Hepatitis B surface antibodies in medical students from a public university in Puebla, Mexico

ABSTRACT

Although preventable with vaccination, Hepatitis B virus (HBV) infection is a major health concern, with ∼400 million people at risk of developing the chronic form of the disease worldwide. The anti-HBV vaccine consists of a recombinant HBV surface antigen (HBsAg), which induces specific anti-HBs antibodies and confers 95% protection for >20 y. The aim of the present study was to analyze the response to HBV vaccination by measuring anti-HBs antibodies in serum samples from medical students of a public university in Puebla, Mexico. HBV infection markers HBsAg and anti-HBs, were also determined. A total of 201 students were included and vaccination coverage was found at 54%. Overall seropositivity for HBsAg, anti-HBc and anti-HBs determined by ELISA was 0.5%, 1.0% and 47%, respectively. Protective levels of anti-HBs >10 mIU/mL were found in 93.2% of subjects vaccinated with 2 or 3 doses and in 40% of those vaccinated with a single dose; while only 4.8% of unvaccinated subjects were anti-HBs positive. The response to the HBV vaccine was different in each participant, despite similar vaccination scheme. A history of blood transfusion/organ transplant or more than 2 sexual partners was significantly associated with anti-HBc positivity, OR = 399 (p = 0.010) and OR = 19.9 (p = 0.044), respectively. HBV immunization coverage was low in our sample compared with reports from countries with similar HBV prevalence, but anti-HBs in vaccinated individuals were in the expected range. It is important to promote HBV vaccination and awareness among medical students, due to their exposure risk.

KEYWORDS:

• anti-HBc
• anti-HBs
• hepatitis B virus
• HBsAg
• recombinant vaccine HBV
• vaccination
• vaccination coverage HBV

Introduction

Hepatitis B virus (HBV) is a health concern because it can lead to chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma. Worldwide mortality is estimated at >235,000 cases of cirrhosis and 328,000 liver cancer deaths annually linked to HBV infection¹ which underscore the importance of infection prevention. Despite the existence of an efficient HBV vaccine since 1986, there are ∼400 million people at risk of developing the chronic form of the disease.¹ The HBV vaccine is composed of a recombinant HBV surface antigen (HBsAg), which induces specific antibodies (anti-HBs) and confers 95% (95% CI 80–100) protection for >20 y.² After primary vaccination, 37% of subjects maintain protective levels of antibodies.

Immunization at an early age is crucial for radically reducing the number of chronic HBV carriers. Thus, in 1997 The World Health Organization (WHO) issued the recommendation that hepatitis B vaccine should be included in national infant and adolescent immunization programs in all countries.³ This vaccination program implemented years ago has decreased the number of young HBsAg carriers in countries with high HBV prevalence.^4,5

Among the population groups at risk for acquiring HBV infection are healthcare workers, in whom vaccination is recommended. The estimated risk for hepatitis B infection in this group is 2–10 times higher than for the general population.⁶ To prevent or reduce HBV infection risk through contaminated blood, it is recommended that healthcare personnel receive immunization against HBV and use personal protective equipment.⁷

The aim of the present study was to evaluate HBV vaccination coverage and to analyze vaccination response by measuring anti-HBs and other serological markers in medical students from a public university in Puebla, Mexico.

Results

Participants and risk factors

The calculated sample size was 97 individuals. The study included 201 medical students, 131 (65.2%) females and 70 (34.8%) males. Age range was between 18 and 30 y old with a mean age of 20.6 y (95% CI 20.3–20.9). A questionnaire was applied to evaluate risk factors for HBV exposure: mother infected with HBV 0 (0.0%), being sexually active 92 (45.9%), having had more than 2 sexual partners 43 (21.4%), being exposed to patient blood or secretions in the clinical practice 63 (32.6%) and history of blood transfusion or organ transplant 1 (0.49%). Concerning vaccination, students were categorized as “vaccinated with 2 or 3 doses” 74 (36.8), “vaccinated with single dose” 35 (17.5%), “unknown vaccination status” 30 (14.9%), “unvaccinated” 62 (30.8%) (Table 1).

