Hepatitis A is an enterically transmitted virus, and infection is common worldwide, particularly in low-income countries and poor health conditions.1 In these regions, there are high levels of circulating hepatitis A virus (HAV), infecting the majority of the population during the first decade of life. The World Health Organization estimates that more than 200 million infections and 30,000 deaths occurred in 2005.2 The infection is usually benign and even asymptomatic in childhood; however, a small number of cases develop acute liver failure and this may result in death. In low-income countries, there is lower intensity exposure to HAV and the age of infection may be delayed to adulthood.3 In these groups, symptomatic or more serious infections are more common. Countries that improve the sanitary conditions of their populations usually show a change in the epidemiological pattern from high to intermediate endemicity. This phenomenon has been described in several countries, including Brazil.4,5

In addition to improving sanitary conditions, universal immunization of children is an important strategy to control the spread of HAV and reduce disease incidence.6 Vaccines against HAV have been available since the 1990s and are generally based on the inactivated virus.7 Two doses of the vaccine are recommended, the second dose being administered six to 18 months following the first dose. The vaccine is highly immunogenic, and almost all vaccinated healthy subjects develop antibodies. On the other hand, the costs of the vaccine are significant, and it was initially meant to be used in high-risk groups, such as health professionals, homosexuals, and persons traveling to areas of high endemicity.2 However, this strategy has not been successful in decreasing incidence rates of HAV infection. Conversely, countries implementing universal immunization for children aged two to five years, such as Israel and the United States, have reported a dramatic decrease in incidence of infection, with herd immunity extending to older population groups.8,9

In Latin America, Mayorga et al. followed up 105 Nicaraguan children immunized with a single dose of HAV vaccine.10 After 7.5 years HAV infection was documented in only one child, which corresponded to an estimated protective effect of 98%. In 2005, Argentina implemented a universal immunization program, providing a single-dose vaccine to children aged 12 months.11 The single-dose strategy took into account the high costs of the vaccine and the development of antibodies by over 90% of vaccinated persons soon after the first dose. Studies assessing the impact of this strategy have shown a substantial fall in incidence of HAV infection.12

Brazil has also experienced a period of epidemiological transition of HAV.13 In 2014, the Ministry of Health of Brazil introduced a similar vaccination program against HAV, based on a single-dose vaccine (document available in http://www.brasil.gov.br/saude/2014/07/vacina-contra-hepatite-a-passa-a-ser-oferecida-pelo-sus; 2016, extracted on 14.03.16). Children aged between 15 and 24 months were targeted by the immunization program.

The single-dose schedule may be a solution for many countries with limited resources. Therefore, it is crucial to assess these programs in countries where they have been implemented. Specifically, it is important to assess whether protection will last and whether these vaccination programs have an impact on the endemic levels of infection.14,15

This study evaluated the single-dose schedule of anti-HAV vaccination adopted by the Brazilian government in a small city in the state of Mato Grosso, Central Brazil. The aim was to estimate the prevalence of anti-HAV antibodies among children who had been vaccinated at least 30 days before.

A total of 277 children were approached for the study. Of these, 12 children were not included for having been vaccinated in private clinics, immunized less than 30 days before being approached, or received a second dose of the vaccine. Out of the remaining 265 subjects, 218 (82.3%) were anti-HAV positive in DBS eluates.

Anti-HAV was further investigated in venous blood samples obtained from 34 out of 47 children who were anti-HAV negative in DBS eluates. Retest was not performed in the remaining 13 DBS-negative children due to parent refusal to consent to another blood sample collection. Therefore, the final study sample consisted of 252 children. Of these, 128 (50.8%) were male, with a mean age of 17 (SD=5) months. The majority of participants lived in urban area of the municipality and 80.4% of them had been vaccinated less than one year prior to the study. The mean elapsed time between vaccination and blood collection was 261.6 (SD=127.3) days. A total of 236 children (93.6%; 95% CI=90.1–96.2%) out of 252 tested anti-HAV positive. Of these, 218 were anti-HAV positive in DBS eluates and 18 were DBS-negative but tested positive in venous blood samples.

Ten children who were anti-HAV positive in DBS eluates had their EIA optical density very close to the cutoff. They were approached again for an antecubital venous blood sample. Nine had the positivity confirmed. In the only case that anti-HAV was not confirmed, the optic density was again very close to the cutoff.

