The seroepidemiology of immunoglobulin G antibodies against pertussis toxin and filamentous hemagglutinin in the east of China during the COVID-19 pandemic

ABSTRACT

This study employed sero-epidemiological methods to estimate the incidence of pertussis within a healthy population located in eastern China. The aim was to gain deeper insights into the epidemiological characteristics and burden of pertussis within the country. Blood samples were collected from healthy individuals in Jiangsu Province between June 2019 and December 2022. The levels of IgG antibodies against pertussis toxin (anti-PT) and filamentous hemagglutinin (anti-FHA) in the serum were quantitatively measured using enzyme-linked immunosorbent assay (ELISA). Additionally, pertussis case data reported in Jiangsu Province were collected from the China Information System for Disease Control and Prevention and compared with the results of this study. In 2022, the reported incidence of pertussis stood at 1.0 per 100,000 individuals, marking the highest rate observed in the past two decades. Among 1,909 patients examined, the geometric mean concentration (GMC) of anti-PT IgG antibody was 20.2 (18.5–21.9) IU/ml, while that of anti-FHA IgG antibody was 27.0 (25.4–28.7) IU/ml. The IgG-PT and IgG-FHA seropositivity rate (>20.0 IU/ml) was highest in the 1 ~ 2 y old group and decreased rapidly to the lowest in the 3 ~ 4 y old group and then increased gradually with age. The estimated rate of pertussis infection based on seroprevalence was approximately 25,625‐fold higher than the reported notification rate in the ≥15 year age group. Our findings highlight decreased immunity post-vaccination, stressing the importance of additional booster shots for adolescents and adults to maintain immunity and reduce severe illness. Additionally, they offer vital guidance for policymakers to enhance immunization strategies.

KEYWORDS:

• Pertussis
• seroepidemiology
• immunological response
• vaccination
• anti-PT IgG
• anti-FHA IgG

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Enhancing the accuracy of seroprevalence studies: Reassessing pertussis infection rates in eastern China during the COVID-19 pandemic

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Introduction

Pertussis, also known as whooping cough, is a highly contagious respiratory disease caused by the bacterium Bordetella pertussis (BP). The disease is most commonly spread through contact with respiratory droplets from an infected person’s cough or sneeze. Pertussis typically manifests with symptoms akin to those of a common cold, including a runny nose, fever, and cough. Pertussis can be particularly severe and even life-threatening in infants and young children. It has a clear epidemic trend worldwide, with outbreaks every 3–5 y.^1,2 In the era without the use of antibiotics and vaccines, the incidence and mortality rate of pertussis were high, especially among children under 5 y of age. The utilization of antibiotics and vaccines has led to significant alterations in the epidemiological characteristics of pertussis. Nonetheless, protective antibodies acquired via vaccination or natural infection may diminish over time, thereby indicating that outbreaks of pertussis can still transpire even in developed nations with relatively high vaccine coverage.^3,4 Despite global vaccine coverage for pertussis components exceeding 85% over the past 5 y, more than 700,000 pertussis cases have been reported worldwide. In 2007, China introduced a combined diphtheria-tetanus-acellular pertussis (DTaP) vaccine containing pertussis toxoid (PT) and filamentous hemagglutinin (FHA) as its main components, administered for basic immunization at three, four, and five months after birth. Subsequently, children receive booster immunization at 18–24 months of age. According to statistics, the full coverage rate of this vaccine exceeds 99.0%,⁵ with 30,027 cases of pertussis were reported in 2019.⁶

Jiangsu Province is a coastal province in eastern China with a population of approximately 88 million. In recent years, the incidence of pertussis in Jiangsu Province has increased, with the number of reported cases in 2022 increasing by 81.2% compared to the previous year (Figure 1). The high DTaP vaccine coverage rate in Jiangsu Province suggests that insufficient vaccination is unlikely to be the primary reason for the increase in incidence. It is noteworthy that the majority of reported pertussis cases in Jiangsu Province were diagnosed clinically, and PCR technology, utilized in laboratory diagnosis, was not included in the national case definition guidelines. Consequently, there may be underreporting of pertussis cases, particularly among teenagers or adults exhibiting atypical symptoms, who significantly contribute to the transmission dynamics of pertussis by potentially serving as sources of infection for infants who have not yet commenced and/or completed the vaccine schedule.⁷ Importantly, unvaccinated infants represent the most vulnerable demographic, with 90% of pertussis-related deaths occurring within this group.^8,9 Consequently, the current Chinese surveillance system faces challenges in accurately reflecting the true epidemiological situation of Jiangsu Province.