Table 1. Demographic data of medical students from a public university in Puebla, Mexico.

Sex	N	%
Female	131	65.2
Male	70	34.8
Total	201	100.0
Risk factor
Mother HBV infected	0	0.0
Sexually active	92	45.9
More than 2 sexual partners	43	21.4
Clinical practice^*	63	32.6
Blood transfusion or organ transplant	1	0.49
Vaccination data
Vaccinated with 2 or 3 doses	74	36.8
Vaccinated with single dose	35	17.5
Unknown vaccination status^**	30	14.9
Unvaccinated	62	30.8
Total	201	100.0

* Having been exposed to blood or secretions of patients.

** No immunization record.

Serological markers and vaccination status

Overall prevalence in the analyzed population was 0.5% (95% CI 0.44–0.56), 1.0% (95% CI 0.87–1.13), and 47.8% (95% CI 40.2–53.8) for HBsAg, anti-HBc and anti-HBs, respectively (Table 2).

Table 2. Association between serological HBV markers and vaccination status in medical students from a public university in Puebla, Mexico.

	Serological marker, n (%)
	HBsAg		Anti-HBc		Anti-HBs
Vaccination status	Positive	Negative	Positive	Negative	Positive	Negative
2 or 3 doses	0 (0.0)	74 (100)	0 (0.0)	74 (100)	71 (95.9)	3 (4.1)
Single dose	0 (0.0)	35(100)	0 (0.0)	35 (100)	15 (42.8)	20 (57.2)
^*Unvaccinated	^*1 (1.6)	61 (98.4)	^*2 (3.2)	60 (96.8)	3 (4.8)	^*59 (95.2)
Unknown	0 (0.0)	30 (100)	0 (0.0)	30 (100)	7 (23.3)	23 (76.4)
Total	1 (0.5)	200 (99.5)	2 (1.0)	199 (99.0)	96 (47.8)	105 (52.2)
Prevalence (95%CI)	0.5 (0.43 – 0.57)		1.0 (0.87 – 1.14)		47.8 (40.2 – 53.8)

* One student was HBsAg (+), Anti-HBc (+) and Anti-HBs (−). *Other was HBaAg (−),

Anti-HBc (+) and Anti-HBs (−)

None of the 74 students “vaccinated with 2 or 3 doses” were positive to HBsAg or anti-HBc. In contrast, 71 (95.9%) were anti-HBs positive. Among the 35 students who were “vaccinated with a single dose,” 15 (42.8%) were anti-HBs positive and 20 (57.2%) were negative. Whereas of the 62 “unvaccinated” students, 1 (1.6%) was HBsAg positive, 2 (3.2%) were anti-HBc positive, 3 (4.8%) were anti-HBs positive and 59 (95.2%) were anti-HBs negative. Among the “unknown vaccination status” group, 7 (23.3%) were anti-HBs positive and 23 (76.4%) were anti-HBs negative (Table 2).

Anti-HBs titers and vaccination status

Overall, 96 (47.8%) medical students were anti-HBs positive. The students “vaccinated with 2 or 3 doses” had anti-HBs titers at different levels. 69 (93.2%) had titers ≥10 mIU/ml, which are considered protective against HBV infection. Fifty six (78.8%) had titers ≥101 mIU/ml and 6 (8.10%) had titers ≥3000 mIU/ml. Among the 35 students “vaccinated with single dose,” 14 (40%) had titers ≥10 mIU/ml, 11 (26.7%) had titers ≥101 mIU/ml and only 2 (2.9%) had titers ≥3000 mIU/ml. In regard to “unvaccinated” students, only 4.8% had antibodies and these were present at relatively high titers (501–1000 mIU/ml). Of the 30 students who had “unknown vaccination status,” 7 (23.3%) were anti-HBs positive and they presented relatively lower titers than the “vaccinated with 2 or 3 doses” group, 3 (10%) had ≥10 ≤ 100 mIU/mL and 4 (13.4%) had ≥101 ≤ 1000 mIU/mL (Table 3)

Table 3. Association of anti-HBs range (mIU/ml) and vaccination status in medical students from a public university in Puebla, Mexico.

