The success of widespread use of conjugate vaccines has resulted in fewer H. influenzae
isolates being submitted to state health laboratories for identification and typing (9
). Consequently, laboratories are likely to perform once-routine methods less frequently. Recent discrepancies between SAST results from state health laboratories and those from the CDC (CDC, unpublished data) led us to study a large number of H. influenzae
isolates from the United States, collected through active surveillance from 1998 to 1999.
We used as our reference method a two-step PCR approach that detects the bexA
gene, whose product is responsible for capsular export, and six sequences that code for each specific capsule type (a to f). These assays, initially developed by Falla et al. (5
), have been shown to be highly sensitive and specific (Turner et al., Abstr. 99th Gen. Meet. Am. Soc. Microbiol. 1999), and with their use we were able to correctly identify all of our H. influenzae
controls. The most common and significant laboratory error with respect to measurable effects of implementing conjugate Hib vaccine was the misidentification of NT strains as those of serotype b (Table ). Only 30% of the isolates identified at state health laboratories as serotype b isolates possessed the necessary capsular genes; consequently, these discrepancies could not be the result of down-regulation of gene expression. These discrepancies might result from the use by laboratories of antiserum specific only for serotype b, instead of the full set of H. influenzae
serotype-specific antisera. This could lead the laboratories to interpret any form of agglutination observed with the serotype b-specific antiserum as a positive and specific reaction. However, if the laboratories tested the same isolate with the full set of serotype-specific H. influenzae
antisera, they could determine whether agglutination occurred with one or more serotype-specific antisera (cross-reaction) or even with the control saline (rough isolate) and thus whether their initial interpretation of agglutination with the serotype b-specific antiserum was incorrect (false positive). The agreement between reported SAST results and PCR results varied from 0 to 79% for H. influenzae
strains of serotypes other than b but was 100% for NT H. influenzae
(Table ). To determine the reasons for the observed discrepancies, we conducted an interlaboratory comparison of the results from three state health department laboratories and the CDC. The participating laboratories performed SAST on the same set of 32 strains, using a CDC-supplied complete set of H. influenzae
type-specific antisera, according to their standard laboratory protocols. Of all the H. influenzae
isolates tested by the three participating laboratories, 94% were correctly serotyped. This represents a significant increase in the proportion of H. influenzae
isolates correctly serotyped by state laboratories, suggesting that standardized reagents and routine quality assurance practices are crucial for obtaining reliable and reproducible SAST results. Consequently, recommendations that laboratories adhere more strictly to quality assurance procedures, including the use of standardized reagents and protocols, should be emphasized. The PCR assays used in this study were more sensitive and specific than SAST and may continue to serve as a method to resolve SAST inconsistencies, but more extensive evaluation of this approach will be beneficial.
Discrepancies between the results of SAST and PCR capsule typing and our finding that two-thirds of the H. influenzae isolates reported to the CDC as those of serotype b were incorrectly serotyped by SAST suggest that the number of actual Hib cases in the United States may be significantly lower than reported and that the national burden of Hib disease may be overestimated. However, since the percentages of SAST results that were incorrect differed substantially among the seven surveillance site laboratories (15 to 66%; median range, 43%), one must be cautious in extrapolating these findings beyond the ABCS sites. Therefore, to determine the extent to which such false-positive laboratory results have contributed to overestimates of the number of Hib cases in the United States, the CDC has initiated a prospective analysis of all H. influenzae surveillance site isolates associated with invasive disease in children less than 5 years old; in this analysis, H. influenzae serotypes will be identified by both SAST and PCR capsule typing. Such monitoring will serve to clarify the prevalence of H. influenzae serotype misidentification. As the burden of Hib disease declines in the United States, it becomes increasingly important to determine accurately the serotypes of all H. influenzae isolates associated with invasive disease, especially since Hib serves as a model for other vaccine-preventable diseases.