Download Clostridium diseases Review Article

Review Article
Indian J Med Res 131, April 2010, pp 487-499
Clinical spectrum & pathogenesis of Clostridium difficile associated
diseases
Chetana Vaishnavi
Department of Gastroenterology, Postgraduate Institute of Medical Education & Research, Chandigarh, India
Received April 23, 2009
Clostridium difficile is the major aetiological agent of antibiotic associated diarrhoea and colitis. The
majority of hospitalized patients infected by C. difficile are asymptomatic carriers who serve as silent
reservoirs for continued C. difficile contamination of the hospital environment. C. difficile associated
disease (CDAD) is a serious condition with mortality up to 25 per cent in frail elderly people. C. difficile
infection may present itself in several forms with both colonic and extracolonic manifestations. Several
factors are involved in determining whether or not a patient develops C. difficile infection. These include
factors related to the pathogen as well as the host. Transmission of C. difficile can be endogenous or
exogenous. Colonization of the pathogen occurs when the gut flora gets disrupted due to various factors.
The main virulence factors for CDAD are the two potent toxins: toxin A and toxin B which share 63 per
cent of amino acid sequence homology and act on small guanosine triphosphate binding proteins. The
emergence of the global hypervirulent C. difficile strain has been a cause of concern. Diagnosis of CDAD
infection can be done by detection of C. difficile toxin in the stool specimen. Vancomycin is the drug of
choice for severely ill patient, whereas metronidazole can be used for mild to moderately ill patients.
Clinical spectrum, the factors precipitating CDAD, pathogenesis, diagnostic assay and treatment of the
disease are reviewed.
Key words Clinical spectrum - Clostridium difficile - diagnosis - pathogenesis - predisposing factors - treatment
carriers who serve as silent reservoirs for continued C.
difficile contamination of the hospital environment6.
However C. difficile-associated disease (CDAD) is
a serious condition with mortality up to 25 per cent
in frail elderly people7. C. difficile is now recognized
as the primary cause of hospital acquired colitis in
patients who receive antibiotics, chemotherapeutics or
other drugs that alter their normal flora.
Introduction
Clostridium difficile, a Gram-positive spore bearing
anaerobic bacteria is the major aetiological agent of
diarrhoea and colitis associated with antibiotics. Hall
and O’Toole1 originally identified the organism as a
component of normal colonic flora of newborn infants.
C. difficile is commonly present in the stools of 5 per cent
of healthy adults usually in low numbers2 and in about
15-70 per cent of infants3-5. The majority of hospitalized
patients infected by C. difficile are asymptomatic
When C. difficile was first discovered, infection by
the organism was regarded primarily as an outcome of
487
488
INDIAN J MED RES, April 2010
antibiotic intake and not as a life threatening disease.
During the recent outbreaks of CDAD in at least 12
hospitals in the entire Estrie region in Quebec, a threefold rise in the incidence of CDAD, and a higher number
of cases involving toxic megacolon, colectomy or death
have been reported8. The mutant hypervirulent strain
was typed as NAP1/BI/027 (North American PFGE
type I/restriction endonuclease analysis BI/ribotype
027)9 and was found to produce greater than 16 times
toxin A and 23 times toxin B in addition to the binary
toxin10. McDonald et al9 found NAP1/BI/027 strain
in eight institutions, in six different States in United
States and it represented more than 50 per cent of the
isolates from five institutions. This global epidemic
strain has also been reported to cause outbreaks in
parts of continental Europe11, Great Britain, The
Netherlands and Belgium12 with increased morbidity
and mortality. The risk for CDAD has increased not
only by usual factors, as pseudomembranous colitis
(PMC), toxic megacolon and perforations in C. difficile
was rare before 2002, but their incidence increased
dramatically after that, particularly due to emergence
of fluroquinolone resistant strains13.
C. difficile is also being reported more frequently
even from non hospital-based settings, such as from
the community14. Domestic as well as wild animals
are probably transmitting this as the same ribotypes
found in them were found to be associated with human
infection. In this review the clinical spectrum, the
factors precipitating CDAD, pathogenesis, diagnostic
assay and treatment of the disease are discussed.
Clinical spectrum
C. difficile, like virtually all bacterial enteric
pathogens, causes a spectrum of clinical conditions15
with both colonic and extracolonic manifestations. The
different manifestations are detailed below:
(a) Colonic manifestation
(i) Asymptomatic carriage: Colonization with C.
difficile is the presence of the organism in a person with
no clinical symptoms like diarrhoea. Asymptomatic
carriage of C. difficile is quite common in hospitalized
patients. Epidemiologic studies have reported that
10-16 per cent of hospital inpatients in high-risk
units become carriers after receiving antibiotics16.
Symptomatic disease is less often seen in carriers,
despite the fact that most of the C. difficile isolates
are toxin producing17. Riggs et al18 suggested that
asymptomatic carriers of epidemic and non epidemic
C. difficile isolates have the potential to contribute
significantly to disease transmission in long-term care
facilities. Asymptomatic carriage can be predicted by
taking into account certain clinical factors such as
recent antibiotic exposure or previous occurrence of
CDAD. Patients with C. difficile colonization and a
serum IgG response to C. difficile enterotoxin usually
become asymptomatic carriers.
(ii) C. difficile diarrhoea: Usually mild to moderate
diarrhoea, sometimes accompanied by lower abdominal
cramps is seen with C. difficile infection. Symptoms
usually begin during or shortly after antibiotic therapy.
Occasionally these may be delayed for several
weeks. C. difficile toxins can be usually detected
from faecal specimens, even though endoscopic and
histologic features may be normal in patients with mild
disease. The diarrhoea resolves with the stoppage of
antibiotics19.
(iii) C. difficile colitis: The most common clinical
manifestation of C. difficile infection is colitis without
pseudomembrane formation. This is a more serious
illness than benign or simple antibiotic-associated
diarrhoea and presents as malaise, abdominal pain,
nausea, anorexia, and watery diarrhoea. Abdominal
pain and cramps in some patients are relieved by
passage of stool19. Sometimes dehydration and a
low-grade fever with a systemic polymorphonuclear
leukocytosis may occur. Levels of lactoferrin released
from the secondary granules of intestinal leukocytes, as
well as other inflammatory markers rise significantly in
patients having advanced CDAD compared to patients
with a milder form of the disease20. Faecal lactoferrin
assay performed simultaneously with the C. difficile
toxin assay can help rule out asymptomatic carriage
of C. difficile21. A nonspecific diffuse or patchy
erythematous colitis without pseudomembrane may be
seen under sigmoidoscopy.
