Rapid laboratory detection of meningococcal disease outbreaks caused by serogroup C Neisseria meningitidis

Journal of Microbiological Methods 67 (2006) 330 – 338 www.elsevier.com/locate/jmicmeth

Rapid laboratory detection of meningococcal disease outbreaks caused by serogroup C Neisseria meningitidis Patrick B. Killoran a,⁎, Janice O'Connell a , Elizabeth A. Mothershed b , Will S. Probert a a b

Microbial Diseases Laboratory, California Department of Health Services, 850 Marina Bay Parkway, Richmond, CA 94804, USA Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA Received 10 December 2005; received in revised form 13 April 2006; accepted 17 April 2006 Available online 5 June 2006

Abstract Molecular subtyping is of significant importance to the recognition of outbreaks of meningococcal disease caused by serogroup C Neisseria meningitidis. We describe the application of multilocus variable number tandem repeat analysis (MLVA) for the molecular subtyping of N. meningitidis and compare its performance to that of pulsed-field gel electrophoresis (PFGE). For MLVA, a multiplex PCR assay targeting five variable number tandem repeat regions was developed and evaluated using a panel of sporadic and outbreak-associated serogroup C N. meningitidis isolates. MLVA was highly reproducible and provided results within 6 h. Overall, the discriminatory power of MLVA was equivalent to that of PFGE. The utilization of MLVA for subtyping N. meningitidis isolates provides a rapid and safer alternative to PFGE for identifying outbreaks of meningococcal disease. As such, it may provide public health officials with timely information that may minimize the spread of outbreak-related cases through prophylaxis. © 2006 Elsevier B.V. All rights reserved. Keywords: Meningococcal disease; Molecular subtyping; Tandem repeats

1. Introduction Neisseria meningitidis is an important human pathogen that causes acute bacterial septicemia and meningitis. In the United States, it most commonly affects children and young adults, with a mortality rate of about 10% (Centers for Disease Control and Prevention, 2005; Schuchat et al., 1997). Prophylaxis through vaccination is effective in preventing meningococcal disease in the U.S. caused by serogroups A,

⁎ Corresponding author. Tel.: +1 510 412 3750; fax: +1 510 412 3706. E-mail address: [email protected] (P.B. Killoran). 0167-7012/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2006.04.004

C, Y, W135, but not serogroup B strains. The epidemiology of meningococcal disease is complicated by the ability of individuals to act as carriers. Asymptomatic carriage of N. meningitidis in the nasopharyngeal mucosa can be as high as 10% in the general population (Caugant et al., 1994) with a progression to serious disease occurring rarely and only with bacterial dissemination to the blood and/or cerebral spinal fluid. The mechanism of action of this dissemination is poorly understood. While the vast majority of infections are sporadic, outbreaks sometimes occur and are usually associated with serogroup C strains in the United States (Centers for Disease Control and Prevention, 2005; Jackson et al., 1995; Rosenstein et al., 2001). Strain typing of isolates

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beyond the serogroup level has provided useful information in determining whether a group of cases truly represents an outbreak or merely represents a change in the number of sporadic cases (Yakubu et al., 1999). Such information can be invaluable when allocating public health resources for mass vaccination campaigns. Unlike sporadic strains, which usually have high genetic variability, outbreak strains can be clonal in nature and can reappear repeatedly over long periods of time and large geographic areas (Achtman, 1995; Caugant, 1998; Morelli et al., 1997). Therefore, outbreak investigations demand a subtyping method that can differentiate closely related but epidemiologically distinct strains of N. meningitidis. Pulsed-field gel electrophoresis (PFGE) has proven to be an important laboratory tool for investigating outbreaks caused by N. meningitidis serogroup C (NMSC) strains in the United States (Popovic et al., 2001). PFGE provides superior discriminatory power when compared to multilocus enzyme electrophoresis (MLEE) (Popovic et al., 2001). However, like MLEE, PFGE is labor-intensive, expensive, and technically demanding. In addition, testing of N. meningitidis by PFGE requires manipulation of high concentrations of a viable pathogen known to be a potential source of laboratory-acquired infections (Centers for Disease Control and Prevention, 2002). In response, our study proposes multilocus variable number tandem repeat analysis (MLVA) as a viable alternative or complement to the use of PFGE for N. meningitidis strain typing. MLVA is a PCR-based subtyping method that targets regions of the bacterial genome that contain tandemly repeated nucleic acid sequences that can vary in copy number from strain to strain. The addition or deletion of repeats can occur through genetic events such as slipped-strand mispairing or unequal crossover events (van Belkum et al., 1998). These variations in tandem repeat copy number can be assessed at multiple loci and compared between bacterial strains to infer genetic relationships. This strategy has been used successfully to develop subtyping schemes for a number of bacterial pathogens with discriminatory power that can approach or exceed PFGE (for a review see Lindstedt, 2005). With this strategy in mind, we have designed a MLVA assay that utilizes multiplex PCR targeting five meningococcal variable number tandem repeat (VNTR) regions for subtyping NMSC. Here, we compare the performance of MLVA and PFGE for subtyping 92 NMSC isolates including 24 isolates representing seven clusters of meningococcal disease.

