Review Article

Clostridioides (Clostridium) difficile

A silent nosocomial pathogen
Ibrahim A. Al-Zahrani, MSc, PhD.

ABSTRACT From the Department of Medical Laboratory Sciences, Faculty of

Applied Medical Sciences, King Abdulaziz University, and from the
‫ما زالت عدوى املطثية العسيرة متثل تهديداً للكثير من أنظمة الرعاية الصحية‬ Special Infectious Agents Unit-Biosafety Level-3, King Fahad Medical
‫ وقد تغير السلوك الوبائي لهذه البكتيريا خالل العشرين‬.‫في جميع أنحاء العالم‬ Research Centre, King Abdulaziz University, Jeddah, Kingdom of
‫ حيث ارتبط هذا التغيير بظهور سالالت شديدة الضراوة ومقاومة‬،‫سنة املاضية‬ Saudi Arabia.
‫للمضادات احليوية من بكتيريا املطثية العسيرة وكذلك بسبب االستخدام‬
‫املفرط للمضادات احليوية مع االفتقار إلى سياسة مثالية الستخدام املضادات‬ Address correspondence and reprint request to: Dr.
،‫ لذلك‬.‫ باإلضافة إلى ممارسات مكافحة العدوى دون املستوى األمثل‬،‫احليوية‬ Ibrahim A. Al-Zahrani, Medical Laboratory Sciences Department,
‫ وخاصة املضادات واسعة املدى‬،‫فإن االستخدام األمثل للمضادات احليوية‬ Faculty of Applied Medical Sciences, King Abdulaziz University,
‫ ميكن‬،‫ مثل نظافة اليدين‬،‫ وتطبيق تدابير بسيطة ملكافحة العدوى‬،‫امليكروبي‬ Jeddah, Kingdom of Saudi Arabia. E-mail: [email protected]
ORCID ID: https://orcid.org/000-0003-3879-5833
،‫ عالوة على ذلك‬.‫أن يقلل بشكل كبير من معدالت اإلصابة باملطثية العسيرة‬
‫فإن االكتشاف املبكر لهذه العدوى وفهم سلوكها الوبائي باستخدام طرق‬
‫معملية دقيقة هو حجر الزاوية لتقليل اإلصابة بعدوى املطثية العسيرة ومنع‬
‫ وعلى الرغم من عدم وجود إجماع على الطريقة املختبرية األفضل‬.‫انتشارها‬
‫ فإن استخدام طريقتني أو أكثر ميكن أن‬،‫لتشخيص عدوى املطثية العسيرة‬
.‫ ويوصى بها‬،‫يحسن دقة التشخيص‬
C lostridioides difficile (also known as Clostridium
difficile [C. difficile]) is the primary cause of
antibiotic-related diarrhea in many healthcare settings,
particularly elderly centers and rehabilitation clinics.1
Clostridioides difficile (C. difficile) infection is still
a threat to many healthcare settings worldwide.
All classes of antimicrobials can potentially contribute
Clostridioides difficile epidemiology has changed to C. difficile infection (CDI) via disrupting the gut
over the last 20 years, largely due to the emergence microbiota, enabling C. difficile to multiply, colonize
of hypervirulent and antimicrobial-resistant the digestive tract, and then infect the host. As a
C. difficile strains. The excessive use of antimicrobials, result, resistance to many antimicrobial agents offers
the absence of optimal antibiotic policies, and C. difficile a selective advantage, enhancing their
suboptimal infection control practices have fueled the survival and transmission.2,3 In addition, the ability of
development of this pressing health issue. The prudent C. difficile spores to withstand common environmental
use of antimicrobials, particularly broad-spectrum cleaning agents and alcohol-based hand disinfection
agents, and simple infection control measures, such increases their survival and enhances their rapid
as hand hygiene, can significantly reduce C. difficile
infection rates. Moreover, the early detection of these transmission in hospital environments.1 Noteworthy,
infections and understanding their epidemiological the epidemiology of C. difficile has shifted in the past
behavior using accurate laboratory methods are the 20 years, essentially due to the emergence of highly
cornerstone to decreasing the incidence of C. difficile virulent and antimicrobial-resistant isolates (namely,
infection and preventing further spread. Although PCR ribotype 027 and PCR ribotype 078).4,5 It is
there is no consensus on the single best laboratory worth noting that both C. difficile ribotypes 078 and
method for the diagnosis of C. difficile infection, the 027 were later discovered in animals such as pigs, cows,
use of 2 or more techniques can improve diagnostic and horses. This discovery further supports the theory
accuracy, and it is recommended. of transmission of C. difficile from animals to humans.6
However, there is insufficient evidence to confirm direct
Keywords: C. difficile, nosocomial infection, C. difficile
infection, PCR ribotype 027, infection control transmission between animals and humans.7 Nowadays,
the detection of CDI presents a major challenge for
Saudi Med J 2023; Vol. 44 (9): 825-835 clinicians and clinical laboratories, as there is no
doi: 10.15537/smj.2023.44.9.20230216 consensus on the best laboratory detection method.8,9
Preventing C. difficile infection requires implementing

