Detection of Salmonella spp. Related Co-Infections Among Children with Diarrheal Diseases in Guangzhou, China

Introduction

Diarrheal disease is the second leading cause of death in children under 5 years of age and was responsible for the deaths of 370,000 children in 2019.^1–3 In China, according to some regional studies, the overall incidence of diarrhea among children under 5 years old is 2.50–3.38 episodes per year for individuals.⁴ Diarrhea is the passage of three or more loose or liquid stools per day, or more frequently than normal for the individual,¹ and is a common symptom of gastrointestinal infection (GI) that is mainly caused by single or multiple bacterial, viral, and parasitic organisms.^3,5–7 The most prevalent bacterial pathogens are Escherichia coli, Salmonella enterica, Campylobacter spp., and Shigella spp, while adenovirus, rotavirus, and norovirus were mainly responsible for viral infections. Infectious diarrhea demonstrates different seasonal distributions.⁴

Salmonella is a rod-shaped, gram-negative bacteria with no capsule and spore, and can be classified into typhoidal Salmonella and non-typhoidal Salmonella (NTS) serovars.⁸ NTS infection is a leading cause of acute gastroenteritis in pediatric patients, especially children aged under 5 years.^9,10 Salmonella infection results in over 30,000 deaths annually, which was estimated by the Chinese Center for Disease Control (CDC) to be the cause of 70–80% of bacterial foodborne illnesses, ranking as one of the two diarrheal agents.^11,12 Zoonotic infection caused by NTS post a significant burden for public health.¹³ A rising trend of Salmonellosis outbreak has been observed since the 1970s with high resistance to quinolone and beta-lactam, such as carbapenem.^14,15 From the nationwide surveillance study, NTS, Shigella, C. jejuni and Y. enterocolitica displayed a “Child-pattern”, with a detection turn-point at 2–5 years.^11,16 However, it remains unclear whether Salmonella-related co-infection patterns influence diarrheal symptoms among children.

The comprehensive detection of gastrointestinal pathogenic microorganisms is challenging because conventional culture methods are time-consuming and serology tests show limited sensitivity.^17,18 The rapid and specific validation of gastrointestinal infectious agents is of great significance for clinical management. To overcome these difficulties, FilmArray™ technology (BioFire Diagnostics, Salt Lake City, Utah, USA) has produced rapid polymerase chain reaction (PCR) multiplexing, which is designed to simultaneously detect the most common gastrointestinal pathogens.^19,20 The FilmArray GI panel (FA) not only offers high sensitivity and specificity for identifying infectious pathogens in different physiological tracts,^21–23 but also provides a comprehensive way to reveal the nature of co-infection patterns among various pathogens.

The distribution of gastrointestinal pathogens, including Salmonella spp., shows significant regional characteristics. However, the distribution and clinical features of Salmonella related diarrhea in Guangzhou remain largely unknown. It is of crucial necessity to analyze the distribution and associated infection patterns of Salmonella among children, which would provide supporting information for promoting the understanding of Salmonellosis and improving bacterial gastroenteritis individualized treatment. The objectives of this study were as follows: 1) to test Salmonella in pediatric patients with diarrheal diseases using the FilmArray GI Panel to assess the overall positive rate of Salmonella; 2) to analyze the prevalence of Salmonella spp. via Film Array GI Panel and retrospective cultural results; and 3) to investigate the co-infection patterns of Salmonella with other enteric pathogens among clinical characteristics to assess the differences in symptoms.

Materials and Methods

Sample Enrollment

All stool samples in this study were obtained from patients as residual specimens of routine hospital testing procedure during January 2022 to December 2023 in Guangzhou, China. A total of 430 diarrheal stool samples from children under five years old were enrolled for FilmArray GI Panel (BioFire Diagnostics, Salt Lake City, UT, USA), and all samples were anonymized (Table S1). Second or subsequent samples from the identical patients or sample without clinical histories implicating infectious causes of diarrhea were excluded. The clinical results of fecal cultures for Salmonella spp. from 1580 patients were collected from the Laboratory Information System (LIS). These data were retrospectively categorized and analyzed to reveal the prevalence and distribution of Salmonella infection. Clinical information exported from LIS only retain gender, age, testing date, laboratory testing results and clinical syndromes. This study was approved by the Medical Ethics Committee of Guangdong Women and Children Hospital.

