INTRODUCTION

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Serology is widely employed for the diagnosis of pertussis, and its use has been recommended (15) as part of the case definition in vaccine efficacy trials. Many serological assays have been developed during the last 20 years, but some lack sensitivity and/or specificity. The enzyme-linked immunosorbent assay (ELISA), using purified pertussis factors such as pertussis toxin (PT) or filamentous hemagglutinin (FHA), has recently been used extensively for epidemiological studies (8). This technique has proven to be reasonably sensitive and specific (1, 3, 13). It is generally concluded that a significant increase in specific anti-PT immunoglobulin G (IgG) or IgA and/or anti-FHA IgG or IgA in paired sera correlates with Bordetella pertussis infection. However, this type of diagnosis of pertussis has been mainly applied to unvaccinated children, with acute-phase serum collected within 14 days after the onset of illness and convalescent-phase serum collected 4 to 8 weeks later. In current practice and in epidemiological studies, such criteria are not always fulfilled: pertussis may be suspected after it is too late to culture the organism or to obtain an acute-phase serum sample, and convalescent-phase sera may be collected more than 8 weeks after the onset of the cough. Moreover, serological responses after infection may be difficult to interpret because of previous vaccination (3). Such conditions are observed in pertussis vaccine trials and will probably occur more often with increasing use of pertussis vaccines.

In the present study, we analyzed the serological response of suspected cases of pertussis infection as part of the Senegal Pertussis Trial (11). Increases in anti-PT IgG and anti-FHA IgG titers between paired sera were detected. However, as previously observed (5, 9), we also detected a significant number of anti-PT IgG and anti-FHA IgG decreases between paired sera. The objectives of this study were (i) to undertake a precise analysis of these decreases, compared with increases, in order to determine whether they might also detect children infected with B. pertussis; and (ii) to study the possible impact of such findings on the interpretation of vaccine efficacy results.

	MATERIALS AND METHODS

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Subjects. The study was based on available serological results for children sampled within the pertussis surveillance system of the Senegal Pertussis Trial and included in the main analysis population.

Serology. (i) Handling of serum specimens. Blood was collected by finger prick on microtainer tubes (Becton Dickinson) and centrifuged, and sera were kept at 80°C in 0.05-ml aliquots in 1.8-ml Nunc tubes. A volume of 0.02 ml was sufficient for two different assays of IgG for PT and FHA.

(ii) Antigens. Purified PT and FHA were obtained from Pasteur Mérieux, Lyon, France, in vials containing, respectively, 200 and 500 µg of purified protein per ml. The antigens were stored at 20°C in 50% glycerol. One batch of each antigen was used throughout the study.

(iii) ELISAs. The main ELISA procedures were as follows. Microtiter plates (Nunc Maxisorp certified) were coated with PT and FHA diluted to 2 µg/ml in bicarbonate buffer (pH 9.6) and phosphate-buffered saline (PBS) (pH 7.4). Coated plates were stored for 1 week maximum.

(iv) Culture. Aspirate specimens were obtained with a suction catheter (Vygon). The aspirates were inoculated in the home (primary plates) and in the laboratory (secondary plates) on Reagan-Lowe agar plates containing 10% defibrinated horse blood and 40 mg of cephalexin per liter. Cultures were inoculated at 36°C and observed regularly during a 7-day period after inoculation. Suspected colonies were identified with antisera specific to B. pertussis and Bordetella parapertussis (Difco Laboratories). Final verification was done with oxidase and urease.

(v) Epidemiological linkage. A suspected case of infection was confirmed as pertussis if the subject was living in the same compound as a child with a culture-confirmed case and if the onset of cough occurred within 28 days either before or after the onset of cough of the reference child.

Data management and statistics. Percentages of antibody changes (increases, decreases, or negatives) were compared by the ² test. Comparisons of means were done by analysis of variance when normal approximation was possible or by the Kruskal-Wallis test, with Epiinfo, version 6 (USD). Significance values were always calculated as two tailed.

	RESULTS

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PT and FHA IgG titer changes. As shown in Table 1, an anti-PT IgG increase was detected in 90 children (20%) and an anti-FHA IgG increase was detected in 147 children (32%). An anti-PT IgG decrease was observed in 113 children (25%), more frequently than an increase and more frequently than anti-FHA IgG decreases, which were observed for 40 (9.4%) children.

Serological results in children with culture-verified pertussis. Eighty children were culture positive among the 460 children with suspected cases of infection who had 8 days or more of cough and who had been sampled for both bacteriology and serology. Children with increases in IgG titers (19%) among those with suspected cases of infection had a positive culture 40% (36 of 89) of the time, whereas only 14% (16 of 113) of the children with a decrease in the IgG titer (24% of suspected cases of infection) were culture positive (Table 2). Among responders, defined as those experiencing either an increase or a decrease in antibody titers, 31% (16 of 52) involved decreases in culture-positive children. This rate was 65% (97 of 150) among culture-negative children. When the sample was restricted to children with 21 days or more of paroxysmal cough, the rate of decreases rose to 35% (28 of 80), and it was then close to the rate of increases (37% [30 of 80]).

Serological results in children with WHO laboratory confirmation criteria. By definition, all 90 children with serologic increases were confirmed by WHO criteria (Table 3). However, 67 (59%) of the serologic decreases were also observed in children with laboratory-confirmed pertussis. This rate was 82% in the subsample of children with 21 days or more of paroxysmal cough. Thus, the majority of serologic decreases observed in this sample were from children with pertussis.

