This study was conducted in the Departments of Pediatrics and Microbiology at University College of Medical Sciences and Guru Tegh Bahadur Hospital, Delhi over a period of 17 months (December 2013-April 2015) after approval from the Ethical Committee of the institute and written informed consent from the parents.

Clinically healthy eligible neonates born consecutively at term gestation were allocated in LBW (1800 to 2499 g) and normal birthweight (2500 to 4000g) groups within 24 hours of birth till desired sample size was reached. Infants born to hepatitis B positive mothers, neonates suffering from sepsis, birth asphyxia, meconium aspiration syndrome, gross congenital anomalies, requiring exchange transfusion and whose families were planning to leave the area before the period of completion of study were excluded.

Sample size was calculated based on the study by Sharma, et al. [7] Wherein indigenous pentavalent vaccine produced anti HBs geometric mean titers (GMT) of 616.7 mIU/mL in healthy term infants. Expecting a difference of 15% in anti-HBs titers between LBW and normal birthweight babies, at 80% power and 5% level of significance, 476 infants were required, equally divided between LBW and normal birthweight groups. Considering an estimated attrition rate of 10%, we planned to recruit 265 infants in each group (total 530 infants).

Pentavalent vaccine consisting of Diphtheria, Tetanus, Pertussis, Hepatitis B, and Haemophilus influenzae type B Conjugate vaccine adsorbed (Serum Institute of India Ltd, Pune) was used. Each dose of 0.5 mL contained Diphtheria Toxoid 25 Lf (30 IU), Tetanus Toxoid 2.5 Lf (40 IU), B. pertussis (whole cell) 16 OU ( 4.0 IU), HBsAg (rDNA) 10 mcg and Purified capsular HiB Polysaccharide (PRP) conjugated to Tetanus Toxoid (carrier protein) 10 mcg.

We collected 2 mL of cord blood in plain sterile vial from placental end and stored at –20ºC after separation of serum. Breastfeeding was initiated within 1 hr after normal delivery; and within 2 hrs in babies delivered through caesarean section. Monovalent recombinant Hepatitis B vaccine (dose 0.5 mL, 10 mcg purified HBsAg), manufactured by Biological E Ltd, India, was administered in the anterolateral aspect of thigh within 24 hours of birth, by a trained staff nurse. BCG and OPV zero dose were also administered at the same time. Birth weight, length and head circumference were recorded at birth by standard methods.

Infants were called at 6 weeks (+2 weeks), 10 weeks (+2 weeks) and 14 weeks (+2 weeks), and 0.5 mL pentavalent vaccine (DTwP-HBV-Hib) was administered by intramuscular injection into the anterolateral aspect of thigh by trained staff nurses. Trivalent OPV was also administered simultaneously. All infants were monitored for 1 hour following immunization for development of any adverse event. Mother/guardian was given a proforma to record the adverse events at home, and was advised to contact telephonically or return back on occurrence of any serious adverse event. The proforma for adverse events was checked at each follow-up visit and minor adverse events such as fever and local tenderness were managed symptomatically. Weight, length and head circumference were recorded at each visit by standard methods. Mothers were counselled to bring the infants 6 weeks after the third dose of pentavalent vaccine and 2 mL venous sample was collected in plain vial; serum was stored at –20ºC.

Serum samples were thawed and anti-HBs titers were estimated using enzyme linked immunosorbent assay (ELISA) based kits (DIA.PRO, Diagnostic Bioprobes Srl, Italy). The calibrators and samples were tested as per the protocol provided with the kit. Validation check was carried out on the controls.

Statistical analysis: The GMTs were calculated by taking the antilog of the mean of the logarithmic transformation of the titers. Antibody titers between the two groups were compared using unpaired student t-test. Proportion of infants developing adverse events following immunization (AEFI) was assessed using chi-square test. The analysis was carried out using SPSS 20.0 software.

A total of 93.1% (443) infants completed follow-up (LBW 94.5%; normal birth weight 91.6%) (Fig. 1). Table I depicts the baseline characteristics of participants in both groups.