	Anti-HBs ranges mIU/mL, n(%)						Anti-HBs, n(%)
Vaccination status	1.0–9.9	10–100	101–500	501–1000	1001–3000	>3000	Positive	Negative	Total
Vaccinated (2 or 3 doses)	2 (2.70)	13 (17.6)	27 (36.5)	6 (8.1)	17 (23.0)	6 (8.1)	71 (95.09)	3 (4.01)	74 (100)
Vaccinated ( single dose)	1 (2.9)	3 (8.6)	6 (17.1)	1 (2.9)	2 (5.8)	2 (2.9)	15 (42.9)	20 (57.1)	35 (100)
Unvaccinated	0 (0.0)	0 (0.0)	0 (0.0)	3 (4.8)	0 (0.0)	0 (0.0)	3 ( 4.80)	59 (95.2)	62 (100)
Unknown	0 (0.0)	3 (10.0)	2 (6.7)	2 (6. 7)	0 (0.0)	0 (0.0)	7 (23.4)	23 (76.6)	30 (100)
Total	3 (1.5)	19 (9.4)	35 (17.4)	12 (6.0)	19 (9.5)	8 (4.0)	96 (47.8)	105 (52.2)	201(100)

Risk factors

Using univariate analysis, we determined that a history of blood transfusion or organ transplant and a history of more than 2 sexual partners were significantly associated with anti-HBc positive status, obtaining the following values OR 399, (95% CI 11.1–1434); p = 0.010 and OR 19.9(95% CI 1.39–406); p = 0.044, respectively. Being sexually active showed a trend but not a statistically significant difference OR 6.05 (95% CI 0.28–128); p = 0.208. Being male OR 1.8 (95% CI 0.11–30.4); p = 1.0 and also being exposed to patients in clinical practice OR 2.2 (95% CI 0.13–35.9); p = 0.529 were not statistically significant (Table 4).

Table 4. Association between risk factors and anti-HBc status in medical students from a public university in Puebla, Mexico.

	Univariate analysis
Gender	Students, n	Anti-HBc (+), n (%)	Odds ratio (95% CI)	p
Male	131	1 (0.76)	1.87 (0.11–30.4)	1.00
Female	70	1 (1.42)
Sexually active
Yes	92	2 (2.17)	6.05 (0.28–128)	0.208
No	109	0 (0.00)
More than 2 sexual partners
Yes	43	2 (4.65)	19.9 (1.39–406)	0.044
No	158	0 (0.00)
Clinical practice
Yes	63	1 (1.58)	2.21 (0.13–35.9)	0.529
No	138	1 (0.72)
Blood transfusion or organ transplant
Yes	1	1 (100)	399 (11.1–1434)	0.010
No	200	0 (0.00)

Discussion

Mexico is considered a low prevalence country for HBV infection. Reported seroprevalence in adults for HBsAg and anti-HBc is 0.21% and 3.3% respectively,⁸ nevertheless, anti-HBV vaccination is important to reduce infection incidence and its subsequent clinical problems.^2-3,5 Anti-HBV vaccination schedules in Mexico have changed multiple times in the last 25 y since the vaccine first became available for health personnel. In 1992, a study evaluated seroconversion for a period of 6 months after a 3 dose anti-HBV vaccination scheme in healthcare workers from Mexico.⁹ Later, in 2003 the scheme was shortened to 2 doses for adolescents and adults¹⁰ and more recently, 3 doses were again recommended and implemented.^11,12

The HBV vaccination in newborns from uninfected mothers was established in Mexico between 1999 and 2001 as the official immunization for children < 5 years of age. Since then, Mexican children complete HBV immunization during the first 6 months of life with 3 doses (at 2 months, 4 months and 6 months) and boosters at 12 years of age.¹³ In 2008 a change was implemented in the immunization scheme for infants (first day of life, 2 months and 6 months and later boosters).¹¹ Infant vaccination has proven effective even in countries with high HBV infection prevalence,^2,5 however it wasn't applied in Mexico when our medical students were born. The high prevalence of “vaccinated with single dose,” “unvaccinated” and “unknown vaccination status” subjects in our study likely derives from the lack of early life HBV immunization. Our subjects were vaccinated during school age or adolescence, and since HBV immunization schemes have changed in the last 25 y, not all of them received 3 vaccine doses. For this reason, we grouped subjects according to the number of doses received. The cases with incomplete vaccination found in the present study are likely also somewhat related to disregard of immunization schedules by parents, since the vaccine has been applied at 2 or 3 doses for several years in Mexico.¹³ Our data highlights the difficulty in completing vaccination schedules in older children / adolescents and the importance of early life immunization.

