Prevalence of toxigenic Clostridium difficile in hospitalized patients in the southwestern province of Saudi Arabia: Confirmation using the GeneXpert analysis

ABSTRACT

Clostridium difficile (Clostridioides difficile) is a leading cause of nosocomial infections in hospitalized patients worldwide. Stool samples were collected from 112 inpatients admitted to different hospitals and were screened for C. difficile GDH + toxin A + B by immunoassay, and all positive samples by immunoassay were processed for molecular detection of C. difficile using the GeneXpert assay. C. difficile strains were detected in 12 (10.71%) out of 112 stool samples using the GDH + toxin A + B immunoassay method and toxigenic C. difficile was confirmed in 5 stool samples using the GeneXpert molecular assay. C. difficile strains were also detected in 7 (8.97%) out of 78 stool samples from intensive care unit patients, 3 (25%) out of 12 stool samples from internal medicine ward patients, 1 (11.11%) out of 9 stool samples from surgery ward patients, and 1 (10%) out of 10 stool samples from isolation ward patients using the GDH + toxin A + B immunoassay method and the toxigenic C. difficile strain was confirmed in 1, 2, 1, and 1 stool samples, respectively, using the GeneXpert molecular assay. Toxigenic C. difficile was confirmed in patients at 4 (51.14%) out of 7 hospitals. In the present study, we also analyzed the clinical information of patients with C. difficile-positive stool samples who were receiving one or more antibiotics during hospitalization. The binary toxin gene (cdt), the tcdC gene, and the C. difficile strain polymerase chain reaction (PCR) ribotype 027 were not detected using the GeneXpert molecular assay among 12 C. difficile-positive samples by immunoassay. This study should aid in the prevention of unnecessary empiric therapy and increase the understanding of the toxigenic C. difficile burden on the healthcare system in the southwestern province of Saudi Arabia.

KEYWORDS:

• Toxigenic and nontoxigenic Clostridium difficile
• nosocomial infection
• Clostridioides difficile
• GeneXpert Molecular assay
• Immunoassay for C. difficile

1. Introduction

Clostridium difficile (Clostridioides difficile) is a Gram-positive bacillus and an anaerobic, spore-bearing bacterium present in the environment that is capable of colonizing and propagating in the human gut. Clostridium difficile infection (CDI) is a frequently acquired nosocomial intestinal infection; increasing severity has been reported, and CDI has a high mortality and morbidity rate. Clinical disease symptoms range from moderate diarrhea to toxic megacolon, colonic perforation, or even death. Asymptomatic transmission of C. difficile might be due to the asymptomatic carriage of C. difficile in the gut [1]. The incidence of C. difficile infection has been increasing, and hypervirulence has emerged (virulence factors such as spore formation, epithelial attachment, germination, and toxin secretion might play important roles in infection). The C. difficile strain polymerase chain reaction (PCR) ribotype 027 (RT027), most commonly known as 027/NAP 1/B1 (PCR ribotype 027/North American pulse-field type 1/restriction endonuclease analysis group BI), has been reported worldwide [2], and its hypervirulence is due to a polymorphism in the tcdB receptor-binding domain, which encodes the hypertoxic and antigenic variable tcdB [3]. This strain demonstrates high-level fluoroquinolone and metronidazole resistance [4] and an increased in vitro sporulation rate [5]. In vitro studies with the RT027 strain demonstrated that these strains produce toxin A approximately 16 times and toxin B 23 times more than control stains [6]. RT027 strains are reported to spread more easily within the hospital because these strains can resist disinfectants, cleaning, and the hospital environment [7]. Epidemic strain RT027 has been reported in Europe and North America [8], and this strain has been associated with severe disease outbreaks in healthcare facilities worldwide. The emergence and fast global spread of hypervirulent strains have led to the application of compulsory national surveillance of CDI in the UK [9] and other countries. However, CDI is not widely detected in Saudi Arabia [10], due to the low sample submission rate, inadequate laboratory diagnostic capacity, and lack of sufficient surveillance systems [7]. The prevalence and disease burden of C. difficile strain RT027 in Saudi Arabia have not been widely studied [10,11], but there was a report of C. difficile strain RT027 in a tertiary care hospital in Riyadh, Saudi Arabia [12]. A two-step or multistep protocol algorithm (GDH + toxin A + B and nucleic acid amplification test (NAAT)) was highly recommended for the detection of CDI [1] by the Infectious Diseases Society of America and the Society for Healthcare Epidemiology of America [10,13]. Since there are no reports on the prevalence of toxigenic C. difficile in the southwestern province of Saudi Arabia, the prevalence of toxigenic C. difficile in hospitalized patients in the southwestern province of Saudi Arabia, as confirmed using the GeneXpert analysis, was determined in the present study. A two-step algorithm for the detection of toxigenic C. difficile in stool [14] was applied as first-step immunoassay screening for glutamate dehydrogenase (GDH) as well as toxin A and toxin B in stool samples, followed by confirmatory molecular analysis [15]. In the present study, we used the CerTest C. difficile GDH + toxin A + B screening immunoassay (CerTest, Zaragoza, Spain) and the GeneXpert C. difficile PCR assay (Cepheid, CA, USA), which detects the toxin B gene (tcdB), binary toxin gene (cdt), and tcdC gene for confirmation.