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2. Materials and methods 2.1. Bacterial strains and nucleic acid extraction N. meningitidis reference strains M7060, M5178, M3045, M7034, and M2578 representing serogroups A, B, C, W135, and Y, respectively, were provided by the Centers for Disease Control and Prevention (CDC). NMSC isolates from California were submitted to the Microbial Diseases Laboratory (California Department of Health Services) for confirmatory testing and serogrouping. All isolates were cultivated on sheep blood agar or chocolate agar plates and incubated at 35 °C and 5% CO2. Serogroup identification was performed by slide agglutination with antisera acquired from Difco Laboratories (Detroit, MI) and Murex Laboratories (Remel, Lenexa, KS). Crude, nucleic acid extracts were prepared by resuspending a 1 μl loopful of overnight growth from a plate into 0.1 ml of TE buffer (10 mM Tris pH 8, 1 mM EDTA). The suspension was heated at 95 °C for 10 min, microfuged for 5 min at 10,000 x g, and the supernatant collected as the nucleic acid extract. In addition, nucleic acid extracts from 26 randomly selected NMSC surveillance isolates representing nine states participating in the Active Core Bacterial surveillance program were provided by the CDC (Popovic et al., 2001). 2.2. VNTR identification and MLVA The genome sequence for the serogroup B N. meningitidis strain MC58 (GenBank accession number AE002098) was downloaded from the NCBI website. The unfinished, assembled genome sequence for the serogroup C strain FAM18 was acquired from the Sanger Institute website (ftp://ftp.sanger.ac.uk/pub/ pathogens/nm/). Both sequences were analyzed with Tandem Repeats Finder version 3.21 (http://tandem.bu. edu/trf/trf.html) and a subset of these tandem repeats was selected for further study (Benson, 1999). Primer sequences encompassing candidate tandem repeat regions were designed with the Primer Select module of Lasergene (DNASTAR, Madison, WI) and are shown in Table 1. Nucleic acid amplification was performed with the QIAGEN Multiplex PCR kit (QIAGEN Inc., Valencia, CA). For multiplex PCR, the reaction mixture consisted of 1× QIAGEN multiplex PCR master mix, 1× Q-solution, primers VNTR2F, VNTR2R, VNTR12F, and VNTR12R at 0.1 μM each, primers VNTR3F, VNTR3R, VNTR8F, and VNTR8R at 0.3 μM each, and primers VNTR4F and VNTR4R

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Table 1 Genome location and characteristics of VNTRs amplified in MLVA for subtyping serogroup C N. meningitidis VNTR Genome a Genome Repeat Repeat Forward primer location size (nt) copies

Reverse primer

Amplicon size (nt) b

2 3 4 8 12

5′-ATGCCTGCCGCTCATATAAAGAAC 5′-AAGCGGCGGGAATGACGAAGAGTG 5′-TCGGTTACGCGTTTTGAAGTTTTG 5′-AAGGGAATCTGATGCCGTCTGAAA 5′-GCCGAGTCTGCCGCTTCTGC

104 532 165 361 232

C C B B C

1844470 1043724 863508 1572486 1408985

13 37 6 18 21

4.8 8.1 5.3 5.4 3.8

5′-GATGTCGAGGGCTGTACCGTATT 5′-CCCCGATAGGCCCCGAAATACCTG 5′-GGTGTACGCCGATAAAGGGTTTTT 5′-GTTTGCCGCTGCTTTGTTGTCTTT 5′-TGGCGGCATCTTTCATTTTGTCTG

nt, nucleotides. a Serogroup B genome sequence from strain MC58 (GenBank accession number AE002098); serogroup C genome sequence from strain FAM18 (www.sanger.ac.uk/Projects/N_meningitidis/seroC/). b Amplicon size predicted from the respective genome sequence.

