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Henkle, Emily MPH, PhD*; Steinhoff, Mark C. MD*†; Omer, Saad B. MB BS, PhD; Roy, Eliza DCh§; Arifeen, Shams E. MB BS, DrPH§; Raqib, Rubhana PhD§; McNeal, Monica MS; Breiman, Robert F. MD; Moss, William J. MD*; Zaman, K. MB BS, PhD§

The Pediatric Infectious Disease Journal: February 2011 - Volume 30 - Issue 2 - p 170-173
doi: 10.1097/INF.0b013e3181f63c39
Brief Reports

We evaluated infant sera from an immunization trial in Bangladesh to assess influenza hemagglutination inhibition antibody titer increases in 131 unimmunized infants from birth to 6 months. We detected 31 serologically defined infections. Combined with 10 additional rapid test-proven influenza cases, the minimal estimated incidence was 31 of 100 infants (95% CI: 24–41). These data suggest a high burden of influenza in young infants in tropical South Asia.

From the *Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD; †Global Health Center, Cincinnati Children's Hospital Medical Centre, Cincinnati, OH; ‡Global Health, Epidemiology, and Pediatrics, Rollins School of Public Health, Emory University, Atlanta, GA; §International Centre for Diarrheal Disease Research, Dhaka, Bangladesh; and ¶Centers for Disease Control and Prevention, Atlanta, GA.

Accepted for publication August 5, 2010.

The Mother's Gift project was supported by the Bill and Melinda Gates Foundation, United States Agency for International Development cooperative agreement HRN-A-0096–90006–00; Wyeth Pharmaceuticals Inc, the Thrasher Research Fund, Aventis Pasteur, ICDDR,B, and the Bloomberg School of Public Health at Johns Hopkins University. ICDDR,B, acknowledges with gratitude the commitment of donors to the Centre's research efforts. Ms. Henkle was supported by the Department of Health and Human Services, National Institutes of Health, National Eye Institute Training Grant Number EY 07127 Clinical Trials Training Program in Vision Research.

Research support from Merck, the Bill and Melinda Gates Foundation, and USAID (to R.F.B); research support from the Bill and Melinda Gates Foundation, USAID, the Thrasher Fund, Wyeth, Glaxo Smith-Kline, Sanofi-Aventis, and Merck, and lecture fees from GlaxoSmithKline and Sanofi-Aventis (to M.C.S.). No other potential conflict of interest relevant to this article was reported.

Registered with NCT 00142389.

Address for correspondence: Mark C. Steinhoff, MD, Children's Global Health Center, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave, MLC 2048, Cincinnati, OH 45229–3039. E-mail:

Young infants 0 to 6 months of age have high rates of hospitalization from influenza and its sequelae, as reported in high income regions.1 The cumulative incidence of influenza virus infection in 0 to 6 month old infants in the United States has been estimated as 12%.2 Historically, influenza has not been considered a frequent or serious illness of infants in the tropics. However, there is limited information on infection rates in infants in tropical regions, where influenza has a perennial pattern with year-round circulation of multiple subtypes and seasonal peaks in incidence.3 Recent clinic surveillance in Bangladesh reported that 10% of pneumonias in children <5 years of age are associated with influenza infection, but provided no information specific to young infants.4 Trivalent influenza vaccine is recommended in some high income regions for infants >6 months of age, but is not licensed for those <6 months.5

The Mother's Gift study was a randomized maternal vaccine trial conducted in Dhaka, Bangladesh in 2004 and 2005. Maternal immunization with influenza vaccine reduced rapid test confirmed influenza infection by 63% and all febrile respiratory illness by 29% in infants 0 to 6 months of age.6 We analyzed prospectively collected infant sera in the Mother's Gift trial to estimate the incidence of serologically defined influenza virus infection in early infancy in Bangladesh.

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A full description of the Mother's Gift trial has been published,6 with a summary provided here. Three hundred forty pregnant women in Dhaka were randomized to receive polysaccharide pneumococcal vaccine (Pneumovax) or trivalent inactivated influenza vaccine (Fluarix). The influenza vaccine contained the World Health Organization (WHO) southern hemisphere recommendation for 2004: A/Fujian/411/2002-like (H3N2), A/New Caledonia/20/99-like (H1N1), and B/Hong Kong/330/2001-like strains.6 Of the 316 infants followed to 6 months of age in the trial,6 we report serologic values from the 157 infants of mothers who received pneumococcal vaccine. None of the mothers and infants included in this analysis received influenza vaccine.