2. Materials and methods

Patients admitted in different wards in seven different general hospitals in the southwestern province of Saudi Arabia were studied. Clinical information and informed consent were obtained from all randomly selected patients (male and female without symptoms of diarrhea) with different age groups before the collection of stool samples. Each patient was given a sterile container and asked to collect a stool sample. Stool samples were collected from 112 inpatients admitted to different hospitals in the southwestern province of Saudi Arabia. All stool samples were screened for C. difficile GDH + toxin A + B (CerTest) by immunoassay immediately after collection. Using a stool collection tube, four drops of each of the collected stool samples were dispensed into three circular windows (GDH, toxin A, and toxin B) according to the manufacturer’s instructions. The results were recorded after 10 min. A result was considered negative if there was a green line in all three circular windows (GDH, toxin A, and toxin B). A result was considered positive if there was a green – red line in any of the circular windows (GDH, toxin A, and toxin B).

All positive samples, according to the immunoassay, were processed for molecular detection of C. difficile using the GeneXpert assay. Direct qualitative detection of toxin-producing C. difficile from all positive stool specimens was performed with a rapid automated in vitro diagnostic test, which detects the tcdB gene, cdt gene, and tcdC gene (tcdCA117; the one-base pair deletion at nucleotide position 117 within the gene encoding the negative regulator of toxin production). The extraction, multiplex PCR, and detection steps were performed in separate chambers. Each chamber contained one time-use cartridge; the chamber contained all the reagents needed for the identification of the C. difficile gene targets. The GeneXpert C. difficile PCR assay was performed according to the manufacturer’s instructions. A re-analysis of samples with errors, invalidity, or no test outcome was retested once. After 45 min, all results were recorded. Analyte results for the tcdB gene (positive or negative), the cdt gene (positive or negative), and the tcdC gene (positive or negative) were noted.

The GeneXpert assay presumptively detects sequences found in the hypervirulent strain (RT027 or genotype-related RT027). In addition, the assay includes an internal sample processing control in the PCR assay. This will ensure sufficient processing of the target bacteria and the existence of inhibitors in the PCR assay.

3. Results

3.1. Epidemiological data

1. C. difficile strains were detected in 12 (10.71%) out of 112 stool samples subjected to the GDH + toxin A + B immunoassay method. The toxigenic C. difficile strain was confirmed in five stool samples using the GeneXpert molecular assay (Table 1).

   Table 1. Demographic characteristics of asymptomatic carriers of Clostridium difficile among hospitalized adult patients, Jazan province, Saudi Arabia.

