Incidence rates and clinical Symptoms of Salmonella , Vibrio parahaemolyticus , and Shigella infections in China , 1998 – 2013

Introduction: The etiological and clinical characteristics of patients with infectious diarrhea have changed during the last decade in Shanghai. Methodology: The records of 29,210 patients with infectious diarrhea in the outpatient department of the Jinshan Hospital (Shanghai, China) between January 1998 and December 2013 were analyzed. Results: A total of 2,849 samples were positive for intestinal pathogenic bacteria including Vibrio parahaemolyticus (2,489; 84.0%), Salmonella spp. (235; 8.3%), and Shigella spp. (125; 4.4%). V. parahaemolyticus infections are mainly characterized by abdominal pain, nausea, and vomiting, whereas Shigella spp. infections can, in addition, induce fever. In contrast, Salmonella infections can produce all of these symptoms but in a smaller percentage of patients. During the 16-year study, both the number of patients and the positive infection rate declined. Notably, the rate of infections by V. parahaemolyticus decreased while the detection rates of Salmonella spp. increased year by year from 2006 on with the introduction of a new detection method. Conclusions: Salmonella has been identified as the third-most frequent cause of diarrhea from 1998–2006, as the second-most frequent cause from 2006–2010, and as the most frequent cause from 2011–2013, which was mainly due to a sharp decrease of V. parahaemolyticus infections in 2011–2013. Salmonella strains collected in 2011–2013 showed high susceptibility to imipenem (100%) and meropenem (100%), whereas susceptibilities for ampicillin (39%) and piperacillin (40%) were low.


Introduction
Infectious diarrhea is a frequently occurring worldwide disease.According to a World Health Organization (WHO) report, the incidence of infectious diarrhea is high in the developing world [11].Pathogens capable of causing infectious diarrhea are numerous and include viruses, bacteria, fungi, and parasites.Shigella, Salmonella, pathogenic Escherichia coli, and Vibrio parahaemolyticus are the main bacterial pathogens that cause infectious diarrhea.According to the data on foodborne disease outbreaks in some parts of China, monitored by the National Foodborne Disease Monitoring Network, a total of 5,770 events of food poisoning were detected in 162,995 patients between 1992 and 2001, 38.5% of which were caused by microorganisms (accounting for 50.9% of the total).Acute gastroenteritis caused by V. parahaemolyticus ranked first in these bacterial food poisoning incidents, and those caused by Salmonella were ranked third [12].However, data collected in recent years have suggested that the incidence of infections caused by V. parahaemolyticus has declined, while the incidence of those caused by Salmonella has increased [14,15,24].Various serious outbreaks of foodborne Salmonella infection have been reported worldwide [8][9][10]23].
V. parahaemolyticus is mainly found in seafood and often causes foodborne diseases due to the consumption of raw or cooked shellfish, lobster, or fish.The clinical symptoms include nausea, vomiting, abdominal pain, and watery diarrhea; these can be reduced by altering the diet and through better health education [5,6].Adult patients with a Shigella infection show obvious symptoms of fever and diarrhea, and the bacteria are sensitive to chemical disinfectants and antibiotics, which makes diagnosis relatively straightforward [1].However, congeneric Salmonella are widespread in the natural world and grow well over a wide environmental temperature range (7°C-45°C).The infection risk is related to serum type and a person's existing health condition, with the elderly, children and those immunocompromised at the highest risk.Salmonella mainly causes typhoid, paratyphoid, acute gastroenteritis, and extra-intestinal inflammation (bacteremia, cholecystitis, pyelonephritis, etc.) [2,7].Thus, Salmonella infections produce both digestive tract and systemic symptoms, and their various serum types make a rapid diagnosis difficult.Therefore, it is of obvious clinical interest to master the epidemiological and clinical characteristics of Salmonella to facilitate rapid diagnosis.Here, we report intestinal infections caused by Salmonella in a 16-year period in our hospital in China, together with an analysis of its clinical and epidemiological characteristics.

Patient data
Data from 29,210 patients with infectious diarrhea in the outpatient department of Jinshan Hospital (Shanghai, China) from January 1998 to December 2013 were retrospectively analyzed, and included the results of stool cultures and clinical characteristics.Diarrhea cases were defined according to a five-step evaluation: 1.Does the patient really have diarrhea?Beware of fecal incontinence and impaction; 2. Rule out medications as a cause of diarrhea (drug-induced diarrhea); 3. Patient has had more than one loose stool, or clinically significant vomiting, over a two-week period with no underlying non-infectious cause, followed by a symptom-free period of three weeks which was considered as acute diarrhea, otherwise more than four weeks as chronic diarrhea [4]; 4. Categorize the diarrhea as inflammatory, fatty, or watery; 5. Consider factitious diarrhea [22].The 2,849 patients' samples that were positive for Shigella, Salmonella, or V. parahaemolyticus were selected, and their epidemiological characteristics and clinical and laboratory data further analyzed.