Knowledge about HBV immunization coverage among young people is relevant to guide health strategies. This coverage was calculated in the present study at 54.3% in medical students including those who received 1, 2 or 3 vaccine doses. However, coverage decreased to 36.8% if only students who received 2 or 3 doses are considered (Table 1). In a recent study among healthcare personnel in a Mexican hospital, 52% reported at least 1 vaccination during their professional life and only 5.5% completed the HBV vaccination schedule.¹⁴ These values are low since other countries with similar HBV prevalence to Mexico have reported higher coverage: 78.7% in Germany ¹⁵ and 92.2% in France,^16,17 in both cases among health students . In countries with intermediate prevalence (2–8%), variable coverage rates have been reported: 75.7% and 60.7% in medical students from Brazil,^18,19 whereas in high prevalence (>8%) countries, coverage has been reported to be 30.5% among health students from Laos²⁰ and 91.6% in dentist students from Iran.²¹

Failure to complete the vaccination scheme is a current problem worldwide. Several studies have shown different success levels in completing the 3-doses vaccination schedule, 22.8% in France,¹⁶ 5.5% in Brazil,⁷ 9.5% in Laos²⁰ and 16.7% in Iran.²¹ In this study we detected 17.5% of students “vaccinated with single dose” and 14.9% who had “unknown vaccination status” (Table 1). This status was also detected in 7.7% of German students and in 5.1% of Iranian students.^15,21

After vaccination, seroconversion rates are important; however, specific anti-HBsAg titers are a major index associated with protection. Anti-HBsAg titers are considered protective at ≥10 IU/ml.^22-24 The percentage of students “vaccinated with 2 or 3 doses” and who presented anti-HBs ≥10 IU/mL were 93.2%, which was higher than that found in other studies of students similarly vaccinated with 2 and 3 doses (77% and 83%),¹⁸ (87.5% and 71.4%).⁷ Five (6.8%) vaccinated medical students were anti-HBs negative or had titres < 10 mIU/mL, and thus can be considered non-responders to vaccination.²⁵ Alternatively these students could have responded initially and their anti-HBs levels could have declined with age,²⁵ but this wasn't evaluated in our study. In our study, anti-HBs were detected in 42.9% of subjects “vaccinated with single dose” (Table 3). Other studies have reported similar data for anti-HBs positivity: 33%¹⁷ and 50%.⁷ These divergent values all indicate that the humoral and cellular response to HBV vaccine was different in each participant, although they were subject to the same vaccination schedule.²⁶

In the “unvaccinated” students group, 4.8% were anti-HBs positive, which could be explained by an unregistered vaccination or may be due to past or present HBV infection. These “unvaccinated” students had levels of anti-HBs at 501–1000 mIU/mL (Table 3), and they were negative to anti-HBc (IgG/IgM) or HBsAg by CMIA, whereas the rest of the group (95.2%) had no antibodies.

Additionally, among the “unvaccinated,” anti-HBs negative students, we detected 2 cases that were positive for 1 or both markers of infection: One was positive for HBsAg and anti-HBc, but negative for anti-HBs, indicating a probable active infection; another student was HBsAg negative, anti-HBc positive and anti-HBs negative, suggesting a past or ongoing infection (Table 2). Both were referred to the Family Medicine Unit. Before the study these students did not know their serological marker status and we conclude that their positivity for anti-HBc was present before being included in this study. Both belonged to the group of “unvaccinated” and determination of anti-HBs was negative, thus anti-HBc positivity was likely unrelated to vaccination.