FilmArray GI Panel

All stool samples were tested using a commercially available multiplex PCR system, FilmArray Gastrointestinal (GI) Panel (BioFire Diagnostics, Salt Lake City, UT, USA). The FilmArray GI Panel was capable of simultaneously detecting and identifying nucleic acids from multiple bacteria, viruses, and parasites in stool samples, including Campylobacter (C. jejuni/C. coli/C. upsaliensis), Clostridium difficile (C. difficile) toxin A/B, Plesiomonas shigelloides, Salmonella, Vibrio (V. parahaemolyticus/V. vulnificus/V. cholerae), Yersinia enterocolitica, enteroaggregative Escherichia coli (EAEC), enteropathogenic Escherichia coli (EPEC), enterotoxigenic Escherichia coli (ETEC), Shiga-like toxin-producing Escherichia coli (STEC) stx1/stx2, Shigella/Enteroinvasive Escherichia coli (EIEC), Cryptosporidium, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, Adenovirus F 40/41, Astrovirus, Norovirus GI/GII, Rotavirus A, Sapovirus (Genogroups I, II, IV, and V). According to FilmArray GI Panel Instruction, two process controls (RNA Process Control and PCR2 Control) were used in each essay and positive results indicated successful assay.

Salmonella Identification

Isolation and identification of Salmonella spp. was conducted according to previous research. Stool samples were plated and incubated onto XLD (Xylose Lysine Desoxycholate) agars at 36°C for 18–48h. The suspicious colonies (transparent, round, with or without black centers that reflect H₂S production) were further identified by MALDI-TOF-MS (Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrum).

Statistical Analysis

All data on clinical characteristics were imported into Microsoft Excel. GraphPad Prism 8 was used to display the figures. Statistical analyses, including Chi-square test or Fisher’s exact test, were performed using the SPSS, version 26.0.

Results

Gender and Age Demographics of Patients

For the FilmArray GI Panel, 430 children aged <5 years with diarrheal diseases were enrolled in this study (Table 1), including 184 (42.79%) females and 246 (57.21%) males. Among them, 72 (39.13%) and 86 (34.96%) were positive for Salmonella spp. No statistically significant difference was observed between the sexes in the detection of Salmonella spp. (χ² = 0.788, P = 0.375). The majority of stool samples were collected from children under 12 months of age (235/430), followed by 1–3 years old (161/430), and over 3 years old (34/430). The positivity rates of Salmonella spp. among the three age groups were 37.87% in children aged <12 months, 38.51% in children aged 1–3 years, and 20.59% in children aged >3 years. Moreover, 1580 patients who underwent fecal culture for Salmonella spp. were categorized and analyzed. Statistical differences were observed between children aged <12 months and those aged >3 years using both the FA (χ² = 3.866, P = 0.049) and culture methods (χ² = 6.481, P = 0.011). Among all stool specimens, 178 were subjected to both Salmonella detection methods.

Table 1 Gender and Age Distribution of Patients with Salmonella Spp. Detection

Prevalence of Salmonella spp. in Monthly and Age Distribution

Either FA or culture detection of Salmonella spp. was defined as a positive result for Salmonella spp. prevalence. In the monthly distribution of Salmonella spp., the highest positive rate was observed in September (42.51%, 71/167), and the lowest rate was 8.49% (9/106) in February (Figure 1A). The positive rate increased from April to July, whereas a remarkable decline was observed from September to December, followed by a peak (Figure 1A). The positive detection rate of Salmonella spp. was similar among the three age groups. In accordance with the results in Table 1, the positivity rate in children aged >3 years was likely to be lower than in the other two age groups (Figure 1B).

