This study was conducted in collaboration between the Sustainable Sciences Institute, the Nicaraguan Ministry of Health, the University of California, Berkeley (UCB), the University of Michigan, Ann Arbor (UM), and the US Centers for Disease Control and Prevention (CDC). The study was approved by the Institutional Review Boards (IRBs) of the Nicaraguan Ministry of Health, UM, and UCB. The CDC’s IRB relied on the UCB IRB for approval. Written informed consent was obtained from a parent/guardian of all participants.

The Nicaraguan Influenza Birth Cohort Study is an ongoing prospective cohort study based at the Health Center Socrates Flores Vivas (HCSFV), a primary health care facility run by the Nicaraguan Ministry of Health. The HCSFV serves a population of approximately 61,500, in the low-to-middle income District II of the city of Managua. For inclusion in the study, a participant must (1) have been ≤4 weeks of age at enrollment, (2) lived in the HCSFV catchment area, (3) had no plan to leave the study area in the next 2 years, and (4) have been willing to attend the HCSFV for all medical needs. Infants with immunocompromising conditions and premature infants who required ≥4 weeks of hospitalized care directly following birth were excluded. Infants were recruited when parents/guardians brought them to their first well-baby appointment or through house visits. Participants were enrolled in the study until the age of 2 years, unless they voluntarily withdrawn before reaching that age. Recruitment into the study began in September 2011. Every month, approximately 20–24 children were enrolled.

At enrollment, socio-economic, risk factor, and breastfeeding questionnaires were administered by study personnel. Participants’ health status was followed through parent self-reporting on a standardized symptom diary and medical visits at the HCSFV. Parents/guardians were instructed to fill out a simple daily symptom diary for the infant collecting information on the presence of fever, cough, and runny nose. Diaries were collected weekly from the participant’s home by a study field worker who was instructed to refer participants with current symptoms to the HCSFV. Parents/guardians were encouraged to seek medical care for all participant illnesses at the HCSFV and to present early in case of a febrile illness. Infants were identified as study participants at chart reception and routed to the study area of the HCSFV. Study physicians were available to all participants 24 hours/day, 7 days/week, and information pertaining to clinical visits was systematically recorded on a standardized case report form. Children requiring hospitalization were transferred to a hospital by study staff.

Every year in March/April, length and weight of healthy infants were measured and questionnaires completed at enrollment were re-administered by study staff to examine risk factors for influenza virus infection and outcome. A blood sample of 3 ml was collected from infants aged 6 months or older in March/April and banked for future analysis.

Respiratory samples were collected from children presenting with 1) influenza-like illness (ILI), defined as fever (≥37.8°C) or history of fever, and rhinorrhea and/or cough;¹² 2) fever or history of fever without a defined focus; or 3) severe respiratory symptoms (stridor, nasal flaring, wheezing, chest indrawing, and/or central cyanosis) regardless of the presence of fever. Participants identified >96 hours after symptom onset were not sampled. Study staff obtained oropharyngeal swabs from infants <6 months as this is the least invasive sampling method. Combined nasal and oropharyngeal swabs were obtained from older infants. After sample collection and depending on the severity of the illness, participants were asked to return to the HCSFV for at least one follow-up visit. Daily occurrence of fever and respiratory symptoms, as observed by the physician or as reported by the parent, were recorded in a separate form. Pneumonia and severe pneumonia were clinically assessed using the WHO’s Integrated Management of Childhood Illnesses (IMCI) guidelines.¹³

Swab specimens were placed in viral transport media, stored at 4°C, and transported within 72 hours to the National Virology Laboratory at the National Center for Diagnosis and Reference (CNDR) of the Nicaraguan Ministry of Health. RNA was extracted (QIAamp Viral RNA Mini Kit, Qiagen) and tested by real-time reverse-transcription polymerase chain reaction (RT-PCR) for influenza A and B using primers, probes and protocols supplied by the CDC. Samples positive for influenza A, were subtyped for seasonal influenza A H1N1pmd09 and H3N2, and lineage-specific PCR for both influenza B Victoria and Yamagata were run for influenza B positive samples.¹⁴

Follow-up time was calculated as the time between enrollment and the infant’s first birthday or withdrawal from the study. For those lost to follow-up, person-years were calculated as the time between enrollment and last contact with study personnel, plus one-half the time between the last contact and the date recorded as lost to follow-up. A Poisson distribution was used to calculate 95% confidence intervals (CIs) for incidence rates. Incidence rate ratios (IRRs) were calculated using generalized estimating equations (GEE) with a Poisson distribution and robust standard errors. Statistical analyses were performed in STATA 13 (StataCorp LP).

Enrollment began on September 8, 2011 and continued through July 15, 2013 with approximately 20 newborns, aged 4 weeks or less, enrolled monthly. Most (97%) of the parents/guardians invited to participate in the study agreed to enroll their children.

This report focuses on the first year of life. All infants born on or before July 15, 2013 were included in this analysis (n=518). A total of 441 (85%) infants, were followed until their first birthday, while 77 (15%) were withdrawn. Forty-six families moved outside of the study area and were lost-to-follow-up. Six infants died while enrolled in the study, 5 with severe pneumonia, which was accompanied with heart and/or lung congenital malformations in two of the five deaths. On average, the time of participation in the study was 0.9 years (10.5 months) per child. Fifty-eight (11%) women were vaccinated against influenza during pregnancy (Table 1). A total of 23 (4%) children were vaccinated between 6 months and 1 year of age.

