Serological evaluation of immunity against Corynebacterium diphtheriae in the Peruvian adult population

ABSTRACT

The reemergence of diphtheria outbreaks in Latin America calls for an assessment of the level of immune protection in adults. This study estimated IgG levels against Corynebacterium diphtheriae in individuals immunized and unimmunized with dT or DTaP boosters over the past 10 years. A cross-sectional study was conducted in 127 adults from Lima and Callao, divided according to recent vaccination history. Serum IgG levels were measured using an indirect ELISA (SERION ELISA® classic) and classified into four categories according to protection. The Mann-Whitney U test was used for comparative analysis. 26.8% showed no protection, while 62.2% showed certain or long-term protection. Among those vaccinated, 92.9% achieved protective levels, compared to 67.7% of those unvaccinated (p = .001). The 40–49 age group had the highest proportion of unprotected individuals (33.3%). Vaccination with dT or DTaP boosters was associated with protective IgG levels. A significant proportion of adults lacked immunity, especially among those who did not receive a booster, highlighting the need to strengthen immunization strategies in adults.

Introducción

Diphtheria is an acute infectious disease caused by Corynebacterium diphtheriae, a Gram-positive bacillus that produces an exotoxin responsible for the most severe clinical manifestations. This toxin induces local tissue damage and can lead to severe systemic complications, such as myocarditis, neuropathies, and even death, especially in the absence of timely treatment.¹ The main route of transmission is respiratory, through droplets expelled by infected individuals; however, cutaneous forms and cases due to contact with contaminated objects have also been documented.²

Although diphtheria was considered a controlled disease in Peru for several decades thanks to national vaccination programs, sporadic outbreaks have been reported in recent years, highlighting the persistence of gaps in vaccination coverage and population immunity. These outbreaks have primarily affected vulnerable populations, such as children and unvaccinated individuals. The most affected areas are often associated with logistical barriers, misinformation, and resistance to vaccination, especially in remote regions or those with limited access to health services.³

Immune protection against diphtheria is primarily assessed by measuring IgG antibodies specific for diphtheria toxin. These antibodies neutralize the toxin and prevent its pathogenic action. IgG levels ≥ 0.1 IU/mL are considered to confer sufficient protection, although higher values are associated with longer-lasting immunity.⁴

The introduction of the diphtheria vaccine into expanded immunization programs has significantly reduced the overall incidence of the disease. However, several studies have documented that antibody titers tend to decline over time after primary immunization, underscoring the need for booster doses every 10 years, especially in adults.⁵ In Latin America, vaccination efforts have significantly reduced autochthonous cases over the past two decades.⁶ However, the recurrence of outbreaks in different regions of the world reveals the persistent vulnerability of populations where vaccination coverage is low or where periodic booster administration is not guaranteed.⁷

In low- and middle-income countries, such as Peru, logistical, economic, and sociocultural factors limit the systematic implementation of booster strategies in adults.⁸ The reemergence of diphtheria in October 2020, after more than 20 years without indigenous notifications, exposed these weaknesses. The first case was detected in an unvaccinated child in Lima, and subsequent cases were identified in different regions, leading the Ministry of Health (MINSA) to issue a national epidemiological alert. In response, MINSA launched immunization campaigns using diphtheria-tetanus toxoid (dT) and acellular pertussis (dTpa) vaccines, targeting primarily children over five years of age and adults with no recent history of booster vaccination.^3,9 This outbreak coincided with the COVID-19 pandemic, which significantly impacted health services, including vaccination programs, likely increasing the number of susceptible individuals.^10,11

In Peru, the childhood immunization program includes a primary series of the pentavalent vaccine (DTwP-HB-Hib), which protects against diphtheria, tetanus, whole-cell pertussis, hepatitis B, and Haemophilus influenzae type b. This vaccine is administered at 2, 4, and 6 months of age. Subsequently, booster doses of DTwP are administered at 18 months and 48 months of age. As an additional measure to protect newborns against pertussis and indirectly contribute to immunity against diphtheria, the Peruvian Ministry of Health incorporated Tdap vaccination in pregnant women between 27 and 36 weeks of gestation from 2019 to 2022, as part of the gestational immunization program (ViP).¹²

In this context, serological evaluation of IgG antibodies against C. diphtheriae is presented as a key tool for monitoring population immunity, identifying vulnerable groups, and guiding more effective booster policies. Unlike the exclusive analysis of vaccination records, serology allows direct estimation of the actual level of immune protection, considering both the vaccine response and its eventual decline over time.¹³ The present study aimed to determine the levels of IgG antibodies against C. diphtheriae in individuals immunized and non-immunized with dT or DTaP vaccines over the last 10 years, in a population treated at a national referral hospital in Peru. This information is essential to strengthen seroepidemiological surveillance and optimize prevention strategies against the reemergence of vaccine-preventable diseases such as diphtheria.

