Clostridioides difficile
Topics
• Natural history
• Prevalence
• Introduction to C. difficile
• Pathogenesis and disease manifestations
• Risk factors
• Clinical features
• Logarithmic approach to lab diagnosis
• Methods of Lab diagnosis
• Antimicrobial resistance in C difficile
• Antimicrobial Susceptibility testing
• Treatment
• Prevention
Natural history
• Pseudomembranous colitis was first described in 1893.
• Clostridium difficile was first isolated from the stool of a healthy
infant by Hall and O’Toole in 1935. Named it Bacillus difficilis.
• 1970- Prospective study conducted in patients of developing
diarrhea after taking clindamycin.
• In 1974- Hafiz characterized the organism and described GDH
component.
• Te-Wen Chang discovered the cytotoxin assay for C difficile.
• In 1978, Bartlett et al. published a sentinel paper linking C difficile
toxin production to PMC in humans
How serious is the threat?
How serious is the threat?
How serious is the threat?
Reeference: Antibiotic Resistant
Threats 2019, Centre for Disease
Control
C. difficile- the formidable nosocomial
pathogen
• Anaerobic, gram positive, spore forming bacillus
• Soil, GI tract of humans and animals
• Exists in 2 forms- vegetative form and spores
• Route of transmission- Ingestion of spores of toxigenic C. difficile
• Colonizes 70% of infants in the first year of life
• Reduces to 6% in the second year and to 2% thereafter
Pathogenesis and Disease Manifestation
Contact with
Decreased Alteration of
spores of toxin + +
immunity normal colonic
producing
microbiota
strain
Pathogenesis and Disease Manifestation
CodY
Pathogenesis and Disease Manifestation
• Shrinkage and
Hexakisphosphat death of cells
e Binds to Rho and • Disruption of
intestinal
other GTPases epithelial barrier
• Release of pro-
inflammatory
cytokines
Pathogenesis and Disease Manifestation
THE BINARY
TOXIN
• Disruption of
cytoskeleton
• Colonization by
microtubule formation
• Increased disease
severity
Pathogenesis and Disease Manifestation
HYPERVIRULENT STRAIN
OF C DIFFICILE
• Ribotype 027 and 078
• Toxinotype III, ST-1,
B1/NAP1
• Fluoroquinolone resistance
• Higher sporulation rate
• Rapid internalization
• 20 fold increase in toxin
production
• Enhanced toxicity
• Binary toxin production
Pathogenesis and Disease Manifestation
Contact with
Decreased Alteration of
spores of toxin + +
immunity normal colonic
producing
microbiota
strain
Pathogenesis and Disease Manifestation
Bile Acid Transformation
Direct Antagonism
Alteration of
normal colonic
Stimulation of innate immune response
microbiota
Competition for nutrition
Risk factors
1. Age >64 years
2. Malnourishment
3. Co-morbid conditions e.g. hypertension, Diabetes mellitus
4. Recent hospitalization
5. Immunocompromised individuals
6. Antibiotic exposure
7. Use of proton pump inhibitors
8. Use of non- selective NSAIDs
9. Inflammatory bowel disease
10. Invasive gastro-intestinal procedures
11. Patients with hepatic and pancreatic diseases
12. Chronic kidney disease
Culprit antibiotics
Third generation cephalosporins
Carbapenems
Fluoroquinolones
Aminoglycosides Aminoglycosides
Fluoroquinolones
Third genera-
Car- tion
bapen- cephalospori
ems ns
Clinical Manifestations
Diarrhoea defined as watery, non-bloody,
Diarrhoea/Dysentry frequency of 3 or more stools over 24
hours
MILD
Pseudomembranous
Evidence of colitis
colitis Leucocytosis upto 15000 cells/ul
Serum creatinine upto 1.5x normal
MODERATE
Toxic megacolon
Evidence of colitis
Leucocytosis more than 15000 cells/ul
Death Serum creatinine > 1.5x normal
Temperature > 40C
SEVERE
Serum albumin <2.5 mg/dl
Extra-intestinal manifestations
• Rare and occur in hospitalized patients who often have severe comorbidities
• Mean incidence of 0.2 per 100 000 person-years
