Evaluation of pulsed-field gel electrophoresis in epidemiological investigations of meningococcal disease outbreaks caused by Neisseria meningitidis serogroup C

Abstract

Since 1990, the frequency of Neisseria meningitidis serogroup C (NMSC) outbreaks in the United States has increased. Based on multilocus enzyme electrophoresis (MEE), the current molecular subtyping standard, most of the NMSC outbreaks have been caused by isolates of several closely related electrophoretic types (ETs) within the ET-37 complex. We chose 66 isolates from four well-described NMSC outbreaks that occurred in the United States from 1993 to 1995 to evaluate the potential of pulsed-field gel electrophoresis (PFGE) to identify outbreak-related isolates specific for each of the four outbreaks and to differentiate between them and 50 sporadic isolates collected during the outbreak investigations or through active laboratory-based surveillance from 1989 to 1996. We tested all isolates collected during the outbreak investigations by four other molecular subtyping methods: MEE, ribotyping (ClaI), random amplified polymorphic DNA assay (two primers), and serotyping and serosubtyping. Among the 116 isolates, we observed 11 clusters of 39 NheI PFGE patterns. Excellent correlation between the PFGE and the epidemiological data was observed, with an overall sensitivity of 85% and specificity of 71% at the 95% pattern relatedness breakpoint using either 1.5 or 1.0% tolerance. For all four analyzed outbreaks, PFGE would have given public health officials additional support in declaring an outbreak and making appropriate public health decisions.

FIG. 1

Designations, molecular characterization, and PFGE analysis (with NheI) of chromosomal DNAs of 35 NMSC isolates collected during an investigation of a meningococcal disease outbreak in Texas in 1995. The PFGE patterns of isolates SP2835 and SP3039 were identified as indistinguishable using the 1.5% tolerance, but based on visual differences in spacing between the bands, isolate SP3039 was defined as distinct and was given a pattern designation of H46N06.0031 (asterisk). ID, isolate identification number; ST/SST, serotype and serosubtype; RT, ribotype; RAPD, RAPD type (with two primers).

FIG. 2

Designations, molecular characterization, and PFGE analysis (with NheI) of chromosomal DNAs of 12 NMSC isolates collected during an investigation of a meningococcal disease outbreak in New Mexico in 1994. The PFGE patterns of isolates SP1530 and SP1531 were identified as indistinguishable using the 1.5% tolerance, but based on visual differences in spacing between the bands, isolate SP1531 was defined as distinct and given a pattern designation of H46N06.0038 (asterisk). ID, isolate identification number; ST/SST, serotype and serosubtype; RT, ribotype; RAPD, RAPD type (with two primers).

FIG. 3

Designations, molecular characterization, and PFGE analysis (with NheI) of chromosomal DNAs of 23 NMSC isolates collected during an investigation of a meningococcal disease outbreak in Arizona in 1993 to 1994. The PFGE patterns of isolates SP638 and OA160 were identified as indistinguishable using the 1.5% tolerance, but based on visual differences in spacing between the bands, isolate OA160 was defined as distinct and given a pattern designation of H46N06.0037 (asterisk). ID, isolate identification number; ST/SST, serotype and serosubtype; RT, ribotype; RAPD, RAPD type (with two primers).

FIG. 4

Designations, molecular characterization, and PFGE analysis (with NheI) of chromosomal DNAs of 20 NMSC isolates collected during an investigation of a meningococcal disease outbreak in California in 1993. ID, isolate identification number; ST/SST, serotype and serosubtype; RT, ribotype; RAPD, RAPD type (with two primers).

FIG. 5

Designations, molecular characterization, and PFGE analysis (with NheI) of chromosomal DNAs of 26 NMSC isolates collected from sporadic cases of meningococcal disease through active laboratory-based surveillance, 1989 to 1996. The PFGE patterns of isolates SU680 and SU810 were identified as indistinguishable using the 1.5% tolerance, but based on visual differences in spacing between the bands, isolate SU810 was defined as distinct and given a pattern designation of H46N06.0039 (asterisk). ID, isolate identification number. GA, Georgia; CT, Connecticut; TN, Tennessee; CA, California; OK, Oklahoma; MN, Minnesota; MO, Missouri; MD, Maryland; OR, Oregon.

FIG. 6

Dendrogram showing relatedness by PFGE analysis (with NheI) of 116 NMSC isolates collected during investigations of four meningococcal disease outbreaks in Texas, New Mexico, Arizona, and California and from sporadic cases of meningococcal disease collected through the active laboratory-based surveillance, 1989 to 1996. Pairs of isolates (SP2835 and SP3039, SP1530 and SP1531, SP638 and OA160, and SU680 and SU810) were identified as having indistinguishable PFGE patterns using the 1.5% tolerance. Based on visual inspection, differences in spacing between the bands were observed, and therefore the additional patterns H46N06.0031, H46N06.0037, H46N06.0038, and H46N06.0039, respectively, were established (asterisks). $, isolates on the smaller dendrograms identified as follows: OA147, pattern H46N06.0001; SP153, pattern H46N06.0001; SU785, pattern H46N06.0004; SU2656, pattern H46N06.0004. Their designations as given in Fig. 4 and 5 were used throughout the analysis. ID, isolate identification number.