Abstract
Influenza may cause severe complications for pregnant women. In this study antibody response against 2009 H1N1 influenza virus in pregnant women was investigated. This seroprevalance cross sectional and questionnaire based study was conducted using a convenient sampling method. Blood samples of pregnant women were checked for antibodies against 2009 H1N1 influenza virus using hemagglutination inhibition assay. An antibody titer level of ≥ 1:40 dilution was considered as the protective level. 167 (43.60%) of 383 pregnant women who participated in this study had protective antibody levels against this virus. 62 (35.63%) of 3rd trimester, 79 (46.74%) of 2nd trimester, and 21(52.50%) of 1st trimester pregnant women were immune respectively (χ2for trend = 8.20, p < 0.004). Lack of protective antibody level was significantly seen more in pregnant women of 3rd trimester of pregnancy (OR = 2.37, CI = 1.09–5.18). Pregnant women with higher education (OR = 1.67, CI = 1.02–2.73) and those with history of anemia (OR = 2.09, CI = 1.18–3.68) had more immunity. Older women (OR = 0.95, CI = 0.91–0.99) and those with history of psychological diseases (OR = 0.19, CI = 0.05–0.70) had less immunity. Vaccination of pregnant women, especially those who are in the higher trimesters of pregnancy, older, or less educated, against the 2009 H1N1 influenza virus should be continued.
Introduction
In April 2009 the first case of the new swine influenza (later renamed as 2009 H1N1 pandemic) was diagnosed.Citation1 In June 2009, the World Health Organization (WHO) announced that the H1N1 pandemic alert had increased from phase 5 to phase 6.Citation1 In August 2010, the WHO announced that “the H1N1 influenza virus had moved into the post-pandemic period but localized outbreaks of various magnitudes are likely to be continued.”Citation2
At least 18,449 deaths with an estimation of 61 (ranging from 43–89) million cases of influenza like illness and 274 (ranging from 195–403) thousands hospital admissions occurred because of the 2009 H1N1 influenza pandemic.Citation1,Citation3 The frequencies of patients with underlying illnesses who were admitted to hospitals or expired, respectively, because of the 2009 H1N1 outbreak were reported as follows: asthma (28%, 17%), chronic obstructive pulmonary disease (COPD) (8%, 22%), diabetes mellitus (15%, 19%), chronic vascular disease (excluding hypertension) (14%, 21%), morbid obesity (16%, 8%), neurocognitive disorder (7%, 11%), and neuromuscular disorder (7%, 8%). Such frequencies for pregnant women were 6% and 6%, respectively.Citation1
Therefore, one year after the announcement of the post-pandemic stage of the 2009 H1N1 influenza infection and considering the remaining uncertainties about its future outbreaks, and the achieved experiences, it is surely important to promote and apply preventive strategies against Influenza. Pregnant women are among the high-risk groups and are more prone to influenza and its complications' induced morbidity and mortality because of the anatomical and physiological changes and relative immune suppression.Citation4-Citation9 There are many reports about the adverse outcomes of the 2009 H1N1 pandemic in pregnant women and/or in their fetuses or newborns.Citation4,Citation10 Therefore, according to the Center for Disease Control and Prevention (CDC) and other authorities, pregnant women are among the top priorities for influenza vaccination.Citation11,Citation12 On the other hand, resources are limited when it comes to the accessibility of the Influenza vaccine, especially in epidemic seasons and in developing countries like Iran. Two years after the 2009 pandemic, the question is whether the strategy of the 2009 H1N1 virus vaccination for pregnant women should be continued or most of them have been immunized sufficiently by natural exposures to this virus since the beginning of the mentioned pandemic. To the best of our knowledge, until reporting the results of this study there are only few studies that have determined the humoral immunity of pregnant women against the 2009 H1N1 influenza virus and nearly all of them were conducted at the beginning, during and/or immediately after the end of the 2009 H1N1 pandemic.Citation7,Citation9
We performed this study to answer the above questions by detecting the antibody level and humoral immunity status of pregnant women against the 2009 H1N1 Influenza virus and its associated factors in Shiraz, southern Iran.