Figure 1. The number of reported pertussis cases, incidence and pertussis vaccination coverage in Jiangsu province, 2007–2022.

PT and FHA were two major virulence factors secreted by BP, serving as key components of acellular pertussis vaccines. Anti-PT and anti-FHA antibodies serve as commonly used indicators in pertussis serological surveillance, facilitating the assessment of population immunity, monitoring pertussis epidemiology, and investigating outbreaks. Additionally, FHA, as another virulence factor produced by pertussis, was utilized as a primary component in many acellular pertussis vaccines and is considered crucial for protection.¹⁰ However, until now, little is known about the seroprevalence of anti-FHA IgG, with only three studies conducted on the detection of anti-FHA IgG in the entire population of China^11–13 (including the pre-survey conducted by our research group in 2018). Furthermore, the non-pharmaceutical interventions (NPIs) implemented during the 2020 COVID-19 pandemic have altered the epidemiology of many respiratory infections and significantly impacted vaccine uptake. This study aims to reassess the seroprevalence of pertussis among different age groups in Jiangsu Province during the COVID-19 pandemic by measuring the concentrations of anti-PT and anti-FHA antibodies in the serum of the general population. This will provide further insights into the disease burden of pertussis in China and offer evidence to optimize immunization strategies.

Methods

Pertussis surveillance

Pertussis is classified as a Class B infectious disease in China. All diagnosed cases by hospital staff must be recorded in the National Notifiable Disease Reporting System (NNDRS), a web-based computerized reporting system. The number of cases is determined from the date of onset, relying on laboratory testing, epidemiological contacts, or clinical criteria. The incidence rate is calculated as the number of cases per 100,000 individuals, utilizing population data provided by the National Bureau of Statistics of China as the denominator.

Subject selection and sample collection

From June 2019 to December 2022, an age-stratified sampling method was employed in six counties of Jiangsu Province. Participants were randomly selected from each county across the following age groups: 0–1 y, 2–3 y, 4–6 y, 7–12 y, 13–16 y, 17–20 y, 21–30 y, 31–40 y, 41–50 y, and >50 y. Blood samples of 3–5 ml were collected from each participant, followed by serum separation. Subsequently, the samples were cryopreserved and shipped to the Jiangsu Provincial Center for Disease Control and Prevention for testing. Immunization history for participants aged over 14 was based on self-reported recall, while information for children under 14 was obtained from the Jiangsu Provincial Immunization Management Information System.

The study had been approved by the Ethics Committee of the Jiangsu Provincial Center for Disease Control and Prevention, and written informed consent was provided by the individual or the child’s parents. Participants were asked to anonymously fill out a questionnaire that included personal information such as gender, age or date of birth and whether they had ever been diagnosed with pertussis

Laboratory testing

Anti-PT IgG and anti-FHA IgG antibodies of BP were quantitatively detected using ELISA, with the reagents sourced from Zhengzhou Yite Biotechnology Co., Ltd. The WHO international standard for human pertussis antiserum (NIBSC 06/140) was utilized for determination. Per the manufacturer’s instructions: the minimum level for detecting anti-PT IgG was 5.0 IU/ml, with concentrations of anti-PT IgG and anti-FHA IgG antibodies below 20.0 IU/mL considered negative. Concentrations equal to or exceeding 20 IU/mL are considered positive and indicative of immune protection. An anti-PT IgG concentration ≥80.0 IU/mL was considered to indicate a recent infection if the subject had not received the pertussis containing vaccine within the previous year.

Statistical analysis

Data analysis was conducted using Microsoft Excel and SPSS V.22.0 (IBM Corp, Armonk, NY) software. Geometric mean concentrations (GMCs) and antibody seropositivity rates, along with their corresponding 95% confidence intervals (95% CI), were calculated and stratified by region, sex, and age. Analysis of variance (ANOVA) was utilized to compare GMCs, with statistical significance set at a two-tailed p-value of .05. Multiple comparisons were conducted using the Student-Newman-Keuls q test (SNK-q test). In cases of unequal variance, a corrected F-test was employed. The Pearson χ2 test was utilized to analyze differences in serum prevalence among various regions, sexes, and age groups, with significance set at p < .05.