(iv) Pseudomembranous colitis (PMC): Finney22 was the
first to describe PMC as a post-operative complication
of gastroenterostomy. PMC is the classic manifestation
of full-blown C. difficile colitis and is accompanied by
similar, but often more severe symptoms than those
observed in colitis. The classic pseudomembranes,
which are raised yellow plaques ranging from 2-10 mm
in diameter scattered over the colorectal mucosa are best
revealed by sigmoidoscopic examination. White blood
cell counts of 20,000 or greater and hypoalbuminaemia
of 3.0 g/dl or lower may be observed in severely ill
patients23. Most patients with PMC have involvement
of the rectosigmoid area. However, colonoscopy
is required because as many as one third of patients
Vaishnavi: C. difficile pathogenesis
have pseudomembranes limited to the more proximal
colon24. In patients with hypoalbuminaemia or acquired
immunodeficiency syndrome a neutrocytic ascites with
low serum to ascites albumin gradient may occur25, 26
with ascites being the only presenting manifestation of
PMC.
(v) Fulminant colitis: C. difficile infections may
present as fulminant colitis in approximately 3 per
cent of patients and account for most of the serious
complications including perforation, prolonged ileus,
megacolon and death27. Patients with fulminant
colitis complain of severe lower quadrant or even
diffuse abdominal pain, diarrhoea, and distension
and some of them may exhibit high fever, chills and
marked leukocytosis. Diarrhoea may occur as a usual
symptom, but may be minimal in patients with ileus
as a consequence of which secretions accumulate
in the dilated, atonic colon. Severe protein-losing
enteropathy may result in hypoalbuminaemia. A patient
with toxic megacolon has a dilated colon with signs
and symptoms of severe toxicity that include fever,
chills, dehydration and high white blood count. On
plain abdominal radiograph the patient may also have
dilated small intestine with air-fluid levels mimicking
an intestinal obstruction or ischaemia or pseudoobstruction28. The risk of perforation and precipitation
of megacolon28 deters a barium enema examination.
However, computed tomographic scan of the abdomen
is most valuable in severe cases and those localized
to the proximal colon may reveal colonic distension,
thickening, pericolonic inflammation, or free air29, 30.
Signs and symptoms of bowel perforation may
be present in some patients with fulminant C. difficile
infection. Further morbidity and mortality can be
prevented in patients with fulminant C. difficile
colitis by aggressive diagnostic and therapeutic
interventions27. Though the risks of perforation are
generally uncommon, limited flexible sigmoidoscopy
or colonoscopy may be performed at the bedside31.
C. difficile infection has also been reported to be
involved in the exacerbation of ulcerative colitis32.
Hookman & Barkin33 observed that fulminant colitis
is reported more frequently during outbreaks of C.
difficile in patients with inflammatory bowel disease
(IBD) and carries higher mortality than those without
underlying IBD.
(vi) Recurrent CDAD: Recurrent CDAD is a difficult
clinical problem due to repeated recurrences of the
manifestation. The pathophysiology is not quite clear
and may be due to persistently altered faecal flora. Repeat
489
antibiotics may subsequently be unable to suppress C.
difficile overgrowth. Alternatively, impaired immune
response may also be responsible. It has been estimated
that approximately 15-20 per cent of patients treated
for CDAD, relapse following successful therapy34. This
condition is manifested by the sudden re-appearance of
diarrhoea and other symptoms usually within a week of
stopping treatment with vancomycin or metronidazole.
Patients who relapse once are at greater risk of further
relapses. McFarland et al35 reported a relapse rate of as
high as 65 per cent in patients who had suffered two
or more previous relapses. Relapse is generally not
related to antibiotic resistance because in some patients
re-infection can occur with the same or different strain.
The small bowel and the appendix may also act as
reservoirs of C. difficile spores that enter the colon and
result in relapse36.
The basis for variability in response to C. difficile
infection is not entirely clear, but host factors appear
to be more important than bacterial virulence factors37.
It was suggested that there might be an association
between the C. difficile strains, production of toxins,
and clinical manifestation of the infection38. However,
a few studies where C. difficile was not an epidemic
strain have shown that there was no difference between
strains causing symptomatic cases and asymptomatic
carriage39,40.
(b) Extracolonic features
Recent literature mentions that CDAD is no longer
limited to the colon. C. difficile may infrequently cause
disease in a variety of other organ systems and except
for bowel involvement and reactive arthritis most
of the cases do not appear to be strongly related to
previous antibiotic exposure though they are preceded
by specific or nonspecific gastrointestinal (GI) disease.
Some of the features of extracolonic diseases can be
summed up as follows:
(i) Small bowel: Jacobs et al41 reviewed literature on
extracolonic manifestation of C. difficile and revealed
that small intestinal C. difficile infections seem to be
increasing in incidence. Small bowel CDAD with
formation of pseudomembranes on ileal mucosa may
occur when previous surgery on it has been carried out
and is associated with a high mortality rate. Testore et
al42 examined jejunal specimens from 100 patients who
died without any immediate history of GI symptoms
and mucosal cultures in 3 cases treated with antibiotics
were positive for C. difficile. Boland and Thomson43
presented a case of C. difficile enteritis in a 42 yr old
490
INDIAN J MED RES, April 2010
patient with ileal pouch-anal anastomosis and flexible
endoscopy revealed copious amounts of mucus with
adherent pseudomembranes throughout pouch and
distal small bowel.
Navaneethan & Giannella44 reported that small
bowel involvement is more frequently reported in
IBD patients who have undergone total colectomy or
in patients with ileal-anal anastomosis. The increase
in the number of these patients may actually reflect an
increase in the rising incidence of C. difficile infection
in general or increasing virulence of the infecting
organism.
(ii) Bacteraemia: Like with other colonic bacteria, C.
difficile is also known to cause bacteraemia with about
20 per cent mortality45. Bacteraemia due to C. difficile
has previously been described in 14 patients with
underlying GI processes46. These authors also reported
a unique case of monomicrobial C. difficile bacteraemia
in a young woman with an underlying haematologic
malignancy but without any GI symptoms.
(iii) Reactive arthritis: C. difficile-related polyarticular
kind of reactive arthritis may involve joints of the
knee and wrist in about a 50 per cent of the cases47.
Reactive arthritis begins an average of 11.3 days after
the onset of diarrhoea and is a prolonged illness, taking
an average of 68 days to resolve41. Ducroix-Roubertou
et al48 reported a case of a monoarticular arthritis of the
left knee following PMC in a 45 yr old man, 8 days
after the onset of a C. difficile enterocolitis.
(iv) Miscellaneous entities: Other extracolonic
manifestations due to C. difficile include cellulitis,
necrotizing fascitis, osteomyelitis, prosthetic device
infections, intra-abdominal abscess, empyema,
localized skin infections, etc.
