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Serotyping Discrepancies in Haemophilus influenzae Type b Disease --- United States, 1998--1999

Since Haemophilus influenzae type b (Hib) conjugate vaccines were introduced in the United States in 1990, the incidence of Hib invasive disease has declined markedly (1,2). The majority of cases of Haemophilus influenzae (Hi) disease are caused by organisms with capsule types other than b or by nontypeable organisms (1). One of the national health objectives for 2010 is to reduce to zero indigenous Hib invasive disease cases in children aged <5 years (objective 14-1c) (3). In 2000, a total of 297 cases of invasive Hi disease were reported in children aged <5 years; serotype b represented 51 (22%) of 236 cases for which serotype information was known (1). This report describes inconsistencies in Hib serotyping between state health departments and CDC; these inconsistencies suggest that the burden of Hib disease might be less than estimated previously. Accurate laboratory information is essential for the accurate assessment of progress toward the elimination of Hib in the United States.

As part of the Emerging Infections Program, CDC conducts active laboratory- and population-based surveillance for Hi disease in eight states through the Active Bacterial Core surveillance (ABCs) system (4). A case of Hi disease is defined as an illness that is clinically compatible with invasive disease, occurring in a resident of a surveillance area, with isolation of H. influenzae from a normally sterile site. Hospital laboratory workers report cases to surveillance staff, who complete case report forms. During 1998--1999, in seven states (population: 26,437,876) of the eight in which surveillance was conducted, all available Hi isolates were sent to state health department laboratories, which performed serotyping by using standardized slide agglutination techniques (5) before forwarding the isolates to CDC for further analysis.

During 1998--1999, a total of 751 sterile-site Hi cases were identified in ABCs sites, and 487 isolates were serotyped at state health departments and sent to CDC, which repeated serotyping by using slide agglutination on a convenience sample of 59 isolates, focusing on isolates reported as being serotype b. CDC and state laboratory serotyping results were discordant for 12 (20%) of these isolates.

To investigate potential reasons for these discrepancies and determine the true capsular type of the isolates, CDC performed capsule typing by polymerase chain reaction (PCR) on a convenience sample of 141 of the 487 H. influenzae isolates serotyped initially by slide agglutination in state health department laboratories, including the 59 isolates tested at CDC by slide agglutination. A PCR procedure was performed to detect bexA, a capsular export gene, and genes specific for capsule types a, b, c, d, e, or f (6). Of 141 isolates tested by PCR, 62 (44%) contained the bexA gene and were identified subsequently as one of the capsule types a, b, d, e, or f (Figure). Slide agglutination serotyping performed at state health department laboratories agreed with PCR capsule typing results for 85 (60%) of 141 isolates analyzed. Of the 56 (40%) remaining isolates, 54 were nontypeable by PCR but had been identified as typeable by slide agglutination serotyping at state health departments. Of the 40 H. influenzae isolates reported to CDC as serotype b, 27 (68%) were nontypeable, and one was serotype f by PCR (Table). Incorrect serotype identification by slide agglutination serotyping differed substantially among the seven state health department laboratories studied (median: 44%; range: 15%--66%).

Reported by: LL LaClaire, MS, ML Tondella, PhD, DS Beall, PhD, CA Noble, NE Rosenstein, MD, T Popovic, MD, Div of Bacterial and Mycotic Diseases and Active Bacterial Core Surveillance/Emerging Infections Program Network, National Center of Infectious Diseases; PL Raghunathan, PhD, EIS Officer, CDC.

Editorial Note:

This report documents frequent discrepancies between the results of H. influenzae slide agglutination serotyping obtained by state health department laboratories participating in the ABCs system and results obtained by CDC. Using PCR capsule typing as the reference standard demonstrates that nontypeable H. influenzae isolates were disproportionately misidentified by slide agglutination serotyping. All 79 isolates that were nontypeable by PCR lacked the bexA capsular export gene, proving that they were unencapsulated organisms and indicating that variable expression levels of capsular polysaccharide were not responsible for the discrepancies (6). As Hib disease declines, state health department laboratories perform slide agglutination serotyping less frequently, which might explain incorrect serotyping of nontypeable H. influenzae isolates.

