BMC Microbiology BioMed Central

Background: The number of scarlet fever occurrences reported between 2000 and 2006 fluctuated considerably in central Taiwan and throughout the nation. Isolates of Streptococcus pyogenes were collected from scarlet fever patients in central Taiwan and were characterized by emm sequencing and a standardized pulsed-field gel electrophoresis (PFGE) method. National weekly report data were collected for investigating epidemiological trends.


Background
Streptococcus pyogenes (Group A streptococcus) is a common pathogen responsible for a number of human suppurative infections, including pharyngitis, impetigo, pyoderma, erysipelas, cellulitis, necrotizing fasciitis, toxic streptococcal syndrome, scarlet fever, septicemia, pneumonia and meningitis. It also causes non-suppurative sequelae, including acute rheumatic fever, acute glomerulonephritis and acute arthritis [1]. Scarlet fever, characterized by a sore throat, skin rash and strawberry tongue, is most prevalent in school children aged four to seven years old. This disease was listed as a notifiable disease in Taiwan until 2007; as such, all cases of scarlet fever had to be reported to the public heath department. According to our records, however, only 9% of the medical centers, regional hospitals and district hospitals in central Taiwan reported cases of scarlet fever to the health authorities between 1996 and 1999. The number of scarlet fever cases is therefore likely to be significantly underreported. Scarlet fever outbreaks frequently occur in young children at day-care centers, kindergartens and elementary schools [2,3] and also occur in adults upon exposure to contaminated food [4].
Genotyping bacterial isolates with various methods is frequently used to compare the genetic relatedness of bacterial strains and provides useful information for epidemiological studies. In a previous study, we used emm (gene of M protein) sequencing [5], vir typing [6] and pulsed-field gel electrophoresis (PFGE) typing to analyze a collection of streptococcal isolates from scarlet fever patients and used these data to build a DNA fingerprint and emm sequence database for long-term disease surveillance [7]. Vir typing has since been abandoned in our lab because it has lower discriminatory power than PFGE and the protocol is difficult to standardize with conventional agarose gel electrophoresis. In contrast, the PFGE protocol for S. pyogenes has been standardized in our laboratory, and a second enzyme, SgrAI, has been found to replace SmaI for analysis of strains with DNA resistant to SmaI digestion [7]. Since PFGE is highly discriminative and emm sequencing provides unambiguous sequence information regarding emm type, we adopted these two genotyping methods to characterize streptococcal isolates and build a Streptococcus pyogenes DNA fingerprint and sequence database for the long-term study of scarlet fever and other streptococcal diseases.

Epidemiological trend of scarlet fever
Taiwan is an island country populated by 22.9 million people, most of whom reside in the western region ( Figure  1A). The population in northern, central, southern, and eastern areas is 10.2, 5.7, 6.4 and 0.6 million, respectively. Nationwide information for all notifiable diseases has been systematically collected since 2000. For accurate analysis, the number of confirmed scarlet fever cases was adjusted by multiplying the number of reported cases and the specimen positive rate. The total, adjusted number of confirmed cases throughout the whole country increased from 716 cases in 2000 to 1,258  The profiles of weekly reported cases revealed that scarlet fever was more prevalent in the winter and spring seasons (2 nd -25 th weeks) in 2000-2006. However, there was a remarkable decrease in the number of cases in the 6 th and 7 th weeks ( Figure 1B). This decrease may be due to the long holiday of the traditional lunar New Year and winter break from school, as it is usually from late-January to mid-February (4 th -7 th weeks). The weekly reported number of scarlet fever cases in 2002 was mostly higher than the weekly average from 2000 to 2006 ( Figure 1B). In 2003, except in the 11 th week, the number of weekly reported cases in the first 16 weeks was greater than the average. Furthermore, the number of cases between the 4 th and 9 th weeks was even higher than that in 2002. After the 16 th week, the number of cases in 2003 was below the overall average and was significantly decreased from the 17 th to 24 th week (mid-April to mid-June). A lower level of reported cases lasted until the first half of year 2004. In early 2003, a severe acute respiratory syndrome (SARS) outbreak occurred in Taiwan. There were two stages for  the SARS epidemic: stage I occurred from late-February to mid-April (9 th -16 th week), with scattered sporadic cases, and stage II occurred between mid-April and mid-June (17 th -24 th week), with severe nosocomial infections in several hospitals. The dramatic decline of scarlet fever notifications in 2003 occurred during the stage II period of the SARS epidemic.