Factors precipitating CDAD
The following factors determine whether or not a
patient develops a C. difficile infection:
(a) General factors: An indepth review on established
and potential risk factors for CDAD has recently been
published49. Briefly these include (i) long duration or
multiple antibiotic intake; (ii) the nature of the faecal
flora; (iii) the size of the C. difficile population; (iv)
production of the requisite cytotoxins; (v) the presence
of other organisms that affect toxin expression or
activity, and (vi) the presence of host risk factors,
including advanced age, presence of a nasogastric
tube37, receiving anti-ulcer medication50, severe
underlying illness, prolonged hospital stay, use of
enemas, GI stimulants and stool softeners. Johnson &
Gerding51 derived a model of pathogenesis for infection
with C. difficile. They hypothesized that a patient
admitted to hospital was at negligible risk for infection
until an antimicrobial agent was administered. If during
or after treatment such a patient gets subsequently
exposed to C. difficile, he/she either develops CDAD
or becomes colonized without diarrhoea or potentially
does not get infected at all. But once established as an
asymptomatic carrier, a patient is at decreased risk for
CDAD. Patients are at continuous risk of exposure to C.
difficile during the period of hospitalization and become
vulnerable to infection after they have been exposed to
antimicrobials. The two most important components
essential for CDAD is exposure to antimicrobials
followed by exposure to C. difficile and majority of the
patients do not get ill with these till the third additional
factor related to host immunity, virulence of infecting
C. difficile strain or to type and timing of exposure
come into play.
(b) Specific factors: Immunosuppressive drugs
have also been reported to be associated with the
development of CDAD52,53. Faulty immune response to
C. difficile toxins has been quoted as one of the major
host factors predisposing patients to the development
of symptomatic CDAD54,55. Patients receiving
immunosuppressive drugs are debilitated and therefore
are unable to mount an effective IgG antibody response
against C. difficile toxin A thereby increasing the risk
for CDAD15.
C. difficile colonization is more frequent in
intensive care and oncology units, where broad
spectrum antibiotics and immunosuppression
are wide spread56. Administration of tacrolimus,
an immunosuppressive agent resulted in the
development of CDAD57. Ulcerative colitis patients
unresponsive to corticosteroids58 may require long
time immunosuppressive treatment, which may result
in multiple infections, inclusive of C. difficile59. Five
leukemic patients treated with immunosuppressives,
died from secondary complications of PMC60. As the
use of immunosuppressives increase, the incidence of
CDAD will also rise and may account for as many as
20 per cent of patients of CDAD without prior use of
antibiotics61.
Gastric acid suppressive use due to raised pH
of stomach results in increased risk of CDAD62,63.
Proton pump inhibitors (PPI) may thus contribute
to the pathogenesis of CDAD, due to increased
survival of spores. PPI use was a significant risk
Vaishnavi: C. difficile pathogenesis
factor for development of CDAD in a retrospective
case control study63. However, Pepin et al8 reported
that elevated risk of CDAD with PPI occurred only
in univariate analysis but not so after adjustment for
co-morbidities on multivariate analysis. Kaur et al50
found that BALB/c mice treated with PPI had a higher
experimental colonization with C. difficile, enhanced
myeloperoxidase activity as well as greater level of
epithelial damage, oedema and neutrophilic infiltrates
in the colon as compared to control untreated animals.
Jayatilaka et al64 in a five year study period found that
PPI usage correlated exactly with the overall annual
increased CDAD incidence and believed that the
widespread prescription of PPI could be responsible.
PPI therapy was reported as an independent and the
only risk factor associated with reported as an increased
length of hospital stay in CDAD patients65. Thus the risk
of CDAD in hospitalized patients receiving antibiotics
may be compounded by exposure to PPI therapy.
Administration of cancer chemotherapeutic agents
possessing antibacterial properties may also result
in sufficient disturbance of the intestinal microflora
to allow colonization with C. difficile. Emoto et al66
reported severe CDAD in 6.1 per cent of patients
receiving cisplatin based combination chemotherapy
for ovarian malignancies. Resnik & Lefevre67 described
development of fulminant C. difficile colitis in a 66 yr
old patient with ovarian cancer who received paclitaxel
and carboplatin chemotherapy. Kumar et al68 reported
that 32.7 per cent patients treated with methotrexate or
mesalamine for psoriasis were positive for C. difficile
toxins. Exposure to corticosteroids was also significantly
associated with an increased risk of CDAD relapse69.
Thus the combination of the environmental presence
of C. difficile in health care settings and the number
of people receiving antibiotics, immunosuppressives,
PPI or cancer therapeutics in these settings can result
in frequent outbreaks.
(c) Factors responsible for recent increase in CDAD in
the West: Zerey et al70 demonstrated that the incidence
of C. difficile infection was increasing in surgical
patients in United States and was most prevalent after
emergency operations particularly among patients
having intestinal tract resections.
The increased incidence of nosocomial CDAD
in the West with marked increase in severity of cases
requiring colectomy or ending in death was attributed
to liberal use of fluroquinolones and cephalosporins71.
The NAP1/BI/027 strain poses a great risk as it is also
491
found to be resistant to fluroquinolone. In 2007, severe
cases of CDAD with this epidemic strain was detected in
Germany for the first time and was strongly associated
with receipt of cephalosporins and fluroquinolones in
the 3 month before onset of symptoms72.
Pathogenetic mechanisms
Transmission of C. difficile occurs both
endogenously as in the carrier state and exogenously
through nosocomial source 73. When an individual is
exposed to C. difficile or its spores, an initial disruption
of the normal colonic bacterial flora occurs resulting in
colonization of the organism74 through surface proteins.
Damage to enterocytes due to C. difficile toxins occur
as soon as C. difficile colonizes the intestine resulting
in cytoskeletal changes and the release of fluids and
inflammatory products75. Pathogenic C. difficile
produces two high molecular weight potent toxins - A
and B - which bind to specific receptors on the luminal
aspect of the colonic epithelium76. The role of surface
proteins and toxins of C. difficile in the pathogenesis of
CDAD are detailed below:
(a) Surface proteins of C. difficile: Colonization
process is thought to be a necessary preliminary
step in the course of C. difficile infection. Calabi et
al77 investigated tissue binding of C. difficile surface
layer proteins (SLPs) which are the predominant
outer surface components encoded by slpA gene.
They demonstrated that SLPs play a role both in the
initial colonization of the gut by C. difficile and in the
subsequent inflammatory reaction. Different adhesins
implicated in the colonization process of C. difficile
are (i) flagella, composed of the flagellin Fli C and the
flagellar cap protein Fli D, involved in cell and mucus
attachment (ii) a cell-surface protein with adhesive
properties, Cwp 66 (iii) a fibronectin-binding protein,
Fbp68 and (iv) s-layer protein.
These adhesins are able to induce an immune
response, which could play a role in the defense
mechanism of the host78. Janoir et al79 observed that
cwp84, a surface protein exhibited proteolytic activity
which could contribute to the degradation of the host
tissue integrity and to dissemination of the infection.