Results

Prior to the introduction of the DTaP vaccine in China (1955–2006), the incidence rate of pertussis in Jiangsu Province stood at 41.9 per 100,000 individuals, peaking in 1972 at 237.8 cases per 100,000 people. During the vaccination era, as vaccination coverage steadily improved (with the average coverage rate of the fourth dose of DTaP vaccine exceeding 95.0%, although specific data is not published), the incidence rate gradually declined until a rapid increase was observed after 2016. A total of 484 cases were reported during the period from 2007 to 2016, contrasting with 1489 cases reported from 2017 to 2022 (During the initial phase of the pandemic, which was from January to March 2020, the vaccine coverage rate decreased to around 85%. However, through subsequent supplementary vaccination campaigns by the Chinese government for various vaccines, the coverage rate rebounded to around 99%, according to the data comes from the Jiangsu Provincial Immunization Program Management Information System and historical archival records) (Figure 1). In 2022, the incidence reached its highest level in nearly 20 y, with a reported incidence of 1.0 per 100,000 individuals. WHO data also demonstrate a clear inverse relationship between coverage rates and morbidity. Additionally, approximately 40.0% of cases occur in infants aged between 0 and 1 year, while an increasing number of cases have been observed in individuals over 15 y of age in recent years.

Sociodemographic information and vaccination

The study enrolled a total of 1909 healthy subjects, spanning in age from 0 to 83 y. The male-to-female ratio was 1.02:1 (965 males to 944 females), and the ratio of native to immigrant participants was 4.6:1 (1569 natives to 340 immigrants). Among children aged 1–14, 98.7% (863 out of 874) were confirmed to have received at least one dose of a pertussis-containing vaccine (see Table 1).

Table 1. Distribution of anti-PT IgG and anti-FHA IgG concentration in the study population.

Variables	N	Anti-PT antibodies		Anti-FHA antibodies
		GMC (95% CI) (IU/mL)	p value	GMC (95% CI) (IU/mL)	p value
Age(years)			.000		.000
<1	199	16.1 (12.3–19.9)		17.9 (13.9–22.0)
1–2	316	38.5 (32.4–44.6)		36.1 (31.9–40.4)
3–4	160	11.5 (7.1–15.9)		18.4 (13.4–23.4)
5–6	119	158 (9.8–21.8)		23.1 (17.1–29.1)
7–9	139	14.8 (9.7–19.8)		25.1 (19.1–31.1)
10–14	196	16.2 (11.5–20.8)		28.2 (23.4–33.1)
15–19	232	14.2 (10.8–17.5)		21.6 (17.9–25.3)
20–29	148	13.9 (10.1–17.7)		24.9 (18.8–31.1)
30–39	151	19.9 (13.8–26.1)		28.6 (21.8–35.5)
40–49	123	20.9 (14.9–26.9)		34.8 (27.9–41.7)
≥50	126	26.5 (18.8–34.2)		36.6 (28.7–44.5)
Sex			.222		.333
Male	965	19.5 (17.2–21.7)		26.6 (24.3–28.8)
Female	944	20.9 (18.5–23.4)		27.6 (25.2–29.9)
Vaccination history			.053		.008
Yes	1151	21.3 (19.0–23.6)		25.6 (23.6–27.6)
No	76	26.5 (16.8–36.2)		38.1 (27.3–48.9)
Unkown	682	17.6 (15.2–20.1)		28.2 (25.4–31.1)
Registered population			.077		.712
Native	1569	20.9 (19.0–22.8)		27.2 (25.4–28.9)
Migrant	340	16.9 (13.3–20.7)		26.4 (22.4–30.4)
Total	1909	20.2 (18.5–21.9)		27.0 (25.4–28.7)

CI, confidence intervals.

Prevalence of IgG-PT and IgG-FHA

The GMC of anti-Pt IgG antibody and anti-FHA IgG antibody in 1909 healthy subjects were 20.2 (18.5–21.9) IU/ml and 27.0 (25.4–28.7) IU/ml, respectively. Both GMCs were found to be associated with age (p < .01). The distribution of GMC among ages showed that the GMC level of anti-pt IgG antibody in the 1-2 year old group and the ≥50 year old group was higher [38.5 (32.4–44.6) IU/ml and 26.5 (18.8–34.2) IU/ml]. There was a significant correlation between the GMCs of anti-PT IgG and anti-FHA IgG (r = 0.835, p < .05). The GMC of anti-FHA IgG exhibited a pattern similar to that of anti-PT IgG antibody, with the second peak observed in the 10-14 year age group (28.2 IU/ml). The change in anti-FHA IgG levels across different age groups mirrored that of the PT group, albeit with slight variations observed in individual groups (see Table 1). As shown in Figure 2, the seropositivity rate of IgG-PT (>20.0 IU/ml) was highest (39.9%) in the 1-2 year-old group, decreased rapidly to the lowest in the 3-4 year-old group, and then gradually increased with age.