Results
A total of 383 pregnant women with a mean age of 27 ± 5.28 y (ranging from 16 to 42 y) participated in this study. Other demographic characteristics of these women are shown in . Two hundred ninty-nine (78.1%) pregnant women were referred to Hafez Hospital and 84 (78%) to Zeinabieh Hospital affiliated to Shiraz University of Medical Sciences. Twenty-three (6%) women had received influenza vaccination (including the 2009 H1N1 influenza strain) since the beginning of the 2009 H1N1 pandemic, and 62 (16.1%) women stated that they had contracted influenza like illness symptoms (ILI) in the same period (). Other OB related information is also shown in . shows the history of various diseases as well as immunocompromising conditions in the participants.
Table 1. Associated factors with positive titer (> 1/40) of antibody against the 2009 H1N1 influenza virus antigen in pregnant women referred to obstetric hospitals of Shiraz, southern Iran. (n = 383)
Table 2. History of different diseases in pregnant women referred to obstetric hospitals of Shiraz, southern Iran. (n = 383)
One hundred seventy-four (45.4%) women were in their third trimester of pregnancy; 169 (44.1%) and 40 (10.4%) women were in their second and first trimesters of pregnancy, respectively ().
Table 3. Significant associated factors, using univariate analysis, to detect protective level (> 1/40) of antibody against 2009 H1N1 influenza virus antigen in pregnant women referred to obstetric hospitals of Shiraz, southern Iran
There was a significant difference between the different trimesters of pregnancy regarding protective antibody levels ( and ). One hundred sixty-seven (43.6%) of pregnant women had protective antibody levels (≥ 1/40) against 2009 H1N1 pandemic, but in this group of 162 (42.3%) whose gestational ages were defined for us, 62 (38.3%) belonged to 3rd trimester, 79 (48.8%) to 2nd trimester, and 21(12.9%) to 1st trimester pregnant women respectively. Lack of protective antibody level was significantly seen more in pregnant women who were in the 3rd trimester of pregnancy (OR = 2.37, CI = 1.09–5.18) as reported in . Moreover, pregnant women with a higher education level had higher protective antibody levels (χ2for trend = 10.3, df = 2, p < 0.002, OR = 3.33, CI = 1.42–7.91 for women with > 12 y of education, ). Also, pregnant women with a history of psychological illness had less protective antibody levels against the 2009 H1N1 influenza virus compared with pregnant women who did not have such a history (OR = 0.19, 95% CI = 0.05–0.70) ().
Figure 1. Error bar showing of 95% CI of natural logarithm of antibody titer against 2009 H1N1 influenza virus in different trimester pregnant women referred to Shiraz University Obstetric hospitals, southern Iran, according to protecive level of antibody (≥ 1/40).
Figure 2. Reversed ratio of antibody titer against 2009 H1N1 influenza virus measured by Hemagglutination assayin 383 pregnant women, Shiraz-southern Iran (Error bars: 95% Cl).
Another finding was the statistically significant difference between the proportion of pregnant women with anemia and those without anemia regarding protective antibody levels against the 2009 H1N1 (OR = 2.09, 95% CI = 1.18–3.68, ). Univariate analysis did not show any significant relationship in the protective antibody levels against the 2009 H1N1 influenza virus antigen with respect to the following data of the participants: age, place of residence, occupational status, household members, hospital of reference, time elapsed from the last pregnancy, number of pregnancies, number of fetuses in the present pregnancy, BMI before pregnancy, BMI of the first trimester pregnant women, history of contracting influenza or being vaccinated against it during the last two years before this study, and history of having chronic diseases (except for psychological illnesses or anemia) (p > 0.05, ).
Table 4. Logistic regression model (Forward Wald Method)* of factors related to protective level of antibody against the 2009 H1N1 influenza virus antigen, in pregnant women referred to university obstetric hospitals of Shiraz, southern Iran
However, after considering variables with a p value ≤ 0.25 (trimester of pregnancy, age, education, occupation, household members, BMI of the 1st trimester pregnant women, number of fetuses in the present pregnancy, history of contracting influenza during the two years after the beginning of the 2009 H1N1 pandemic, history of having asthma, hypertension, anemia, and psychological illness) in the logistic model (Forward method), the result showed that the first trimester of pregnancy (OR = 2.70, CI = 1.17–6.22), age (OR = 0.95, CI = 0.91–0.99), more than 12 y of education (OR = 3.54, CI = 1.44–8.68), having psychological illness (OR = 0.19, CI = 0.05–0.70), and anemia (OR = 2.09, CI = 1.18–3.68), had a significant association with protective antibody levels against the 2009 H1N1 influenza virus ().