(b) Role of C. difficile toxins: The main virulence
factors for CDAD are the two potent toxins - toxin
A and toxin B that share 63 per cent of amino acid
sequence homology. Both the toxins induce mucosal
injury and colitis as seen by neutrophil infiltration,
which is a prominent feature of CDAD34.
492
INDIAN J MED RES, April 2010
Toxin A is an enterotoxin that causes haemorrhage
and fluid secretion in the intestines of rodents whereas
toxin B is a cytotoxin detectable by its cytopathic
effects in tissue culture2. However, both toxins affect
the cytoskeletal features, even though their activity
differs in potency. Toxins A and B have been shown by
nucleotide sequencing to be located in close proximity
to each other80 within a 19.6 kbp pathogenecity locus
(PaLoc) encoded by two separate genes (tcdA and tcdB)
on the same chromosome. C. difficile can be divided
into 24 toxinotypes based on the changes in both toxin
genes81. Some toxinotypes possess a third kind of toxin
known as the binary toxin82 described elsewhere in this
review.
The toxins get transported into the cytoplasm where
these act on small guanosine triphosphate binding
proteins known as the Rho proteins. The Rho proteins
are associated with actin polymerization, maintenance
of the cytoskeletal architecture, and the cell movement83,
84
. A severe inflammatory reaction in the lamina propria
with the formation of micro-ulcerations of the colonic
mucosa that is covered by a pseudomembrane occurs
due to the activity of the toxins85.
(i) Toxin A - Toxin A is a 308-kDa lethal enterotoxin
and minute quantities can stimulate fluid secretion in
animal intestinal loops86 similar to cholera toxin though
the mechanism of action is quite different87. Toxin A
causes extensive damage to the epithelial lining of the
intestine and accounts for nearly all of the GI symptoms.
The villus tips of the epithelium are initially disrupted
followed by damage to the brush border membrane.
Katyal et al88 reported a disturbance in the intestinal
brush border membrane enzymes in CDAD patients.
Denuding of the mucosa eventually is accompanied by
extensive neutrophil infiltration resulting in massive
inflammation. The fluid response is partly an outcome
of the damage to the intestinal epithelium. Toxin A also
acts as a cytotoxin resulting in disruption of the tight
junctions of the intestinal epithelium and might be an
important mechanism of toxin A enterotoxicity89.
Toxin A also elicits the production of various
cytokines and neurokinins, the biological reactants,
which are believed to be playing an important role in
pathogenesis90. Purified toxin A has potent effect on
human colonic epithelial cells as seen in vitro. Toxin
A initially induces cell rounding which results in
detachment of the cell from the basement membrane,
followed by apoptosis. Toxin A also brings about a
rapid loss of resident cells such as macrophages, T cells
and eosinophils and induces changes in the shape of
adherent polymorphonuclear leukocytes. At least two
pathophysiologic pathways are involved in changes
in the epithelial cell barrier via glycolysation of the
Rho proteins. These are (i) disaggregation of actin
microfilaments leading to epithelial cell destruction
and opening of tight junctions, and (ii) early release
of proinflammatory cytokines from intestinal epithelial
cells probably via activation of mitogen-activated
protein kinase91. The spherical cells become thin and
rope like with rearrangement of F-actin cytoskeleton
into aggregates92. Thus the toxins alter the actin
cytoskeleton, cause epithelial cell damage and result in
increased permeability of the tight junctions. A severe
acute necro-inflammatory reaction is produced by toxin
A in the intestine resulting in activation of mast cells,
vascular endothelium, and immune cells91.
(ii) Toxin B - Toxin B is also a very large and
potent cytotoxin with 279 kDa molecular weight. It
causes a number of non specific in vitro responses in
mammalian cells. This includes disorganization of the
actin filaments, loss of intracellular potassium, decrease
in the level of protein and nucleic acid synthesis93.
Under normal physiological conditions toxin B by
itself cannot cause damage or a fluid response in
intestinal loops94 probably due to its inability to bind
to the specific carbohydrate receptors on the intestinal
brush border membrane. After toxin A has bound to
the receptor initiating the damage, toxin B joins in and
gains access to the underlying tissue as supported by
animal experimentations94.
The cytotoxic activity of toxin B is similar to
that of toxin A, but is 1000-fold more potent than the
former. There is formation of neurite-like retraction
fibers resulting in partial detachment of cells. Next the
cell-spanning stress fibers disappear and the remainder
of the actin filaments accumulates in the perinuclear
space95,96. Both toxins disrupt the function of the Rho
family of protein97. Decreased transepithelial resistance
and increased flux of paracellular marker such as
mannitol and raffinose98 indicate the disruption of the
tight junction.
(iii) Binary toxin - Another toxin, which is an iotalike toxin, was described from the C. difficile strain
CD19699 and has been named binary toxin CDT. Binary
toxin contains both toxins A and B and is a product of
both toxin genes (cdtB for the binding component and
cdtA for the enzymic component). It is located on the
chromosome outside the PaLoc and is actin-specific
Vaishnavi: C. difficile pathogenesis
ADP-ribosyltransferase toxin that could be acting
synergistically. It has not been found to be essential
for eliciting C. difficile associated colitis. Binary toxin
CDT is produced by most of C. difficile isolates with
mutations in the tcdA and tcdB genes100 and up to 2 per
cent A-B- strains of C. difficile are estimated to produce
only binary toxin CDT101.
Diagnosis by toxin assay: C. difficile toxins can be
detected in the faecal samples by several methods as
mentioned below:
(i) Tissue culture: Tissue culture can detect as little
as 1.0 pg of toxin B making it the most sensitive test
available and has therefore been regarded as the gold
standard in laboratory diagnosis of C. difficile toxin.
Toxin identification can be confirmed with C. difficile
antitoxin or antitoxin against C. sordellii, which
produces the cross-reacting toxins102. Commercial tests
recommend a final dilution of 1:40 to 1:50 of the stool
sample103. Cell lines that may be used include Vero,
Hep 2, Chinese hamster ovary, HeLa cells and MRC-5
lung fibroblasts.
Many disadvantages accompany tissue culture
technique because it is the least controlled test.
Specimens may at times cause nonspecific cell
rounding that is neutralized not only by the specific
antitoxin but also by neutral serum. The addition of
too much faecal material to the tissue culture well
can cause false positive reactions. The maintenance
of cell cultures is also very difficult. The procedure
is cumbersome, expensive and time consuming and
requires a well-developed laboratory. Sometimes a
non specific cytopathic effect can also occur in a few
of the cases due to a viral agent or another bacterial
enterotoxin such as that of C. perfringens, rendering
any interpretation difficult. False negatives can occur
in stored samples due to several reasons such as (i)
toxin degrading enzymes, (ii) delay in transportation
of sample, (iii) medication of the patient, and because
(iv) some cell lines are less sensitive than others to
the cytopathic effect of the toxin. Infact, a negative
cytotoxicity assay does not completely rule out C.
difficile as the cause of diarrhoea.