Figure 2. Distribution of the IgG-PT and IgG-FHA seropositivity rate in different age groups.

Pertussis infection

Table 2 and Figure 3 presented the estimated infection rates for 1394 subjects aged over 3 years, with 73 subjects (5.2%) exhibiting IgG-PT levels >80.0 IU/ml, indicative of recent infection. The estimated rate of infection was found to increase with age. Between 2019 and 2022, the pertussis notification rate among the population in Jiangsu Province was 4.3 per 100,000 individuals in the 5-6 year age group and 0.2 per 100,000 individuals in the ≥15 year age group. A comparison of the notification rate with the results of this study revealed that the estimated rate of pertussis infection based on seroprevalence was approximately 25,625 times higher than the reported notification rate in the ≥15 year age group.

Figure 3. Estimated infection rates (IgG-PT levels >80 IU/ml) for subjects of different ages.

Table 2. Estimated underreporting by age group in China in 2019–2022.

Age Group	N. of Subjects	Anti ‐ PT ≥ 80IU/mL	Estimated Rate (%)	China Rate of Notification (n/100,000)	Excess Incidence *
3–4	160	6	3.8	2.2	1728
5–6	119	6	5.0	4.3	1175
7–9	139	6	4.3	2.8	1550
10–14	196	7	3.6	0.7	5492
≥15	780	48	6.2	0.2	25625

* Number of times the study incidence exceeded the reported incidence.

Discussion

This study elucidates the epidemiological characteristics of pertussis in Jiangsu Province spanning the last 70 years. Following the introduction of vaccines, the reported incidence of pertussis experienced a gradual decline; however, since 2016, a rapid resurgence has been observed. This increase was particularly notable during the COVID-19 pandemic, coinciding with enhancing disease surveillance in certain regions.^14,15 This circumstance potentially contributed to the escalation in reported cases. Nevertheless, the issue of underreporting pertussis cases persists, leaving the true disease burden uncertain.^16,17 Consequently, this cross-sectional study aimed to explore PT and FHA antibody levels in healthy children and adults across eastern China. Additionally, it sought to estimate pertussis susceptibility rates across various age cohorts, utilizing anti-PT antibody levels as the primary indicator.

In this study, the overall IgG-PT serum positivity rate among participants was 25.4%, which was lower than the rates reported in studies conducted in Zhejiang, China (29.8%)¹² and among healthcare workers in Spain (31.2%).¹⁸ This discrepancy may be attributed to various factors influencing pertussis antibody levels and duration, such as age, vaccine type, and immunization history. Chinese children are mandated to receive four doses of the pertussis vaccine before reaching the age of two, ensuring high vaccine coverage rates. Our findings indicate that anti-PT levels in the 1–2 year age group [38.5 (32.4–44.6) IU/mL] surpassed those observed in other age cohorts. Nonetheless, pertussis vaccine-induced antibodies typically exhibit short-term efficacy, with levels declining to 0%-20.0% within roughly 10 years.^19,20 Consequently, recognizing this risk and the concomitant waning of immunity, numerous countries have implemented pertussis booster vaccination programs targeting pre-school children, adolescents, or adult populations. In Singapore,²¹ a booster dose of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (Tdap) has been administered to school children at 10 to 11 y of age since 2008. Additionally, Australia²² administers two extra doses of DTaP vaccine at ages 4 and 11–12, aiming to safeguard vaccinated individuals and mitigate the risk of pertussis transmission from adults to infants. Hence, to mitigate the pertussis burden, it is advisable to implement booster doses of the vaccine targeting older children, adolescents, and adults across China.

This study revealed no significant disparity in gender-specific serum prevalence rates, consistent with findings from studies conducted in Taiwan.²³ Findings also showed a GMC of anti-PT IgG antibodies in the 50-year age group at 26.5 (18.8–34.2) IU/mL. The elevated seroprevalence and GMC of anti-PT IgG in this age bracket might be attributed to recent natural BP infections.²⁴ Individuals within this demographic are prone to underlying conditions like chronic obstructive pulmonary disease (COPD)²⁵ or asthma, potentially heightening their vulnerability to pertussis infection. Moreover, individuals within the 50-year-old age bracket are prone to exposure to other bacteria or viruses inducing symptoms akin to pertussis, such as Bordetella parapertussis or Chlamydophila pneumoniae. Such exposure may foster cross-reactive antibodies, potentially elevating the GMC of anti-PT IgG antibodies. Furthermore, age itself may serve as a contributing factor to the elevation of anti-PT IgG antibody GMC, as age-related alterations in the immune system can impact antibody responses.²⁶ However, further research is needed to confirm this. Despite our study revealing a lower GMC of anti-PT IgG antibodies among individuals with a history of immunization compared to those without [21.3 (19.0–23.6) IU/mL vs. 26.5 (16.8–36.2) IU/mL], no statistical significance was observed between the two groups (p > .05).