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 C. difficile nosocomial infection ... Al-Zahrani

various measures that include using contact precautions, Pathogenesis and virulence factors. Clostridioides
maintaining good hand hygiene, carrying out efficient difficile causes human infections that vary from
environmental cleaning, using sporicidal cleaning mild (usually diarrhea) to severe infections, such as
agents, and practicing antimicrobial stewardship.1,10 PMC. Clostridioides difficile is transmitted via the
Therefore, this review article highlights the significance oral-fecal route, and its spores are highly resistant
of C. difficile in hospital-acquired infections and to harsh conditions such as heat, disinfectants, and
provides valuable information that may contribute to antimicrobials.19 Spores can also provide additional
a better understanding of C. difficile epidemiology and protection for this obligate anaerobic organism from an
appropriate diagnostic methods, as well as efforts to oxygenated environment outside the host.19 Because of
control its infection. the ability of C. difficile to produce spores, gastric juice
does not affect the bacterium and spores can reach the
History. Clostridioides difficile is spore-forming, an intestines, then turn into vegetative cells, and colonize
obligate anaerobic Gram-positive bacilli bacterium. the large intestines.20 Antimicrobials, in particular broad-
These bacteria are found in nature (soil), and they spectrum antimicrobials, can disrupt the gut microbiota
usually inhabit the gastrointestinal tract of young and thus encourage C. difficile to grow and thrive,
animals and humans without causing disease form spores, and produce toxins. Toxigenic isolates of
(approximately 20% in some healthcare centers for C. difficile usually produce 2 types of exotoxins: the
the elderly and rehabilitation centers and up to 70% first is toxin A (TcdA), which is an enterotoxin and the
of healthy human neonates). Clostridioides difficile
second is toxin B (TcdB), which is cytotoxic. Both toxins
was first identified in 1935 in the fecal microbiota
affect mucous membranes of the colon, while TcdB is
of healthy newborns.8,11-13 Because the organism did
not appear to be associated with a human infection a key virulence factor causing CDI.3,21-23 Additionally,
during that time, it was disregarded until the advent C. difficile produces other virulence determinants that
of the era of antibiotic treatment. Pseudomembranous contribute directly and indirectly to the virulence of
colitis (PMC) was reported before the antibiotics era this pathogen. These determinants include colonization
and attributed to Staphylococcus aureus (S. aureus), and adherence factors (namely, surface proteins, flagella,
which was called “Staphylococcal enterocolitis’’. Hence, and fimbriae), biofilm formation and bacterial spread
oral vancomycin was prescribed as an effective option factors (namely, proteolytic enzymes).3,24 The clinical
to treat this disease. Beginning in the 1970s, Tedesco manifestations and the severity of CDI depend on the
et al14 found that an increase in severe diarrhea cases patient’s characteristics (namely, age and underlying
was associated with clindamycin treatment, which was diseases); the common symptoms include diarrhea,
called “clindamycin colitis’’. At that time, researchers abdominal pain, vomiting, fatigue, and loss of appetite.
were unable to culture S. aureus from the stool of the In severe cases, clinical features can be life-threatening,
patients, and they became discouraged. Since the end including fatal PMC, colon perforation, septic shock,
of the 1970s, many studies have reported the presence kidney failure, and death.19,25
of cytotoxin in stool specimens of pseudomembranous Antimicrobial resistance. Nowadays, antimicrobial-
colitis patients.15,16 In 1978, Bartlett et al17 published resistant C. difficile is a primary concern for the
the first article linking PMC disease and C. difficile healthcare community. Furthermore, the emergence of
cytotoxin production. The mortality rates associated C. difficile infection is usually associated with antibiotic
with CDI were not significant until the end of the use.26 Although C. difficile is a spore-forming bacterium
20th century. Nevertheless, at the beginning of the capable of surviving during antimicrobial therapy, it is
21st century, with the intersection of epidemiological also resistant to several antibiotics, particularly broad-
factors, the health status of the hosts, the extensive use spectrum antimicrobials such as aminoglycosides,
of antimicrobials, the emergence of highly virulent tetracyclines, erythromycin, clindamycin, penicillin,
C. difficile isolates, and the rates of CDI increased in cephalosporins, and fluoroquinolones.26 Fortunately,
terms of frequency and severity. Since then, CDI has fidaxomicin and vancomycin are still effective against
gained wide attention in the medical community in C. difficile, the former is recommended as the first-line
terms of treatment and laboratory diagnosis.18 choice for treating initial and first recurrence CDI,
while the latter is used for severe CDI.27-29 Moreover,
multidrug resistance is a common feature of many
Disclosure. Author has no conflict of interests, and the clinical C. difficile isolates, and this is attributed to the
work was not supported or funded by any drug company. accumulation of resistance due to extensive exposure
to frequently used antimicrobials.2,30 Antimicrobial