Distribution of emm types among isolates collected in central Taiwan
For

Distribution of prevalent emm clones over time
In this study, a cluster of strains (as defined by PFGE types) having a common emm type and sharing higher PFGE pattern similarity than others with different emm types were considered to belong to a common emm clone. The stIL103 strain is an exception to this, as it shared high PFGE pattern similarity with the cluster of emm1 strains and was therefore considered to be part of the emm1 clone. Based on the groupings made by the PFGE patterns, six major emm (emm1, emm4, emm6, emm12, emm12* and emm22) clones were identified and are shown in Figure 2.  (Table 1), and 115 to 273 isolates were collected each year for genotyping. The number of isolates genotyped was adjusted to the number of annual confirmed cases to investigate the association of the fluctuation of scarlet fever cases and the relative prevalence of the emm clones. As shown in Figure [10]. In our study, the most common emm types in 427 isolates collected in the same time period in central Taiwan were emm12 (35.6%), emm1 (34.2%), emm4 (18.5%), emm6 (7.5%) and emm11 (0.9%). stIL103 was present in northern Taiwan, but it was not found in the central region during the same time period. Thus, the distribution and frequency of emm types appear to be geographically varied even in such a small country. The geographic variation in the prevalence of emm clones may explain why the epidemiological trend of scarlet fever in 2006 in central Taiwan was different from that in the whole country.
The major emm types were further discriminated into a number of PFGE types, and clustering analysis of the PFGE patterns suggests that the emm1, emm6 and emm4 strains belong to a single clone. The emm12 strains belong to two major clones and two singletons, and emm22 strains belong to one major clone and one singleton (Figure 2). Thus, six emm clones caused most (96.5%) of the scarlet fever cases in central Taiwan  emm12* emm12 emm4 emm1 emm6 emm22 Other time period. The fluctuation of scarlet fever cases was associated with the shuffling of the prevalent emm clones (Figure 4). The finding that only a few prevalent M (emm) types caused most occurrences of scarlet fever in a specific location in a given year period, as well as the shuffling of predominant M types, has been observed in many epidemiological studies in the early 20 th century [11]. During major epidemics of streptococcal infections in previous years, only a few serotypes predominated, and the strains were rich in M protein, encapsulated and were highly virulent [11]. Type-specific immunity was important for preventing re-infection with the same M type. It is thought that the shuffling of predominant M types is due to the type-specific immunity, leading to the decline of infections with certain M types and the emergence of other virulent M types. In the present study, the prevalence of the emm12*, emm1 and emm6 clones both increased and decreased within one year. In contrast, the emm12 and emm4 clones persisted throughout the seven year period. This phenomenon may be due to the fact that the emm12 and emm4 clones produced less M protein and were less virulent than the emm12*, emm1 and emm6 clones.
The PFGE study also indicates that each of the six emm clones has one predominant PFGE type, except for the emm4 clone, which has two major PFGE types ( Figure 2 [12] have shown that resistance to SmaI cleavage is due to the presence of a DNA methyltransferase gene, which is carried on a mobile chimeric element that has transposon-and bacteriophagelike characteristics. This mobile element may explain the high genetic diversity among the SmaI-resistant strains that emerged in such short period of time. The fluctuation of scarlet fever cases between 2000 and 2006 may be partially explained by the shuffling of several prevalent emm clones. However, the dramatic drop in reported cases in 2003 is difficult to explain. In early 2003, Taiwan was badly hit by a severe SARS outbreak. The SARS epidemic in Taiwan had two distinct stages, with the beginning in the late-February (the 9 th week) and the second ending in mid-June [13]. The stage I epidemic occurred from late-February to mid-April (the 9 th to 16 th week) and consisted of only scattered, sporadic cases, with most of the patients having recently traveled to China. In this stage, the disease did not cause much panic and the level of scarlet fever remained high. In stage II (from mid-April to mid-June or the 17 th to 24 th week), several clusters of infection occurred via intra-hospital or inter-hospital transmission. Enormous panic spread over the whole country after an outbreak of nosocomial infection was confirmed on the 22 nd of April. The disease was subsequently transmitted to several hospitals and spread from the North to the South. The number of scarlet fever cases dropped remarkably during this period. Because a large portion of the SARS infections was associated with hospitals, fear of SARS drove people out of hospitals and public places. This fear and the change of people's behavior may have significantly reduced the number of outpatients and the transmission of many infectious diseases, including scarlet fever. In fact, the SARS outbreak had a long-term effect on the occurrences of scarlet fever. After the SARS epidemic, the number of weekly scarlet fever reports was often lower than the overall average until the first half of 2004.