(ii) Counter immunoelectrophoresis: This method for
detection of toxin is expensive, cumbersome, and lacks
the required levels of sensitivity and specificity for
satisfactory diagnostic test.
(iii) Latex agglutination test: Commercially available
latex agglutination test (LAT) is rapid, but is
493
unaffordable for routine use because of the high cost per
test. Moreover commercially available LAT is known
to detect a non toxic marker antigen for C. difficile and
therefore frequently results in false positive reactions.
Now a days commercial LAT is less frequently available
and has been replaced by enzyme-immunoassays.
(iv)
Enzyme
immunoassays:
Enzyme-linked
immunoassays (ELISA) are commercially available to
detect either toxin A alone or both toxins A and B in stool
specimens. ELISA has sensitivity and specificity ranges
of 50 to 90 per cent and 70 to 95 per cent respectively.
About 100 to 1000 pg of toxins must be present for the
test to be positive. The advantage of using ELISA is
because of the lesser time required for the test. But the
high cost per single test may necessitate batching of
samples. A high percentage of indeterminate readings
may also occur.
Most ELISA have a sensitivity of more than 80 per
cent compared to that of tissue culture assay. However,
ELISA that detect only toxin A may miss out on toxins
from A-B+ isolates resulting in wrong interpretation.
Thus, ELISA that detect both toxin A and B are
recommended to detect these atypical isolates. Such
tests will also take care of specimens containing low
levels of toxin A and B.
(v) Dot immunobinding assay: The test is carried out on
the surface of individual membrane cassette employing
the principle of enzyme immunoassay (EIA). Stool
supernatant is made to pass through a filter onto the
membrane and then made to react to mouse monoclonal
antibody to C. difficile toxin. Appropriate enzyme
conjugate and substrate are added to visualize the blue
coloured dot. Sometimes the presence of excessive
amount of debris can cause difficulty in interpretation
of the results.
(vi) Rapid membrane tests: These are lateral flow
devices with coloured conjugates or flow through
formats that require multistep processing. Such tests
utilize peroxidase tagged antibodies and a wash step
followed by the addition of a substrate. The sample
preparation for these tests requires centrifugation or
filtration. These tests have sensitivity in the range of
60 to 89 per cent. These tests are toxin A specific and
therefore do not detect A- B+ isolates.
(vii) Polymerase chain reaction: The polymerase chain
reaction (PCR) technique is used to detect enterotoxin
or toxin B gene in isolates or faeces and has sensitivity
similar to cytotoxin testing. The advantage of PCR
494
INDIAN J MED RES, April 2010
is the rapidity of the test. But PCR needs appropriate
infrastructure and technical expertise, and is timeconsuming. Most assays target only one of the two
genes, potentially missing isolates carrying only one of
them. Feces may contain PCR inhibitory components,
which can cause difficulties in the assay. Moreover,
PCR detects even minute number of C. difficile genome
copies present even in healthy individuals thereby
overemphasizing the aetiology.
(viii) Immunochromatography assay: This technique
is a single test EIA for detection of toxins A and B
in faecal samples. It can be done within 20 min and
without any requirement of pre-treatment.
(ix) Loop mediated isothermal amplification: Loop
mediated isothermal amplification (LAMP) is a rapid
and simple method for detecting toxin B gene in stool
samples as well as in isolates. Detection of tcdB by
LAMP from overnight cultures in cooked meat medium
could be an alternative method of diagnostic testing at
clinical laboratories without special apparatus. Even
though the technique is easier to perform it is not as
sensitive as the PCR104.
Therapy and management
Withdrawal of the antibiotic therapy that
precipitated the disease or at least changing antibiotic
regimens results in early resolution of the diarrhoeal
symptoms even in some cases of established PMC.
Fluid replacement and electrolyte balance maintenance
is important. About 25 per cent of patients respond
within a few days to these simple measures105. In case
of non response, they should be treated with specific
antimicrobial therapy, which is crucial to prevent the
progression of C. difficile pathogenesis. The drug of
choice for seriously ill patients is oral vancomycin
because it has no side effect and is not absorbed by
the intestine. Diarrhoea generally resolves over
an average of 5 days even though up to 50 per cent
relapse rate may occur after vancomycin treatment106.
Vancomycin administered in the form of capsules, help
to camouflage its bitter taste. However, in seriously
ill patients, oral suspensions help to achieve high
concentrations in the colon more quickly. Vancomycin
can also be administered by nasogastric tube, a long
intestinal tube, by enema, or by direct instillation
through a colostomy or ileostomy in patients too
ill for oral therapy. Intravenous administration of
vancomycin to patients unable to take oral medication
can be done with the hope that some of the drug will
reach the colonic lumen through the inflamed mucosal
surface. Other desperate measures in patients who
continue to do poorly are cecostomy or colectomy.
Some patients develop a series of relapses, thereby
extending the illness. Use of vancomycin in routine is
discouraged because it is expensive and there is a risk
of development of vancomycin-resistant enterococci.
Oral metronidazole can be used in place of
vancomycin and is favoured because it is less expensive.
Randomized trials show excellent initial responses
in approximately 95 per cent of patients treated with
metronidazole107. But, the disadvantage of the drug
is the near complete absorption such that the levels
achieved in the colon are virtually nil. Resistance to
metronidazole has been found in some isolates of C.
difficile. Metronidazole therefore is used for patients
with mild or moderate illness but should not be used
for critically ill patients.
Antibiotics that are active against C. difficile include
ampicillin, bacitracin, fusidic acid and teicoplanin,
which have been tried with little success. C. difficile
also shows good in vitro susceptibility to various
antimicrobials, such as rifaximin, ramoplanin and
nitazoxanide and these could be used in future trials.
Antimicrobial treatment also kills the normal bacterial
flora thereby causing the disease to recur. Once the
colon has been injured, it seems to be more susceptible
to reinfection. Nelson108 suggested that in order to
improve the patient’s clinical condition and prevent the
spread of C. difficile infection to other patients if one
does decide to treat, one should choose the antibiotic
that brings both symptomatic and bacteriologic cure
and that teicoplanin appears to be the best choice.
Limited success can be achieved by administration of
intravenous gamma globulin. Probiotics like Lactobacilli
species or Saccharomyces boulardii can also be used
to replace the pathogenic C. difficile flora. Even rectal
instillation of fresh stool in saline from living related
donors and rectal instillation of mixed broth cultures
of stool flora have been tried to replenish the normal
flora109. Binding agents like cholestyramine, Isabgol
husk and tolevamer may also bind to administered drugs
and delay elimination of C. difficile toxins2.