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resistance of C. difficile is multifactorial and ascribable bacterial DNA transcription by targeting the β-subunit
to the acquisition of resistance genes, alteration in of DNA-dependent RNA polymerase (RpoB).26
the antimicrobial target, gene mutation, and biofilm Clostridioides difficile has also developed resistance to
formation.2,26 Many studies have shown that the rate of rifamycins, with high levels of resistance being reported
antimicrobial resistance in C. difficile varies from study in several studies. Krutova et al43 reported that 65%
to another one (Table 1). Although C. difficile isolates of C. difficile isolates were rifampicin-resistant. Very
resistant to metronidazole and vancomycin are still recently, Mutai et al36 determined the prevalence of CDI
rare, some isolates have decreased susceptibility to both and evaluated antimicrobial resistance among C. difficile
antimicrobials.31,32 Metronidazole and vancomycin cases in Kenya and found that 91.5% of C. difficile
resistance mechanisms in C. difficile are not fully isolates were resistant against rifampicin. The primary
understood. However, recent evidence suggests that resistance mechanism to rifamycins is mutations in
metronidazole resistance may arise as a result of the β-subunit of the rpoB gene.26,34 Although there is
alteration in metabolic pathways (namely, the activity evidence that biofilm formation plays a vital role in the
of nitroreductases), DNA repair, and biofilm formation, antimicrobial resistance of C. difficile, more research is
while the vancomycin resistance is attributed to changes needed to clarify its specific contribution mechanism.
in amino acid of peptidoglycan biosynthesis-associated Clostridioides difficile’s genome. Its genome is
proteins (Table 1).2,26,33 Biofilm may also contribute to ~ 4.3 Mbp in length with a G + C content of 29%.
resistance to high concentrations of vancomycin.34 A Furthermore, a circular plasmid of 7,881 bp with a
multicenter study from the United States of America G + C content of 27% was identified. Clostridioides
revealed that approximately 36% of C. difficile isolates difficile contains a large pan-genome with 9000 coding
are resistant against clindamycin.35 Nevertheless, some sequences (CDS) and a large proportion of mobile
countries in Europe, Asia, and Africa have a higher rate genetic elements (MGEs, 11% in C. difficile strain
of resistance to clindamycin, 80.3% in Kenya, 74% 630).44,45 However, C. difficile has an ultra-low level of
in Spain, 73.3% in China, and 65% in Poland.36-39 core genome (between 16-24%), while other bacterial
Clostridioides difficile isolates also showed a relatively species have more than 60%.45-48 Many CDS identified
high rate of resistance against erythromycin (range: in C. difficile’s genome are responsible for its adaptation
13-100%).26,32,36 Clindamycin and erythromycin to the gastrointestinal tract and survival in the harsh
belong to the macrolide-lincosamide-streptogramin environment through endospore formation.49 Mobile
B family, which act on the ribosomal 50S subunit to genetic elements of C. difficile are mainly carried on
inhibit protein synthesis.32 Many bacterial pathogens conjugative transposons and insertion sequences that
can resist clindamycin and erythromycin by modifying encode antimicrobial resistance, 2 key virulence factors,
their ribosomal target or active efflux. Erythromycin toxins A (TcdA) and B(TcdB), and the production
resistance in C. difficile is commonly due to ribosomal of surface structures.49,50 These mobile elements are
methylation encoded by the ermB gene.32,40 In addition, exchanged with high frequency and contribute to
C. difficile is resistant to most cephalosporins, with a the diversity and plasticity of the C. difficile genome.
resistance rate between 11-100%. This resistance The genomic plasticity and variability of C. difficile
might be due to the production of β-lactamases and contribute to its extraordinary capacity to adapt to
modified penicillin-binding proteins (PBPs).2,26,30 different growth conditions for a long time in the gut
Fluoroquinolones (namely, ciprofloxacin) are broad- and recurrent infection.49
spectrum antimicrobials associated with CDI. Global Clostridioides difficile colonization. It is defined
studies on the antimicrobial resistance of C. difficile as the presence of an organism or its toxins without
showed that 77-99% of isolates were resistant to any symptoms of CDI, which can vary from mild
ciprofloxacin.41 Fluoroquinolones resistance in symptoms (usually diarrhea) to severe infections such
C. difficile is due to alteration in DNA gyrase subunits as PMC.51,52 The ingestion of C. difficile spores is
GyrA or GyrB.26,36,42 Clostridioides difficile’s resistance to the first stage of colonization; the spores can survive
tetracyclines varies between different countries ranging in gastric acid and reach the colon to germinate
from 2.4-62.7%.26,30,32 Resistance to tetracycline is into vegetative cells. Colonization of C. difficile is
achieved by generating a ribosomal protective protein prevented by the fecal microbiota barrier, which can
(TetM) and an active efflux pump system.2,26,42 Rifamycin be disrupted by antimicrobial use.20 The incidence of
and fidaxomicin have been recommended as alternatives C. difficile colonization varies depending on various
to treat recurrent CDI associated with treatment failure factors, such as the host, pathogen, and environment.53
of metronidazole and vancomycin.30 Rifamycins inhibit Previous hospitalization, antimicrobial exposure