From August 2004 through October 2005, cohorts of study mothers and their infants were followed from birth to 6 months by weekly home visit or telephone interview to record symptoms of febrile respiratory illness. Influenza A/B virologic testing in symptomatic infants was performed using a rapid detection test (RDT; Zstat, ZymeTx, Oklahoma City, OK). Influenza antibodies against the 3 vaccine strains were assayed using a standard hemagglutination inhibition (HAI) assay7 on infant sera collected at birth (cord), 10 weeks, and approximately 22 weeks of age.

We calculated the half-life of passively transferred HAI antibody, assuming exponential decay, among the infants from the full cohort with decreasing antibody values. We used the average calculated half-life of HAI antibody to estimate the expected antibody concentration at 10-week intervals. We defined serologic infection as a 4-fold increase in the observed titer compared with the expected declining titer, expressed as a later/earlier antibody ratio of ≥1 for sera obtained at least 10 weeks apart (Fig. 1). To account for the known variation in the HAI assay, we also specified that if the earlier titer was ≤1:10, the later titer should be ≥1:20. Incidence was calculated using Poisson regression with robust confidence intervals. The estimates are expressed as cumulative incidence per 100 infants and infections per 100 infant-months of observation.



The project protocol was approved by the Institutional Review Boards of International Centre for Diarrheal Disease Research, Bangladesh and the Bloomberg School of Public Health, Johns Hopkins University, and all mothers provided written informed consent.

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The HAI data were available from 92% of 157 study infants. There was no difference in baseline demographic or birth characteristics comparing infants with and without samples available for analysis.

The estimated half-life of HAI antibody ranged from 33 to 45 days for the 3 virus subtypes in the 2 infant age groups. The overall weighted average half-life was 38 days, weighted by the number of sera tested for each influenza antigen. Thus, it would take about 76 days or 10.5 weeks for antibody titers to decline 2 half lives (a decline to 25% of the initial value). An antibody ratio of ≥1 during a 10 week interval is approximately equivalent to a ≥4-fold increase over the expected declining titer.

For the serologically defined incidence analysis, we excluded 14 (9.7%) infants because the planned study 22-week samples were collected at ≥28 weeks of age. Among the evaluated 131 infants, 29 experienced 31 serologically defined influenza virus infections with vaccine subtypes (23.7/100 infants, 95% CI: 17.0–30.0), of which 6 (4.6/100 infants) had an absolute 4-fold increase (Table 1). Significantly more infections occurred after 10 weeks than before; 25 versus 6, respectively (P < 0.001). Two infants had 2 infections each with different subtypes, all between 10 and 22 weeks. The serologically defined incidence of influenza was 4.2 infections per 100 infant-months (95% CI: 3.0–5.9). Serologically defined infections were primarily A/Fujian (29 infections, 93%), with one each of A/New Caledonia and B/Hong Kong subtypes.



Our original report described 16 RDT cases (10.2%) in the 157 infants.6 Among the 131 included in this report, 15 (11.5%) had a positive RDT. Of these, 5 (33%) had concurrent serologically defined A/Fujian subtype infections that were considered the same infection. One infant had 2 infections: a serologically defined infection before 10 weeks of age plus an RDT-proven infection at 22 weeks of age. Adding the 10 distinct RDT-proven infections resulted in a total of 41 influenza infections and a cumulative incidence of 31.3 of 100 0 to 6 month old infants (95% CI: 23.6–41.4). The overall incidence rate of infection in 0 to 6 month old infants was 5.6 of 100 infant-months (95% CI: 4.2–7.4) (Table 1).

We explored the relationships between serologically defined influenza virus infection, RDT-proven influenza, and reported respiratory illness with fever.6 Of 12 infants with RDT-proven influenza by 22 weeks, 4 (25%) had serologic evidence of infection. Including data up to 22 weeks, 75.9% of 29 infants with serologically defined influenza virus infection reported any respiratory illness with fever compared with 52.0% of 102 with no serologically defined infection (P = 0.02).