Methods

Study design

An observational, descriptive, cross-sectional study was conducted to determine the levels of IgG antibodies against Corynebacterium diphtheriae in adults with and without a history of recent immunization with dT or DTaP vaccines.

Population and sample

The population consisted of 241 people aged between 18 and 70 years, divided into two groups: 127 subjects from the Nova Salud Polyclinic (Callao) – including outpatient attendees and staff members, who constituted the primary study group – and 114 students from the Professional School of Medical Technology of the San Fernando Faculty of Medicine (National University of San Marcos), whose samples were used for validation and comparison of the test results. Data collection took place between April and June 2022.

Non-probability convenience sampling was used, driven by the availability of volunteers in a context still limited by the aftermath of the COVID-19 pandemic. The absence of a consolidated vaccination registry system, along with widespread ignorance of immunization history, prevented the application of random or stratified sampling methods.

People aged 18 to 70 were included. The non-immunized group consisted of those who had not received a booster dose with either dT or DTaP vaccines in the previous 10 years. The immunized group included only those who reported having received at least one booster dose between three months and ten years prior to sample collection. People with a recent history of C. diphtheriae infection, primary or secondary immunodeficiencies, immunosuppression due to pathological or pharmacological causes, or active immunosuppressive treatment were excluded. Likewise, five (5) samples were excluded that did not meet the quality and integrity criteria necessary for serological analysis, due to evident hemolysis.

A post hoc power calculation was performed, considering only participants with certain or long-lasting protection levels. With a sample of 127 participants, a protection ratio of 90.6% in vaccinated participants and 33.3% in unvaccinated participants, and a significance level of 5%, a power of 99.9% was obtained, indicating a high capacity to detect significant differences between both groups.

Procedures

Prior to the start of data collection, the corresponding institutional authorization was obtained, and an awareness campaign on diphtheria prevention and the importance of vaccination was conducted to promote informed voluntary participation. Participants were informed about the study’s objectives and ethical implications, and they provided their written informed consent.

A demographic form prepared by the researchers was administered and completed in person with the support of the technical team. Basic sociodemographic information, vaccination history, and relevant clinical data were collected.

Blood samples were obtained by venipuncture in the antecubital fossa using tubes with separating gel (Vacutainer®, yellow cap). These were transported under biosecurity measures and stored at 4°C until processing.

Definition of antitoxin levels

The quantification of IgG antibodies against Corynebacterium diphtheriae was performed using the indirect ELISA technique, using the commercial SERION ELISA® classic Diphtheria IgG kit. This method detects the presence of specific IgG antibodies in serum or plasma by binding the antibody to an antigen previously fixed to the ELISA plates. Detection is carried out with an anti-IgG enzyme conjugate linked to alkaline phosphatase, using para-nitrophenyl phosphate as a substrate. The results were read by spectrophotometry at a wavelength of 405 nm.

Sample processing was automated using the SERION Immunomat™ system, which allows simultaneous analysis of up to four ELISA plates. This system has a lower detection limit of 0.05 IU/mL and an upper limit of 2.00 IU/mL, according to the manufacturer’s specifications.

For the interpretation of immunity levels, the qualitative classification proposed by the manufacturer of the commercial kit (SERION ELISA® classic Diphtheria IgG) was used, which was adapted to the analytical capacity of the equipment used. According to these guidelines, titers <0.05 IU/mL were considered absent protection; values between 0.05 and 0.09 IU/mL as minimal protection; levels between 0.1 and 0.99 IU/mL as secure protection; and concentrations ≥1.00 IU/mL as indicative of long-lasting protection. It should be noted that a value ≥0.1 IU/mL is internationally considered the minimum threshold for protection, but this more detailed classification was useful to better discriminate the immune profiles in our sample.

Statistical analysis

The data were coded and refined in Microsoft Excel, calculating measures of central tendency and dispersion. They were then exported to RStudio® for inferential analysis, table generation, and visualizations. Mean comparison and correlation tests were applied, with p < .05 considered significant.

Ethical considerations

The research was conducted in accordance with the ethical principles of the Declaration of Helsinki, ensuring respect for autonomy, confidentiality, and informed consent. Participants were able to withdraw from the study at any time without consequences. The protocol was approved by the Ethics Committee of the Faculty of Medicine of the National University of San Marcos.