   Variable	C. difficile strains were detected in stool sample by immunoassay (C. difficile GDH + toxin A+B) (%)	Confirmation of toxigenic C. difficile by GeneXpert molecular assay among 12 C. difficile-positive stool samples by immune assay	Total number of stool samples (%)
   Total number	12 (10.71)	5	112
   Sex
   Male	8 (10.66)	5	75 (66.96)
   Female	4 (10.81)	0	37 (33.04)
   Duration of hospital stay
   More than 1 week	1 (5)	0	20 (17.85)
   More than 2 weeks	1 (3.33)	0	30 (26.78)
   More than 3 weeks	1 (7.69)	1	13 (11.60)
   More than 4 weeks	9 (18.36)	4	49 (43.75)
   Age group (years)
   18–34	2 (14.28)	1	14 (12.5)
   35–51	1 (10)	1	10 (8.92)
   52–67	4 (12.12)	1	33 (29.46)
   Over 68	5 (9.09)	2	55 (49.10)
   Ward
   ICU	7 (8.97)	1	78 (69.64)
   Internal medicine	3 (16)	2	12 (10.71)
   Orthopaedic	0	0	3 (2.67)
   Surgical	1 (11.11)	1	9 (8.03)
   Isolation	1 (10)	1	10 (8.92)
   Hospitals
   Hospital 1	3 (12)	2	25 (22.32)
   Hospital 2	3 (6.81)	1	44 (39.28)
   Hospital 3	0	0	14 (12.5)
   Hospital 4	1 (33.33)	1	3 (2.67)
   Hospital 5	1 (17)	0	5 (4.46)
   Hospital 6	0	0	5 (4.46)
   Hospital 7	4 (16)	1	16 (14.28)

2. C. difficile strains were detected in 1 (5%) out of 20 stool samples from patients who were hospitalized for more than 1 week and in 1 (3.33%) out of 30 patients who were hospitalized for more than 2 weeks using the GDH + toxin A + B immunoassay method. Furthermore, C. difficile strains were detected in 1 (7.69%) out of 13 patients who were hospitalized for more than 3 weeks and in 9 (18.36%) out of 49 patients who were hospitalized for more than 4 weeks using the GDH + toxin A + B immunoassay method and the toxigenic C. difficile strain was confirmed in 1 and 4 stool samples, respectively, using the GeneXpert molecular assay (Table 1).

3. C. difficile strains were detected in 2 (14.28%) out of 14 patients aged 18–34 years, 1 (10%) out of 10 patients aged 35–51 years, 4 (12.12%) out of 33 patients aged 52–67 years, and 5 (9.09%) out of 55 patients aged over 68 years using the GDH + toxin A + B immunoassay method and the GeneXpert molecular assay detected the toxigenic C. difficile strain in 1, 1, 1, and 2 stool samples, respectively (Table 1).

4. C. difficile strains were detected in 7 (8.97%) out of 78 stool samples from intensive care unit (ICU) patients, 3 (25%) out of 12 stool samples from internal medicine ward patients, 1 (11.11%) out of 9 stool samples from surgery ward patients, and 1 (10%) out of 10 stool samples from isolation ward patients using the GDH + toxin A + B immunoassay method and the toxigenic C. difficile strain was confirmed in 1, 2, 1, and 1 stool samples, respectively, using the GeneXpert molecular assay (Table 1).

According to the GeneXpert molecular assay, toxigenic C. difficile was detected in 2 stool samples from hospital 1, 1 from hospital 2, 1 from hospital 4, and 1 from hospital 7 (Table 1).

3.2. Detection of C. difficile in stool samples by GDH + toxin A + B by immunoassay

GDH was detected in 12 (10.71%) out of 112 stool samples, whereas toxin A and toxin B were detected in 5 (4.46%) out of 112 stool samples (Table 2).

Table 2. Detection of C. difficile in stool samples by GDH + toxin a + B by immunoassay.