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Table 1 - Mechanism of action and resistance of different antimicrobials and resistance rates against Clostridioides difficile.

Antimicrobials Targets Putative resistance mechanism (S) Resistances (%) Refrences

Alterations in some metabolic pathways, biofilm
Metronidazole Bacterial DNA 0-20.25 32,33,36,41
formation
Binding to D-alanyl-D-alanine residues Mutations in peptidoglycan biosynthesis-required
Vancomycin 0-41 32,33,36,41
precursor of peptidoglycan proteins, biofilm formation
Rifampicin Bacterial DNA-dependent RNA polymerase Mutations in rpoB 0-91 32,36,41
Alterations in ribosomal target or active efflux
Clindamycin 50s ribosomal subunit of bacteria 0-100 26,32,33,36,41
pumps
Alterations in ribosomal target or active efflux
Erythromycin 50s subunit of the bacteria 0-100 26,32,33,36,41
pumps
Tetracycline resistance protein (tetM) and the active
Tetracycline 30S ribosomal subunit of bacteria 0-62.7 26,32,33,36,41
efflux pumps
Moxifloxacin Bacterial DNA gyrase Alteration of the drug target 0-100 32,33,41
Blocking the ribosome by binding to
Fusidic acid Mutations in fusA 0-40 41
“factor G”
Alteration of the drug target and the active efflux
Ciprofloxacin Bacterial DNA gyrase 23-42 33,41
pumps