A total of 23,654 symptom diaries from 517 (99.8%) of the 518 study infants were collected by field workers, with most (96%) collected in the week following their completion. The average number of medical visits per participant was 13 (range: 0–50), and 504 (97%) participants attended at least one medical visit. The total number of medical visits attended was 6,552 with 4,577 (70%) initial visits (i.e., medical visits for a new illness) and 1,975 (30%) follow-up visits.

A total of 1,286 influenza-like illnesses (ILI) were identified using the symptoms diaries completed by the parent/guardians of study participants and 1,053 ILI events were medically-attended. Three hundred and ninety-eight (77%) participants experienced at least one medically-attended ILI event. The average number of medically-attended ILI events per infant was 2.0 (range: 0–12). The incidence of medically-attended ILI was 229.6 cases per 100 person-years and was similar in both genders (Table 2). The medically-attended ILI incidence was higher in infants aged ≥6 months when compared to younger infants (Table 2). Of the 1,053 medically-attended ILI cases, 1,021 presented in the first 96 hours from symptom onset and thus met the testing definition. Of these, 1,019 (99.8%) had a respiratory sample collected immediately after presentation. Moreover, an additional 165 samples were collected from infants presenting with fever without a defined focus and 29 from infants with severe respiratory symptoms in the absence of fever.

Of the 1,213 collected samples, 74 (6%) tested positive for influenza by RT-PCR. Three infants had two respiratory samples that tested positive for the same influenza virus type/subtype within 14 days, and were considered a single influenza infection, leaving a total of 71 influenza positive cases. Of the 518 infants, 68 (13%) tested positive at least once, 3 children experienced two infections. Of the influenza cases (n=71), 49 were attributed to influenza A and 22 to influenza B. Of the 49 influenza A cases, 12 were H1N1pdm09 and 37 were H3N2. All influenza B cases tested were positive for the Victoria lineage. Influenza occurred predominantly during June–November in 2012 and June–October in 2013 (Figure 1).

Seven (10%) influenza cases did not meet the ILI definition and presented with fever without a defined focus. No infant with severe respiratory symptoms in the absence of fever tested positive for influenza. Three (0.6%) participants experienced two influenza infections during their first year of life. Two participants exhibited two influenza A infections (in both cases, one H1N1pdm09 and one H3N2), and one experienced an influenza A H1N1pdm09 infection and an influenza B infection. The mean number of days between infections was 49 (range: 24–268).

The incidence of laboratory-confirmed influenza illness among children meeting the testing definition was 15.5 cases per 100 person-years (95% CI: 12.2, 19.5) (Table 2). Consistent with the ILI data, influenza incidence was higher in children aged 6 months or older. This increased incidence in infants aged 6 months or older compared to younger infants was significant for both influenza A (IRR: 1.87, 95% CI: 1.04, 3.37) and influenza B (IRR: 2.65, 95% CI: 1.04, 6.78).

We then compared influenza-positive and -negative cases in terms of clinical signs at presentation. For this analysis, participants who were sampled because they presented with severe respiratory symptoms in the absence of fever were excluded (n=29). The proportion of children who presented with rhinorrhea and nasal congestion was significantly higher in influenza-positive cases (Table 3). On the contrary, the proportion of children with cough, increased respiratory rate, difficult breathing, or loss of appetite was not different in influenza-positive versus influenza-negative cases (Table 3). Clinical symptoms throughout the episodes were also compared using the daily information collected during the initial and follow-up medical consults in the follow-up forms. Follow-up consults were attended by 763 (63%) of the participants who were sampled. The number of days with clinical symptoms was not different in influenza-positive versus influenza-negative cases (See Table, Supplemental Digital Content 1).

We examined the contribution of influenza virus infection to severe respiratory outcomes. A total of 241 pneumonia cases were identified in the study, yielding an incidence of 52.6 cases per 100 person-years (95% CI: 46.3, 59.6). Eight (3%) had a positive influenza test at presentation or in the 28 days prior to onset of pneumonia and were considered influenza-associated. These 8 influenza-associated pneumonia cases were distributed as follows: 4 presented with an initial diagnosis of pneumonia and were influenza-positive at presentation (time between symptom onset and presentation: 1 day for all 4 cases); 2 presented with ILI, tested positive for influenza, and developed pneumonia during follow-up (time between ILI and pneumonia onset for both cases: 3 days); and 2 presented with ILI, tested positive for influenza, and developed pneumonia after the initial illness resolved (time between ILI and pneumonia onset: 16 and 18 days). Further, 1/60 (1.7%) severe pneumonia and 1/81 (1.2%) hospitalizations for respiratory causes were influenza-associated. The influenza-associated hospitalization was due to severe pneumonia. The incidence of influenza-associated pneumonia and hospitalization was 1.7 (95% CI: 0.9, 3.5) and 0.22 (95% CI: 0.03, 1.55) cases per 100 person-years, respectively (Table 2). None of the 6 infants who died during the course of the study had an influenza infection in the month before their death.