CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Sept. 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.
NOTES
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 Clı́nico, Caja de Seguro Social de Panamá,1 Servicio de Inmunologı́a, Hospital
Nacional de Niños “Dr. Carlos Sáenz Herrera,”2 and Instituto Clodomiro Picado,
Facultad de Microbiologı́a, Universidad de Costa Rica,3 San José, Costa Rica
Received 14 August 2000/Returned for modification 4 January 2001/Accepted 17 May 2001
We evaluated children (15-months old and older) with recurrent upper respiratory tract infections and nor-
mal 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.
Infections of the upper respiratory tract in children are a 1999 were included. All patients had normal serum immuno-
main reason for visits with pediatricians, and these patients globulin concentrations. Recurrent infection was defined as
show a high morbidity rate. In some of these children, such follows: (i) recurrent otitis, three episodes in 6 months or four
infections have a recurrent pattern (2). At present, there are episodes in 1 year; (ii) sinusitis and/or recurrent bronchopneu-
no uniform criteria to define recurrent infection, but several monia, two episodes in 6 months or three episodes in 1 year or;
repeated infections in a year should be considered to constitute (iii) recurrent rhinopharyngitis, four episodes in 6 months or
recurrent infection (9). eight episodes in 1 year. Children were excluded from the
In recent years, an immunodeficiency characterized by the study if they had a primary immunodeficiency, a chronic pul-
selective inability to respond to polysaccharide antigens has monary illness, or a structural congenital malformation. Once
been described (5, 6). This defect is suspected in patients who the patients were identified, parents were asked for written
suffer from recurrent upper respiratory tract infections, espe- consent. The study was approved by the hospital’s investigation
cially those caused encapsulated bacteria, and who have nor- committee.
mal serum immunoglobulin levels (9, 12). The absence of an- Two blood samples were collected from each patient. The
tibody response to bacterial polysaccharides, in the presence of first sample was collected before the application of the Act-
a normal response to protein antigens, is a common charac- HIB vaccine (Pasteur-Mérieux), and the second was collected
teristic of these patients (1). 4 to 6 weeks later. Both serum samples, pre- and postvaccina-
Different laboratory methods that determine specific anti- tion, were stored at 20°C until analysis.
bodies toward bacterial polysaccharide antigens have been de- ELISA. Haemophilus influenzae type b (Hib) vaccine conju-
veloped (8, 13). However, immunoenzymatic techniques such gated to diphtheria toxoid (Hib TITER; Lederle Laboratories)
as enzyme-linked immunosorbent assay (ELISA) are used was used as an antigen to coat ELISA plates (Immulon 2;
most often due to their simplicity and sensitivity. As an alter- Dynatech Laboratories). To determine the optimal minimum
native, special plastic surfaces for the covalent attachment of concentration of antigen for coating, the following concentra-
polysaccharides, or the conjugation of polysaccharides to pro- tions were tested: 1.0, 0.5, 0.25, 0.12, 0.06, and 0.03 g/100
tein carriers, have been utilized (10, 13).
l/well. The antigen was diluted in coating buffer (Tris, 0.05 M;
In this investigation three ELISA methods were standard- NaCl, 0.15 M; pH 9.0) and adsorbed onto the wells of micro-
ized to detect children with polysaccharide-specific immuno-
deficiency in a population of patients with recurrent respira- titer plates during incubation at room temperature overnight.
tory tract infections. After rinsing the plates five times with this buffer, the wells
Population. All children older than 15 months of age with were blocked with 1% bovine serum albumin (BSA) in FALC
recurrent respiratory tract infections who had been referred to buffer (Tris, 0.05 M; NaCl, 0.15 M; ZnCl2, 20 M; MgCl2, 1
the Immunology Outpatient Clinic at National Children’s Hos- mM; pH 7.4) for 30 min. Then, the plates were decanted and
pital in San José, Costa Rica, between October 1998 and June different dilutions (100 l/well) of international reference anti-
Hib sera (70 g of anti-Hib/ml, lot 1983; Food and Drug Ad-
ministration, Washington, D.C.) starting at 1:50 were added.
Dilutions were prepared in FALC buffer containing 1% BSA.
* Corresponding author. Mailing address: Servicio de Inmunologı́a,
Hospital Nacional de Niños “Dr. Carlos Sáenz Herrera,” San José, After 2 h at room temperature, the plates were rinsed five
Costa Rica. Phone and fax: (506) 223-51-25. E-mail: aabdelnour@hnn times with FALC buffer; goat anti-human immunoglobulin G
.sa.cr or alberto68@latinmail.com. (IgG)–alkaline phosphatase, diluted 1:5,000 in FALC buffer–
1012
VOL. 8, 2001 NOTES 1013
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.
1% BSA was added; and the plates were incubated for 2 h at tract infection and normal serum immunoglobulin concentra-
room temperature. After washing, 100 l of a 1-mg/ml con- tions. We evaluated 12 patients because to meet the criteria for
centration of p-nitrophenyl phosphate in substrate buffer was selection for the study population, children needed to receive
added to all wells. Absorbance readings at 410 nm were deter- a booster vaccine and be at least 15 months old.
mined using a Dynatech MR5000 microplate reader. All sam- This population is not uniform because, at the time the study
ples were assayed in triplicate. was done, the Costa Rican Social Security System did not
Pre- and postvaccination samples were analyzed at four dif- provide Hib vaccination.
ferent dilutions, with threefold serial dilutions starting at 1:100. The inconvenience of using ELISA methods in the detection
Dilutions were prepared in FALC buffer–1% BSA. The back- of antipolysaccharide antibodies lies in the difficulties encoun-
ground was established with wells without antigen, processed tered when these antigens are absorbed to the solid phase.
identically to a patient sera. Conjugation techniques sometimes result in alterations of
Hib vaccine (0.05 g) conjugated to diphtheria toxoid was the antigen’s native structure (3–5). We utilized a commercial
selected at an optimal concentration for coating microtiter preparation of PRP conjugated to a carrier protein, which has
plates, and a 1:300 dilution was selected as optimal for IgG been used for vaccination and which has shown good immu-
antibody determinations. nogenicity (9). This antigen was efficient in detecting anti-PRP
An antibody response to polyribosyl ribitol phosphate (PRP) IgG in children receiving an H. influenzae vaccine that was
was considered positive if absorbance readings of the postvac- based on PRP conjugated to a different carrier protein. In spite
cination serum sample were at least double the absorbance of the small number of patients, it was proven that a specific
readings of the prevaccination sample when absorbance read-
ings were higher than 0.1. For statistical comparisons, the Stu-
dent 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 eval-
uated. 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 slight-
ly (Fig. 2). All patients produced anti-tetanus toxoid IgG an-
tibodies in high concentrations after immunization (Fig. 3). FIG. 2. Levels of anti-diphtheria toxoid IgG antibodies, pre- andpostvaccination, in serum of children with recurrent respiratory infec-
This investigation evaluated polysaccharide-specific immu- tion and normal serum immunoglobulin concentrations, Asterisk indi-
nodeficiency in children showing recurrent upper respiratory cates a significant (P  0.05) increase from prevaccination level.
1014 NOTES CLIN. DIAGN. LAB. IMMUNOL.
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 im-
mune system to polysaccharide antigens.
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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