Results

A total of 127 serum samples were analyzed. Most participants were women (67%), and the most represented age group was young adults aged 18 to 39 years (56%). Regarding IgG antibody levels against Corynebacterium diphtheriae, 26.8% of participants did not present serological protection, while 11% had minimal protection, 50.4% had safe protection, and only 11.8% reached levels considered long-term protection. Immunized participants mostly presented safe (36.2%) and long-term (9.4%) protection, while among non-immunized participants, the absence of protection predominated (23.6%) (Table 1).

Table 1.

Distribution of IgG antibody levels against Corynebacterium diphtheriae according to age group, sex and immunization (n = 127).

		Protection status
Variables	n (%)	No protection <0.05 UI/ml (%)	Minimal protection 0.05–<0.1 UI/ml (%)	Safe protection 0,1–<1,0 UI/ml (%)	Long-term protection ≥1,0 UI/ml (%)	p*
Total	127 (100%)	34 (26.8%)	14 (11.0%)	64 (50.4%)	15 (11.8%)
Age (years)						.031
18–39 (Young adult)	71 (56.0%)	19 (26.8%)	10 (14.1%)	35 (49.2%)	7 (9.9%)
40–49 (Middle aged)	15 (11.8%)	5 (33.3%)	0 (0.0%)	9 (60.0%)	1 (6.6%)
50–64 (Senior)	39 (30.6%)	10 (25.6%)	4 (10.3%)	20 (51.3%)	5 (12.8%)
65–70 (Elderly)	2 (1.6%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	2 (100%)
Sex						.902
Male	42 (33.0%)	10 (23.8%)	5 (11.9%)	21 (50%)	6 (14.3%)
Female	85 (67.0%)	24 (28.2%)	9 (10.6%)	43 (50.6%)	9 (10.6%)
Immunization (volunteers)						.001
Yes	64 (50.3%)	4 (6.2%)	2 (3.1%)	46 (71.9%)	12 (18.8%)
No	63 (49.6%)	30 (47.6%)	12 (19.0%)	18 (28.6%)	3 (4.8%)

*Chi squared. The classification of serological protection levels is based on the qualitative ranges proposed by the manufacturer of the SERION ELISA® classic Diphtheria IgG kit (Institut Virion/Serion GmbH): No protection (< 0.05 IU/mL), Minimal protection (0.05–0.09 IU/mL), Safe protection (0.1–0.99 IU/mL) and Long-term protection (≥1.00 IU/mL).

When performing the bivariate analysis of antibody levels, it was observed that there were no statistically significant differences between men and women (p = .76), although the median was slightly higher in men (0.18 IU/mL) than in women (0.16 IU/mL). However, a significant difference was found according to immunization status (p = .001), where vaccinated individuals showed considerably higher median antibody levels (0.36 IU/mL) than unvaccinated individuals (0.06 IU/mL) (Table 2).

Table 2.

Bivariate analysis between antibody levels, sex and immunization status.

	Antibody levels (UI/mL)
	Median	Interquartile range	p*
Sex			.76
Male	0.18	0.052–0.559
Female	0.16	0.049–0.468
Vaccination			.001
Yes	0.36	0.152–0.721
No	0.06	0.049–0.187

*U de Mann Whitney.

Discussion

The results of this study show that a considerable proportion of adults evaluated do not have serological levels of protection against diphtheria toxin. In particular, the group of adults between 40 and 49 years of age had the highest proportion of unprotected individuals, which could be due to the lack of booster doses during adulthood. Furthermore, a statistically significant difference was observed in antibody levels between those who received boosters and those who did not, reinforcing the importance of maintaining active vaccination schedules beyond childhood. This finding is especially relevant in the Peruvian context, where there is no systematic booster strategy for adults and where serological surveillance is scarce.

Although diphtheria is no longer a serious threat in many regions of the world thanks to routine vaccination programs, it remains a latent risk to public health. Its reappearance in the form of sporadic outbreaks, coupled with increased migratory movements, highlights the need for constant epidemiological surveillance.¹⁴ This risk is particularly high in densely populated urban areas, where vaccination rates are low or irregular and where disease transmission can occur more rapidly.¹⁵

In Latin America, studies on immunity to Corynebacterium diphtheriae are limited. Previous studies in Colombian pregnant women, such as those in Rivera-Santamaría, reported protection levels above 95%. According to the National Repository of Health Information (Reunis), 23% of pregnant women in the country have not received the DTPa vaccine.¹⁶ This difference highlights significant gaps in maternal vaccination coverage, with potential consequences for community and neonatal transmission of the disease.