Parenteral administration of C. difficile toxoid vaccine
might protect high-risk individuals against CDAD by
development of antibody response.
Indian experience with CDAD
The prevalence of C. difficile-associated colitis is
global and the incidence varies considerably from place
Vaishnavi: C. difficile pathogenesis
to place. In India, the studies on C. difficile-associated
diarrhoea have been limited, probably due to the lack of
technology and the difficulty in culturing the pathogen.
Available reports from India estimate a prevalence of
15-30 per cent of paediatric and adult patients taking
antibiotics110-114. Gupta & Jadav115 reported 25.3 per
cent isolation of C.difficile from diarrheal patients of
all age groups. Niyogi et al116 reported C. difficile in 8.4
per cent and cytotoxin in 7 per cent of faecal samples
in children between 0-14 yr of age. C. difficile was the
only pathogen in 7.3 per cent of patients with acute
diarrhoea whereas 3.1 per cent control children without
diarrhoea harboured the organism. Niyogi et al117
isolated C. difficile in 11 per cent hospitalized patients
with diarrhoea and 2.9 per cent non diarrhoeic controls;
87 per cent isolates produced cytotoxin even though
the diarrhoeic patients had no history of antibiotic use.
Bhattacharya et al118 investigated 233 patients with acute
diarrhoea and isolated C. difficile as a sole pathogen
from 7.3 per cent, of which, 82.4 per cent produced
cytotoxin. In another study Niyogi119 reported that of
the 43 C. difficile isolates, 100 per cent were inhibited
by low concentrations of metronidazole, penicillin G,
tetracycline and ampicillin, but were highly resistant
to gentamycin, trimethoprim, sulphamethoxazole,
nalidixic acid, cycloserine and cefotaxime.
Kochhar et al120 demonstrated that infectious
agents like C. difficile were responsible for some of
the exacerbations in ulcerative colitis patients without
any history of recent exposure to antimicrobial drugs
or hospitalization. Vaishnavi et al112 reported 30 per
cent positivity for C. difficile toxin in hospitalized
patients of all age group receiving single to multiple
antibiotics for various ailments, but only in 7 per cent
of samples from patients not receiving antibiotics.
When only adult population were investigated, the
positivity for C. difficile toxin was 19.4 per cent in
the antibiotic receiving hospitalized patients121. Kang
et al122 reported that C. difficile-associated diarrhoea
was more common in the post transplantation period in
India than in developed countries.
Gogate et al123 observed that C. difficile was an
important pathogen for antibiotic associated diarrhea
in children of age group 5-12 yr. Vaishnavi et al124
reported the association of C. perfringens with antibiotic
associated diarrhoea either by itself or in synergy with
C. difficile infection. Balamurugan et al125 reported
overgrowth of C. difficile in the stool of Indian patients
with ulcerative colitis compared to healthy controls
using real time PCR. A decrease in the number of C.
495
difficile positive cases has been reported during a 5 year
study period and attributed the reduction to stringent
surveillance and an improved antibiotic policy adopted
in the hospital126.
Conclusions
C. difficile associated disease is a growing
nosocomial and public health problem. Hospitalized
patients receiving antibiotics for their ailments are
at great risk of acquiring CDAD. Clinical suspicion
is more important than ever before because stool
assays for diagnosing CDAD are not widely available.
Wherever available it is fraught with inherent problems
and therefore diagnosis may be missed or delayed.
Several measures significantly reduce the incidence
of CDAD. Infection control procedures that should
be followed to prevent spread of the disease include
environmental hygiene, washing hands with ordinary
soap and water or using 0.03 per cent Triclosan and
isolating patients with CDAD. Environmental cleaning
should be done with phenolic disinfectant. Isolated
patients should have equipment dedicated to them to
reduce cross-contamination. Infection control plays a
key role in controlling CDAD outbreaks. The spores
of C. difficile can survive in the hospital environment
for months, providing a reservoir for new infections.
Active and aggressive surveillance activity is the
key to reduce incidence. Monitoring should enable
the development and implementation of policies and
procedures that minimize the risk of this nosocomial
pathogen. Preventing C. difficile infection offers
a potentially significant improvement in patient’s
outcomes, as well as a reduction in hospital costs and
resource expenditures.
References
1.
Hall IC, O’Toole E. Intestinal flora in newborn infants with
description of a new pathogenic anaerobe. Am J Dis Child
1935; 49 : 390-402.
2.
Fekety R, Shah AB. Diagnosis and treatment of Clostridium
difficile colitis. JAMA 1993; 269 : 71-5.
3.
George RH, Symonds JM, Dimock F, Brown JD, Arabi Y,
Shinnagawa, N et al. Identification of Clostridium difficile as
a cause of pseudomembranous colitis. BMJ 1978; 1 : 695.
4.
Larson HE, Price AB, Honour P, Borriello, SP. Clostridium
difficile and the etiology of pseudomembranous colitis. Lancet
1978; 1 : 1063-6.
5.
Falson E, Kaijder B, Nehls L, Nygreu B, Svedham A.
Clostridium difficile in relation to enteric bacterial pathogens.
J Clin Microbiol 1980; 12 : 297-300.
6.
Johnson S, Gerding DN. Clostridium difficile. In: May Hall
GC, editor. Hospital epidemiology and infection control, 3rd ed.
Philadelphia: Lippincott Williams &Wilkins; 2004. p. 623-32.
496
INDIAN J MED RES, April 2010
7.
Crogan NL, Evans BC. Clostridium difficile: an emerging
epidemic in nursing homes. Geriatr Nurs 2007, 28 : 161-4.
8.
Pepin J, Saheb N, Coulombe M, Alary M, Corriveau M,
Authier S, et al. Emergence of fluoroquinolones as the
predominant risk factor for Clostridium difficile-associated
diarrhoea: a cohort study during an epidemic in Quebec. Clin
Infect Dis 2005; 41 : 254-60.
9.
McDonald LC, Killgore GE, Thompson A, Owens RC,
Khazakova Jr, Sambol SV, et al. An epidemic, toxin genevariant strain of Clostridium difficile. N Engl J Med 2005;
353 : 2433-41.
10. Warny M, Pepin J, Fang A, Killgore G, Thompson A, Brazier
J, et al. Toxin production by an emerging strain of Clostridium
difficile associated with outbreaks of severe disease in NorthAmerica and Europe. Lancet 2005; 366 : 1079-84.
11. Kuijper EJ, Coignard B, Tull P. Emergence of Clostridium
difficile associated disease in North America and Europe. Clin
Microbiol Infect 2006; 12 : 2-18.
12. Cloud J, Kelly CP. Update on Clostridium difficile associated
disease. Curr Opin Gastroenterol 2007; 23 : 4-9.