Serological results as a function of timing of the samples. In the present study, the decreases in IgG titers between acute- and convalescent-phase sera might have been due to a long interval between collection of sera. The median interval was not significantly longer when decreases were observed (48 days) than when increases were observed (42 days) (P = 0.07). For 76% of the children, this interval was between 4 and 8 weeks, the interval generally used in other studies.

Serological results as a function of vaccination status. Because of the results presented above, it was anticipated that anti-PT IgG titers might decrease more rapidly in the weeks following infection in vaccinated children compared to that in unvaccinated children because of a more rapid rise in antibodies related to the anamnestic response following vaccination. As seen in Table 4, increases were more frequently observed among unvaccinated children and decreases were more often observed among vaccinated children. This difference was more pronounced in the subsample of children with paroxysmal cough. When only increases and decreases were considered in Table 4 and since they might both have represented positive serological results, it could be calculated from the full sample that decreases represented 64% of the positive results among vaccinees and only 27% of those among unvaccinated children. This difference was highly significant (P < 0.00001). A similar analysis of the two groups showed comparable rates of decreases in the AC group (65%) and in the WC group (62%). When the sample was restricted to those children with paroxysmal cough, comparable rates were observed. When the analysis was restricted to the classical delay between convalescent- and acute-phase sera of more than 27 days and less than 57 days, similar rates of decreases were observed: 62 and 59% in the AC and WC groups, respectively, compared to 31% among unvaccinated children (data not shown).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Evolution of anti-PT IgG antibody titers after exposure to pertussis according to vaccination status. Values are shown as means ± standard errors.

	DISCUSSION

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This study was based on data from the Senegal Pertussis Trial, and therefore the data were not collected specifically to address its objectives. Therefore, the design was not optimized for all aspects. For example, the group of nonvaccinated children was not as comparable to the vaccinated children as were the two groups of vaccine recipients, which had been randomized. Unvaccinated children were younger, before the age at vaccination, or their parents refused vaccination. Also, the study of the kinetics of antibodies lacked balance in terms of the sizes of the classes, as reflected by the standard deviation. However, the design of the trial was unique in providing serologic results within an active surveillance system, with a large number of suspected and confirmed cases of infection determined by other techniques, including the use of a large number of vaccinated patients. Also, serologic testing was performed in the same laboratory, according to standard procedures, and was blind to vaccination status. External quality assurance was performed through an international collaborative initiative, and our results fit well with those of the reference laboratory (7). Therefore, these results provide some insight into the high proportion of decreases in antibodies in paired sera to PT and/or FHA. These decreases were observed over a wide span of time between the onset of cough and when the acute-phase sample was obtained, but with a marked positive trend over time of acute-phase sampling, associated with an inverse trend for increases in paired sera. These decreases were observed in children with pertussis confirmed by other laboratory-based methods or with typical clinical whooping cough. Furthermore, this observation is in agreement with that of a study reported recently from Sweden (5), in which the authors recommended considering decreases in antibodies for the diagnosis of pertussis. They stressed the importance of the timing of blood sampling with respect to the onset of cough, which may be insidious and reported late by parents. However, while this is also observed in our study, another important independent factor is the different kinetics of titer rises observed between vaccinated and unvaccinated children.

Those results were observed in a rural population of a developing country, and they could be thought to express a particular antibody response due to such factors as low nutritional status, high general infectious background, and intense exposure to the diseases. This might well be true. However, study vaccines have been found to induce immunogenicity comparable to that in other settings (12), and a recent comprehensive study of the same population failed to show an impaired immune response among children (10). Decreases have also been observed in vaccine failures for measles (14). Thus, it is likely that our observation strengthened earlier ones (9, 13) and has general relevance.

These results suggest that decreases as well as increases might be taken as serologically positive diagnoses. This could have a major impact, since, in the present study, depending on whether or not decreases were taken into consideration for positive pertussis diagnoses for either PT or FHA (Table 1), the final positive results rose from 157 (34%) to 266 (58%) cases of infection.

In recent vaccine trials, judgments were based on cases of infection including not only bacteriology but also serology and epidemiological contact with a culture-confirmed case. Vaccine efficacy may be biased if the antibody response differs between vaccinated and unvaccinated children. Even when preacute-phase sera are available in specific settings or as part of research protocols in vaccine trials (2), this bias can be observed because of the rapid decline in the antibody response in vaccinated children. Thus, nonuse of decreases when absolute efficacy is being measured (i.e., when unvaccinated children are part of a trial) will cause an underestimation of cases of infection among vaccinated children and an overestimation of vaccine efficacy. Similarly, if two vaccines are to be compared, the relative vaccine efficacy could also be biased if the serologic responses between vaccine groups are not similar. This is not supported by our results for the vaccines under study. The magnitude of the bias depended on and was minimized by the proportion of cases confirmed by bacteriology or by epidemiological contact.

Our data suggest that vaccinated children have an earlier, lower, and briefer antibody response when the disease occurs. If the response to exposure among unvaccinated children is considered a primary immune response and the response among vaccinated children is considered a secondary immune response, the lower and briefer antibody response observed among vaccinated children is atypical. Comparable observations were reported for tetanus (4) and leishmaniasis (6). This "negative phase" may be related to an increase in the avidity of the antitoxin produced during maturation of the immune response. It may also reflect less-severe disease among vaccinated children, rather than temporary immunodeficiency associated with vaccine failure, and deserves further investigation.

In conclusion, this study supports an earlier recommendation that a significant decrease in PT or FHA should be added to the diagnostic criteria. A differential antibody response in vaccinated and unvaccinated children was established, which should be considered in vaccine efficacy studies so as to avoid an overestimation of absolute vaccine efficacy.