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We are not aware of a published, prospective serologic study of influenza incidence rates in young infants in Asia. Our unique prospective serologic and RDT data show a substantial burden of influenza in this region in young infants. We found that 31 of 100 (95% CI: 23.6–41.4) infants at 0 to 6 months were infected over a 15-month calendar period. Three infants (2.3%) each had 2 separate influenza virus infections detected before 6 months of age.

By comparison, in the United States, Glezen et al found an average annual influenza incidence of 12.4 of 100 infants aged 0 to 6 months, using culture and serology in Houston from 1975 to 1985.2 Since they only assessed antibodies to the 2 predominant influenza virus strains each year, this is likely an underestimate. Infection in the Texas study was often asymptomatic, similar to our study. Both observations suggest that studies relying on symptoms or clinic visits of young infants as indicators for influenza testing will miss a significant proportion of infections in these infants. Our data in young infants suggests that prospective serology more than doubles the number of influenza virus infections compared with clinic surveillance of febrile illness (Table 1).6

A similar serologically defined infection methodology was used to assess H1N1 influenza virus infection by Reuman et al in a natural history study of 18 US infants followed until age 3 to 6 months during the 1978–1979 season.8 A 4-fold rise over expected titer was seen in 28% of 18 infants of nonimmune mothers, similar to our serologically defined incidence of 24%. The 33 to 45 day half-life of maternally acquired infant antibodies we observed was similar to a US maternal immunization study reporting infant influenza antibody half-lives of 40 to 50 days, using a non-HAI ELISA assay.9

One of the strengths of this serologic analysis was the careful follow-up and sampling of nearly all infants in the study cohort, which allowed detection of a substantial number of asymptomatic influenza virus infections. However, the 10+ week interval between sera tested for HAI antibody results made it challenging to associate serologically defined infection with the weekly reported symptoms and illness. These serologic data are a minimal estimate for several reasons. We only tested for antibodies to the 3 influenza vaccine subtypes and did not assess infection with other influenza subtypes.4 Further, some infants with higher maternal antibody concentration may have had an attenuated antibody response to infection. Finally, the RDT test that we used has low sensitivity,6 suggesting that at least 30% of true cases were missed. Both these factors indicate these data are a minimal estimate of the true rate of infection. The discrepancy between RDT and serologically detected influenza cases likely represents infections with influenza subtypes not included in the HAI serologic testing panel.

Young infants are a high-risk group for influenza infection, illnesses, and sequelae which can lead to substantial parent/caretaker absenteeism.1,6 We observed a minimal influenza virus infection rate of 31 of 100 (95% CI: 23.6–41.4), which suggests a sizeable burden of influenza illness in this tropical setting. These data are part of a growing body of literature that suggests influenza illness in infants and children may also be a neglected tropical disease.4,6

Infants in tropical and subtropical regions comprise more than 60% of the 136 million annual global births,10 and many are exposed to influenza virus infection year-round.3,4 Additional studies of the incidence of influenza in young infants in Asian and other tropical regions should be carried out to better define the burden of preventable influenza illness in this vulnerable group. Maternal antenatal immunization with influenza vaccine is a proven and implementable strategy to protect both pregnant women and their young infants in tropical regions.6

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The authors thank Dr. David Sack for his invaluable assistance in the project. The authors also thank the ICDDR,B field and data management team for their efforts, and they express their gratitude to the families who participated in the project. Special thanks to Justin Lessler for figure graphics.

Dr. Steinhoff had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Steinhoff, Zaman, Roy, Arifeen, Breiman; Acquisition of data: Roy, Zaman, Arifeen, Raqib, McNeal; Analysis and interpretation of data: Henkle, Steinhoff, Zaman, Roy, Altaye, Omer, Moss, Arifeen; Drafting of the manuscript: Henkle, Steinhoff, Moss; Critical revision of manuscript for important intellectual content: Steinhoff, Omer, Moss, Zaman, Roy, Breiman, Raqib, McNeal; Statistical analysis: Henkle, Steinhoff; Obtaining funding: Steinhoff; Administrative, technical, or material support: Arifeen, Zaman, Steinhoff, McNeal; Study supervision: Zaman, Steinhoff.

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10. United Nation's Children's Fund. State of the World's Children 2009. New York: UNICEF; 2008.

influenza; infants

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