Discussion
By looking at the influenza virus pandemics in the 20th century such as the 1918 (H1N1), 1957 (H2N2) and 1968 (H3N2)Citation12 and the epidemics of seasonal influenza,Citation9 we observe that pregnant women have been more vulnerable to death because of influenza and its complications compared with the general population.
In the recent 2009 H1N1 pandemic, pregnant women and their fetuses or newborns were among the high risk groups with respect to the higher rate of hospitalization, intensive care unit (ICU) admission, and morbidity or mortality compared with the general population.Citation5,Citation6,Citation10,Citation13,Citation14 Therefore authorities declared that the most effective and the only way to eliminate uncertainties regarding possible future waves of the 2009 pandemic influenza A (H1N1) is vaccination against it.Citation11 Based on recommendations, pre-vaccination surveillance of the immune status of different risk groups may help to prioritize which groups should be vaccinated.Citation8
We found that only 43.6% of the pregnant women had protective levels (titer ≥ 1/40) of Antibody against the 2009 H1N1 influenza virus antigen and more than half were not sufficiently immunized against this virus. This result shows that pregnant women had significantly less protective antibody titer levels than the general population.Citation7 Our findings could be supported by the evidences shown in other study about attenuated humoral immune response in pregnant women compared with before pregnancyCitation9 while contradictory report also exist.Citation7
Pregnant women’s antibody response to the 2009 H1N1 influenza virus that was detected in this study is higher than the result of another study in Australia. In the latter study, HI titers ≥ 1/40 against the 2009 H1N1 virus were found in 4.5% (95% CI, 2.4–8.3%) and 10.2% (95% CI, 4.1–17.1%) of the 21–45 y-old pregnant women, before and after the circulation of the 2009 H1N1 pandemic virus, respectively.Citation15 Another report from Australia showed that pregnant women (50%) developed less protective levels of antibody titer (≥ 1/40) than non-pregnant age-matched women (88%) and the total population (86%) ≥ 2 weeks after their 2009 H1N1 infection confirmation by real time PCR. 9In that study no statistically significant correlation was found between seroprotection and the age of the participants.
According to another study done by Moghadami and colleagues in December 2009 (during the peak of the 2009 H1N1 influenza pandemic) in Shiraz, elder age groups (60–64 y olds) had higher antibody levels, while youths (20–24 y olds) had lower antibody levels. There were no differences regarding other demographic characteristics or history of influenza 6 mo before conducting the mentioned study.Citation7 A study done in the initial stages of the 2009 H1N1 pandemic showed that unvaccinated individuals older than 60 y had a higher level (34%) of pre-existing antibody against the 2009 H1N1 compared with unvaccinated younger individuals (4%),Citation16 assuming the previous exposures of older age groups to this type of virus (such as the 1918 influenza pandemic).Citation16,Citation17 The CDC proved that 33% of people older than 60 y had pre-existing antibodies against the mentioned virus in general population in the US.Citation18
The differences regarding the protective antibody level against the 2009 H1N1 influenza virus among various studies including our study may be related to the different study period (according to the 2009 H1N1 pandemic), sample size, sampling method, age range of participants, history of prior seasonal influenza vaccination (considering the cross reactivity among the 2009 H1N1 and seasonal H1N1 documented in another study,Citation9 and possibly other reasons that need further investigation. One common finding among all these studies is that at least 40% of pregnant women do not have sufficient immunity against the 2009 H1N1 influenza virus. Moreover in our study, it was shown that fewer pregnant women in their third trimester had protective antibody levels against the 2009 H1N1 influenza virus compared with women in other trimesters of pregnancy. Up to the time of writing this paper, we did not find any other study that compared pregnant women in different trimesters of pregnancy or different gestational ages with respect to 2009 H1N1 antibodies.