S.No	GDH (glutamate dehydrogenase) positive (%)	Toxin A positive (%)	Toxin B positive (%)	Total number of stool samples
1	12 (10.71)	5 (4.46)	5 (4.46)	112

3.3. Identification of toxigenic C. difficile using the GeneXpert assay

Toxin B gene (tcdB) and toxigenic C. difficile were detected in 5 (41.66%) out of 12 C. difficile-positive samples by immunoassay, whereas the binary toxin gene (cdt), tcdC gene, and C. difficile strain RT027 were not detected using the GeneXpert analysis among the 12 positive samples (Table 3).

Table 3. Identification of toxigenic C. difficile and hypervirulent C. difficile strain RT027 by the GeneXpert assay.

S. No.	Serial number of stool sample collected	SPC (Result)	Toxin B gene (tcdB)	Binary toxin gene (cdt)	tcdC gene	Toxigenic C. difficile	Hyper virulent C. difficile strain RT027
1	1	NA	Positive	Negative	Negative	Positive	Negative
2	4	NA	Positive	Negative	Negative	Positive	Negative
3	5	NA	Positive	Negative	Negative	Positive	Negative
4	28	Pass	Negative	Negative	Negative	Negative	Negative
5	38	NA	Positive	Negative	Negative	Positive	Negative
6	40	Pass	Negative	Negative	Negative	Negative	Negative
7	56	Pass	Negative	Negative	Negative	Negative	Negative
8	82	Pass	Negative	Negative	Negative	Negative	Negative
9	83	Pass	Negative	Negative	Negative	Negative	Negative
10	85	Pass	Negative	Negative	Negative	Negative	Negative
11	93	Pass	Negative	Negative	Negative	Negative	Negative
12	111	NA	Positive	Negative	Negative	Positive	Negative

SPC, sample processing control; tcdC, the one-base pair deletion at nucleotide position 117 within the gene (tcdCA117).

4. Discussion

CDI is a severe and often deadly nosocomial intestinal infection caused by the Gram-positive bacterium C. difficile. The current study aims to investigate the prevalence of characteristics of the hypervirulent C. difficile strain PCR ribotype 027 (RT027) in the southwestern province of Saudi Arabia, where limited data on CDI exist.

In the present study, stool samples were collected from hospitalized patients and screened for C. difficile using an immunoassay. Positive samples were further analyzed using the GeneXpert molecular assay to confirm the presence of toxigenic C. difficile strains, including RT027.

There is a paucity of data on the incidence of CDI in Saudi Arabia. There were two studies conducted in the eastern region of Saudi Arabia, one in 2010 and the other in 2019 in Dhahran. In 2010, 913 stool samples were screened using an enzyme immune assay (EIA) for C. difficile, and the incidence rate was reported at 4.6% [18]. In a re-conducted study in 2019, 10995 stool samples were screened between 2001 and 2018 using NAAT, and the incidence rate was reported at 5.2% [19]. The prevalence of C. difficile (nontoxigenic and toxigenic) in hospitalized patients was analyzed using two-step testing algorithms as reported in a previous study [15]. In the present study, toxigenic C. difficile was detected in 5 out of the 12 C. difficile-positive (immunoassay) stool samples using the GeneXpert molecular assay. This result is in line with previous reports from the eastern region of Saudi Arabia [19]. Overall, the prevalence of C. difficile was 10.71% in stool samples subjected to the GDH + toxin A + B immunoassay method (Table 1). One recently published study reported an 8.4% incidence rate based on EIA from King Abdulaziz University-affiliated hospital, Jeddah [10]. In another study in the eastern province of Saudi Arabia, 146 stool samples were collected from patients with diarrhea and tested for C. difficile toxin (EIA), and the incidence rate was reported at 13.7% [20]. Asymptomatic carriers of C. difficile constitute an important source for continuous transmission, especially in hospital populations [1]. The presence of known risk factors for antibiotic use and colonization may lead to infection [15]. In the current study, we also analyzed the clinical information of patients with C. difficile-positive stool samples who were receiving one or more antibiotics during hospitalization.