(cephalosporins), use of proton-pump inhibitors, risk factors.59,60 These changes have led to the emergence
immunosuppressive drugs, and the presence of of new hypervirulent isolates of C. difficile, such as the
antibodies against toxin B are the most commonly risk fluoroquinolone-resistant PCR ribotype 027/North
factors linked with C. difficile colonization.54,55 Although American pulsed-field gel electrophoresis type I (NAPI)
the colonization rate by C. difficile is still low among strains in North America and PCR ribotype 078 strain
healthy adults, it increases significantly in individuals in Europe.4,5 Although PCR ribotype 027 was linked
with previous hospitalization, particularly among to severe cases and higher mortality, other C. difficile
rehabilitation center patients. Moreover, underlying ribotypes have also been responsible for many outbreaks
diseases also contribute to the high percentage of worldwide.59 The hypervirulence of PCR ribotype 027/
asymptomatic C. difficile colonization.52 Unlike adults, NAPI strains was attributed to the polymorphisms in
a high rate of C. difficile colonization has been reported the receptor-binding domain of tcdB, resulting in a
in infants and neonates.56 The colonization process hypertoxic form of TcdB. Additionally, these strains
involves many factors, including immune evasion of produce a binary toxin that is linked to higher clinical
the host defenses and adhesion to the host mucous severity.61 Noteworthy, the earliest report of the PCR
membrane, usually facilitated by surface proteins. ribotype 027 was from a Parisian hospital in 1985, and
These surface layer proteins (SLPs) of C. difficile play the next record was from a Minneapolis hospital, on
a crucial role in their adherence to the mucus layer a non-epidemic strain specified BI-1 in 1988.62,63 A
in the intestine.24 Moreover, flagella of C. difficile are few years later, many CDI outbreaks with severe cases
important for colonization and host invasion.57,58 were reported in hospitals across the United States and
Although the relationship between CDI and C. difficile in Canada.63,64 The initial reports of PCR ribotype 027
colonization is not fully understood, the former is not outbreaks in Europe came from the Netherlands in
directly associated with the development of the latter.52 2005 and England in 2006, and afterward, there were
Noteworthy, colonization by toxigenic strains can several reports from various other European countries.65
increase the risk of developing CDI compared to being Nevertheless, the emergence of this clone and its rapid
colonized by non-toxigenic strains.51 global dissemination remained unknown until a global
Molecular epidemiology of C. difficile. Until the end set of C. difficile ribotype 027 clinical isolates collected
of the last century, C. difficile infection was a leading between 1985-2010 were sequenced. Genomic analysis
cause of antimicrobial-associated diarrhea among the revealed that 2 different epidemic lineages of ribotype
elderly in healthcare settings. However, over the last 027 (FQR1 and FQ2) with a similar fluoroquinolone
20 years, there has been a dramatic increase in reports of resistance mutation, had emerged, and disseminated
C. difficile infection, describing a high number of cases in North America within a short period.66,67 The
with considerable changes in clinical manifestations, 2 lineages showed different spread patterns. One spread
including more severe cases, many outbreaks, and novel throughout North America, Chile, Switzerland, and