The lower immune response of pregnant women in their third trimester against the 2009 H1N1 influenza in our study is consistent with evidence showing that immune response of pregnant women to viruses in the last months of pregnancy may be attenuated. 19We also showed that younger women had higher immunity against the 2009 H1N1 influenza virus than older women. This finding is against other reportsCitation7,Citation16 and may be related to the higher exposure of these groups to the influenza virus since the beginning of the 2009 H1N1 influenza pandemic. Also pregnant women with higher educational level were more immune than less educated pregnant women.
We also found that a significantly lower proportion of pregnant woman with a history of psychological diseases had protective antibody levels against the 2009 H1N1 influenza virus compared with pregnant women without such history. Our assumption is that psychological diseases and/or drugs may play an important role in this regard. However, the reasons should be clarified more in studies with larger sample sizes of pregnant women with such history. Moreover, pregnant women with anemia had more protective levels of antibody against H1N1 but this result alone should not be taken into decision making. Although the uptake rate of the 2009 H1N1 included seasonal vaccine among pregnant women in our study was poor and lower than some other countriesCitation20,Citation21 nearly all those studies showed low 2009 H1N1 influenza vaccination in pregnant women and concluded that education is necessary in order to overcome barriers against influenza vaccination among these groups.Citation20-Citation22 Our study also had some limitations. We included only pregnant women who referred to university affiliated hospitals. Pregnant women referred to private hospitals may have had better access to influenza vaccine because of their better financial status, although we know that they comprise only the minority of pregnant women in our city.
Another limitation was that we studied pregnant women in different trimesters of pregnancy in a cross sectional manner. If we had done a cohort study and followed up each pregnant woman during different trimesters, from before pregnancy to the postpartum period, including the control group, the comparison of immune status among different trimesters or between pregnant women and the general population would have been more accurate. Another point is that the only source of information about having chronic diseases such as psychological diseases or anemia in the pregnant women was verbal history and their self-explanation. Therefore, more details of their diseases or related treatments were not clear for us. So the reliability of this information may not be as high as documented information. However, we recommend further document-based studies with larger sample sizes to determine the immune status of pregnant women with such diseases against the 2009 H1N1 influenza virus. Conclusively, since the humoral immunity of most pregnant women is not high enough against the 2009 H1N1 influenza virus and also because it is not possible to easily determine each pregnant woman’s immunity status against this virus in daily OB clinics or in hospitals, the strategy to encourage pregnant women to receive vaccination against this virus should be continued. Therefore, the vaccination of pregnant women, especially those who are in higher trimesters of pregnancy or those who are older or less educated is strongly recommended. Also, reasons of low uptake rate of influenza vaccination among pregnant women should be determined and overcome.
Patients and Methods
This seroprevalance cross sectional and questionnaire based study was conducted from November 2010 to January 2011 in obstetric (OB) hospitals affiliated to Shiraz University of Medical Sciences in Shiraz, the capital city of Fars province, southern Iran. A convenient sampling method was used. The sample size necessary for this study was 371 pregnant women, calculated using the formula z2Pq/d2, assuming that p = 58.91%, confidence level = 95%, and error = 5%. The prevalence of 58.91% was extracted from the 2009 H1N1 pandemic antibody (Ab) titer defining survey, taken from the general population of Fars province two years before this study.Citation7
There were no exclusion criteria, except for those pregnant women who did not want to participate in this study. There are two major OB hospitals affiliated to Shiraz University of Medical Sciences (Hafez and Zeinabieh) where pregnant women refer to. Initially, three experienced and trained midwives explained the aims of the study to the pregnant women and filled the related informed consent forms and questionnaires. A 5-ml blood sample was taken from each participant. Each woman was privately interviewed and blood samples were taken individually in a separate room in the OB clinics of the above-mentioned hospitals.
The questionnaire consisted of an introductory explanation about the aims of the study and the identity of the researchers, and some questions regarding the demographic characteristics of the participants and obstetrical issues. Histories of contracting influenza or being vaccinated against it, or having chronic diseases were also included in the questionnaire. These histories were based only on information provided by the pregnant women themselves. The content and face validity of the questionnaire were assured by an expert opinion, and its reliability was 0.65, calculated using the Kuder-Richardson Equation 20 (KR-20).
