A multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) method was developed and evaluated for the subtyping of Shigella sonnei isolates. A total of 26 VNTR loci were identified by exploring the repeat sequence loci in the genomic sequences of S. sonnei strains Ss046 and 53G and by testing 536 isolates that had previously been characterized by pulsed-field gel electrophoresis ( The usefulness of MLVA for outbreak investigation was evaluated using 151 isolates from 10 shigellosis outbreaks and 22 PFGE-indistinguishable isolates collected from nine epidemiologically unrelated events in five different countries. The evaluations indicated that MLVA was a powerful typing tool to distinguish isolates for outbreak investigation and that it exhibited a good discrimination of the 22 PFGE-indistinguishable isolates. Single-locus variants did occur during the outbreak; therefore, S. sonnei isolates with MLVA profiles differing at no more than a single locus should be considered part of the same outbreak. The present study suggests that MLVA has the potential to replace PFGE as a standard method of typing S. sonnei isolates for disease surveillance and outbreak investigation.

Background Meningococcal disease is infrequently found in Taiwan, a country with 23 million people. Between 1996 and 2002, 17 to 81 clinical cases of the disease were reported annually. Reported cases dramatically increased in 2001–2002. Our record shows that only serogroup B and W135 meningococci have been isolated from patients with meningococcal disease until 2000. However, serogroup A, C and Y meningococci were detected for the first time in 2001 and continued to cause disease through 2002. Most of serogroup Y meningococcus infections localized in Central Taiwan in 2001, indicating that a small-scale outbreak of meningococcal disease had occurred. The occurrence of a meningococcal disease outbreak and the emergence of new meningococcal strains are of public health concern. Methods Neisseria meningitidis isolates from patients with meningococcal disease from 1996 to 2002 were collected and characterized by serogrouping, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The genetic relatedness and clonal relationship between the isolates were analyzed by using the PFGE patterns and the allelic profiles of the sequence types (STs). Results Serogroups A, B, C, W135, Y, and non-serogroupable Neisseria meningitidis were, respectively, responsible for 2%, 50%, 2%, 35%, 9%, and 2% of 158 culture-confirmed cases of meningococcal disease in 1996–2002. Among 100 N. meningitidis isolates available for PFGE and MLST analyses, 51 different PFGE patterns and 30 STs were identified with discriminatory indices of 0.95 and 0.87, respectively. Of the 30 STs, 21 were newly identified and of which 19 were found in serogroup B isolates. A total of 40 PFGE patterns were identified in 52 serogroup B isolates with the patterns distributed over several distinct clusters. In contrast, the isolates within each of the serogroups A, C, W135, and Y shared high levels of PFGE pattern similarity. Analysis of the allelic profile of the 30 STs suggested the serogroup B isolates be assigned into 5 clonally related groups/ clonal complexes and 7 unique clones. The ST-41/44 complex/Lineage 3, and the ST-3439 and ST-3200 groups represented 79% of the serogroup B meningococci. In contrast, isolates within serogroups A, serogroup W135 (and C), and serogroup Y, respectively, simply belonged to ST-7, ST-11, and ST-23 clones. Conclusion Our data suggested that serogroup B isolates were derived from several distinct lineages, most of which could either be indigenous or were introduced into Taiwan a long time ago. The serogroup A, W135 (and C), and Y isolates, respectively, belonged to the ST-7, ST-11, and ST-23, and the represented clones that are currently the major circulating clones in the world and are introduced into Taiwan more recently. The emergence of serogroup A, C and Y strains contributed partly to the increase in cases of meningococcal disea...