at 0.05 μM each (Table 1). For a PCR reaction containing a single primer set, the final concentration of each primer was 0.2 μM. Finally, 2 μl of nucleic acid extract was added to the reaction mixture for a final volume of 25 μl. Amplification was performed with an ABI Prism 9700 (Applied Biosystems, Foster City, CA) and the following parameters: 95 °C for 15 min, 30 cycles of 94 °C for 30 s, 55 °C for 90 s, and 72 °C for 3 min, followed by a final extension step of 72 °C for 10 min. PCR products were separated on 4% NuSieve 3:1 agarose gels containing ethidium bromide (Cambrex BioScience, Rockland, ME) for 100 min at 100 V in 1× TBE. To facilitate gel normalization, each gel contained at least three replicates of a DNA ladder (BioMarker Ex, BioVentures, Murfreesboro, TN) with size standards ranging from 50 bp to 700 bp. The gels were stained an additional 7 min in 10 μg/ml ethidium bromide and the gel image captured as a TIFF with an AlphaImager 2000 (Alpha Innotech Corporation, San Leandro, CA). Gel and pattern analysis was performed with BioNumerics software (Applied Maths Inc., Austin, TX) by using the Dice coefficient with optimization and tolerance settings of 0.5% and 0.7%, respectively. Cluster comparisons were performed by using the unweighted pair group with arithmetic averaging (UPGMA). 2.3. Pulsed-field gel electrophoresis PFGE was performed as described by Popovic et al. (2001). NheI was used as the primary restriction enzyme for PFGE. However, for some isolates, a second PFGE was performed with the restriction enzyme, SpeI, to further assess relatedness of strains. Gel and pattern analyses were performed as described above but with the optimization parameter of 0.5% and a tolerance setting of 1.5%.

3. Results 3.1. Selection of VNTRs Tandem repeats found within both serogroup B and serogroup C genome sequences were identified by performing a Tandem Repeats Finder search (Benson, 1999). The number of candidate tandem repeats was reduced by considering only those repeats with a copy number greater than three. A further reduction in the number of candidate tandem repeats was achieved by performing BLAST searches (www.ncbi.nlm.nih.gov/ sutils/genom_table.cgi) of the serogroup A, B, and C genome sequences with the candidate tandem repeat sequences. In cases where copy number variability was established between serogroups by the BLAST search, the candidate tandem repeat was selected for further study. Using this approach, 13 tandem repeat segments were selected and primer sets were designed to amplify each of these regions by PCR. Sequence conservation among the serogroups A, B, and C genomes was considered in the design and selection of the primer sequences. Next a test panel of eight serogroup B isolates and eight serogroup C isolates was used to evaluate primer sequence conservation and copy number variability in the candidate tandem repeat segments (data not shown). For each set of eight isolates, two isolates had matching PFGE patterns (NheI and SpeI), whereas the remaining six isolates had distinct PFGE patterns (NheI). Thus, for a candidate tandem repeat segment to be selected it had to be amplified from all isolates, the size of the expected amplification product had to be equal for the isolates with identical PFGE patterns, and it had to demonstrate some size variability among isolates with distinct PFGE patterns. Based on these criteria, five tandem repeat regions (VNTRs 2, 3, 4, 8, and 12; Table 1) were selected for MLVA.

P.B. Killoran et al. / Journal of Microbiological Methods 67 (2006) 330–338 VNTR 2

VNTR 3

VNTR 4

MABCWYABCWY

VNTR 8

ABCWYABCWY

700 500 400 -

VNTR 12

Multiplex

ABCWY

A

B

3 3 8

300 -

8

C

12

12 4

W

3

3

8

8

12

200 -

333

Y

3 8

12 12

4

4

2

2

4

100 -

2

2

2

Fig. 1. Single reaction tube and multiplex amplification of VNTR regions from reference strains of Neisseria meningitidis. Lane A—serogroup A N. meningitidis strain M7060; lane B—serogroup B N. meningitidis strain M5178; lane C—serogroup C N. meningitidis strain M3045; lane W—serogroup W135 N. meningitidis strain M2578; lane Y—serogroup Y N. meningitidis strain M7034. DNA sizing ladder in base pairs is shown in lane M.

