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Open Access Highly Accessed Research article

Rate of decline of antibody titers to pandemic influenza A (H1N1-2009) by hemagglutination inhibition and virus microneutralization assays in a cohort of seroconverting adults in Singapore

Jung Pu Hsu12, Xiahong Zhao2, Mark I-Cheng Chen23*, Alex R Cook2, Vernon Lee24, Wei Yen Lim2, Linda Tan2, Ian G Barr5, Lili Jiang2, Chyi Lin Tan4, Meng Chee Phoon1, Lin Cui6, Raymond Lin6, Yee Sin Leo4 and Vincent T Chow1

Author Affiliations

1 Department of Microbiology, National University Health System, National University of Singapore, Singapore, Singapore

2 Saw Swee Hock School of Public Health, National University Health System, National University of Singapore, 16 Medical Drive, Kent Ridge, 117597 Singapore, Singapore

3 Department of Clinical Epidemiology, Tan Tock Seng Hospital, Singapore, Singapore

4 Department of Infectious Diseases, Tan Tock Seng Hospital, Singapore, Singapore

5 WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, Australia

6 National Public Health Laboratory, Ministry of Health, Singapore, Singapore

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BMC Infectious Diseases 2014, 14:414  doi:10.1186/1471-2334-14-414

Published: 28 July 2014



The rate of decline of antibody titers to influenza following infection can affect results of serological surveys, and may explain re-infection and recurrent epidemics by the same strain.


We followed up a cohort who seroconverted on hemagglutination inhibition (HI) antibody titers (≥4-fold increase) to pandemic influenza A(H1N1)pdm09 during a seroincidence study in 2009. Along with the pre-epidemic sample, and the sample from 2009 with the highest HI titer between August and October 2009 (A), two additional blood samples obtained in April 2010 and September 2010 (B and C) were assayed for antibodies to A(H1N1)pdm09 by both HI and virus microneutralization (MN) assays. We analyzed pair-wise mean-fold change in titers and the proportion with HI titers ≥ 40 and MN ≥ 160 (which correlated with a HI titer of 40 in our assays) at the 3 time-points following seroconversion.


A total of 67 participants contributed 3 samples each. From the highest HI titer in 2009 to the last sample in 2010, 2 participants showed increase in titers (by HI and MN), while 63 (94%) and 49 (73%) had reduction in HI and MN titers, respectively. Titers by both assays decreased significantly; while 70.8% and 72.3% of subjects had titers of ≥ 40 and ≥ 160 by HI and MN in 2009, these percentages decreased to 13.9% and 36.9% by September 2010. In 6 participants aged 55 years and older, the decrease was significantly greater than in those aged below 55, so that none of the elderly had HI titers ≥ 40 nor MN titers ≥ 160 by the final sample. Due to this decline in titers, only 23 (35%) of the 65 participants who seroconverted on HI in sample A were found to seroconvert between the pre-epidemic sample and sample C, compared to 53 (90%) of the 59 who seroconverted on MN on Sample A.


We observed marked reduction in titers 1 year after seroconversion by HI, and to a lesser extent by MN. Our findings have implications for re-infections, recurrent epidemics, vaccination strategies, and for cohort studies measuring infection rates by seroconversion.

Pandemic influenza A(H1N1)pdm09; Declining antibody; Seroconversion; Hemagglutination inhibition assay; Microneutralization assay