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South Korea, while the other was reported broadly across detecting toxins A or B, and nucleic acid amplification
Europe and Australia (Figure 1).66,68 These findings shed test (NAAT; Table 2).
new light on the importance of fluoroquinolone use as Toxigenic culture. It is a 2-step technique based on
a selective pressure in the evolution and spread of the isolating C. difficile from stool and determining whether
FQR1 and FQ2 lineages within healthcare settings.67 the isolated strain is a toxin producer or not. There
Clostridioides difficile ribotype 027 strains have been are many approaches for accomplishing this purpose,
sporadically detected in China and Japan, but the including stool culture on selective and differential
Japanese strains are susceptible to fluoroquinolones, in media under prolonged anaerobic incubation
contrast to the fluoroquinolone-resistant ribotype 027 (~ 48 hours), identifying suspected colonies, and finally,
strains spreading in North America and Europe.60,69 the identified C. difficile isolates are subjected to toxin
Also, in the United States and Europe, CDI clusters testing.8,18 Although there is no consensus on the optimal
are often caused by ribotypes 001, 002, and 014/020.19 method for C. difficile culture from stool, a heat shock
Moreover, ribotypes 017, 018, 014, 002, and 001 are or alcohol treatment before direct plating on a selective
the most prevalent ribotypes in Asian countries (Japan, media (namely, cycloserine-cefoxitin-fructose agar or its
Korea, Hong Kong, Singapore, Taiwan, and China).70,71 variant) is recommended by many investigators.18,81-83
There are few molecular epidemiological studies on This is mainly to inhibit or minimize the growth of stool
C. difficile in Africa and the Middle East. Although organisms and enhance C. difficile spores.18 However,
many epidemiological studies on the prevalence of CDI other studies revealed that using an enrichment broth
in Saudi Arabia have already been carried out72-75, only containing antimicrobials, carbohydrates, lysozyme,
one study characterized the ribotypes of isolated strains; and taurocholate can increase the recovery of C. difficile
they detected the hypervirulent ribotype 027 in 4 elderly without requiring heat shock or ethanol pretreatment.8
patients in Riyadh.76 Furthermore, Al-Thani et al77 Additionally, several chromogenic media have been
found 258, 001, 014, 046, 011, 053, 056, and 107 developed to isolate C. difficile from stool samples.
were the most prevalent ribotypes in Qatar, and the Those chromogenic media showed variable sensitivity,
hypervirulent RT027 was detected in only one isolate. and they include chromID C. difficile TCCA,
Another study in Kuwait showed that ribotypes 139, chromID C. difficile TCCFA, chromID C. difficile
014, 056, 070, 097, and 179 were the most common CDSA, chromID C. difficile CCFA, and chromID
ribotypes.78 A recent study from Iran has revealed C. difficile CDSA.84 Thereafter, identification of
that ribotypes 001 and 126 were the most frequent isolate can be achieved by conventional methods such
ribotypes, and none of the hypervirulent ribotypes as the morphology of colonies, Gram staining, and
(027, 078) were reported.79 Moreover, the genotype of biochemical tests such as latex agglutination for GDH
hypervirulent isolates is not as clearly apparent in the or using Matrix-assisted laser desorption/ionization-
Middle East as it is in Europe and the United States. time of flight (MALDI-TOF) mass spectrometry
This can be attributed to the lack of information on the (MS).84,85 After identification of an isolate, its ability
ribotypes in circulation in the Middle East. Therefore, to produce toxins must be confirmed by testing the
further studies are required to pinpoint the prevalent supernatant for bacterial growth using a cell cytotoxicity
hypervirulent strains in this geographical region.79 neutralization assay or by a more rapid method such
Laboratory detection of C. difficile. Its symptoms as PCR to detect toxin genes.8,18,84,85 Although the TC
vary from asymptomatic colonization, and mild method requires a relatively long time (>2 days), it is the
symptoms to severe infections such as PMC and toxic recommended reference method for detecting toxigenic
megacolon. Clostridioides difficile infection is defined strains of C. difficile and is regarded as the gold standard
by the emergence of more than 3 unformed stools technique in evaluation studies.18,85
within 24 hours and is approved through a laboratory Cell cytotoxicity assay. It is regarded a standard
diagnosis for the presence of toxin-producing C. difficile technique for the detection of C. difficile toxins in a stool
isolate.80 For many clinicians and clinical laboratories, sample. This technique involves incubating stool filtrate
the identification of CDI poses a significant problem, with a proper cell line for 1-2 days and observing any
and there is still no agreement on the one approach cytopathic effects of toxins using a neutralization assay
that is the optimal laboratory detection method.8,9 with an antiserum against C. difficile toxin B or against
Currently, there are many laboratory tests for CDI the Clostridium sordellii toxins, which have similar
detection, including toxigenic culture (TC), cell antigens.18,85 Although CTA has acceptable sensitivity
cytotoxicity assay (CTA), enzyme immunoassay (EIA) and specificity and is inexpensive, it is currently used
for detecting glutamate dehydrogenase (GDH), EIA for only by a small number of laboratories due to its

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Figure 1 - Worldwide distribution of hypervirulent Clostridioides difficile PCR ribotype (RT) 027.