combined VNTRs observed for each reference isolate using single primer sets. 3.2. Reproducibility and stability of MLVA To assess the reproducibility of MLVA, seven replicate nucleic acid extracts of the NMSC reference strain M3045 were prepared and tested by MLVA on three separate occasions. All 21 patterns were found to be identical indicating that the method was highly

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80

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The ability to amplify these five VNTR regions individually and in a multiplex format from five reference strains representing serogroups A, B, C, W135, and Y is shown in Fig. 1. With the exception of VNTR4 for the serogroup A reference strain, VNTRs were successfully amplified from all five serogroups. Variation in amplification product size for each VNTR is also illustrated in Fig. 1. Furthermore, this figure also demonstrates the capacity of the multiplex PCR assay to produce MLVA patterns that are representative of the

SU3688 SU4015 SU4648 SU209 SU239 SU649 SU2656 SU810 SU1441 SU540 SU680 SU6304 SU3689 SU785 SU1171 SU3790 SU980 SU4027 SU4214 SU2708 SU1591 SU2286 SU87 SU2750 SU3686 SU4212

Fig. 2. MLVA pattern relatedness for 26 sporadic serogroup C N. meningitidis isolates collected from nine states.

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100

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B. 60

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A.

04S0202

05S00015

05S00574

05S00380

05S00268

06S00001

05S00436

04S0155

01A4416

04S0463

03S0634

05S00376

04S0602

02A0993

00A4850

02S0150

02S0013

04S0243

02S0150

04S0602

03S0144

05S00016

04S0243

05S00321

05S00092

03S0020

05S00628

04S0065

05S00182

02S0013

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03S0634

02A1997

04S0247

02A4232

05S00182

04S00752

00A4850

01A10562

04S00800

04S0247

05S00178

05S00016

05S00871

05S00321

05S00628

05S00871

03S0420

03S0232

05S00092

04S0155

01A10562

04S0229

01A4416

04S0463

02A1210

05S00015

04S00752

05S00376

01A2294

05S00380

03S0232

06S00001

02A4232

01A2294

03S0144

02A1210

02A1997

05S00214

04S00616

05S00215

05S00574

02A0993

05S00214

04S00616

05S00215

03S0420

05S00268

05S00178

05S00436

04S00800

04S0202

04S0065

Fig. 3. Relatedness of MLVA (A) and PFGE (B) patterns for 42 sporadic serogroup C N. meningitidis isolates representing 32 different geographic regions within California.

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04S0229

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reproducible (data not shown). To establish whether the MLVA pattern for an individual isolate was stable with time, we monitored the MLVA pattern of NMSC strain M3045 subcultured every 2 days for 14 days. Nucleic acid extracts prepared prior to each subculture were found to be identical to one another indicating that the MLVA pattern was stable over this time period (data not shown). 3.3. MLVA of sporadic meningococcal isolates The ability of MLVA to discriminate between different NMSC strains was determined by testing a panel of 26 isolates representing sporadic meningococcal cases that were previously characterized by PFGE and were collected between 1989 and 1996 as part of a multistate active surveillance program coordinated by the CDC (Popovic et al., 2001). The dendrogram of MLVA patterns for these isolates is shown in Fig. 2. Twenty different MLVA patterns were observed. In comparison, PFGE performed by Popovic et al. (2001) yielded 21 patterns among this same group of 26 isolates. Using Simpson's index of diversity (Hunter and Gaston, 1988), the discriminatory power of MLVA and PFGE were 0.975 and 0.982, respectively. To evaluate the ability of MLVA to discriminate between strains more representative of our test population, we selected a panel of 42 NMSC isolates from sporadic meningococcal cases reported from 32 different counties within California over the last 5 years. Twenty-five MLVA patterns were observed among this panel of isolates (Fig. 3A). PFGE performed by our laboratory on these isolates produced 23 patterns (Fig. 3B). The Simpson's Index of diversity for this set of isolates was 0.965 for MLVA and 0.953 for PFGE. 3.4. MLVA of meningococcal clusters The capacity of MLVA to define clusters of meningococcal cases caused by apparently identical isolates of NMSC was also investigated. A panel of 13 isolates representing five epidemiologically linked clusters was tested by MLVA and PFGE (Table 2; clusters B, C, D, E, and F). Eleven additional isolates representing two clusters (A and G) were identified through active laboratory based surveillance by PFGE and were included in this study (Table 2). For these two clusters, geographically and temporally linked isolates with matching PFGE patterns by two enzymes (NheI and SpeI) were considered part of the same cluster. Overall the cluster designations defined by MLVA, based on 100% pattern similarity, closely matched those