There was a higher rate of antibody response among female (96.8%) than in male (90.6%) participants, although this difference was not statistically significant (p=0.068). Age and living place (urban/rural) were not associated with anti-HAV status. Furthermore, no association was found between anti-HAV positivity and variables of interest, such as prematurity, low birth weight, comorbidities, and time between DBS blood sample collection and elution. Few participants (9.8%) reported mild vaccine-associated adverse events (fever, pain or local redness). These individuals had lower anti-HAV rate (81.8%) than participants who did not report these events (95.0%); this finding was statistically significant (p=0.036). There was also an association between presence of anti-HAV with concomitant administration of other vaccines on the same day of HAV vaccination (95.6%) compared to participants who received only the HAV vaccine (85.7%; p=0.019) (Table 1).

The associations identified in univariate analysis were adjusted by multiple logistic regression, including variables with p<0.2. The final model included 210 individuals. The presence of anti-HAV was associated only with having received another vaccine concurrently (p=0.050) (Table 2).

This study identified 93.6% positivity for anti-HAV after a single-dose HAV vaccination, as adopted by the National Immunization Program of Brazil. This rate is comparable to those found in previous studies that used a single-dose.7,8,11,12 Although no assays have been performed to assess cellular immune response in the present study, other authors have shown that the CD4+ T cell, CD8+ T cell, and B memory cell responses against HAV are associated with the development of antibodies.16,17 Therefore, it seems that the strategy adopted in Brazil is achieving its initial targets.

The use of finger pricking for blood samples was a practical solution, well accepted by the community, and avoided loss of participants. EIA performed on samples obtained by DBS have proved useful for the detection of anti-HAV antibodies after both natural infection and vaccination. Sensitivity and specificity ranging from 62.5% to 89.6% and 97.5% to 100%, respectively, compared to the standard method of EIA using serum.16,18,19 Since the detection of anti-HAV using DBS is less sensitive than detection using venous blood serum, we got a second blood sample from venipuncture to increase sensitivity.19 Anyway, the DBS technique achieved good levels of detection and over 82% of participants did not require another blood sample collection.

Ten weakly positive samples were retested using serum. Reproducibility was 90%. In one case, it was not possible to confirm positivity for anti-HAV antibodies. Of note, the two samples taken were very close to the cut off, despite more than a year separating the two blood collections. Consequently, non-confirmation of the second test may be due to a small decrease of antibody titers over time.

In this study, it was not possible to retest all 47 individuals who had tested negative with the DBS technique. However, retesting 34 of these children showed that 18 (53%) of them were anti-HIV positive. For ethical reasons, children with two negative tests received a second dose of the vaccine. This is not a practice to be adopted in primary care unless further studies show that single dose vaccination is not as effective as two doses. It is anticipated that some individuals will not seroconvert after one dose of vaccine.2 This does not indicate a need for retesting the entire population or a flaw in the strategy adopted. Routine retesting of all vaccinated individuals would make the program expensive and would be of little practical advantage. Even those who have not seroconverted may be protected by herd immunity if the strategy is successful.

The analysis of the demographic and epidemiological aspects did not identify any determinant factor for anti-HAV seropositivity, except for the administration of more than one vaccine on the same day. However, the statistical significance was borderline (p=0.05), and the logistic model had little explanatory power (pseudo-R2=0.13). The small number (only 16) of non-seroconverted individuals may have contributed to this. As this association does not seem very plausible, it may have occurred randomly. A robust review involving more than 50,000 subjects showed no interference in the HAV vaccine immunogenicity in those who received other vaccines simultaneously.20

A limitation of the study was that the participants were not randomly included. Therefore, the sample was increased by twofold to reduce chances of bias and increase the study power. A further limitation is that the anti-HAV status was unknown before vaccination. Therefore, it is not possible to determine whether antibodies in anti-HAV positive individuals were induced by the vaccine or natural infection. In a randomized study performed over the last decade and including major cities in the Central region of Brazil, the authors reported an anti-HAV prevalence of 32% in children aged between 5 and 9 years.21 The prevalence among children aged between 1 and 3 years is likely to be considerably lower. Therefore, it is plausible to postulate that the vast majority of anti-HAV positive subjects developed these antibodies as a consequence of vaccination.

Finally, this survey showed that following the introduction of HAV vaccination in a small town in Brazil, more than 90% of children developed antibodies. This is the first evidence suggesting that the strategy adopted in Brazil using a single-dose of the vaccine may be sufficient for mass immunization. Nonetheless, it is too early to assess whether the immune response elicited will be persistent and whether the strategy will lead to decrease the endemic levels of illness in the country. Further studies are required to evaluate the single-dose strategy effectiveness.

The authors declare no conflicts of interest.