the TVIP. The association between scores on the TVIP and the RL scale of the MSEL provides preliminary support for the construct validity of this translated and adapted version of the MSEL.

Abstract Background Diagnosing ZIKV infection in children in dengue (DENV) endemic regions is challenging. The kinetics and effects of maternal Ab on infant infection responses remain unknown, as do the ND effects of ZIKV acquired in early life. Methods This is a population-based prospective cohort study in infant–mother pairs and children <5 years in a rural DENV endemic area of Guatemala evaluating the incidence and ND outcomes of postnatally cquired ZIKV infection. Subjects were followed 1 year for symptoms of flavivirus-like illness (FLI), serologic and virologic evidence of ZIKV or DENV infection and ND outcomes. ZIKV and DENV neutralizing antibodies (NAb) were measured at enrollment and longitudinally. Subjects were classified as ZIKV- or DENV-infected based on NAb. Specimens from acute illnesses were tested for viral RNA by rRT-PCR. ND was assessed at enrollment and longitudinally using an adapted Mullen Scales of Early Learning (MSEL). Results In total, 1,371 subjects (374 children 1–5 years, 500 infants, 497 mothers) were enrolled from June 2017 to July 2018. Among 1,335 evaluable subjects, 7.6% (101) had serologic evidence of recent ZIKV infection (NAb >500 or >100 and DENV-neg); 13.2% (176) were DENV-pos only; 44.8% (598) were ZIKV-neg (NAb15 −<100) or low (<500) ZIKV and DENV NAb, suggesting prior flavivirus infection or cross-reactivity (Figures 1 and 2). ZIKV infection alone was more prevalent in children 1–5 years, while infants’ serologic pattern was similar to that of their mothers. One child ZIKV seroconverted. In 391 FLI episodes (67 children 1–5 years and 215 infants with fever, rash, myalgia, and conjunctivitis; 109 mothers with fever, myalgia, and joint pain), acute DENV infections, but not ZIKV, were identified by rRT-PCR. MSEL scores were similar to US norms in infants <12 m (composite mean 94.8, SD 11.9), but lower in children 1–5 years in all domains (mean 75.1, SD 17.4, P < 0.0001) (Figure 3). Conclusion Serologic evidence of recent ZIKV infection, but no acute ZIKV, was documented in young children in Guatemala. Infant seroreactivity derives from prior maternal infection and DENV cross-reactivity. Observed substantial differences in ND scores between infants and children 1–5 years challenge the ability to isolate the potential effects of ZIKV infection in early life. NIAID Contract HHSN272201300015I Task Order HHSN27200013. FMM and EJA Co-PIs. Disclosures Flor M. Munoz, MD, Biocryst: Grant/Research Support; CDC: Research Grant; Moderna: Other Financial or Material Support, Safety Monitoring Board Member/Chair; NIH: Research Grant; Novavax: Research Grant; UP to Date: Author and Editor: Royalties, Other Financial or Material Support.

Abstract Background The impact of early post-natal Zika virus (ZIKV) infection on neurodevelopment (ND) is unknown. A prospective study of post-natal ZIKV infection in rural Guatemala (ZIKV study) enrolled a cohort of children ages 1–5 years, including children previously enrolled in a dengue virus (DENV) study during the 2015–2016 ZIKV epidemic. We evaluated ND outcomes by age and ZIKV infection status. Methods Subjects enrolled in the ZIKV study June 2017–April 2018 underwent ND testing using the Mullen Scales of Early Learning (MSEL) at baseline and 12 months later. ZIKV/DENV-1/2 FRNT50 was performed on enrollment and on banked serum samples from the 2015 to 2016 subset. ZIKV serostatus and MSEL scores were correlated using multiple linear mixed models, adjusted for age and gender when appropriate, to evaluate their association. Geolocation was used to explore clustering of ZIKV serostatus and MSEL score. Results We enrolled 183 children (43% female, mean age 3.2 years). Of these, 38 (21%) were classified as ZIKV-positive (+), 111 (61%) ZIKV-negative (-), 31 (17%) ZIKV-possible, and 3 (2%) ZIKV-indeterminate. ZIKV(+) cases and higher composite MSEL scores clustered in more densely populated areas (Figure 1). ZIKV(+) serostatus was associated with higher MSEL composite (increase in log score 0.09, P = 0.003) and subdomain scores: fine motor (0.13, P = 0.011), visual reception (0.15, P = 0.002), receptive language (0.09, P = 0.041), gross motor (0.14, P = 0.09), and expressive language (0.09, P = 0.058) (Figure 2). Of the 78 children (43%) with 2015–2016 samples, 46 (59%) remained ZIKV(−), 16 (21%) seroconverted from ZIKV(−) or possible/indeterminate to ZIKV(+), and 16 (21%) were indeterminate when enrolled in the ZIKV study. ZIKV seroconversion was associated with higher composite (0.13, P = 0.02) MSEL scores compared with ZIKV(−). Conclusion In this exploratory analysis, post-natal ZIKV infection was not associated with adverse ND outcomes in children age 1–5 years. Overall, ZIKV(+) status was associated with higher average ND scores than ZIKV(−), and scores decreased with age for most children, independent of ZIKV status. The correlation of ZIKV(+) status and higher MSEL scores may be confounded by geographic-related factors or other confounders. NIAID Contract HHSN272201300015I Task Order HHSN27200013 (Co-PIs: FMM & EJA). Disclosures Flor M. Munoz, M.D, Biocryst: Grant/Research Support; CDC: Research Grant; Moderna: Other Financial or Material Support, Safety Monitoring Board Member/Chair; NIH: Research Grant; Novavax: Research Grant; UP to Date: Author and Editor - Royalties, Other Financial or Material Support.

The ongoing Zika virus (ZIKV) epidemic in Latin America presented a unique opportunity to develop a neurodevelopmental assessment protocol for children in a lower middle-income country. Although studies of neurodevelopment in young children have taken place in many diverse global settings, we are not aware of any study that has provided a high level of detail about how a measure was selected and then specifically translated and adapted in a low-resource setting. Here, we describe considerations in measurement selection and then the process of translation and adaptation to assess neurodevelopmental outcomes of infants and young children with postnatal exposure to ZIKV in rural Guatemala. We provide a framework to other research teams seeking to develop similar assessment models across the globe.

Background . Rotavirus vaccination ef fi cacy in low-income countries is variable and frequently lower than in high-incomecountries, and our understanding ofthe factors underlying these phenomena is incomplete. As a sub-analysis of a clinical trial for treatment of acute diarrhea, we examined the impact of demographic and socioeconomic factors on risk of rotavirus acquisition in young children in Guatemala. Using a multiplex PCR assay for stool pathogens we analyzed the in fl uence of stool and distribution) on rotavirus acquisition. Methods . Children months with acute diarrhea (<72 hours) enrolled between March 2015 and January 2016 in two sites (1 rural, 1 urban). Rotavirus vac- cination is routine in Guatemala but uptake is not universal. Stool samples collected at enrollment were analyzed by