Conclusion
The occurrences of scarlet fever in central Taiwan between 2000 and 2006 were primarily caused by six emm clones: emm12 (40.0%), emm4 (23.2%), emm1 (16.3%), SmaIresistant emm12* (10.3%), emm6 (3.8%) and emm22 (2.9%). Each emm clone had predominant PFGE genotype(s), and most minor genotypes within an emm clone emerged and quickly disappeared. The large fluctuation in the number of scarlet fever cases during this time period can be attributed to the shuffling of several prevalent emm clones and to a SARS outbreak in 2003.

Epidemiological data and bacterial strains
Scarlet fever was a notifiable disease in Taiwan until 2007; hospitals and clinics were obligated to report confirmed or suspected cases to the county public health department via a web-based Notifiable Diseases Reporting System established by the Taiwan CDC in 2000. The hospitals and clinics that reported scarlet fever cases were asked to provide throat swab specimens or S. pyogenes isolates to the regional laboratories of the Taiwan CDC for bacterial examination and genotyping. Confirmed cases were those in which S. pyogenes was isolated from the specimens. The number of annual confirmed cases detected through the Notifiable Diseases Reporting System was adjusted by multiplying the number of reported cases and the rate of positive specimens. S. pyogenes isolates used for characterization in this study were obtained directly from hospitals located in central Taiwan through the Notifiable Diseases Reporting System or were recovered from throat swab specimens collected from hospitals and clinics through the Notifiable Diseases Reporting System and the Sentinel Physician Active Reporting System.

emm typing
The procedure developed by Beall and colleagues [5] was used to prepare the emm DNA fragments from S. pyogenes isolates for sequencing. The amplified DNA amplicons and primer 1, 5'-TATT(C/G)GCTTAGAAAATTAA-3', were sent to a local biotech company (Mission Biotech Corp. Taipei, Taiwan) for DNA sequencing. The 5' emm sequences (at least the first 240 bases) were subjected to a BLAST comparison with those in the emm database (http:/ /www.cdc.gov/ncidod/biotech/strep/strepindex.htm; accessed on April 20 th , 2009) to determine emm type.

PFGE analysis
S. pyogenes isolates were subjected to PFGE analysis using a previously described protocol [7]. All of the isolates were analyzed by SmaI digestion. Isolates with DNA resistant to SmaI digestion were analyzed with SgrAI. PFGE patterns were recorded using a Kodak digital camera system (Kodak Electrophoresis Documentation and Analysis System 290; Kodak; Rochester, NY, USA) with 1792 × 1200 pixels. The digital PFGE images were then analyzed using BioNumerics software version 4.5 (Applied Maths, Kortrijik, Belgium) and the DNA pattern for each isolate was compared using the computer software. A unique PFGE pattern (genotype) was defined if it contained one or more DNA bands different from the others. The genetic relatedness among isolates is presented in a dendrogram built by clustering the PFGE patterns. The clustering analysis was performed using the UPGMA algorithm provided in the BioNumerics software and the value of Dice predicted similarity of two patterns at settings of 1% optimization and 0.7% position tolerance.