labor-intensive, long turnaround time and lack of this test lacks specificity and should be combined with
harmonization (cell type, dilution of stool specimen, another method (namely, toxin detection test).87
and incubation period).18,85,86 Nucleic acid detection techniques. Nucleic acid
Enzyme immunoassays. It uses specific monoclonal amplification assays (NAATs) are mainly based on
or polyclonal antibodies to detect toxins A and B, and real-time PCR, and there are several commercial
GDH antigens. Many commercial EIAs are readily platforms targeting a diversity of genes, involving
available, quick to produce results, and simple to use. tcdA, tcdB and 16S ribosomal RNA (rRNA).85,87 Of
For many years, EIAs that detect toxins A or toxin B were note, the first FDA-approved NAAT platform was
among the most detective tests for C. difficile isolates. not accessible in the United States until 2009, despite
They include solid-phase microwell, lateral flow NAATs for detecting C. difficile in stool first appearing
membranes, immunoassays in chromatographic in the literature in the early 1990s.8,18,85 Nucleic acid
cassettes, and chemiluminescent immunoassays.8,18,85 amplification assays are less sensitive than TC but
However, EIAs are not sensitive enough to identify more sensitive than toxin-based EIAs for C. difficile
toxigenic isolates of C. difficile compared with detection. However, depending on the prevalence of the
techniques such as toxigenic culture or cytotoxicity disease and the assay’s limit of detection, the positive
assays.85,87 Recently, an ultrasensitive immunoassay predictive value of NAATs for CDI ranges from low
for toxin A and toxin B was developed by Song et al88 to moderate.87 Nucleic acid amplification assays, on
that can serve as a standalone diagnostic test for CDI. the other hand, only identify the existence of toxin
Thereafter, many ultrasensitive immunoassays were genes and, consequently, the ability of C. difficile to
introduced for the detection of C. difficile toxins. Those produce toxins. Consequently, the major drawback of
assays can detect disease-specific markers at extremely NAATs is that they can also identify carriers of toxigenic
low concentrations with good accuracy.89 However, C. difficile who have no symptoms in addition to CDI
these ultrasensitive immunoassays require further cases.90 Moreover, genetic variation in the tcdB or tcdA
validation.8,89 Glutamate dehydrogenase immunoassays genes is another potential issue (Table 2), which might
identify a metabolic enzyme that is highly conserved lead to false-negative results. Therefore, the European
and abundant in all C. difficile isolates. Since GDH Society of Clinical Microbiology and Infectious
is found in both toxigenic and nontoxigenic isolates, Diseases guidelines advise against using NAAT as a

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single test to diagnose C. difficile and instead suggest the fecal-oral route, and old age, antimicrobial therapy,
using it as a screening test.85 Ultimately, NAATs are still and hospitalization are the most significant risk factors
more expensive than other detection methods such as associated with CDI.25 Non-antimicrobial medications
toxigenic culture or EIAs.8 such as acid-suppressing drugs (namely, proton pump
Infection control of C. difficile. Clostridioides difficile inhibitors and histamine-2 receptor antagonists) have
is considered the primary cause of nosocomial infections also been linked to an increased risk of CDI.93 In
and gastroenteritis-associated deaths.1 In the United addition to morbidity and mortality rates, CDI adds
States alone, C. difficile accounts for approximately considerable financial burdens to healthcare systems.
500,000 healthcare infections and approximately Therefore, C. difficile has gained global attention from
29,000 deaths every year.91,92 The European Centre the healthcare community.94 Healthcare workers also
for Disease Prevention and Control has reported that
play a role in transferring these bacteria among patients
approximately 124,000 patients in the European Union
and between different units.
develop CDI each year.19 In addition, C. difficile has
been linked to many outbreaks, some of which are Clostridioides difficile is endemic to many hospital
caused by new, highly virulent strains (namely, RT027 departments, elderly centers, and rehabilitation clinics,
[NAP1/BI]) that leads to more severe disease and worse especially under poor hygiene conditions, as well as with
patient outcomes. Furthermore, C. difficile spores the use of broad-spectrum antimicrobials. Therefore,
increase their ability to survive in hospital environments the best way to prevent CDI is to apply barrier
for a long time and to resist common environmental precautions such as effective hand washing before
cleaning agents and standard alcohol-based hand and after contact with all patients and wearing gloves,
disinfection; together, these factors contribute to the and the appropriate personal protective equipment.1
rapid spread of C. difficile in hospitals.1 Although However, the use of alcohol-based hand gels alone does
C. difficile initially appeared as a hospital-associated not destroy C. difficile spores.87 In addition, effective
infection, its epidemiology is constantly changing, and cleaning and disinfection of the hospital environment
currently, one-third of C. difficile cases are community- and medical instruments such as rectal thermometers,
acquired infections.92 This pathogen spreads mainly via colonoscopy devices, toilet chairs, handles, and any