13. Riley TV. Epidemic Clostridium difficile. Med J Australia
2006; 185 : 133-4.
14. Dial S, Kezouh A, Dascal A, Barkun A, Suissa S. Patterns
of antibiotic use and risk of hospital admission because
of Clostridium difficile infection. Can Med Assoc J 2008;
179 : 767-72.
15. Kyne L, Warny M, Qamar A, Kelly CP. Association between
antibody response to toxin A and protection against recurrent
Clostridium difficile diarrhoea. Lancet 2001; 357 : 189-93.
16. Johnson S, Kent SA, O’Leary KJ, Merrigan MM, Sambol SP,
Peterson LR, et al. Fatal pseudomembranous colitis associated
with a variant Clostridium difficile strain not detected by toxin
A immunoassay. Ann Intern Med 2001; 135 : 434-8.
17. Shim JK, Johnson S, Samore MH, Bliss DZ, Gerding, DN.
Primary symptomless colonisation by Clostridium difficile
and decreased risk of subsequent diarrhoea. Lancet 1998;
351 : 633-6.
18. Riggs MM, Sethi AK, Zabarsky TF, Eckstein EC, Jump RL,
Donskey CJ. Asymptomatic carriers are a potential source
for transmission of epidemic and nonepidemic Clostridium
difficile strains among long-term care facility residents. Clin
Infect Dis 2007; 45 : 992-8.
19. Farrell RJ, LaMont JT. Pathogenesis and clinical manifestations
of Clostridium difficile diarrhea and colitis. Curr Top Microbiol
Immunol 2000; 250 : 109-25.
20. Steiner TS, Flores CA, Pizarro TT, Guerrant RL. Fecal
lactoferrin, interleukin-1beta, and interleukin-8 are elevated
in patients with severe Clostridium difficile colitis. Clin Diagn
Lab Immunol 1997; 4 : 719-22.
21. Vaishnavi C, Bhasin D, Kochhar R, Singh K. Clostridium
difficile toxin and faecal lactoferrin assays in adult patients.
Microbes Infect 2000; 2 : 1827-30.
22. Finney JM. Gastroenterostomy for cicatrizing ulcer of the
pylorus. Bull John Hopkins Hosp 1893; 4 : 53-5.
23. Gebhard RL, Gerding DN, Olson MM, Peterson LR, McClain
CJ, Ansel HJ, et al. Clinical and endoscopic findings in
patients early in the course of Clostridium difficile-associated
pseudomembranous colitis. Am J Med 1985; 78 : 45-8.
24. Tedesco FJ. Treatment of recurrent antibiotic-associated
pseudomembranous colitis. Am J Gastroenterol 1982; 77 :
220-1.
25. Zuckerman E, Kanel G, Ha C, Kahn J, Gottesman BS,
Korula J. Low albumin gradient ascites complicating severe
pseudomembranous colitis. Gastroenterology 1997; 112 :
991-9.
26. Jafri SF, Marshall JB. Ascites associated with antibioticassociated pseudomembranous colitis. South Med J 1996;
89 : 1014-7.
27. Rubin MS, Bodenstein, LE, Kent KC. Severe Clostridium
difficile colitis. Dis Colon Rectum 1995; 38 : 350-4.
28. Tedesco FJ, Barton RW, Alpers DH. Clindamycin associated
colitis. A prospective study. Ann Intern Med 1974; 81 : 42933.
29. Drapkin MS, Worthington MG, Chang TW, Razvi SA.
Clostridium difficile colitis mimicking acute peritonitis. Arch
Surg 1985; 120 : 1321-2.
30. Yankes JR, Baker ME, Cooper C, Garbutt JCT. Appearance
of focal pseudomembranous colitis. J Comput Assist
Tomogr 1988; 12 : 394-6.
31. Bartlett JG. Clostridium difficile: clinical considerations. Rev
Infect Dis 1990; 12 : S243-51.
32. Vaishnavi C, Kochhar R, Bhasin, DK, Thennarasu K, Singh
K. Simultaneous assay for Clostridium difficile and fecal
lactoferrin in ulcerative colitis. Trop Gastroenterol 2003;
24 : 13-6.
33. Hookman P, Barkin JS. Clostridium difficile associated
infection, diarrhea and colitis. World J Gastroenterol 2009;
15 : 1554-80.
34. Kelly CP, Pothoulakis C, LaMont JT. Clostridium difficile
colitis. N Engl J Med 1994; 330 : 257-62.
35. McFarland LV, Surawicz CM, Greenberg RN, Fekety R,
Elmer GW, Moyer KA. A randomized placebo-controlled trial
of Saccharomyces boulardii in combination with standard
antibiotics for Clostridium difficile disease. JAMA 1994;
271 : 1913-8.
36. Mahajan LA, Hupertz V, Mahajan, S, Lisa F, John D. The
appendix: A possible reservoir for Clostridium difficile. Am J
Gastroenterol 2006; 101 : S392.
37. Bignardi GE. Risk factors for Clostridium difficile infection.
J Hosp Infect 1998; 40 : 1-15.
38. Pantosti A, Cerquetti M, Gianfrilli PM. Electrophoretic
characterization of Clostridium difficile strains isolated from
antibiotic-associated colitis and other conditions. J Clin
Micobiol 1988; 26 : 540-3.
39. McFarland LV, Elmer GW, Stamm WE, Mulligan ME.
Correlation of immunoblot type, enterotoxin production,
and cytotoxin production with clinical manifestations of
Clostridium difficile infection in a cohort of hospitalized
patients. Infect Immun 1991; 59 : 2456-62.
40. Cheng SH, Lu JJ, Young TG, Perng CL, Chi WM. Clostridium
difficile-associated disease: comparison of symptomatic
infection versus carriage on the basis of risk factors, toxin
production and genotyping results. Clin Infect Dis 1997;
25 : 157-8.
Vaishnavi: C. difficile pathogenesis
41. Jacobs A, Barnard K, Fishel R, Gradon JD. Extracolonic
manifestations of Clostridium difficile infections. Presentation
of 2 cases and review of the literature. Medicine (Baltimore)
2001; 80 : 88-101.
42. Testore GP, Nardi F, Babudieri S, Giuliano M, Di Rosa R,
Panichi G. Isolation of Clostridium difficile from jejunum:
identification of a reservoir for disease? J Clin Pathol 1986;
39 : 861-2.
43. Boland E, Thompson JS. Fulminant Clostridium difficile
enteritis after proctocolectomy and ileal pouch-anal
anastomosis. Gastroenterol Res Pract 2009; 2008 : 1-5.
44. Navaneethan U, Giannella RA. Thinking beyond the colonsmall bowel Involvement in Clostridium difficile infection.
Gut Pathog 2009; 19 : 7.
