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Clinical and Diagnostic Laboratory Immunology, September 2001, p. 1012-1014, Vol. 8, No. 5
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.5.1012-1014.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Serum Antibody Response to Polysaccharides in
Children with Recurrent Respiratory Tract Infections
Alberto
López-Yap,1
Arturo
Abdelnour,2,*
Bruno
Lomonte,3 and
Oscar
Porras2
Laboratorio Clínico, Caja de Seguro
Social de Panamá,1 Servicio de
Inmunología, Hospital Nacional de Niños "Dr.
Carlos Sáenz Herrera,"2 and
Instituto Clodomiro Picado, Facultad de
Microbiología, Universidad de Costa
Rica,3 San José, Costa Rica
Received 14 August 2000/Returned for modification 4 January
2001/Accepted 17 May 2001
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ABSTRACT |
We evaluated children (15-months old and older) with recurrent
upper respiratory tract infections and normal levels of
immunoglobulins in serum for specific polysaccharide immunodeficiency
using an enzyme-linked immunosorbent assay method. Results showed that of 12 patients vaccinated with Act-HIB vaccine, one did not develop specific antibodies to Haemophilus influenzae type b,
demonstrating that such immunodeficiency is present in Costa Rican children.
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TEXT |
Infections of the upper respiratory
tract in children are a main reason for visits with pediatricians, and
these patients show a high morbidity rate. In some of these children,
such infections have a recurrent pattern (2). At present,
there are no uniform criteria to define recurrent infection, but
several repeated infections in a year should be considered to
constitute recurrent infection (9).
In recent years, an immunodeficiency characterized by the selective
inability to respond to polysaccharide antigens has been described
(5, 6). This defect is suspected in patients who suffer
from recurrent upper respiratory tract infections, especially those
caused encapsulated bacteria, and who have normal serum immunoglobulin
levels (9, 12). The absence of antibody response to
bacterial polysaccharides, in the presence of a normal response to
protein antigens, is a common characteristic of these patients (1).
Different laboratory methods that determine specific antibodies toward
bacterial polysaccharide antigens have been developed (8,
13). However, immunoenzymatic techniques such as
enzyme-linked immunosorbent assay (ELISA) are used most often due
to their simplicity and sensitivity. As an alternative, special plastic
surfaces for the covalent attachment of polysaccharides, or the
conjugation of polysaccharides to protein carriers, have been utilized
(10, 13).
In this investigation three ELISA methods were standardized to detect
children with polysaccharide-specific immunodeficiency in a population
of patients with recurrent respiratory tract infections.
Population.
All children older than 15 months of age with
recurrent respiratory tract infections who had been referred to the
Immunology Outpatient Clinic at National Children's Hospital in San
José, Costa Rica, between October 1998 and June 1999 were
included. All patients had normal serum immunoglobulin concentrations.
Recurrent infection was defined as follows: (i) recurrent otitis, three episodes in 6 months or four episodes in 1 year; (ii) sinusitis and/or
recurrent bronchopneumonia, two episodes in 6 months or three episodes
in 1 year or; (iii) recurrent rhinopharyngitis, four episodes in 6 months or eight episodes in 1 year. Children were excluded from the
study if they had a primary immunodeficiency, a chronic pulmonary
illness, or a structural congenital malformation. Once the patients
were identified, parents were asked for written consent. The study was
approved by the hospital's investigation committee.
Two blood samples were collected from each patient. The first sample
was collected before the application of the Act-HIB vaccine (Pasteur-Mérieux), and the second was collected 4 to 6 weeks later. Both serum samples, pre- and postvaccination, were stored at
20°C until analysis.
ELISA.
Haemophilus influenzae type b (Hib) vaccine
conjugated to diphtheria toxoid (Hib TITER; Lederle Laboratories) was
used as an antigen to coat ELISA plates (Immulon 2; Dynatech
Laboratories). To determine the optimal minimum concentration of
antigen for coating, the following concentrations were tested: 1.0, 0.5, 0.25, 0.12, 0.06, and 0.03 µg/100 µl/well. The antigen was
diluted in coating buffer (Tris, 0.05 M; NaCl, 0.15 M; pH 9.0) and
adsorbed onto the wells of microtiter plates during incubation at room temperature overnight. After rinsing the plates five times with this
buffer, the wells were blocked with 1% bovine serum albumin (BSA) in
FALC buffer (Tris, 0.05 M; NaCl, 0.15 M; ZnCl2, 20 µM; MgCl2, 1 mM; pH 7.4) for 30 min. Then, the plates were
decanted and different dilutions (100 µl/well) of international
reference anti-Hib sera (70 µg of anti-Hib/ml, lot 1983; Food and
Drug Administration, Washington, D.C.) starting at 1:50 were
added. Dilutions were prepared in FALC buffer containing 1% BSA.
After 2 h at room temperature, the plates were rinsed five times
with FALC buffer; goat anti-human immunoglobulin G (IgG)-alkaline
phosphatase, diluted 1:5,000 in FALC buffer-1% BSA was
added;
and the plates were incubated for 2 h at room
temperature. After
washing, 100 µl of a 1-mg/ml concentration of
p-nitrophenyl phosphate
in substrate buffer was added to all
wells. Absorbance readings
at 410 nm were determined using a Dynatech
MR5000 microplate reader.
All samples were assayed in
triplicate.