In the present study, C. difficile strains were detected in 1 (5%) out of 20 stool samples from patients who were hospitalized for more than 1 week and in 1 (3.33%) out of 30 patients who were hospitalized for more than 2 weeks using the GDH + toxin A + B immunoassay method. Additionally, C. difficile strains were detected in 1 (7.69%) out of 13 patients who were hospitalized for more than 3 weeks and in 9 (18.36%) out of 49 patients who were hospitalized for more than 4 weeks using the GDH + toxin A + B immunoassay method and the toxigenic C. difficile strain was confirmed in 1 and 4 stool samples, respectively, using the GeneXpert molecular assay. Although the relationship between symptom onset and spore exposure is unexplained on most occasions, asymptomatic carrier contribution to transmission has been demonstrated; one-third of diagnosed CDI individuals had recent contact (within 4 weeks) with a symptomatic patient, whereas the other one-third had recent contact with only an asymptomatic C. difficile carrier [21]. Larger studies with a multidimensional approach are required to completely understand the effect of carriage and/or asymptomatic colonization on transmission in healthcare settings. Additionally, further alternative studies to determine the source of CDI in the hospital framework are required, with a target on untested and asymptomatic patients [1].

A clinical infection may develop in a person when C. difficile spores germinate and impact the production of toxins within the gut lumen [17]. TcdA (toxin A) and TcdB (toxin B) exotoxins disturb colonic epithelial cells and stimulate the release of proinflammatory chemokines and cytokines [1]. In the present study, it was found that 10.71% of the stool samples tested positive for C. difficile using an immunoassay. Among the positive samples, 41.66% were confirmed to contain toxigenic C. difficile strains through molecular analysis. However, the specific virulence factors associated with RT027, such as the binary toxin gene (cdt), the tcdC gene, and RT027 itself, were not detected in any of the positive samples. Additionally, in the present study, the tcdB gene and toxigenic C. difficile were detected in 5 (41.66%) out of 12 C. difficile-positive samples by immunoassay (Table 3). In contrast with the current results, Meenakshi Singh et al. [22] reported a 54.6% positivity rate for toxigenic C. difficile, and among the toxigenic strain distributions, 62.42% of toxigenic C. difficile samples were from patients of different age groups (2–45 years old), and 37.58% were from geriatric patients (above 45 years old). In the present study, C. difficile strains were detected in 2 (14.28%) out of 14 patients aged 18–34 years, 1 (10%) out of 10 patients aged 35–51 years, 4 (12.12%) out of 33 patients aged 52–67 years, and 5 (9.09%) out of 55 patients aged over 68 years using the GDH + toxin A + B immunoassay method and toxigenic C. difficile was confirmed in 1, 1, 1, and 2 stool samples, respectively, using the GeneXpert molecular assay (Table 1). Comparison of the epidemiology of CDI between various countries is difficult because of the various approaches to sample collection and diagnosis. In previous studies, the prevalence of strains (toxigenic and nontoxigenic) differed due to the fact that the studies were conducted over various time durations, in various geographical locations, and among different groups of the population (adults and/or children) [23].

CDI is commonly reported in the ICU, and severely ill patients are at significant risk for mortality and morbidity from this bacterial infection. C. difficile RT027 strain may further increase the risk in patients [23,24]. In the present study, C. difficile strains were detected in 7 (8.97%) out of 78 stool samples from patients in the ICU, 3 (25%) out of 12 stool samples from patients in the internal medicine ward, 1 (11.11%) out of 9 stool samples from patients in the surgery ward, and 1 (10%) out of 10 stool samples from patients in the isolation ward using the GDH + toxin A + B immunoassay method and the toxigenic C. difficile strain was confirmed in 1, 2, 1, and 1 stool samples, respectively, using the GeneXpert molecular assay. The prevalence of C. difficile strains was more prevalent in the stool samples from the medical ward than in the stool samples from the ICU ward, which may be due to patients staying in the medical ward for prolonged periods [25]. Predrag et al. [22] also detected C. difficile (toxigenic and nontoxigenic) in 14% of samples from ICU patients in western Europe. In our study, we collected 78 (69.64%) samples from ICU patients because previous studies reported that up to 40% of patients develop diarrhea after admission to the ICU [16].