Figure 1 Prevalence of Salmonella spp. detected by FA and culture method. (A) Monthly distribution of Salmonella spp. positive rate from January 2023 to December 2024. (B) Distribution of Salmonella spp. in children (female vs male) aged under 12 months, 1–3 years and over 3 years respectively.

Prevalence of All Enteric Pathogens Detected by FilmArray

Of all the stool samples, 79.3% had at least one type of enteric pathogen (Table 2). The overall positive rate of Salmonella spp. was the highest among all the bacterial pathogens, which was detected in 36.74% (158/430), followed by EPEC (19.77%, 85/430), C. difficile (19.53%, 84/430), and Campylobacter (12.56%, 54/430). Among enteric viral pathogens, adenovirus contributed to the majority of infections (20.23%, 87/430). The proportions of bacterial single infection (Table 3), viral single infection, bacterial–bacterial infection, viral–viral infection and bacterial–viral infections were 24.19% (104/430), 10.00% (43/430), 18.60% (80/430), 3.02% (13/430), and 23.49% (101/430), respectively.

Table 2 Frequency of Enteric Pathogens Detected by FA

Table 3 Infection Patterns of Enteric Pathogens

Co-Infection Patterns of Salmonella spp

Among the stool samples positive for Salmonella spp., 35.44% (56/158) were positive for Salmonella spp., whereas 37.34% (59/158) of stool samples were positive for both Salmonella spp. and other enteropathogenic bacteria. Moreover, 30 stool samples (18.99%) tested positive for Salmonella spp. and enteric viruses. A total of 13 (8.23%) stool samples were positive for Salmonella spp. and bacterial-viral co-infection. In the Salmonella mixed infection, Salmonella plus EPEC (12.66%, 20/158), Salmonella plus C. difficile (8.86%, 14/158) and Salmonella plus adenovirus (6.33%, 10/158) were the first three highest compositions. Salmonella, C. difficile and EPEC combination was detected in 4.43% (7/158) of all stool samples. Salmonella plus Norovirus, Salmonella plus EAEC were detected in 4.43% (7/158) and 2.53% (4/158) of the stool specimens, respectively (Table 4).

Table 4 Infection Patterns of Salmonella Spp. Detected by FA

Clinical Symptoms in Different Salmonella Co-Infection Patterns

The percentage of female patients with Salmonella single infection, Salmonella plus C. difficile, Salmonella plus EPEC, and Salmonella plus adenovirus were 42.86%, 35.71%, 40.00%, and 50.00%, respectively (Table 5). Similarly, most of the patients in the four groups were under 1 year of age, and 80.00% of the children in Salmonella plus EPEC group were under 12 months of age. Clinical symptoms of fever were observed in 67.86% (38/56) of Salmonella single infections, 71.43% (10/14) of Salmonella infections plus C. difficile, 60.00% (12/20) of Salmonella infections plus EPEC, and 50% of Salmonella infections plus adenovirus. Vomiting symptoms were recorded in 14.29% of patients with Salmonella single infections. All patients in the four groups had diarrhea, whereas the blood stools were recorded in 42.86% of Salmonella single infections and in 42.86% of Salmonella plus C. difficile. Notably, mucus stools were recorded in Salmonella plus C. difficile at a high rate of 92.86% (13/14), which was significantly higher than that in Salmonella infections alone (P < 0.05).

Table 5 Symptom Distribution of Different Salmonella Infection Patterns

Discussion

Salmonellosis is responsible for one of the major causes of diarrhea, and salmonellosis outbreaks have post a great threat to worldwide developing regions, especially China.¹⁴ Salmonella spp. is primarily transmitted through contaminated foods such as eggs, milk, meat, and drinking water.^12,24 Although children’s consumption of nutritious foods has increased, their immature immune systems heighten susceptibility to Salmonella infections. This study found the high Salmonella positive rate among children with gastroenteritis, which was lower than the finding of Truong J et al and Spina A et al,^25,26 for the Salmonella infection is likely to be associated with climate, regions and living habit. Notably, co-infection with Clostridium difficile correlated with exacerbated diarrhea severity, highlighting the necessity to enhance pediatric dietary hygiene management and reinforce preventive strategies against gastrointestinal infections. Furthermore, healthcare institutions should optimize rapid pathogen detection technologies to facilitate comprehensive enteric pathogen screening in pediatric populations.