Much of the available evidence comes from Europe and Asia, so this study provides valuable information by evaluating diphtheria IgG antibody levels in Peruvian adults. It was found that 26.8% did not have protective titers. In Bulgaria, 59.3% of adults had protective titers. Italy showed lower protection the older the study subjects became, with the percentage reaching 82.9% in the group over 85 years of age.^17,18

The progressive decline in antibody levels is not solely due to aging, but also to the lack of booster doses, which are not regularly included in national vaccination schedules for adults in many countries, including Peru. This situation is aggravated by the lack of updated or digitalized vaccination records.¹⁹ A study in Vietnam found that mean levels of diphtheria antitoxoid antibodies were 0.07 IU/ml, and these levels were associated with age and history of diphtheria vaccination, decreasing as the years passed without a booster.²⁰ It is crucial to maintain high routine vaccination coverage for children and promote booster doses for adolescents and adults every 10 years.

While the number of vaccinated and unvaccinated individuals was similar, the levels of protection differed significantly. This difference highlights the effectiveness of vaccination, which was also evident in Vietnam. During 2015–2018, seven schools in rural Vietnam experienced diphtheria outbreaks. Low vaccination coverage and overcrowding in dormitories may have contributed to the outbreaks.²¹

It is important to understand the underlying reasons for low vaccination coverage in certain groups. Research conducted in Spain has identified lack of time, low-risk perceptions, and exclusion from prioritized campaigns as the main barriers to vaccination.²² In addition, other social factors that affect vaccine acceptance include low socioeconomic status, lack of health insurance, multiparity, and the circulation of misinformation.^23,24 In the Peruvian context, these difficulties can be amplified by the logistical limitations of the health system and the spread of misinformation through social media, which represents a significant challenge to achieving adequate and sustained vaccination coverage.²⁵

The lower rate of seroprotection observed in our adult population differs from that reported in studies from other countries, and this may be attributed to several factors. First, Peru lacks a systematic policy for administering 10 year boosters to adults, unlike European countries where these doses are integrated into occupational health or geriatric care programs.^19,26 Second, several studies have documented that diphtheria antibody titers progressively decline with age in the absence of boosters, consistent with our findings of lower protection in adults over 40 years of age.²⁷ Third, the populations evaluated also differed: studies such as the one from Colombia focused on pregnant women, a priority group with high Tdap coverage, while our study included adults in general.²⁸ Finally, differences may also be influenced by inequalities in health infrastructure, logistical barriers, and the lack of regular reinforcement campaigns in remote regions of Peru.

This study has some limitations. Because it is a cross-sectional design, it is not possible to establish causal relationships between the presence of antibodies and vaccination or exposure history. Furthermore, it should be noted that information on vaccination history in this study was self-reported, without verification through official records, due to the absence of a consolidated digital immunization system for adults in the country. This limitation could introduce recall bias or misclassification between vaccinated and unvaccinated individuals. We recognize that this aspect may have influenced the coverage estimate and, therefore, the interpretation of seroprotection levels, which highlights the importance of strengthening vaccination registration and monitoring systems in adults. This study should be interpreted as exploratory. Although it was designed with a cross-sectional approach, the sample was obtained by convenience and included diverse subgroups (outpatients, healthcare personnel, and students), without a defined sampling frame. This heterogeneity limits the possibility of extrapolating the findings to a specific population. In this sense, the results allow us to identify preliminary trends and possible vulnerable groups, but they should not be considered representative of the general adult population of Lima and Callao. Another limitation of the study was the limited availability of demographic variables, as only data on age, sex, and recent vaccination history were collected. This was due to the exploratory nature of the study and the need to reduce the participation burden to facilitate voluntary recruitment during a still-sensitive health context. The lack of other variables, such as socioeconomic status or occupation, limits the possibility of exploring other potential associations and should be considered when interpreting the results. No multivariate regression analysis was performed due to the limited number of independent variables collected (age, sex, and recent vaccination history), which restricted the possibility of constructing a model that would offer additional or robust conclusions beyond the bivariate analysis.

Conclusions

Many vaccinated individuals presented protective antibody levels, while a significant proportion of unvaccinated individuals did not reach the minimum protection threshold. This finding highlights the need to strengthen vaccination strategies in adults. Furthermore, it was observed that immunity levels vary with age, being lower in middle-aged adults, likely due to the lack of recent boosters.

Overall, the results highlight the importance of maintaining up-to-date vaccination schedules and administering timely boosters, especially among young and middle-aged adults. They also demonstrate the value of serological monitoring as a tool for guiding more effective and targeted immunization policies.

Biography

Sofia E. Romero, Dr. HS, is a professor at the Universidad Nacional Mayor de San Marcos (UNMSM). Her work has contributed significantly to increasing the rate of investigations among students at the School of Medical Technology, mostly in emerging infectious diseases.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).