Reference System for Characterization of Bordetella pertussis Pulsed-Field Gel Electrophoresis Profiles
Reference System for Characterization of Bordetella pertussis Pulsed-Field Gel Electrophoresis Profiles
July 2004
Received 13 February 2004/ Returned for modification 20 March 2004/ Accepted 31 March 2004
Abdolreza Advani,1 Declan Donnelly,1 and Hans Hallander1*
Journal of Clinical Microbiology, July 2004, p. 2890-2897, Vol. 42, No. 7
Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden1
Pulsed-field gel electrophoresis (PFGE) has been used as an epidemiological tool for surveillance studies of Bordetella pertussis since the early 1990s. To date there is no standardized procedure for comparison of results, and therefore it has been difficult to directly compare PFGE results between laboratories. We propose a profile-based reference system for PFGE characterization of B. pertussis strain variation and to establish traceability of B. pertussis PFGE results. We initially suggest 35 Swedish reference strains as reference material for PFGE traceability. This reference material is deposited at the Culture Collection of the University of Gothenburg, Gothenburg, Sweden. Altogether, 1,810 Swedish clinical isolates from between 1970 and 2003 were studied, together with the Swedish Pw vaccine strain, six reference strains, and two U.S. isolates. Our system provides evidence that profiles obtained by using only one enzyme, i.e., XbaI, give enough data to analyze the epidemiological relationship between them. Characterization with one enzyme is far less labor intensive, yielding results in half the time than when a two-enzyme procedure is used. Also, we can see that there is a correlation between PFGE profile and pertactin type. One common PFGE profile, BpSR11 (n = 455), showed 100% prn2 and 100% Fim3 when analyzed for pertactin type and serotype. On the other hand, strains with the same profile may express various serotypes when isolated over longer periods of time. Subculturing of the same isolate eight times or lyophilization caused no change in PFGE profile.
Whooping cough caused by Bordetella pertussis is a worldwide disease. The organism continues to circulate even in populations where high vaccine coverage of infants and children is achieved (8, 16). Although the mortality and morbidity are substantially reduced, peaks of disease appear every 3 to 5 years (2, 6, 10). Waning immunity and changed epidemiology are often referred to as explanations of this phenomenon (10, 28).
In 1998 Mooi et al. (19), focusing on the polymorphism of pertactin and pertussis toxin, suggested a vaccine-driven evolution as the reason for the emergence of new subtypes in The Netherlands. Recent Dutch isolates were shown to be distinguishable from strains that had been used for the production of vaccines. These same findings were reproduced in many other countries (10, 12, 14, 22, 29).
Acellular pertussis vaccines were introduced nationwide in Sweden in 1996, 17 years after the withdrawal of whole-cell pertussis vaccine from the childhood immunization schedule (20). In October 1997 a whooping cough surveillance program started, including collection of strains from Swedish laboratories for clinical microbiology.
Pulsed-field gel electrophoresis (PFGE) is used as an epidemiological tool for surveillance studies of B. pertussis strain variation over time and for identification of outbreak-associated isolates (4, 5, 7, 14, 22, 24, 29). It is of particular interest to monitor the bacterial population and its possible influence on vaccine effectiveness after the introduction of acellular vaccines. With the aid of PFGE, it has been possible to identify isolates that are epidemiologically related which were previously indistinguishable from each other by other typing methods (13). It is also possible to type an endemic flora of closely related organisms frequently recovered from infected patients for which no direct or epidemiological linkage can be demonstrated (25).
One problem with PFGE results is that the direct and precise comparison of profiles from different laboratories is difficult because of variation in technique and terminology. Currently there is no standardized protocol for performing PFGE or criteria for analyzing the fragment patterns. Protocol harmonization on a European level and even an international level is necessary for interlaboratory comparison of prevalent and emerging new variants.
In 1999 European research groups working together proposed a reference methodology for epidemiological typing of B. pertussis (18). The PFGE protocol proposed in that methodology suggested a two-enzyme system, i.e., XbaI and SpeI, for chromosomal DNA cleavage. Based on the profiles obtained with each of these two enzymes, Weber et al. (29) identified six main groups of B. pertussis, i.e., groups I, II, III, IV, IV?, and V. Reference strains were recommended for strain characterization at the group level (18, 29). Many recent studies have put forward the idea of a one-enzyme system, primarily using XbaI (4, 5, 7, 14, 22, 24). In many of these studies, references to internationally available type strains were lacking (4, 5, 7, 14).
The aims of this study were to establish traceability of B. pertussis PFGE results, as follows: (i) to describe a profile-based reference system, i.e., a reference method and reference materials with Swedish clinical material; (ii) to examine other characteristics such as toxin, pertactin, and serotype for these profiles; (iii) to study the stability of the relationship between isolates and PFGE profile; and (iv) to make key isolates available by depositing them in a culture collection bank.
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