Using standardized procedures and quality control reduced the number of discrepancies. For example, when three state health department laboratories conducted H. influenzae serotyping after receiving standardized reagents and protocols, >95% of slide agglutination serotyping results agreed with slide agglutination serotyping and PCR capsule typing results performed by CDC (7). Slide agglutination serotyping performed by CDC correlated 100% with PCR capsule typing results. These results indicate that slide agglutination serotyping remains a valid and reliable method. To improve reproducibility, laboratories should adhere to standard H. influenzae slide agglutination serotyping procedures. Compared with slide agglutination, the PCR approach appears sensitive and specific. Because the PCR approach might resolve serotyping inconsistencies, further evaluation of this approach might be beneficial.

In this study, of 40 H. influenzae isolates reported to CDC during 1998--1999 as serotype b, 28 (70%) were identified incorrectly by slide agglutination serotyping. Discrepancy rates varied substantially among the seven state health department laboratories. Consequently, these findings cannot be extrapolated beyond the ABCs sites. During October 2002--September 2003, CDC requests state health department laboratories to send all H. influenzae isolates associated with invasive disease among children aged <5 years, along with the surveillance forms, to CDC for slide agglutination serotyping and PCR capsule typing to confirm H. influenzae serotypes. Additional information is available from CDC's Meningitis and Special Pathogens Branch, telephone 404-639-1380.

As the burden of Hib disease declines in the United States, determining the serotypes of H. influenzae isolates associated with invasive disease becomes increasingly important. Accurate laboratory information is essential to assess progress toward Hib elimination and to monitor the emergence of replacement Hi disease associated with other serotypes.

Acknowledgments

This report is based on data contributed by L Gelling, MPH, P Daily, MPH, G Rothrock, MPH, California Emerging Infections Program; A Reingold, MD, School of Public Health, Univ of California, Berkeley; D Vugia, MD, Div of Communicable Disease, California Dept of Health Svcs. NL Barrett, MS, J Hadler, MD, Epidemiology Program, Connecticut Dept of Public Health. MA Pass, MS, Maryland Emerging Infections Program; LH Harrison, MD, Dept of Epidemiology and Medicine, Univ of Pittsburgh. J Razeq, PhD, B Callahan, J Roche, MD, G Thomas, Maryland Dept of Health and Mental Hygiene. J Rainbow, MPH, J Besser, MS, SK Johnson, MT, CA Lexau, MPH, R Lynfield, MD, R Danila, PhD, H Hull, MD, Minnesota Dept of Health. N Spina, MPH, S Zansky, PhD, Div of Epidemiology; J Hibbs, PhD, M Maupin, D Morse, MD, Wadsworth Center, New York State Dept of Health. KR Stefonek, MPH, PR Cieslak, MD, MA Kohn, MD, L Stauffer, MT, B Taylor, MS, Oregon State Health Div. B Barnes, W Schaffner, MD, Dept of Preventive Medicine, Vanderbilt Medical Center; A Craig, MD, Tennessee Dept of Health. G Ajello, MS, M Berkowitz, M Reeves, PhD, K Robinson, S Schmink, MS, B Plikaytis, MS, Div of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, CDC.

References

  1. CDC. Progress toward elimination of Haemophilus influenzae type b invasive disease among infants and children---United States, 1998--2000. MMWR 2002;51:234--7.
  2. CDC. Progress toward eliminating Haemophilus influenzae type b disease among infants and children---United States, 1987--1997. MMWR 1998;47:993--8.
  3. U.S. Department of Health and Human Services. Healthy people 2010, 2nd ed. With understanding and improving health and objectives for improving health (2 vols). Washington, DC: U.S. Department of Health and Human Services, 2000.
  4. Schuchat A, Hilger T, Zell E, et al. Active Bacterial Core Surveillance of the Emerging Infections Program Network. Emerg Infect Dis 2000;7:92--9.
  5. Popovic T, Ajello G, Facklam R. Laboratory manual for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. Geneva, Switzerland: World Health Organization, 1999.
  6. Falla TJ, Crook DW, Brophy LN, Maskell D, Kroll JS, Moxon ER. PCR for capsular typing of Haemophilus influenzae. J Clin Microbiol 1994;32:2382--6.
  7. LaClaire L, Tondella ML, Beall D, et al. Identification of Haemophilus influenzae serotypes by standard slide agglutination and PCR-based capsule typing. [Abstract]. Presented at the International Conference on Emerging Infectious Diseases, Atlanta, Georgia, July 2000.


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