In the present work we identified a prevalence of 0.5% (95%CI 0.43-0.57) for the serological marker HBsAg. In Mexico the prevalence of this marker has been reported at 0.14% (95%CI 0.05-0.27) in the age group of 20–29 y old (general population, not limited to health-related students or personnel).⁸ Our students had similar HBsAg prevalence to that reported in Brazilian medical students 0.5%,¹⁹ and different to that reported in Colombian and Taiwanese medical students at 0.15% and 1.7%, respectively.^27,28

Overall anti-HBc prevalence in our study was 1.0% (95%CI 0.87-1.13). A prevalence of 0.5% (95%CI 0.2-0.9) has previously been reported in Mexico in the group age 20 to 29-y old subjects, again in population not limited to the health area.⁸ In another study young people including children between 11 and 29 y old living in rural areas with a high migration rate, had a prevalence of anti-HBc of 22.7%.²⁹ In both studies, a positive relationship between age and prevalence was found.^8,29 Absence of infant vaccination apparently has a negative impact on young people in marginalized rural areas as evidenced by this high prevalence of anti-HBc serological marker of 22.7% reported in 2011.²⁹

No other report of anti-HBs prevalence in young people or health related students has been reported in Mexico because they are already considered a group at risk for HBV infection and/or it is assumed that they have received a complete immunization scheme.

The present study is novel in that it analyzes a population exposed to diverse immunization schemes. We study protective levels of anti-HBs in this population that is currently substantial, composed of young people that have a long life expectancy, that will soon begin working as health professionals, but did not have the benefits of HBV immunizations as newborns and may not be aware of the danger posed by their incomplete HBV immunization. We provide data to know the dimensions of partial HBV protection in this population. Plus, many students have been exposed to known risk factors for acquiring HBV infection and we explored those associations between the anti-HBc serological marker which suggests a past or ongoing HBV infection and history of several risk factors in our subjects. Using univariate analysis, we determined that history of blood transfusion or organ transplant OR = 399; p = 0.010 was significantly associated with anti-HBc positive status. This mode of HBV transmission should be preventable with adequate blood bank screening. However, the genetic variability of HBV may explain the low sensitivity and specificity of immunoassays used in diagnostic laboratories and blood banks in Mexico because they are designed based on genotypes D and A whereas the prevailing genotype in Mexico is the newly identified genotype H.³⁰ On the other hand, a nucleic acid test for HBV is used in blood banks with a dual purpose, first to detect blood donors who are in the acute infection phase and are thus HBsAg negative and anti-HBc negative; the second, to detected occult hepatitis B virus infection in donors who are HBsAg negative and anti-HBc positive, both with extremely low viral load.^31,32

A history of more than 2 sexual partners OR = 19.9; p = 0.044 was significantly associated with anti-HBc positive status. HBV transmission may be due to exposure to biological fluids, e.g., unprotected sex practices.³³ Being sexually active showed a trend but not a statistically significant difference in its association with anti-HBc positive OR = 6.05; p = 0.208. This may be explained because the mean age in our study was 20 y and a positive correlation between age and detection of anti-HBc has been reported that it is doubled starting at 25 y in Mexico.^8,29 Thus our subjects may still be young to detected correlation between sexual activity and anti-HBc positivity. Risk factors such as male gender and being exposed to patients in clinical practice were not significantly associated with anti-HBc positivity (Table 4). This may be related to the fact that only 32% of medical students had clinical experience, or could be due to protected clinical practices. These students mentioned having used personal protection devices and no history of accidents was reported. In another study with medical students a total of 23.6% reported having been somehow exposed to blood or secretions, although reported vaccination coverage was at 75.7%.¹⁸ In our study 2 medical students were anti-HBc positive, but this was unrelated to clinical practices.

Further studies, not limited to students and health personnel, are needed to assess the impact of lack of infant anti-HBV immunization. However, health students are a group of special interest since they will be future health professionals and studies like the one reported here can help determine the need for programs toward adequate HBV immunization coverage in population not vaccinated in the first 6 months of life.