One hundred seventy-nine Streptococcus pyogenes isolates recovered from scarlet fever patients from 1996 to 1999 in central Taiwan were characterized by emm, Vir, and pulsed-field gel electrophoresis (PFGE) typing methods. The protocols for Vir and PFGE typing were standardized. A database of the DNA fingerprints for the isolates was established. Nine emm or emm-like genes, 19 Vir patterns, and 26 SmaI PFGE patterns were detected among the isolates. Among the three typing methods, PFGE was the most discriminatory. However, it could not completely replace Vir typing because some isolates with identical PFGE patterns could be further differentiated into several Vir patterns. The prevalent emm types were emm4 (n ‫؍‬ 81 isolates [45%]), emm12 (n ‫؍‬ 64 [36%]), emm1 (n ‫؍‬ 14 [8%]), and emm22 (n ‫؍‬ 13 [7%]). Some emm type isolates could be further differentiated into several emm-Vir-PFGE genotypes; however, only one genotype in each emm group was usually predominant. DNA from nine isolates was resistant to SmaI digestion. Further PFGE analysis with SgrAI showed that the SmaI digestion-resistant strains could be derived from indigenous strains by horizontal transfer of exogenous genetic material. The emergence of the new strains could have resulted in an increase in scarlet fever cases in central Taiwan since 2000. The emm sequences, Vir, and PFGE pattern database will serve as a basis for information for the long-term evolutionary study of local S. pyogenes strains.Streptococcus pyogenes causes a variety of human diseases ranging from relatively mild skin infections to severe invasive diseases, such as acute rheumatic fever, glomerulonephritis, puerperal sepsis, necrotizing fasciitis, meningitis, and streptococcal toxic shock syndrome (9,29). Among the diseases caused by this pathogen, scarlet fever, characterized by a strawberry tongue, skin rash, and sore throat, is most prevalent in schoolchildren aged 4 to 7 years. In Taiwan, scarlet fever is a notifiable disease, with 150 to 230 confirmed cases per year from 1996 to 1999. Since only 9% of the medical centers, regional hospitals, and district hospitals in central Taiwan had ever reported cases to the health authorities from 1996 to 1999, the number of scarlet fever cases should be severely underreported. Scarlet fever outbreaks are not uncommon at daycare centers, kindergartens, and elementary schools (5, 16). However, the molecular epidemiology of this disease has not been well studied in Taiwan.Analysis of the clonal relationships between clinical isolates from patients by various typing methods is a practical approach to elucidation of the epidemiology of a disease. To date, a number of phenotyping and genotyping methods have been described for S. pyogenes (1, 3, 10, 13, 18-20, 22, 26, 27, 30, 33). Among these methods, M serotyping has been taken as the "gold standard" for the characterization of S. pyogenes strains, in light of its importance to streptococcal virulence. However, application of M serotyping is restricted due to the lack of a comprehensive set...

No abstract

Shigella sonnei contains numerous IS1 elements. The existence of polymorphisms in the length of the inter-IS1 spacer is a basis for the development of a PCR-based method for the subtyping of S. sonnei strains. The usefulness of inter-IS1 spacer typing (IST) was evaluated and compared with that of pulsed-field gel electrophoresis (PFGE) by characterization of S. sonnei isolates from epidemiologically nonrelated cases and outbreaks and of isolates that were indistinguishable by PFGE and that were collected from independent infection events. IST was less discriminatory than PFGE, with discriminatory indices of 0.96 and 0.63, respectively, but was able to compensate for the drawbacks of PFGE. PFGE exhibited a high level of discriminatory power for S. sonnei isolates; however, PFGE was also, at times, too discriminatory, which was a disadvantage in constructing the clonal relationships among strains circulating over a period of months or years. Furthermore, IST provided greater subtyping information for isolates indistinguishable by PFGE. The present study indicates that IST is more useful than PFGE for investigating the genetic relationships among S. sonnei strains circulating over a longer time span and also for discriminating certain strains which are indistinguishable by PFGE.Shigella sonnei is the most commonly isolated species among the four Shigella species in industrialized countries (8, 10) and also the predominant species responsible for travel-associated shigellosis (7). Shigellosis is relatively infrequent and is a designated notifiable disease in Taiwan, meaning that all shigellosis cases must be reported and that the Shigella isolates must be sent to the Centers for Disease Control of Taiwan (Taiwan CDC). During the period from 1993 to 2004, 61 to 1,357 (average, 388) cases of shigellosis cases were confirmed annually in Taiwan, a country with 23 million people (3). In Taiwan, S. sonnei was responsible for most of the large shigellosis outbreaks in the industrialized western area (13) and was frequently associated with imported infections (11). In contrast, S. flexneri was mainly circulating among the aboriginal tribes in the mountainous areas (5).Analyses of Shigella isolates by molecular subtyping methods can provide useful information on the outbreak and can help to trace the transmission and spread of the pathogen (6, 12). To date, several DNA-based methods have been developed for the subtyping of S. sonnei (14)(15)(16)18). Among the subytping methods, pulsed-field gel electrophoresis (PFGE) has been standardized and used in the laboratories of "PulseNet," a national molecular subtyping network for food-borne disease surveillance in the United States (19). The standardized PulseNet PFGE protocol (4) has been adopted by the Taiwan CDC and since 2003 has been used for the routine subtyping of Shigella species. PFGE has proven to be a powerful tool in our laboratory and others (1, 6) for discriminating Shigella strains for investigation of the outbreak. However, in our experience, PFGE is, in fact, at ...