Sample collection and analysis
Patients were asked to defecate into a clean bedpan; any stool with purulent blood or mucus feces were sampled with sterile bamboo and placed into a culture bottle for testing.Bacteria were separated by incubating the stool sample in SS (used for the separation of Shigella and Salmonella, Meixiang Company, Shanghai, China)and TCBS (thiosulfate, citrate, bile salts, sucrose, agar culture medium was used for the separation of V. parahaemolyticus (Meixiang Company, Shanghai,China).Suspicious colonies were selected for pure culture after 6-10-hour culture at 35°C.Traditional methods were used for bacteria identification before the year 2000, which included enrichment cultivation, selective separation culture, and biochemical identification.After 2000, pure culture colonies were analyzed using an automatic bacteria identification instrument (VITEK-2 Compact, bioMerieux, Marcy l'Etoile, France) to identify the pathogens.
Serotyping was achieved at the Jinshan branch of the Chinese Center for Disease Control and Prevention in Shanghai.O and H antigens were characterized using commercial antisera (S&A Reagents Lab, Bangkok, Thailand), and phase 1 flagellar and phase 2 flagellar antigen agglutination was done using commercial antisera (Bio-Rad, Marnes-la-Coquette France).Reagents were purchased via Ningbo Tianrun Biological & Pharmaceutical Co., Ningbo, China.The serotypes were identified according to the Kauffmann-White scheme [16].

Statistics
SPSS version 10.0 software was used for data analysis.A Z test was used for a large sample size and a Mann-Whitney test was used if heterogeneity of variance was evident.A p value < 0.05 was considered to be statistically significant.

General patient data
A total of 29,210 patients with acute diarrhea as their first symptom visited the intestinal outpatient department over 16 years.In this study, 26,395 stool samples were tested, with a total test rate of 90%.Of these patients, 2,849 (10.8%) were found to be pathogen positive (Table 1), including 2,489 (84.4%) cases of V. parahaemolyticus, 235 (8.3%) cases of Salmonella, and 125 (4.4%) cases of Shigella (Table 2).In addition, 3 cases of V. alginolyticus and 2 cases of E. coli were diagnosed but omitted for analyses in this study because of their low frequencies.The youngest and oldest patient with a Salmonella infection was 6 months and 83 years of age, respectively, with the average patient age being 35.6 ± 12.8 years.According to monthly records, Salmonella infections mainly occurred between April and November and were responsible for 79.6% of all infections.The highest rate was between May and September, which accounted for 83.4% of all cases.

Distribution and changes in the three main intestinal pathogens with time
Before 2003, more than 2,000 patients with an intestinal infection visited the hospital each year, but during the last 10 years, the number of patients attending the clinic steadily reduced, year by year, with a concomitant gradual decline in the positive detection rate of pathogenic bacteria.There was an average of 2,153 patients per annum during the first 8 years, with an average positive detection rate of 13.1%.In the last 8 years, there was an average of 1,142 patients, with an average positive detective rate of 6.4%.Both of these averages significantly decreased by about 50% (p = 0.0003, p = 0.0006, respectively) (Table 1).It is important to note that the positive detection rate of V. parahaemolyticus declined year by year and in contrast, the positive rate of Salmonella detection rose, particularly from 2006 on (Table 2).

Clinical features of Salmonella infection
In 235 cases of Salmonella infection, 57.9% of patients had abdominal pain, 50.6% had bouts of diarrhea > 5 times a day, 26.0% presented with Drug sensitivity tests (Table 5) were performed on 111 cases of Salmonella infections over three years.The results showed that the drugs that Salmonella was sensitive to were mainly imipenem, meropenem, cefotaxime, cefoperazone, levofloxacin, and ciprofloxacin.
Only those patients with severe clinical symptoms or elderly patients were given anti-infection treatment.It is prohibited to administer quinolone drugs for patients under the age of 18, thus imipenem, meropenem, cefoperazone, or cefotaxime sodium were prescribed.Quinolone or cephalosporins were only given to adult patients.

