Evaluation of Methodology for Serotyping Invasive and Nasopharyngeal Isolates of Haemophilus influenzae in the Ongoing Surveillance in Brazil

Abstract

To assess the magnitude of discrepant results obtained by routine Haemophilus influenzae serotyping, 258 isolates, collected by the epidemiological surveillance system in Brazil from individuals with invasive diseases or carriage, were evaluated by two slide agglutination (SlAg) methods: SlAg method 1, by which strains were initially screened with a serotype b-specific antiserum, and SlAg method 2, by which strains were tested against all serotype-specific antisera in parallel. Investigators comparing results of the two SlAg methods with those obtained by capsule type-specific PCR were blinded to the method used. The serotype prevalence rates found by the three methods were significantly different, involving discrepancies mainly between serotype b and noncapsulated (NC) isolates. For invasive isolates (n ‫؍‬ 131), the overall agreement rate between SlAg method 1 or 2 and PCR was 68.0 or 88.3%, respectively, whereas for colonizing isolates (n ‫؍‬ 127) the corresponding rate was 46.5 or 94.2%, respectively. SlAg method 2 improved the ascertainment of serotypes over that obtained with SlAg method 1, demonstrating good correlation with PCR. Use of the polyvalent antiserum as a screening reagent for SlAg for invasive and colonizing isolates showed poor discriminatory power, with a sensitivity of 65.8% and a specificity of 91.7%. We stress the importance of using a well-standardized SlAg methodology and suggest that reference laboratories should utilize PCR routinely to confirm SlAg results and to check all nonspecific SlAg reactions and apparent NC isolates by SlAg in order to provide reliable data on the prevalence of H. influenzae serotypes in the H. influenzae type b vaccine era.

Comparison of two slide agglutination serotyping methods and PCR-based capsule typing for the characterization of Haemophilus influenzae serotypes

“…This result is in accordance with those reported by La Claire et al (7) and Bokerman et al (1). However, as previously reported (1), the use of all antisera in parallel (SAST 2), significantly improved capsule type agreement with PCR.…”

“…The first PCR reaction detected the bexA gene responsible for the transport of capsular material; its presence or absence determines whether an isolate is encapsulated or nonencapsulated and therefore, nontypeable (NTHi). The second PCR assay using capsule type specific primers (a to f) is then used to determine the capsule types of the isolates (1,4,7). The aim of the present study was to verify the discrepancy between slide agglutination methods and the PCR method in the identification of the serotypes of Hi isolates from healthy carriers of the bacteria.…”

“…Recently however, cases of misidentification of Hi serotypes by this method have been reported (1,7). To help to overcome this mishap, a SAST method (SAST 2) has been developed in the Adolfo Lutz Institute (1).…”

Impacto da vacinação contra o Haemophilus influenzae b na redução de meningites, Goiás

“…If the specificity of case definition is low, when the actual incidence decreases, the predictive value of diagnostic tests also decreases, and the proportion of false-positive H. influenzae diagnoses increases. Fur-thermore, a recent study 2 of H. influenzae isolates obtained from different Brazilian regions showed discordant results between routine serotyping (latex agglutination) and the PCR technique, considered as the gold standard. The results of this study ratify the need for a reevaluation of the laboratory procedure protocols used in H. influenzae serotyping.…”

Real-Time PCR for Determining Capsular Serotypes of Haemophilus influenzae

“…Our study showed that both the real-time and the gel-based PCR assays were more sensitive than the slide agglutination test for capsular typing of H. influenzae, as reported by others (1,6,13). Among our strains, 6% were Hib by real-time PCR, 5.4% were Hib by gel-based PCR, and 3% were Hib by serotyping.…”