Given that the gastrointestinal tract is home to a large number of microorganisms, co-infection is a common enteric pathogen in diarrheal infections. The high burden of coinfection with multiple enteric pathogens in children with diarrhea should be given more attention.²⁷ The FilmArray GI Panel is an efficient and sensitive method for detecting enteric pathogens including the most prevalent bacteria, viruses, and parasites, which have a significant potential for improving the detection of important enteric microorganisms. From our data, the overall positivity rates of enteric pathogens and co-infections were higher than those reported elsewhere.^28,29 A key possible factor is that children under five years of age are more vulnerable to infectious diarrhea than children over five years of age, especially in developing countries.

In this study, children co-infected with Salmonella spp. and C. difficile displayed a high frequency of fever and mucus stools compared with Salmonella single infection. It has been reported that children coinfected with rotavirus and toxin-producing C. difficile have more severe clinical characteristics and are more vulnerable to severely dehydrated.³⁰ However, Shafiq M et al found that co-infection with other pathogens did not affect the severity of C. difficile infection or cause treatment failures.³¹ Moreover, although co-infection with Campylobacter and Salmonella was observed at low levels, it was estimated that co-culture with Salmonella spp. significantly promoted the survival of Campylobacter jejuni under aerobic conditions.³² Based on various gastrointestinal infection patterns and distinguished clinical symptoms, it would be reasonable to apply individualized treatments to the pediatric patients. Assessment of multiple possible infectious etiologies may contribute to better pharmacological management of acute gastroenteritis among children, whereas the strategies of the pathogenesis of concurrent infections require further investigation. Interactions between different enteropathogenic microorganisms are poorly understood. Therefore, it is reasonable to reveal and interpret the underlying mechanisms involved in pathogenic co-infections using multi-pathogen detection technology.

It was widely reported that age is a risk factor associated with Salmonella incidence, for the younger children were more vulnerable to infections. As estimated in a nationwide study, 34% of diarrheal patients infected with non-typhoid Salmonella are under five years of age.³³ In this study, the overall positive rate of Salmonella spp. among children under 5 years of age was 36.74% by the FilmArray GI Panel and 25.19% by the culture method, which may be attributed to the difference in sample size and patient source. Based on the surveillance network, the most prevalent pathogen in Beijing is Shigella spp., followed by Vibrio parahaemolyticus, Salmonella spp. and EPEC.³⁴ In another study of pathogens in children under five years of age with acute gastroenteritis in Guangzhou, China, the most prevalent pathogen was Salmonella spp.,³⁵ which is consistent with the results of this study. Taken together, the prevalence of Salmonella spp. may vary across regions, detection methods, and patient sources.

There are some limitations to this study, including: 1) The sample size in this study was relatively small and the samples were not evenly distributed in each month, so the prevalence of Salmonella spp. detected by the FilmArray GI Panel was not representative enough. 2) Not all stool samples for the FilmArray GI Panel were simultaneously assessed using the conventional culture method, and the comparison of the two assays was limited. 3) The FilmArray GI Panel could not distinguish between Salmonella active infections and dead bacteria. 4) The FilmArray GI Panel could not determine whether the detected pathogens were pathogenic or enteric-colonized. 5) The strains and serotypes of Salmonella spp. could not be classified using the current FilmArray GI Panel.

In conclusion, this study showed that Salmonella is the predominant pathogen detected in diarrheal diseases in children under five years of age in Guangzhou, China. Furthermore, these results showed a high frequency of co-infection with Salmonella and other enteric pathogens, mainly Clostridium difficile, Enteropathogenic Escherichia coli, and adenoviruses. This study provides a comprehensive understanding of the clinical characteristics and outcomes of the potential interactions between Salmonella and other enteric microorganisms.