Discussion
Our hospital provides the main medical service and health screening facilities of the local and surrounding areas for sick people.Many patients come to the intestinal outpatient department with diarrhea as the main symptom of their ailment.After medical data analysis of intestinal infectious diarrhea patients over the last 16 years, we found that both the number of patients and the pathogen positive separation rates were significantly decreased by about 50% (p = 0.0003, p = 0.0006), which might be attributed to improved hygiene when preparing food and to the widely publicized diet and health information in recent years (Table 1).
Specifically, the incidence of infectious diarrhea caused by V. parahaemolyticus declined over the 16 years, while the incidence of diarrhea caused by Salmonella has risen year by year, a change mainly reflected in the composition ratio.Until 2010, the actual number of isolated V. parahaemolyticus strains was still much higher than the number of isolated Salmonella strains, and our results also show that V. parahaemolyticus was the most common pathogen of infectious diarrhea in Jinshan Hospital, with abdominal pain, diarrhea, nausea, and vomiting being the main symptoms, which may also be accompanied by dehydration.Before 2006, infections due to Shigella were ranked second of the infectious diarrheal diseases without typical tenesmus and mucous bloody purulent stools.This was different from the traditional Shigella infection, suggesting that whether a Shigella infection exists should be confirmed in patients who do not exhibit the typical symptoms of a Shigella infection.After 2006,  Salmonella infections ranked second, with abdominal pain, diarrhea, and fever being the main symptoms, which often occurred 12 to 24 hours after eating infected food.Typically, symptoms included mainly a watery stool was excreted 3 to 30 times daily, which occasionally contained mucous or bloody purulent.These symptoms were accompanied by moderate fever and/or chills, symptoms which lasted for three to five days in most adults, and even longer in old or weak patients.Severe dehydration could occur in patients suffering from serious vomiting and diarrhea.However, although the clinical manifestation of Salmonella infection in this study was in line with reports in the domestic literature [19,21], the symptoms of abdominal pain, nausea, and tenesmus could not be used to distinguish a Salmonella from a Shigella infection.
Salmonella infections occurred all year round, were easy to contract from April to November, and occurred with a high incidence from May to October, consistent with previous reports of Salmonella infections in this region [3,13,18] [25], which is in line with our data until 2010, but contrary to our findings of 2011 because Salmonella was the major cause of acute diarrhea during this period in our study until 2013, which might be due to the fact that in our study, small, region-limited Salmonella outbreaks have been detected.However, the percentage of V. parahaemolyticus cases in acute diarrhea patients gradually decreased from 2006-2011.Seafood is mainly responsible for the spread of V. parahaemolyticus; therefore, the incidence of infections can be reduced by educating people about the absolute need to follow strict hygiene guidelines when preparing seafood.
In contrast, Salmonella infections can be spread through the fecal/oral route in a number of ways (e.g., water, flies, mosquitos), and previous papers [8][9][10]23] have reported that outbreaks of Salmonella infection could be attributed to the consumption of unclean food contaminated by Salmonella.In the present study, the suspected unclean food, which may have caused Salmonella infections, mainly included ice cream, watermelon, seafood, and meat.
Diverse resistance patterns of various Salmonella strains to different antibiotics were found in our clinical study, which may be due to the different medical treatment expertise of doctors [17,20].Nagshetty et al. [17] reported that the percentage of Salmonella Typhi isolates resistant to ciprofloxacin in India was 4.2%, while the susceptibility of Salmonella to ciprofloxacin in the present study was 79.7%.

Conclusions
New automated diagnosis techniques since 2006 have led to increased detection rates, particularly of Salmonella infections.Until 2006, V. parahaemolyticus was identified as the main diarrhea-causing bacteria followed by Shigella and Salmonella, which changed then from 2006-2010 to V. parahaemolyticus followed by Salmonella and Shigella.During the period of 2011-2013, Salmonella was identified as the most frequent diarrhea pathogen followed by V. parahaemolyticus and Shigella, which was mainly due to a sharp decline of V. parahaemolyticus infections, indicating that seafood as the major source of V. parahaemolyticus has been processed under improved hygienic conditions.

Table 1 .
Number of patients with intestinal infections and the separation rate of the three main intestinal pathogens

Year Sample number (n) Patients who were infected (n) Positive rate (%)
P refers to the statistical significance of the means of patient numbers and the positive rate between the first 8 years and the last 8 years of the 16-year study.

Table 2 .
Distribution of the three main intestinal pathogens

Table 3 .
Comparison of the clinical symptoms of Salmonella, Vibrio parahaemolyticus and Shigella infections.
P refers to the statistical analysis between symptom proportions of Salmonella and Shigella infections.

Table 4 .
Serotype distribution of 235 salmonella strains

Table 5 .
Sensitivity analysis of 113 Salmonella strains, detected in 2011-2013, to common antibiotics

Antibiotics Sensitivity Number of sensitive strains Sensitivity rate (%)
. During the period 1998 to 2005, Salmonella infections were sporadically detected throughout the year, with one to two cases being diagnosed yearly, but no Salmonella infections were detected in 2003 and 2005.Since second half of 2006, significantly more Salmonella infections were detected, which might be attributed to improved identification methods.Zhang et al. reported that the frequency of V. parahaemolyticus infections was highest, followed by Shigella and Salmonella for infectious diarrhea cases in Shanghai from 2006-2011