Pre- and postvaccination samples were analyzed at four different
dilutions, with threefold serial dilutions starting at 1:100.
Dilutions
were prepared in FALC buffer-1% BSA. The background
was established
with wells without antigen, processed identically
to a patient
sera.
Hib vaccine (0.05 µg) conjugated to diphtheria toxoid was selected at
an optimal concentration for coating microtiter plates,
and a 1:300
dilution was selected as optimal for IgG antibody
determinations.
An antibody response to polyribosyl ribitol phosphate (PRP) was
considered positive if absorbance readings of the postvaccination
serum
sample were at least double the absorbance readings of the
prevaccination sample when absorbance readings were higher than
0.1. For statistical comparisons, the Student
t test was
utilized.
A
P value of <0.05 was considered statistically
significant.
During the 9-month study period, 12 patients were included, 8 male and
4 female, with ages between 15 and 44 months (median,
22 months). One
patient had received three previous doses of Hib
vaccine, and another
one had been vaccinated twice. The rest of
the patients had never been
vaccinated against
Hib.
Patient analysis.
In this investigation, 12 children were
evaluated. One patient did not show an IgG antibody response after
vaccination (Fig. 1). Anti-diphtheria
toxoid IgG antibody analyses demonstrated that postvaccine levels
decreased or remained similar to prevaccine levels in 11 patients. In
the one remaining patient, postvaccine antibody levels increased
slightly (Fig. 2). All patients
produced anti-tetanus toxoid IgG antibodies in high concentrations
after immunization (Fig. 3).
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FIG. 1.
Levels of anti-PRP IgG antibodies to Hib pre- and
postvaccination, in serum of children with recurrent respiratory
infection and normal serum immunoglobulin concentrations. Patient 1 had
three doses and patient 4 had two doses of Act-HIB vaccine, prior to
the study. Asterisks indicate significant (P < 0.05)
increases from prevaccination levels.
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FIG. 2.
Levels of anti-diphtheria toxoid IgG antibodies, pre-
and postvaccination, in serum of children with recurrent respiratory
infection and normal serum immunoglobulin concentrations, Asterisk
indicates a significant (P < 0.05) increase from
prevaccination level.
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FIG. 3.
Levels of anti-tetanus toxoid IgG antibodies, pre- and
postvaccination, in serum of children with recurrent respiratory
infection and normal serum immunoglobulin concentrations. Asterisks
indicate significant (P < 0.05) increases from
prevaccination levels.
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This investigation evaluated polysaccharide-specific immunodeficiency
in children showing recurrent upper respiratory tract
infection and
normal serum immunoglobulin concentrations. We evaluated
12 patients
because to meet the criteria for selection for the
study population,
children needed to receive a booster vaccine
and be at least 15 months
old.
This population is not uniform because, at the time the study was done,
the Costa Rican Social Security System did not provide
Hib
vaccination.
The inconvenience of using ELISA methods in the detection of
antipolysaccharide antibodies lies in the difficulties encountered
when
these antigens are absorbed to the solid phase. Conjugation
techniques
sometimes result in alterations of the antigen's native
structure
(
3-5). We utilized a commercial preparation of PRP
conjugated to a carrier protein, which has been used for vaccination
and which has shown good immunogenicity (
9). This antigen
was
efficient in detecting anti-PRP IgG in children receiving an
H. influenzae vaccine that was based on PRP conjugated to a
different
carrier protein. In spite of the small number of patients, it
was proven that a specific immunodeficiency to polysaccharides
of
bacterial origin, detected by the absence of antibodies to
polysaccharide antigens of bacterial origin, is a pathology present
in
Costa Rican children; thus, it may be a cause of recurrent
infections
of the upper respiratory tract in this population.
The low percentage
of detection of this specific immunodeficiency
to the PRP
polysaccharide of Hib is similar to that observed in
countries such as
Spain (
7) and Brazil (
11).
In addition, the present study verified that the tetanus protein can be
used as an indicator of an adequate immune response
to T-dependent
antigens (
1). This is because the minimal concentration
present in the Hib TITER vaccine was sufficient to stimulate the
immune
system of the evaluated children without the need for vaccinating
them
separately with tetanus
toxoid.
We did not check the patients' IgG subtypes before doing these ELISAs
because a method was not available and clinical history
does not
coincide with children having an IgG subclass
deficiency.
Since the causes of the specific immunodeficiency to the PRP
polysaccharide of Hib are still unknown, we recommend that this
study
be repeated when these patients are more than 6 years old,
in order to
determine whether the cause of this immunodeficiency
is retardation in
the maturation of the immune system to polysaccharide
antigens.
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ACKNOWLEDGMENTS |
We thank the German Academic Exchange Service (DAAD) for supporting
this study and Carl Frasch (Food and Drug Administration) for providing
international reference anti-Hib sera.
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FOOTNOTES |
*
Corresponding author. Mailing address: Servicio de
Inmunología, Hospital Nacional de Niños "Dr. Carlos
Sáenz Herrera," San José, Costa Rica. Phone and fax:
(506) 223-51-25. E-mail: aabdelnour{at}hnn.sa.cr or
alberto68{at}latinmail.com.
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Clinical and Diagnostic Laboratory Immunology, September 2001, p. 1012-1014, Vol. 8, No. 5
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.5.1012-1014.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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