The infection rate estimated from serological results markedly surpasses the reported rate of infection. IgG-PT levels signify exposure to pertussis rather than manifest clinical onset. Numerous clinical manifestations of pertussis cases in children resemble those of other respiratory infections, with underreporting of atypical symptoms more prevalent among adolescents and adults. A seroepidemiological investigation in Denmark prompted researchers to estimate an adult incidence rate 4613 times greater than that reported by the surveillance system.²⁷ An examination of adults (aged 50 years) in the United States revealed estimated incidence rates substantially exceeding those reported by physicians based on clinical findings.²⁸ Additionally, the findings of this study demonstrated that estimated incidence rates in the age group ≥15 years surpassed reported incidence rates by a factor of 25,625. To enhance comprehension of the immunity imparted by vaccines against pertussis, this study also assessed serum anti-FHA antibodies. Anti-FHA levels in the entire study population [27.0 (25.4–28.7) IU/mL] surpassed those reported in prior studies conducted in Henan¹¹ and Zhejiang provinces,¹² China. Furthermore, a correlation between anti-FHA and anti-PT was observed, albeit with the titer of anti-FHA IgG seemingly higher than that of PT. The antibody response to FHA is not exclusive to pertussis bacterial infection. Unlike PT-specific anti-PT IgG, which is generated following exposure to pertussis bacteria, anti-FHA IgG can be elicited not only by pertussis bacteria but also by human respiratory pathogens such as Bordetella parapertussis, influenza hemophilus, and pneumococcal pathogens.¹²

This study also had some limitations. Firstly, the study did not prospectively monitor the decline of antibody levels following vaccination. Secondly, owing to limitations of the vaccine information system, the immunization history of individuals aged over 15 years remained unclear. Thirdly, the cutoff value of 20.0 IU/mL utilized to indicate seropositivity is somewhat arbitrary and may not constitute the optimal threshold for all populations, possibly resulting in an inaccurate assessment of the susceptible population. Nevertheless, the novelty of this article stems from being the inaugural study to assess pertussis seroprevalence across various age groups in Jiangsu Province subsequent to the COVID-19 pandemic. Additionally, it examines the reporting of pertussis cases amid the COVID-19 pandemic. These findings contribute to a deeper comprehension of how age impacts antibody responses and pertussis susceptibility, alongside post-COVID-19 pandemic pertussis epidemiology, thereby furnishing evidence for optimizing immunization strategies to prevent and control pertussis.

Conclusion

In conclusion, our study revealed new insights into the epidemiology of pertussis post-COVID-19 pandemic by assessing the pertussis seroprevalence among different age groups in Jiangsu Province. Our research demonstrated for the first time that vaccination against pertussis is effective in boosting anti-PT IgG and anti-FHA immunity. However, immunity levels decline shortly after vaccination, and an extra booster dose for adolescents and adolescents was highly recommended to enhance immunity and protect against severe disease. Furthermore, our study, being the first of its kind on pertussis seroprevalence in the general population of Jiangsu, China, provides valuable insights for policymakers in refining future pertussis immunization strategies. Moving forward, it is imperative to conduct longitudinal studies to monitor vaccine efficacy and immunity persistence over time, as well as explore innovative approaches to enhance pertussis immunization coverage and effectiveness.

Author contributions

All authors participated in the design, implementation, analysis, and/or interpretation of the study. All authors were involved in drafting the manuscript or revising it critically for important intellectual content. All authors provided final approval of the manuscript.

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Jiangsu of the Jiangsu Provincial Center for Disease Control and Prevention (JSJK2019-A004-02). Informed consent was obtained from all subjects involved in the study.

Acknowledgments

We would like to thank the participants of the project and the staff of the Centers for Disease Control and Prevention in the six counties for their dedication and contribution to the project.

Disclosure statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Additional information

Funding

This manuscript was funded by the Jiangsu Health Development Research Center Open Project [grant number JSHD2022043], the Jiangsu Provincial Geriatric Health Research Project [grant number LKM2023005], Suqian Sci&Tech Program [grant number SY202312] and Jiangsu Provincial Health Commission Preventive Medicine and Hematology Prevention Research Project [grant number Ym2023099].