Toxigenic C. difficile prevalence in patients varied significantly between the regions in our study. Toxigenic C. difficile was detected in patients from 4 (51.14%) out of 7 hospitals.

GDH-positive but stool-toxin-negative samples are regularly reported in previous studies [26] similar to the present study.

The present study shows toxigenic strains of C. difficile prevalence in hospitalized patients in the southwestern province of Saudi Arabia, which has not been reported previously. Nontoxigenic C. difficile strains do not produce toxin or induce disease because these strains do not carry the pathogenicity locus (PaLoc), and via horizontal gene transfer, PaLoc may transform nontoxigenic producers into toxin producers, and toxin production leads to macroscopic and microscopic changes to the colonic epithelium in a person with CDI [1].

This study highlights the importance of understanding the prevalence of toxigenic C. difficile strains in healthcare settings to improve CDI prevention and management. By identifying the presence of toxigenic strains, unnecessary empiric therapy can be avoided, leading to better patient outcomes. The study provides valuable insights into the burden of toxigenic C. difficile in the southwestern province of Saudi Arabia and emphasizes the need for further research in this region to combat CDI effectively.

5. Conclusion

This study contributes to the understanding of toxigenic C. difficile impact on the healthcare system in the southwestern province of Saudi Arabia; the presence of toxigenic C. difficile was confirmed using the GeneXpert molecular assay. Binary toxin gene (cdt), tcdC gene, and C. difficile strain RT027 were not detected using the GeneXpert molecular assay among 12 C. difficile-positive samples by immunoassay. This study will help in the prevention of unnecessary empiric therapy and increase the understanding of the toxigenic C. difficile burden in the southwestern province of Saudi Arabia.

6. Limitations

Owing to the limited number of samples and those positive for C. difficile, it is difficult to generalize the findings to all of Saudi Arabia. Hence, it is recommended to conduct a similar study with a large number of samples to draw a final conclusion regarding the prevalence of toxigenic C. difficile in hospitalized patients in the southern province of Saudi Arabia.

Abbreviations

GDH	=	glutamate dehydrogenase
RT027	=	ribotype 027
tcdC	=	one-base pair deletion at nucleotide position 117 within the gene encoding the negative regulator of toxin production
tcdB	=	toxin B gene
cdt	=	binary toxin gene
PaLoc	=	pathogenicity locus
CDI	=	Clostridium difficile infection
SPC	=	sample processing control
ICU	=	intensive care unit
UK	=	United Kingdom

Availability of data and materials

All the data supporting the conclusion are available in Tables 1, 2 and 3.

Authors contributions

Conceptualization, methodology, software, validation, and writing: VKB and NK; resources, investigation, review, and editing: AA, AMM, and MUA; investigation and sample collection: KAMZ and AHAG. All authors read and approved the final manuscript.

Ethics approval and consent to participate

This study was approved by the Deanship of Scientific Affairs and Research, Jazan University, Jazan, Saudi Arabia. Informed written consent was obtained from all participants in the study before sample collection.

Acknowledgments

We thank the dean and the staff of the Faculty of Applied Medical Sciences for their cooperation and help in conducting this research.

We also thank Lalitha Shree Basode, Data analyst, Zarb School of Business, Hofstra University, Hofstra, New York, USA for data analysis.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

The authors would like to thank the Deanship of Scientific Research, Jazan University. This research is part of the research group funding program at Jazan University. The authors would like to thank the Vector-Borne Diseases Research Group, Grant No. RG-2-1.