Changing trends and serotype distribution of Shigella species in Beijing from 1994 to 2010

Shigella species are a common cause of acute diarrheal disease in China. In this study, we characterized the changing trends and serotype distribution of Shigella species in Beijing from 1994 to 2010. A total of 5999 Shigella strains were isolated and serotyped from the 302nd Hospital in Beijing. The annual number of Shigella isolates reached a peak (n = 1192; 19.84%) in 1996 and then decreased annually, reaching the lowest point (n = 24; 0.41%) in 2010. S. flexneri 2a and S. sonnei were the most frequently isolated Shigella, with their respective isolates making up 53.3% and 27.6% of the total. Isolates of S. flexneri 4c, 4a, and x made up 3% respectively of the total isolates. Significant decreases in percentage of S. flexneri over time were observed. S. sonnei surpassed S. flexneri 2a as the predominant serotype in 2000. Most isolates were recovered from July to September; 13.6% of the isolates were recovered from children aged 0 to 5 years, and 16% were recovered from those aged 21 to 25 years. S. flexneri 2a and 5 were recovered mostly from males (33.41%, p < 0.001; and 0.46%, p < 0.001%; respectively), whereas S. flexneri 2b and 6, and S. sonnei were most often isolated from females. Continuous monitoring of Shigella showed that all 4 species and 27 serotypes were present in Beijing, China, during the study period. The emergence of S. sonnei and the overall decreasing isolation rate of Shigella in Beijing can potentially aid in the development of vaccine and control strategies for shigellosis in the city.


Introduction
Shigella species are a common cause of acute diarrheal disease worldwide, with an estimated 167 million cases per year and resulting in approximately 1.1 million deaths; 97.6% of the cases occur in developing countries [1]. According to the Chinese National Infectious Disease Internet Reporting System, the annual incidence of shigellosis in China made it rank in the top three of the most notable infectious diseases for four consecutive years (2005 to 2008), with close to 500000 cases of shigellosis per year (http://www.moh.gov.cn); this number is now widely believed to be underestimated [2].
Shigellosis is caused by four species, S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. Shigella species can be identified by serotyping with group-specific antigens; serotyping is based on structural differences within the O-antigen repeating unit of lipopolysaccharide [3]. A total of 47 serotypes of Shigella have been recognized, including 15 for S. flexneri, 13 for S. dysenteriae, 18 for S. boydii, and a single one for S. sonnei [4]. The distribution of species and serotypes of Shigella is heterogeneous over time and place [5].
The World Health Organization has made the development of a safe and effective vaccine against S. flexneri [1,[6][7][8], but the vaccine effectiveness depends on the distribution patterns of local species and serotypes, because only type-specific immunity has been demonstrated in humans [9][10][11][12] and moreover cross-serotype protection is controversial [11,13].
According to a previous multicenter study of Shigella diarrhea in six Asian countries, S. flexneri is the most common species in Bangladesh, China, Pakistan, Indonesia, and Vietnam; whereas S. sonnei is predominant in industrialized countries [14]. Two recent reports have indicated that S. flexneri 2a is the most frequently isolated Shigella organism in China [15,16]. However, these reports may not be generalizable for the whole China; the time period of these studies is short, and surveillance is performed only in less-developed areas of China.
Little is known about the distribution of Shigella serotypes in Beijing, the political, educational, cultural, and economic center of China with a population of over 30 million. The present study describes the trends in Shigella species and their serotypes isolated from patients with diarrhea in a national infectious disease hospital in Beijing, China, from 1994 to 2010.

Study sites and settings
The location was a clinical diagnostic center at the 302nd Hospital of the People's Liberation Army in Beijing, China. The 302nd Hospital is the largest infectious disease teaching hospital in Beijing, China, with 1300 beds and receiving more than 36400 patients annually. From January 1994 to December 2010, fresh stool specimens were collected from patients with diarrhea and clinically suspected dysentery. The specimens were submitted to the microbiology laboratory of the 302nd Hospital. All experimental research have been performed with the approval of ethics committee of 302nd Hospital of the People's Liberation Army, with reference number 2004013D.

Bacterial isolation
Samples were cultured for Shigella by streaking diarrheal stools directly onto Salmonella-Shigella agar (Tian Tan Biologic Technology Company, Beijing, China) and incubating for 24 h at 37°C. Shigella-like colonies were selected and subcultured on Kligler iron agar (Qingdao Hope Biol-Technology Co., Ltd., Shandong, China). Except for S. flexneri 6 and S. boydii 14, Shigella spp. produce an alkaline slant and an acid butt but do not produce gas or H 2 S. As a species, Shigella organisms are characteristically nonmotile and lack the enzyme lysine decarboxylase.

Serotyping
Serologic identification was performed by slide agglutination with polyvalent somatic (O) antigen grouping sera, followed by testing with available monovalent antisera for specific identification of serotypes according to the manufacturer's instructions (Denka Seiken, Japan). Only one Shigella isolate per patient per diarrheal episode was included in the analysis.