• Prosthetic joint infections, osteomyelitis, spondylodiscitis, splenic abscess,
brain abscess, pericardial fluid, pleural empyema, C. difficile bacteremias,
abdominal aneurysms, wounds particularly decubitus ulcers, perineal
necrotizing fasciitis
• Infections in the abdominal area result either from intestinal perforation after
infection or leakage after surgery
• Wound infections may result from contamination by feces
Laboratory diagnosis
METHOD SENSITIVITY SPECIFICITY
Toxigenic culture 90-100% 98-100%
Cell culture cytotoxicity 80-90% 99-100%
neutralization assay
Toxin immunoassay 65-85% 95-100%
Glutamate dehydrogenase 58-68% 80-96%
detection
Molecular platforms for 92-97% 100%
direct detection
Adjunctive tests ---------- ----------
Alogrithmic approach to lab diagnosis
Screening using GDH assay
Positive Negative
Sample reported as
Confirmatory test
negative
Enzyme Nucleic acid
Immunoassay amplification test
References: Diagnosis of Clostridium difficile infection: An ongoing conundrum for clinicians and clinical laboratories
cmr.asm.org; July 2015
Evaluating the Logarithmic approach
Advantages Disadvantages
• Better sensitivity • High turn around time for GDH
• Cost reduction positive samples
• Convenient approach for • Need for inventory, training,
laboratories without equipment quality control and maintenance
of competency of various assays
Culture
• Gold standard method for C. difficile detection in fecal
specimens
• Isolating organism from fecal specimen
• Determine whether isolated strain is toxigenic or not
• Anaerobic agar with selective and differential agents
• Pre- reduce the medium prior to inoculation
• Incubate under anaerobic conditions for minimum 48 hours
• Indicates toxin production only in vitro
C. difficile on CCFA
• Large
• Circular
• Yellow
• Spreading
• Translucent
• Horse barn odour
C. difficile on CCFA
Fluorescence shown by colonies of Clostridioides difficile when grown on
CCFA and exposed to UV light.
C. difficile on Brucella Blood Agar
Flat, translucent colonies of C. difficile on BBA; sensitive to metronidazole
Other media used
Cycloserine cefoxitin Cycloserine cefoxitin mannitol broth Cycloserine cefoxitin
fructose broth with taurocholate and lysozyme egg yolk agar
Other media used
Chrom Agar
Trypticase soy agar with 5% sheep
blood
Identification
Gram Stain- Gram positive bacilli with oval subterminal spores
Biochemical tests
Gelatin liquifaction
Fermentation of fructose,
cellobiose, mannitol, mannose
Esculin hydrolysis
Cell culture cytotoxic neutralization assay
Preparation of stool
filtrate
Applied on monolayer
of appropriate cell line
Incubate for 24-48
hours
Observe for cytopathic
• Degradation of toxin due to
effects
delay
• Cell lines used
Neutralisation assay
• Pretreatment of patient performed with
• Fecal filtrate preparation C.difficile/C.sordelli
antiserum
Toxin immunoassay
• Use monoclonal or polyclonal antibodies directed against C. difficile toxins.
• Commercially available as immunochromatographic/ lateral flow membrane
immunoassays or microwell assays
• Detect both toxin A and B
• Advantage- low cost per test
• Disadvantage- Poor sensitivity
Hetererogeneity in performance
Abundant false positive results
According to SHEA/IDSA, toxin immunoassays should no longer be used
as stand alone tests for diagnosis of C. difficile
Glutamate dehydrogenase detection
• Metabolic enzyme coded by gluD
• Produced by both toxigenic and non-toxigenic strains
• Important as a screening test
ADVANTAGES DISADVANTAGES
Minimal hands-on time Cross reactivity with GDH of
C.sordelli
Rapid TAT for negative results Inadequate as a diagnostic test
Regional or geographic differences Freeze thawed stool samples may
do not affect assay performance affect yield erroneous results.