In conclusion, we found that HBV vaccination coverage in students who received 1,2 or 3 doses was 54.3%. This percentage decreases to 35.32% if only students who received 2 or 3 doses were included. The prevalence value for HBsAg, anti-HBc and anti-HBs was 0.5%, 1.0% and 47%, respectively. Ninety-three percent of students vaccinated with 2 or 3 doses reach protective anti-HBs levels ≥10 IU/mL. Our results indicate that anti-HBs response is different in each individual, even with similar vaccination schemes. It is necessary to improve the immunization programs and to increase health students' awareness of vaccine preventable diseases, considering they are an at-risk population.

Materials and methods

Participants

Medical students from the Benemérita Universidad Autónoma de Puebla (BUAP) were invited to participate in the study between July and December 2013. The inclusion criteria were: medical students, female and male, age > 18 ≤ 30 y, with or without. Vaccination data from the National Health Card. Subjects were excluded if they had a diagnosis of autoimmune disease, and eliminated if they had insufficient sample of serum for all assays, or their samples were hemolysed or lipemic, and if they did not answer the questionnaire. Students received an information sheet (informed consent together with questionnaire). Basic information from each student was included (age, sex, risk factors for acquiring HBV infection, and vaccination data from the National Health Card). Sample size (n) was calculated according to the formula.³⁴

${\displaystyle n=(n\prime /(1+n´/N)}$where n′ is calculated as ${\displaystyle s^2/V^2,s^2=p(1-p)}$and ${\displaystyle V^2}$ is the square of standard error. N is the number of health sector students at BUAP; ${\displaystyle p}$ is the prevalence (%) calculated in previous studies reporting anti-HBs in serum of students.^18,27 The substitution in this formula was carried out with the following values:

$N=3765,se=0.05(standarderror),andp=\mathrm{0.5.}$${\displaystyle s^2=0.5\left(1-0.5\right)=0.25}$${\displaystyle V^2={\left(se\right)}^2={\left(0.05\right)}^2=0.0025}$${\displaystyle n\prime =s^2/V^2=0.25/0.0025=100provisionalsamplesize=samplevariance}$${\displaystyle n=n\prime (1+n\prime /N)=100(1+100/3765)=100\left(0.97\right)=97}$

Two hundred and one were students who wanted to participate because they are aware of the risk factors for acquiring HBV infection and wanted to know if they had protective levels of anti-HBs and status for serology markers HBsAg and anti-HBc.

Serologic testing

Samples were first analyzed with ELISA kits EIA-389 for anti-HBc (qualitative) and EIA-5166 for HBsAg (confirmative) from DRG Diagnostic Inc., (Marburg, Germany). Anti-HBc was considered positive when the sample OD₄₅₀ was ≤ COV (COV = average negative control OD₄₅₀ x 0.5). The HBsAg was considered a true positive when ratio OD₄₅₀ C / N >2.

All positive samples were further confirmed with chemiluminescent microparticle immunoassays (CMIA) approved for diagnostic use from Abbott (Wiesbaden, Germany), ARCHITEC HBsAg 6C36 84-5655/R8 and ARCHITEC anti-HBc II (IgG/IgM), 8L44 48-1810/R1. Sample was reactive if S/CO > 1.0 for anti-HBc and if concentration values ≥ 0.05 IU/mL for HBsAg.

Anti-HBs were determined with (quantitative) EIA-4135 ELISA kit from DRG Diagnostics Inc. (Marburg, Germany). Anti-HBs levels were considered positive if > 1.0 mIU/mL and protective when ≥ 10 mIU/Ml.^22-24

Statistical analysis

For continuous variables, averages, frequencies and percentages were calculated. Univariate analysis was performed using χ²or Fisher's exact test to assess the association of nominal anti-HBc variables and risk factor/anti-HBs. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA);

p < 0.05 was considered statistically significant.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Tomas Martín Vargas-Maldonado, who facilitated the ARCHITEC HBsAg and anti-HBc tests and Dr. Paulina Cortés-Hernández for critical review and suggestions to this article.

Funding

This work was supported by SEP (SEP-23-005 F-PROMEP-39/Rev-03), CONACYT (SALUD-2011-1-161867 [FIS/IMSS/PROT/1038]) and Funds for Scientific Infrastructure from the IMSS (CTFIS/10RD/12/2011).