Pregnant women with a gestational age (documented by their last menstrual period or pelvic sonography) of up to 14 weeks, 14+1-28 weeks, and equal or more than 28+1 weeks were categorized as being in their first, second, or third trimesters of pregnancy, respectively.Citation23 The body mass index (BMI) was calculated as weight (kg) divided by the square of height per meter (m2).Pregnant women in their first trimester were classified as underweight, normal, overweight, and obese if their BMI was below 19.8, 19.8–26, 26.1–29, or more than 29, respectively.Citation24
Blood samples were transferred quickly to the Sub-national Influenza Diagnosis Laboratory of Shiraz University of Medical Sciences, keeping cold chain standards. The samples were centrifuged in the first 4 h for 10 min with a speed of 3,000 rpm, and then the sera were separated and stored at -25°C to be tested.
The Ethics Committee of the Health Policy Research Center (HPRC) affiliated to Shiraz University of Medical Sciences approved this study based on the mentioned protocol.
Antigen preparation
The influenza virus was isolated from throat swabs of patients with confirmed H1N1 infection using real time polymerase chain reaction (RT-PCR) and primers suggested by the WHO. Madin-Darby canine kidney (MDCK) cell monolayer was grown in Dulbecco’s modified Eagle’s medium (DMEM) (Stigma) supplemented with 10% fatal bovine serum (FBS) (Gibco), 100 unit/ml penicillin G and 100 µg/ml streptomycin and infected with the pharyngeal specimen, and incubated at 34°C for 4–5 d.Citation25 Upon the development of a cytopathic effect, viral presence was shown using the hemagglutination assay and RT- PCR using specific 2009 H1N1 influenza primer Taqman® Probes (influenza A, swine influenza A) (CDC protocol for RT-PCR) for H1N1 influenza A.
Hemagglutination inhibition assay (HIA)
In our study, HI assay was applied using the Suing Method.Citation26 The preserved sera of the pregnant women were inactivated at 56°C for 30 min. Non-specific inhibitors and auto hemagglutinins were then removed. Doubling dilutions of sera (resulting in dilutions from 1:10 to 1:1280) were made in 96 V shaped microplates. Twenty-five µL of a solution containing four hemagglutination antigen units was added to each sera dilution and incubated at 37°C for one hour. Then, 50 µL of guinea pig erythrocytes (0.5% v/v) was added to each well and the results were recorded after one hour at 4°C. The hemagglutination antibody titer of each serum sample was the inverse of the last dilution where cells were not agglutinated. The antibody titer of 1/40 or more was considered as the protective level against the 2009 H1N1 influenza virus.Citation18,Citation27,Citation28
Data analysis
The data were analyzed using SPSS software, version 11.5 (SPSS) and software for epidemiological information, version 6 (EPI 6). The accuracy of the entered data was assured by randomly selecting and checking completed questionnaires with their corresponding data in SPSS software. The relationship of each demographic characteristic, OB related variables, history of having chronic diseases, history of contracting influenza or vaccination against it with protective antibody levels against the 2009 H1N1 Influenza virus antigen was determined separately by independent t, Chi square, Chi square for Trend and Fischer’s exact tests, as appropriated. Then, the simultaneous relation of the above variables with the protective antibody titer (as the dependent variable) was investigated by including the more important variables (with p value ≤ 0.25) in the logistic regression model (Forward method). A P value of < 0.05 was considered significant.
| Abbreviations: | ||
| HI | = | hemagglutinin |
| N | = | nuraminidase |
| WHO | = | World Health Organization |
| CDC | = | Center for Disease Control and Prevention |
| CI | = | confidence interval |
| OR | = | odds ratio |
| BMI | = | body mass index |
| RT-PCR | = | real time polymerase chain reaction |
| OB | = | obstetric |
| Ab | = | antibidy |
| MDCK | = | Madin-Darby canine kidney |
| DMEM | = | Dulbecco’s modified Eagle’s medium |
| FBS | = | fatal bovine serum |
| HIA | = | hemagglutination inhibition assay |
| SPSS | = | Statistical Program for Social Sciences |
| EPI | = | epidemiological information |
| HPRC | = | Health Policy Research Center |
| ILI | = | influenza like illness |
Acknowledgments
We would like to thank Hafez and Zeinabieh hospitals' OB clinics and sub-national influenza laboratory staff at Shiraz Medical School that supported this study.
Disclosure of Potential Conflicts of Interest
No conflicts of interest were disclosed.
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