Low cost per test
Molecular diagnosis
First nucleic acid amplification test (BD GeneOhm C Diff assay) for C. difficile
detection in stool samples was approved by FDA in 2009
NAAT started in 1990s
Most sensitive method for diagnosis
Conventional PCR targeting tcdA, tcdB and 16SrRNA genes
Concerns with NAATs- (i) differentiation of the carrier state from active infection;
(ii) potential for laboratory contamination
(iii) changes in test performance over time
(iv) cost-effectiveness
(v) the impact on infection control
(vi) their performance in pediatric patients.
Further development- Real time PCR, quantitative real time PCR
Adjunctive tests
Adjunctive tests and
biomarkers
Fecal lactoferrin Fecal calprotectin Cytokine analysis
Adjunctive tests
Fecal calprotectin
Fecal lactoferrin
Calcium binding Cytokine analysis
Iron binding protein
protein present in Elevated IL-1b and IL-8
found in neutrophils
cytosol of neutrophils levels in moderate to
Released following
Resistant to bacterial severe disease.
neutrophil activation
degradation Levels reduce on
Concentration
Levels between 50-150 resolution of the
proportional to
ug/g of stool indicate disease
number of
infection with enteric No commercial assay
neutrophils recruited
pathogens available for
Useful in assessing
Low sensitivity to be measurement of
severity of C. difficile
even used as a stand cytokine levels in stool
infection
alone screening test
How serious is the threat?
Antibiotic resistance in C. difficile
• Spores survive the antimicrobial therapy
• Recent statistics based on 30 antimicrobial susceptibility
studies - reveal that resistance to clindamycin (8.3% to
100%), cephalosporins (51%), erythromycin (13% to
100%), and fluoroquinolones (47%)
• 98% of ribotype 027 strains were resistant to moxifloxacin
• Ribotype 078 showed resistance to ciprofloxacin,
erythromycin, imipenem, and moxifloxacin according to the
CLSI
References: breakpoints
Update on Antimicrobial Resistance in Clostridium difficile: Resistance Mechanisms and Antimicrobial Susceptibility Testing
Zhong Peng, Dazhi Jin, Hyeun Bum Kim, Charles W. Stratton, Bin Wu, Yi-Wei Tang, Xingmin Sun
Published in Journal Of Clinical Microbiology, 2017
Antibiotic resistance in C. difficile
• Resistance-associated genes harbored in the bacterial chromosome
• Mobile genetic elements (MGEs)
• Alterations in the antibiotic targets of antibiotics and/or in metabolic
pathways in C. difficile
• Biofilm formation.
Antibiotic resistance in C. difficile
The Rates of C. difficile clinical isolates resistant to Metronidazole were
reported to be 0.11% (based on the CLSI breakpoint), 13.3% (based on the CLSI
breakpoint), and 18% (based on the EUCAST breakpoint) in Europe in 2011 to
2012, in the United States (Texas) in 2007 to 2011, and in Israel in 2014,
respectively
several alterations
in metabolic
pathways e.g.
nitroreductases,
iron uptake, and
DNA repair
Antibiotic resistance in C. difficile
Resistance to Vancomycin- ribotype 027 isolates, was 47% in
Israel based on the EUCAST breakpoint
A US-based national sentinel surveillance study- 17.9% of C.