Table 2 - Simplified comparison of various laboratory detection methods for the detection of Clostridioides difficile infection.

Techniques Criteria Advantages Limitations References

Simple Simple
Specimen Turnaround Sensitivity Specificity
to to
type time (%) (%)
perform interpret
Due to its high sensitivity,
it is recommended as
Long turnaround time; need
a reference method; it
TC Good Good Stool 2-7 days High Low to be combined with another 8,18,85
allows performing strain
method
typing and antimicrobial
suitability test
Has acceptable sensitivity Labor-intensive, long
Stool
CTA Fair Good 48-72 hours High High and specificity, and it is turnaround time, and lack of 18,85,86
filtrate
inexpensive harmonization
Lacks specificity, high false-
EIA for
Rapid Quick to produce results, positive rate, and should be
detecting Good Good Stool Low Low 8,18,85,87
2-6 hours and simple to use used in combination with
GDH
another method
Insensitive enough in the
EIA for detection of toxin-producing
detecting Rapid Quick to produce results, isolates of C. difficile
Good Good Stool Low Moderate 8,18,85,87
toxins A 2-6 hours and simple to use in comparison to other
or B techniques such as TC or
CTA. High false-negative rate
Identify carriers of toxigenic
C. difficile who have no
Rapid Low/ symptoms. Genetic variation
NAAT Good Good Stool High Rapid and highly sensitive 8,85,87,90
2 hours moderate in tcdB or tcdA genes might
lead to false-negative results.
Expensive
TC: toxigenic culture, CTA: cell cytotoxicity assay, GDH: glutamate dehydrogenase, NAAT: nucleic acid amplification test, EIA: enzyme immunoassay

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 C. difficile nosocomial infection ... Al-Zahrani

potential source of C. difficile are very important.1,10 control practices have fueled the development of this
Moreover, asymptomatic individuals of C. difficile are a pressing health issue. Therefore, the prudent use of
crucial source of pathogen spread to other patients who antimicrobials, particularly broad-spectrum agents, and
are more susceptible to the infection.95 Therefore, the the application of simple infection control measures,
early detection of carriers is vital to ensure rapid patient such as washing hands with soap, can significantly
treatment and to avoid the further spread of C. difficile.8 reduce the rates of C. difficile infection. Moreover, the
Additionally, restricting the use of antimicrobials early detection of these infections and an understanding
(particularly broad-spectrum forms that disrupt the of their epidemiological behavior based on accurate
balance of gut microbiota, promote C. difficile growth, laboratory diagnostic methods are the cornerstones to
and cause infection) is considered highly significant way decreasing the prevalence of C. difficile infection and
to reduce C. difficile infections.96 preventing its further spread. Although there is no
Fecal microbiota transplantation as a treatment agreement on the single best laboratory method for the
option for CDI. Fecal microbiota transplantation (FMT) diagnosis of C. difficile infection, the use of 2 or more
is the introducing of a liquid solution containing fecal techniques can improve diagnostic accuracy. Molecular
genotyping of C. difficile isolates is also an important
matter from a healthy individual into the recipient’s
step in controlling C. difficile outbreaks to recognize the
intestinal tract to alter his microbiota and confer
differences between sporadic and epidemic strains, and
benefit. The process typically involves selecting a donor
to monitor their epidemic spread.
who has no family history of autoimmune diseases or
cancer and then screening for any possible blood-borne Acknowledgment. The author gratefully acknowledge AME for
and fecal pathogens.97 It is not a modern method; it English language editing.
was utilized in the 4th century in China to treat several
maladies, including diarrhea.97 In the 1950s, FMT References
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