From the Departments of Pediatrics, Cerrahpasa Faculty of Medicine, University of Istanbul, Istanbul, Turkey; *Department of Public Health, Cerrahpasa Faculty of Medicine, University of Istanbul, Istanbul, Turkey; and Department of Microbiology, Istanbul Faculty of Medicine, University of Istanbul, Istanbul, Turkey.

Reprint requests: Yücel Tastan, Ahmet Kutsi Tecer Cad No: 4/11, Merter, 340 10, Istanbul, Turkey. Fax: (0212) 586 15 95

Manuscript received: March 10, 1999;
Initial review completed: April 26, 1999;
Revision accepted: October 13, 1999

Despite the decline in the number of cases of disease due to Haemophilus influenzae type b (Hib) in the vaccine era, it still continues to be a major cause of severe morbidity and mortality world-wide among children under five years of age. The incidence of disease varies from one country to another, depending on geographical, genetic and social factors. In developing countries, as opposed to industrialized countries, most of the cases are seen in the first12 months, half of which occur in the first 6 months of life(1,2). In Turkey, the incidence of invasive Hib disease is not known and the vaccine is still not included into the National Immunization Program. The aim of this study was to evaluate the natural immunity to Hib in healthy children under 13 months of age.

This longitudinal study was performed between December 1995 and May 1997, in the well-child clinic, Department of Pediatrics, Cerrahpasa Faculty of Medicine. This clinic serves a heterogeneous population of approxi-mately 10 million in Istanbul. Sixty-four full term healthy infants (39 males, 25 females), with no apparent congenital anomalies, chronic illnesses or immunodeficiencies were enrolled in the trial at 1 month of age during their routine well-baby visists after obtaining oral consent from the parents. No infant was either vaccinated against Hib or had received any immunoglobulin preparation or blood product. They had received BCG vaccine at birth, hepatitis B vaccine at 0, 1 and 6 months of age, diphtheria, tetanus toxoids, pertussis and oral poliovirus vaccines at 2, 4, and 6 months of age. Demographic and medical data were obtained through personal interview with mothers. At a mean age of 1.5, 2.5, 6.5 and 13 months blood samples were drawn from infants, and sera were stored at –20°C until analysis.

Serum capsular polysaccaride specific IgG antibody (anti-PRP) concentrations were determined at the department of Microbiology by using commercially available ELISA kits (BIND-A-ZYMETM MK 016), as recommended by the manufacturer. The lowest and the highest limits of detection for anti-PRP antibody were 0.10 mg/ml and 1 mg/ml, respectively. For the description of antibody concentrations, the values below the limits of detection were excluded and data was analyzed by geometric means (GM) as logarithmic transformation normalized the data. Comparisons between continuous variables were made using Mann-Whitney U test.Proportions were compared by Fisher’s exact and c2 test, as appropriate. The Friedmann test was used for the repeated measures and the Spearman test was used for correlation investigations.

Sixty-four infants were enrolled in the study at 1.5 months of age. Forty-nine infants returned at 2.5 months, 54 at 6.5 months and 46 at 13 months of age. The mean number of persons per household was 3.6. Eighty per cent of the families were covered by social security. Fifty per cent of the infants had one or more than one sibling. Sixty-four per cent of the siblings were older than 6 years of age. All of the infants were in home care.Most of the infants (95%) were exclusively breast-fed in the first 4 months of life. Between the first 1.5-2.5 months, 2.5-6.5 months and 6.5-13 months, 18%, 40% and 80% of infants, respectively had otitis media or upper airway infection. During the study period none of the children had invasive Hib disease.

Table I shows the GM anti-PRP titers and the percentage of infants with anti-PRP concentration ³0.15 mg/ml. This level was accepted as protective level in unimmunized children and produce a short-time protection(3). The total anti-PRP concentrations showed spontaneous increases ³0.15 mg/ml in 18.2% of the infants (8 of 49) at 2.5 months, in 14.8% of the infants (8 of 54) at 6.5 months, in 19.5% of the infants (9 of 46) at 13 months of age while their antibody level was £0.15 mg/ml previously. However, some of the infants who had protective level of antibody at younger ages showed a decrease in antibody level below 0.15 mg/ml at later ages. Thus the percentage of the infants having anti-PRP antibody at protective level did not change significantly between 1.5 to 13 months of age. Neither the percentage of infants with protective antibody level at each age group nor the percentage of infants with spontaneous increase of antibody level were significantly related to the number of siblings and their ages, feeding type or the number of otitis media and upper airway infections during the intervening period.

Table I - Anti-PRP Specific IgG Antibody Titers Measured by ELISA in Healthy Turkish Children

  There was no significant difference among various age groups.

This longitudinal study investigated naturally acquired anti-PRP in healthy children during the first year of life in Turkey, a country with unknown incidence of invasive Hib diseases. The results demonstrated that a high percentage of infants from 1.5 months to 13 months of age had an anti-PRP concentration considered to be protective and their GM anti-PRP titers were also higher than the protective level. Serum anti-PRP in the first few months of age were considered to be maternally acquired and were expected to decrease with time(1,2). In this study, at 6.5 months of age GM anti-PRP titers and the percentage of infants having anti-PRP at protective level showed a decline, but they were not significantly different from those at other ages.

In some populations with either high (Gambia) or low (Finland) incidence of invasive Hib disease in the children under one year of age, the GM anti-PRP titers, unlike our results, are below protective levels(4–6). In Finland, anti-PRP concentrations were not detected to be within protective level in almost all children under 6 months age(5). In Gambia, 60% of infants born to mothers immunized with Hib Conjugate vaccine and 26% of infants of unvaccinated mothers had protective anti-PRP concentration at two months of age(4). In our study the percentage of infants with protective anti PRP antibody level was approximately similar to those found in Gambian infants whose mothers received the Hib vaccine during pregnancy.

The results obtained from our study suggest that the majority of the infants have high concentrations of maternally transferred anti-PRP and have also acquired the natural immunity to Hib at an early period of life. This study population consisted of middle socio-economic class, who did not live in corwded conditions nor attended day care centers. Thus they were not in a high risk group for Hib exposure. On the other hand, no significant correlation was found between the percentage of infants having anti-PRP at protective level of the percentage of infants with spontaenous increase of antibody level and recent exposure to upper airway infections and otitis media. These results could be related to the relatively small number of subjects in our study group. Otherwise, prominent spontaneous anti-PRP increases may be due to corss reaction or Hib colonization(7). Unfortunately this study was not designed to evaluate the colonization of Hib or the rectal carriage of Escherichia coli K 100, an organism with a capsule antigenically similar to Hib(8).

Lately WHO has recommended the world-wide inclusion of Hib conjugate vaccines in infant-immunization programs(9). It must be considered how such an expensive vaccine can be made available to populations with limited resources.

In conclusion, infants in Turkey may be at risk of exposure to Hib infection at an early age and further research directed to the epidemiology of Hib diseases should be done to prioritise routine vaccination against Hib.

This study was supported by a grant from Ms. Pasteur Merieux Connaught.

Contributors: YT, MA and OI coordinated the study (particularly its design and interpretation) and YT and MA drafted the paper.YA will act as the guarantor for the paper. FT, AA and MA participated in the data collection, and FT also helped in drafting the paper. EE helped in interpreting the data. DY and SB performed the analysis of Anti-PRP.

Competence interests: None stated.
Funding: Pasteur Merieux Connaught.

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