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Table 2 Clusters of serogroup C N. meningitidis cases identified by either epidemiological investigation or laboratory surveillance using pulsedfield gel electrophoresis Cluster designation

Isolate identifier

Date of collection

Method of cluster identification

A

01A0599 01A0600 01A1410 01A1964 01A0443 01A0516 01A1887 01A1929 03S0183 03S0184 01A2725 01A2854 00A9405 00A9406 00A9473 00A3113 00A3114 00A2137 00A3380 00A4585 00A5524 00A5526 00A5614 00A5625

1/17/01 1/17/01 2/13/01 3/13/01 1/19/01 1/6/01 3/13/01 3/13/01 4/19/03 4/20/03 4/9/01 4/16/01 12/10/00 12/11/00 12/14/00 4/26/00 4/19/00 3/18/00 4/28/00 6/13/00 7/15/00 7/4/00 7/14/00 7/21/00

PFGE a

B

C D E

F G

Epi-linked

Epi-linked Epi-linked Epi-linked

Epi-linked PFGE a

a Matching patterns between isolates determined independently using 2 restriction enzymes (NheI and SpeI).

determined by PFGE and epidemiological investigation (Fig. 4). However, MLVA yielded an outlier for two clusters (A and G) identified through active laboratory surveillance (Fig. 4A). In cluster A, isolate 01A-1410 was distinct from isolates 01A-0599, 01A-0600, and 01A-1964; and in cluster G isolate 00A-4585 was distinct from isolates 00A-2137, 00A-3380, 00A-5524, 00A-5526, 00A-5614, and 00A-5625. In both instances, the outlier displayed greater than 90% pattern similarity to its respective cluster pattern. There were also two instances where PFGE results did not correlate perfectly with MLVA and epidemiological data. The three cases associated with cluster E attended the same high school and had disease onset dates within the same week. The corresponding isolates (00A-9405, 00A-9406, and 00A-9473) yielded an identical MLVA pattern, but produced two different NheI PFGE patterns with the pattern for isolate 00A9473 differing from the 00A-9405 and 00A-9406 patterns by three bands (Fig. 4B). Two different NheI PFGE patterns were also observed for the epidemiologically linked cluster B (01A-0443, 01A-0516, 01A-1887, and 01A-1929) that

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100

80

60

40

20

A. 01A0599 (A) 01A0600 (A) 01A1964 (A) 01A1410 (A) 00A3113 (F) 00A3114 (F) 00A9405 (E) 00A9406 (E) 00A9473 (E) 00A2137 (G) 00A3380 (G) 00A5524 (G) 00A5526 (G) 00A5614 (G) 00A5625 (G) 01A0443 (B) 01A0516 (B) 01A1887 (B) 01A1929 (B) 01A2725 (D) 01A2854 (D) 00A4585 (G) 03S0183 (C) 03S0184 (C)

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50

B. 01A0 599 (A ) 01A0 600 (A ) 01A1 964 (A ) 01A1 410 (A ) 00A2 137 (G ) 00A3 113 (F) 00A3 114 (F) 00A3 380 (G ) 00A4 585 (G ) 00A5 524 (G ) 00A5 526 (G ) 00A5 614 (G ) 00A5 625 (G ) 01A0 443 (B ) 01A0 516 (B ) 01A1 887 (B ) 01A2 725 (D) 01A2 854 (D) 00A9 473 (E ) 01A1 929 (B ) 00A9 405 (E ) 00A9406 (E) 03S0183 (C) 03S0184 (C)

Fig. 4. MLVA (A) and PFGE (B) pattern relatedness for seven clusters of serogroup C N. meningitidis isolates.

was identical by MLVA. All four cases associated with this cluster of isolates attended the same high school and had disease onset within 3 months of each other, but isolate 01A-1929 yielded a NheI PFGE pattern that differed by two bands from the other three isolates. Given the similar PFGE patterns among isolates within the same cluster, it is possible that in both situations such pattern discrepancies are the result of single genetic events.