Statistical analysis
Statistical comparisons were performed using the CHISS software (version 2001, Yuan YiTang Sci-Tech Co., Ltd., Beijing, China). Categorical data were expressed as percentages and calculated using a chi-square test, and p ≤ 0.05 was considered statistically significant.

Results
From 1994 to 2010, a total of 5999 Shigella isolates were collected from 372 inpatients and 5627 outpatients with diarrhea. All patients acquired diarrheal infection in the community, most likely through the ingestion of contaminated food and water. Cases for which the diarrheal infection was acquired through travel or via sexual contact were excluded from the analysis. Among the 5999 Shigella isolates, 12 were of an unknown subgroup (i.e., either not reported, not further typed, or untypeable); a final total of 5987 isolates were further analyzed in this study.

Subgroup trends
A statistically significant decreasing trend in S. flexneri and an increasing trend in S. sonnei were observed by chi-square analysis (p < 0.01) ( Figure 1). The trends in Shigella spp. isolated from Beijing between 1994 and 2010 are shown in Figure 2. The recording of annual Shigella isolation began in 1994, and the maximum number of isolates was reported in 1996 (n = 1194). The annual total number of isolated Shigella organisms had been decreasing since then, reaching a low point in 2008 (n = 22). This trend may be related to the strict hygiene inspection and adequate sanitation during the 2008 Olympic season [17]. Four peaks were observed during the 17-year collection period. Peak 1 appeared in 1996, with subsequent peaks in 1998 (peak 2, n = 602), 2002 (peak 3, n = 398), and 2004 (peak 4, n = 251). A sudden decrease in Shigella isolation was observed more in 2003 than in 2002 and 2004; one possible explanation is that resources were redirected to identify severe acute respiratory syndrome cases in China in 2003, thereby limiting bacterial diarrheal isolation. It should be noted that as the numbers of observed cases of shigellosis were decreasing, China's per capita gross domestic product (GDP) was increasing ( Figure 3).

Seasonality
Shigella isolates were recovered routinely throughout the study but were frequently recovered in the summer months (June to September; t = 7.83, p < 0.001; Table 2      S. sonnei is shown in Table 3. Adults aged between 21 and 25 years were the most commonly affected group (n = 978; 16.3%), followed closely by children aged less than 6 years (n = 821; 13.6%). S. flexneri 2a and S. sonnei were recovered from patients in each age group, although most infections caused by S. sonnei were found in children (n = 1639; 48%); children aged 0 to 5, 6 to 10, and 11 to 15 years accounted for 17.6% (n = 289), 15.2% (n = 250), and 14.8% (n = 242) of the cases, respectively. S. flexneri 2a occurred frequently in adults, especially those in the 21 to 25 (n = 594; 18.8%) and 26 to 30 (n = 380, 12%) age groups, although a high percentage (n = 400; 12.7%) was found to affect children aged less than 6 years.

Gender
Information about patient gender was known for all of the patients infected with S. flexneri and S. sonnei; distribution was slightly biased toward male patients (n = 3616; 60%; Table 4). Unexpectedly, the ratio for some of the predominant serotypes of S. flexneri and S. sonnei differed in distribution between male and female patients. The most prevalent S. flexneri serotype (2a) was found more frequently in males (63.5%, p < 0.0001) than in females (36.5%). This result suggests that in Beijing, males either have greater exposure or are more susceptible to this subserotype than females. Similarly, S. flexneri 5 affected more males than females (ratio of infected males to females, 28 [46%]:5 [44%]; p < 0.001), although this serotype is not common in Beijing. By contrast, S. flexneri 2b and 6 as well as S. sonnei were more often associated with women than with men (p < 0.003, p < 0.04, and p < 0.0001, respectively).

Discussion
According to a multicenter shigellosis surveillance study that incorporated data from six Asian sites, including China, shigellosis incidence is approximately 100-fold higher in Asia than in industrialized countries [5]. Other reports in China have indicated that shigellosis is even more ubiquitous than previously thought [18,19]. These studies have generally selected underdeveloped areas as surveillance sites, with little current information available on the epidemiologic trends in large cities like Beijing.
To begin to address this knowledge gap, the present study tracked Shigella infections in the largest infectious hospital in Beijing over a period of 17 years (1994 to 2010), thus providing a picture of serotype distribution across seasons, ages, and gender. The changing trend of Shigella in Beijing over time was determined in this work. The results will be useful in providing information that can be used by policy makers to implement control strategies or to predict the efficacy of vaccines for the prevention of shigellosis.
This study also demonstrated the diversity of all four species in recent years; the overall numbers of S. flexneri and S. sonnei have decreased, whereas the numbers of S. dysenteriae and S. boydii have not. S. flexneri 2a, the dominant serotype since 1994, was found to have been overtaken by S. sonnei in 2000; however, S. flexneri remained the dominant species in Beijing until 2006.