Fig. 1 Distribution of participants in the study.

TABLE I Baseline Parameters of Enrolled Infants at Birth  

The median (IQR) cord blood anti HBs levels of 443 infants was 0 (0,0). Minimum level of anti HBs titers observed after 6 weeks of primary immunization with pentavalent vaccine was 40 mIU/mL in both the groups. Maximum anti-HBs titers attained were 280 mIU/mL and 282 mIU/mL, respectively in LBW and normal birth weight groups. Mean (SD) anti-HBs titers were 206.76 (60) mIU/mL and 214 (55.46) mIU/mL, respectively for LBW and normal birthweight infants (P=0.17). Anti-HBs GMTs were 194.76 mIU/mL and 204.2 mIU/mL in LBW and normal birthweight infants, respectively and the difference was not significant (P=0.17).

Table II shows the adverse events observed in 443 infants of the two groups who completed three doses of pentavalent vaccine. Common adverse events were fever, tenderness and induration. All the adverse events resolved with symptomatic management. These adverse events decreased with subsequent doses of immunization.

TABLE II  Incidence of Adverse Events in Infants Receiving Pentavalent Vaccine (N=443)

The assumed difference of 15% in the anti HBs titres between LBW and normal birth weight infants was arbitrary and this could have affected the actual sample size. The other limitation of the study was that the number of infants studied might not address safety and tolerability of the vaccine. A good (93%) follow-up of the infants at 6 weeks after immunization is the strength of the study.

Earlier studies have shown concerns that LBW infants have low levels of T and B lymphocytes and lower vaccine specific IgG responses as compared to normal birth weight babies [8-10]. Studies evaluating the immunogenicity of hepatitis B component of pentavalent vaccine in term infants enrolled at 6 weeks of age have concluded the vaccine to be immunogenic with seroprotection rates ranging from 97% to 100% [11-14]. Studies evaluating different brands of pentavalent (DTwP-HBV- Hib) vaccine reported comparable anti HBs GMT in all infants [7,14,15]. Our study reiterates that pentavalent vaccine is highly immunogenic in infants immunized with monovalent hepatitis B vaccine at birth. Although, all infants in our study achieved seroprotective titers, the anti HBs GMTs observed in our trial were lower than those reported in the above studies in both normal birth weight and LBW infants. Different pharmacological preparations and physical properties of the vaccine; characteristics of ELISA testing kits and ethnicity may be the possible reasons for different immunogenicity and levels of GMTs. However, this difference does not seem to be clinically significant because anti HBs seroprotective titre (>10 mIU/mL) is attained in all children.

There are studies documenting the good immunogenicity of monovalent hepatitis B vaccine in both low birth weight and normal birth weight infants [5, 6, 16, 17]. These results correlate well with our results where term LBW infants attained good immune response and reiterate the fact that pentavalent vaccine is as immunogenic as the separately administered monovalent vaccine. A retrospective cohort study in Nigeria analyzing the immunization records from June 2011 to May 2013 revealed the significant improvement in uptake of vaccines and completion of the schedule when pentavalent vaccine was used as compared to separately administered DPT and Hepatitis B vaccine [18]. The vaccine was also safe and tolerable in these studies.

We conclude that three primary doses of pentavalent vaccine administered along with zero dose of Hepatitis B vaccine at birth achieved comparable seroprotective anti HBs GMT in LBW and normal birth weight infants and that the immunization with pentavalent vaccine appears to be safe.

Contributors: MMAF: Conceptualized the study, supervised the work, and critically reviewed the manuscript and will stand guarantor; CV: collected the data, searched literature, carried out estimation of anti-HBs titers, drafted and analyzed the results; MN: helped in search of the literature and reviewed the manuscript; IK: critically analyzed the results and reviewed the manuscript. All authors approved the final draft.

Funding: Directorate of Family Welfare, Govt. of NCT of Delhi.

Competing Interest: None stated