Overall, the agreement between MLVA and PFGE for this set of 24 isolates was 83%. For the five clusters defined by epidemiologic information, the accuracy of MLVA was 100%. MLVA showed slightly better discriminatory power in differentiating unrelated clusters compared to PFGE because of its ability to distinguish cluster F from other clusters. Excluding isolate 01A-1929, NheI PFGE patterns were identical for all isolates in clusters B, D, F and G while MLVA

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patterns, apart from isolate 00A-4585, were identical for all isolates in only clusters B, D and G. 4. Discussion This study describes the development and implementation of a rapid strain typing method based on MLVA that may assist outbreak investigations by distinguishing between sporadic and outbreak-associated strains of NMSC. The VNTR regions described in this study were selected based on repeat copy number variability found by aligning the corresponding region from the serogroup A, B, and C genome sequences. Screening of these regions against a small panel of serogroup B and C strains for primer sequence conservation and repeat copy number variability further reduced the number of useful VNTRs to five. Amplification of these target regions was simplified by implementing a multiplex PCR assay that included primer sets for all five VNTRs. To validate successful amplification of all five VNTR targets, we routinely include an amplification reaction with NMSC reference strain M3045 as a positive control with each assay. Our MLVA method utilizes a conventional agarose gel electrophoresis system using commercially available precast gels for accurate and reproducible sizing of PCR products. Gel analyses and database management of MLVA patterns are modeled after methods used by the international network of foodborne disease surveillance laboratories, PulseNet, and our MLVA procedure can be readily adapted by these laboratories with very little in the way of additional equipment costs. Higher resolution of VNTR amplification products and faster electrophoresis times may be possible with the use of labeled primers and capillary sequencing systems, but the associated cost may limit the application of this method to only specialized laboratories. Furthermore, primer sequence conservation may allow application of our method for monitoring strain diversity among other invasive serogroups of N. meningitidis. MLVA has several advantages over PFGE for strain typing of bacterial pathogens. MLVA is less labor intensive, safer, cheaper, more rapid, and has equal or better discriminatory power when compared to PFGE (Lindstedt, 2005). Like PFGE, our MLVA method incorporates a band based similarity index for determining pattern relatedness. The absence of a band can influence overall pattern similarity. The absence of a single band was noted for less than 10% of the isolates in our study. Band absence may occur with amplification failure or co-migration of the amplification product

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with another product of similar size. Eight isolates lacked an amplification product for VNTR3 and one isolate lacked an amplification product for VNTR4. Attempts to amplify VNTR3 using different primer sets failed suggesting that this region may have been deleted in these isolates. Co-migration of amplified products was not observed in our study. The discriminatory power of MLVA and PFGE were nearly equivalent in our comparison. MLVA appeared to have slightly better resolution for separating unrelated clusters of meningococcal isolates. Four unrelated clusters of meningococcal isolates that yielded a single PFGE pattern could be separated into two groups based on MLVA patterns. It is noteworthy that three of the meningococcal clusters (B, D, and G) were grouped together by both PFGE and MLVA. This observation suggests that these isolates share a common genetic backbone and that they may represent a particularly virulent clonal group circulating within our population base. Two discordant results between MLVA and PFGE were observed within the clusters identified by active laboratory-based surveillance. These clusters were defined by isolates that were temporally and geographically associated and possessed identical PFGE patterns using two restriction enzymes. The MLVA patterns for isolate 01A-1410 (cluster A) and isolate 00A-4585 (cluster G) differed slightly from other isolates from their respective cluster. Since epidemiological investigations for these two clusters were incomplete, it is possible that these isolates represent sporadic meningococcal cases despite having identical PFGE patterns with other isolates in their corresponding clusters. Alternatively, these isolates would group with their respective clusters by lowering the MLVA pattern similarity cut-off from 100% to 90%. Further investigation with a larger sample size will be necessary to establish a MLVA pattern similarity threshold value. Yazdankhah et al. (2005) have recently described a MLVA method for meningococcal strain typing and found it to be superior to MLEE and MLST, but no direct comparison to PFGE was presented. It is interesting to note that between the two studies, only a single VNTR is shared by both MLVA assays. VNTR3 in our study targets the same tandem repeat region designated as VNTR02 by Yazdankhah et al. (2005). Our study evaluated a new MLVA based subtyping method using 92 isolates of NMSC. Although this scheme may be applicable for other serogroups, we used NMSC isolates because they are most often associated with outbreaks in the United States (Jackson et al., 1995; Rosenstein et al., 2001). Our results using MLVA

