The 2009 pandemic has highlighted the need for timely studies to investigate the extent of age-specific human infection after multiple pandemic waves in different geographic regions (19
). Such studies can estimate total numbers of infections upon which to base more accurate estimates of rates of severe or fatal disease. They may also provide policy makers with a better understanding of the proportion of susceptible persons remaining in populations in order to better predict the public health impact of successive pandemic waves. To better understand the relative benefits of the HI versus the MN assay for the detection of 2009 H1N1 virus-infected persons, we characterized serum antibody responses to 2009 H1N1 virus in rRT-PCR-confirmed cases using both serological assays and confirmed that the titers obtained by either assay were highly correlated. Furthermore, using an additional set of sera from non-2009 H1N1-exposed populations, we assessed the relative sensitivities and specificities of both assays. Although the HI assay alone was more specific for detecting antibody to 2009 H1N1 virus, the MN assay was significantly more sensitive for detecting low-titer seroconversions. Finally, based on our sensitivity and specificity analyses, we identified a combination of threshold titers (MN, ≥40, and HI, ≥20) that provided the highest sensitivity and specificity to identify 2009 H1N1 virus-infected persons <60 years of age and high specificity for adults aged ≥60 years using only convalescent-phase sera and in the absence of demonstrable seroconversion in paired sera. These criteria were used to facilitate the analyses of multiple seroepidemiological investigations conducted in the United States during the first wave of the 2009 pandemic.
The kinetics of antibody responses in rRT-PCR-confirmed 2009 H1N1 cases confirmed that the optimal timing of acute-phase serum collection is within 1 week of symptom onset. Although 90% of cases achieved threshold titers for seropositivity (MN titer of ≥40 and HI titer of ≥20) by day 15 p.s.o., sera collected 22 to 28 days p.s.o. had the highest geometric mean titers, suggesting that this remains the optimal time frame for the collection of convalescent-phase sera. These results are consistent with those of Miller et al. (20
) and Hung et al. (15
), who reported that 11% of confirmed 2009 H1N1 cases in England failed to develop HI antibody titers of ≥32 and 10% of confirmed cases in Hong Kong failed to develop neutralizing antibody titers of ≥40. Similar to our results, Hung et al. (15
) also found that the MN assay detected a higher seroconversion rate (89%) than the HI assay (82%).
The HI assay detects antibodies that bind near the receptor binding site of the viral HA, blocking the interaction of HA with sialic acid receptors on erythrocytes and inhibiting their agglutination. Virus neutralization assays, such as the MN assay, detect antibodies that neutralize the virus by inhibiting viral entry and/or replication in mammalian cells, including antibodies recognizing epitopes within the stem region of HA that block membrane fusion and that are conserved among viruses of different influenza A virus subtypes (26
). Detection of cross-reactive antibodies to the stem region could consequently lower the specificity of the MN assay, particularly in the adult and older adult populations, who presumably have had greater exposure to different influenza A viruses throughout their lifetimes.
Several studies have used an HI titer of ≥40 as a marker of infection with or immunity to 2009 H1N1 virus (20
). This is a reasonable approach for large-volume seroprevalence and seroincidence studies and for optimal rapidity of reporting results following successive pandemic waves. However, our data suggest that this titer threshold may underestimate the numbers of 2009 H1N1 virus-infected individuals. On the other hand, due to its lower specificity in adults aged 40 and over, the use of the MN assay alone may overestimate 2009 H1N1 virus infections in the age group for U.S. populations. Where resources permit, and particularly when studies seek to identify 2009 H1N1 virus-infected individuals rather than population rates, our results suggest that the use of both assays and the combination titer achievements provide optimal sensitivity and specificity. However, it should be noted that while it provides a sensitive and specific serological marker for infection, the combination of titer achievements cannot be correlated with a level of protection against the pandemic virus. Furthermore, once a pandemic virus becomes seasonal, as is now the case for the 2009 H1N1 virus (10
), serological confirmation of human infection will once again require detection of seroconversion by either assay, the gold standard for all influenza virus serodiagnosis.
Our study had several limitations. The age distribution of our rRT-PCR-confirmed cases differed from that of 2009 H1N1 cases based on national estimates (21
): the 0- to 4-year age group was underrepresented, and the 5- to 24-year age group was overrepresented. In addition, we were unable to estimate the sensitivity of the assays and titer cutoffs for adults ≥60 years of age. Furthermore, because individuals 80 years old and older exhibit high frequencies of serum antibodies that cross-react with 2009 H1N1 virus, due to structural similarities that exist between the HA molecules of 2009 H1N1 and 1918-like influenza viruses, the seropositivity criteria developed here cannot be applied to this age group (16
). Efforts to discriminate preexisting serum cross-reactive antibody from 2009 H1N1 virus infection-induced antibodies in this age group are ongoing in our laboratory.
Studies from China and Singapore found little evidence of preexisting 2009 H1N1 virus cross-reactive antibodies in older adults, even those ≥80 years of age, suggesting that there are geographic or other factors that contribute to the presence of preexisting antibody in human populations (3
). These findings support the need for laboratories undertaking seroepidemiological or seroprevalence studies for detection of antibody against novel viruses to individually evaluate the prepandemic age-specific prevalence of cross-reactive antibody in local populations. Furthermore, due to interlaboratory assay variation, caution should be exercised in directly using the seropositivity criteria developed here to identify 2009 H1N1 virus-infected persons based on serological data from other laboratories (25
In summary, we have demonstrated an approach whereby serological criteria can be developed to identify human infections using only convalescent-phase sera when novel influenza viruses first emerge to infect humans. Assessing the relative sensitivities and specificities of serological assays is an important component in establishing threshold titers used to estimate the extent of seropositivity among populations after the first pandemic waves. We believe these studies may provide a strategy to assist with timely serological investigations for future pandemics or outbreaks of novel influenza viruses among humans.