difficile isolates were resistant to vancomycin based on the
EUCAST breakpoint
ribotype 027 strain with reduced susceptibilities to vancomycin and
metronidazole has disseminated across Israel and is now the
most common clinical strain isolated
changes in peptidoglycan
biosynthesis-associated
proteins such as MurG
Antibiotic resistance in C. difficile
Rifamycins Fidaxomicin Tetracycline Chloramphenicol
2.4% to
57% Only one
3.7%
isolate till date 41.67%
Mutations in the tet(M), tet(44
alterations catP gene
β subunit of )and tet(W)
of rpoB might
the rpoB gene
Antimicrobial Susceptibility Testing
Phenotypic methods Molecular methods
• Agar dilution • Whole Genome Sequencing
• Broth dilution • Proteomics
• MIC gradient diffusion
Agar Dilution method
• GOLD STANDARD
• Medium- Brucella agar supplemented with haemin (5ug/ml),
vitamin K (1ug/ml) and 5% laked sheep blood/ Wilkins Chalgren
agar
• Inoculum- 10 5 CFU/spot
• Incubation- 36+/- 1oC anaerobically for 42-48 hours
• Quality control- B. fragilis ATCC 25285
B. thetaiotamicron ATCC 29741
C. difficile ATCC 700057
E. lenta ATCC 43055
References: Clinical and Laboratory Standards Institute guidelines 2020
Agar Dilution method
Advantages Disadvantages
• It is an accurate and high- • Labor and time consuming
throughput assay • Requires skilled and experienced
• Choice of antibiotics tested is microbiology technologists
flexible and can be changed
according to clinical or • Batched and does not readily
investigational needs allow testing of individual
isolates one by one
• Easily established and inexpensive
• Suitable for large numbers of
isolates.
Broth microdilution= Agar dilution?
• Studies indicated that the broth microdilution method has a good
reproducibility of 100% and an agreement of 90 to 95% in
comparison to agar dilution
• easier to perform and more convenient for clinical use than agar
dilution
• multiple antibiotics are measured at one time with low costs
Gradient diffusion- Etest strip
• This assay has been widely used to identify antibiotic susceptibility
profiles for patient care in clinical sets and for surveillance of
antibiotic resistance in molecular epidemiology
• Significant differences in MICs between gradient diffusion agar
dilution method
• Etest is a convenient easy-to-use assay by which multiple
antimicrobials can be measured on a plate at the same time.
• Delivers quantitative results with exact MIC values
• Disadvantage of high cost
Molecular methods
• Rapid and accurate
• Whole-genome sequencing and proteomics have also been applied to study C.
difficile resistance with promising performance
• Direct testing on clinical samples overcomes the problem of difficult isolation
of causative organism from the specimens
• Takes into consideration the presence of more than one strain of the organism
in the same patient
Treatment of C. difficile infections
Treatment
Pharmacological
Non-pharmacological
agents
management
• Vancomycin
• Metronidazole • Fecal Microbiota
• Fidaxomicin Transplant (FMT)
• Nitazoxanide • Surgical
• Rifaximin management
• Immunotherapy
Severity Treatment
Mild to moderate illness Oral Metronidazole 500 mg 8 hourly for 10-14
days
Severe disease Oral Vancomycin 125 mg 6 hourly for 10-14
days
Severe disease with Oral Vancomycin 500 mg 6 hourly + IV
complications Metronidazole 500 mg 8 hourly
Rectal instillation
First recurrence Oral Metronidazole 500 mg 8 hourly for 10-14
days
Second recurrence Vancomycin pulse regimen
Fecal Microbiota Transplant
• Administration of a solution of fecal matter from a donor into the
intestinal tract of a recipient in order to directly change the recipient’s
gut microbial composition
• Treatment of refractory CDI not responding to medical therapy,
fulminating colitis or toxic megacolon
• The mixture can be administered through a nasogastric tube, nasojejunal
tube, esophagogastroduodenoscopy, colonoscopy, or retention enema.
• Average cure rate of 87–90%
Prevention of CDI
1.Isolate and initiate contact precautions for suspected or
confirmed CDI
2.Confirm CDI in patients
3.Perform environmental cleaning to prevent CDI
4.Develop infrastructure to support CDI prevention
5.Engage the facility antibiotic stewardship program
6.Consider use of supplemental interventions
Reference:- https://www.cdc.gov/hai/prevent/cdi-prevention-strategies