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closely matched both PFGE and epidemiological data. MLVA was found to be highly reproducible, inexpensive, safe, rapid, and relatively easy to perform, and would be a useful tool for public health officials in future outbreak investigations of meningococcal disease. Efforts are underway to explore the applicability of MLVA to the typing of meningococci directly from clinical specimens. Acknowledgements The authors wish to thank Deborah Talkington, Susanna Schmink, and Patricia Wilkins of the Centers for Disease Control and Prevention for their advice and technical expertise. They also wish to thank the network of laboratories participating in the Active Bacterial Core surveillance program for providing N. meningitidis reference strains and nucleic acid extracts. Finally, they gratefully acknowledge the Immunization and the Infectious Diseases Branches of the California Department of Health Services for their support. References Achtman, M., 1995. Epidemic spread and antigenic variability of Neisseria meningitidis. Trends Microbiol. 3, 186–192. Benson, G., 1999. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res. 27, 573–580. Caugant, D.A., 1998. Population genetics and molecular epidemiology of Neisseria meningitidis. APMIS 106, 505–525. Caugant, D.A., Høiby, E.A., Magnus, P., Scheel, O., Hoel, T., Bjune, G., Wedege, E., Eng, J., Frøholm, L.O., 1994. Asymptomatic carriage of Neisseria meningitidis in a randomly sampled population. J. Clin. Microbiol. 32, 323–330. Centers for Disease Control and Prevention, 2002. Laboratoryacquired meningococcal disease—United States, 2000. MMWR Morb. Mortal. Wkly. Rep., vol. 51(RR-07), pp. 141–144.

Centers for Disease Control and Prevention, 2005. Prevention and control of meningococcal disease: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Morb. Mortal. Wkly. Rep., vol. 54(RR-07), pp. 1–21. Hunter, P.R., Gaston, M.A., 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J. Clin. Microbiol. 26, 2465–2466. Jackson, L.A., Schuchat, A., Reeves, M.W., Wenger, J.D., 1995. Serogroup C meningococcal outbreaks in the United States—an emerging threat. JAMA 273, 383–389. Lindstedt, B.A., 2005. Multiple-locus variable number tandem repeats analysis for genetic fingerprinting of pathogenic bacteria. Electrophoresis 26, 2567–2582. Morelli, G., Malorny, B., Muller, K., Seiler, A., Wang, J., del Valle, J., Achtman, M., 1997. Clonal descent and microevolution of Neisseria meningitidis during 30 years of epidemic spread. Mol. Microbiol. 25, 1047–1064. Popovic, T., Schmink, S., Rosenstein, N.A., Ajello, G.W., Reeves, M.W., Plikaytis, B., Hunter, S.B., Ribot, E.M., Boxrud, D., Tondella, M.L., Kim, C., Noble, C., Mothershed, E., Besser, J., Perkins, B.A., 2001. Evaluation of Pulsed-field gel electrophoresis in epidemiological investigations of meningococcal disease outbreaks caused by Neisseria meningitidis serogroup C. J. Clin. Microbiol. 39, 75–85. Rosenstein, N.E., Perkins, B.A., Stephens, D.S., Popovic, T., Hughes, J.M., 2001. Meningococcal disease. N. Engl. J. Med. 344, 1378–1387. Schuchat, A., Robinson, K., Wenger, J.D., Harrison, L.H., Farley, M., Reingold, A.L., Lefkowitz, L., Perkins, B.A., 1997. Bacterial meningitis in the United States in 1995. N. Engl. J. Med. 337, 970–976. van Belkum, A., Scherer, S., van Alphen, L., Verbrugh, H., 1998. Short-sequence DNA repeats in prokaryotic genomes. Microbiol. Mol. Biol. Rev. 62, 275–293. Yakubu, D.E., Abadi, F.J.R., Pennington, T.H., 1999. Molecular typing methods for Neisseria meningitidis. J. Med. Microbiol. 48, 1055–1064. Yazdankhah, S.P., Lindstedt, B., Caugant, D.A., 2005. Use of Variablenumber tandem repeats to examine genetic diversity of Neisseria meningitidis. J. Clin. Microbiol. 43, 1699–1705.