45. Daruwala C, Mercogliano G, Newman G, Ingerman MJ.
Bacteremia due to Clostridium difficile: Case report and
review of the literature. Clin Med Case Reports 2009; 2 : 5-9.
46. Libby DB, Bearman G. Bacteremia due to Clostridium
difficile - review of the literature. Int J Infect Dis 2009; 13 :
e305-9.
47. Birnbaum J, Bartlett JG, Gelber AC. Clostridium difficile: an
under recognized cause of reactive arthritis? Clin Rheumatol
2008; 27 : 253-5.
48. Ducroix-Roubertou S, Genet C, Rogez JP, Weinbreck P,
Denes E. Reactive arthritis due to Clostridium difficile. Med
Mal Infect 2005; 35 : 419-21.
49. Vaishnavi C. Established and potential risk factors for
Clostridium difficile infection. Indian J Med Microbiol 2009;
27 : 291-302.
50. Kaur S, Vaishnavi C, Prasad KK, Ray P, Kochhar R.
Comparative role of antibiotic and proton pump inhibitor in
experimental Clostridium difficile infection in mice. Microbiol
Immunol 2007; 51 : 1209-14.
51. Johnson S, Gerding DN. Clostridium difficile associated
diarrhoea. Clin Infect Dis 1998; 26 : 1027-36.
52. West, M., Pirenne, J., Chavers, B Gillingham, K, Sutherland,
DE, Dunn, DL, et al. Clostridium difficile colitis after kidney
and kidney-pancreas transplantation. Clin Transplant 1999;
13 : 318-23.
53. Keven K, Basu A, Re L. Clostridium difficile colitis in patients
after kidney and pancreas-kidney transplantation. Transpl
Infect Dis 2004; 6 : 10-14.
54. Bartlett JG. Clinical practice: Antibiotic associated diarrhoea.
N Engl J Med 2002; 346 : 334-9.
55. Dallal RM, Harbrecht BG, Boujoukas AJ, Sirio CA, Farkas
LM, Lee KK, et al. Fulminant Clostridium difficile: an under
appreciated and increasing cause of death and complications.
Ann Surg 2002; 235 : 363-72.
56. Manian FA, Meyer L. CDAD rates. Infect Control Hosp
Epidemiol 1995; 16 : 63-5.
57. Sharma AK, Holder FE. Clostridium difficile diarrhea after
use of tacrolimus following renal transplantation. Clin Infect
Dis 1998; 27 : 1540-1.
497
virus and Clostridium difficile. Zeitschriff fur Gastroenterologie
2008; 46 : 780-3.
60. Milligan DW, Kelley JK. Pseudomembranous colitis in a
leukemia unit: a report of five fatal cases. J Clinical Pathol
1979; 32 : 1237-43.
61. Chen FC, Woods R. Pseudomembranous panenteritis and
septicaemia in a patient with ulcerative colitis. Aust N Z J Surg
1996; 66 : 565-7.
62. Williams C. Occurrence and significance of gastric
colonization during acid-inhibitory therapy. Best Pract Res
Clin Gastroenterol 2001; 15 : 511-21.
63. Cunningham R, Dale B, Undy B, Gaunt N. Proton pump
inhibitors as a risk factor for Clostridium difficile diarrhoea.
J Hosp Infect 2003; 54 : 243-5.
64. Jayatilaka S, Shakov R, Eddi R, Bakaj G, Baddoura WJ, DeBari
VA. Clostridium difficile infection in an urban medical center:
five-year analysis of infection rates among adult admissions
and association with the use of proton pump inhibitors. Ann
Clin Lab Sci 2007; 37 : 241-7.
65. Nachnani JS, Bulchandani D, Allen MJ. Proton pump
inhibitors are an independent risk factor for an increased
length of hospital stay in patients with Clostridium difficile
infection. Indian J Gastroenterol 2008; 27 : 171-2.
66. Emoto M, Kawarabayashi T, Hachisuga T, Eguchi F, Shirakawa
K. Clostridium difficile colitis associated with cisplatin-based
chemotherapy in ovarian cancer patients. Gynecol Oncol
1996; 61 : 369-72.
67. Resnik E, Lefevre CA. Fulminant Clostridium difficile colitis
associated with paclitaxel and carboplatin chemotherapy.
Internat J Gynecol Cancer 2001; 9 : 512-4.
68. Kumar B, Vaishnavi C, Sandhu K, Kaur I. Clostridium difficile
toxin assay in psoriatic patients. Trop Gastroenterol 2004;
25 : 164-7.
69. Gellad ZF, Alexander BD, Liu JK, Griffith BC, Meyer AM,
Johnson JL, et al. Severity of Clostridium difficile-associated
diarrhea in solid organ transplant patients. Transpl Infect Dis
2007; 17 : 1-5.
70. Zerey M, Paton BL, Lincourt AE, Gersin KS, Kercher KW,
Heniford BT. The burden of Clostridium difficile in surgical
patients in the United States. Surg Infect (Larchmt) 2007;
8 : 557-66.
71. Loo VG, Poirier L, Miller MA, Oughton M, Libman M,
Michaud S, et al. A predominantly clonal multi-institutional
outbreak of Clostridium difficile-associated diarrhea with high
morbidity and mortality. N Engl J Med 2005; 353 : 2442-9.
72. Weiss B, Kleinkauf N, Neumann M, Eckmanns T, Michels H,
Jansen A. Risk factors for Clostridium difficile ribotype 027
infection in Germany: preliminary results of a retrospective
case-control study. 18th European Congress of Clin. Microbial
& Infectious Diseases, Barcelona, Spain Abst 2008. p. 1480.
73. Tabaqchali S, Jumma P. Diagnosis and management of
Clostridium difficile infection. BMJ 1995; 310 : 1375-80.
58. Sohi S, Cohen RD. Management of refractory ulcerative
colitis. Curr Treat Options Gastroenterol 2006; 9 : 234-45.
74. Borriello SP, Barclay FE. Protection of hamsters against
Clostridium difficile ileocaecitis by prior colonisation with
non-pathogenic strains. J Med Microbiol 1984; 19 : 339-50.
59. Arnold C, von Sanden S, Theilacker C, Blum HE. Ulcerous
colitis and infection with cytomegalovirus, herpes simplex
75. Rupnik M, Kato N, Grabnar M, Kato H. New types of toxin
A-negative, toxin B-positive strains among Clostridium
498
INDIAN J MED RES, April 2010
difficile isolates from Asia. J Clin Microbiol 2003; 41 : 111825.
76. Krivan HC, Clark GF, Smith DF, Wilkins TD. Cell surface
binding site for Clostridium difficile enterotoxin: evidence for
a glycoconjugate containing the sequence Gal alpha 1-3 Gal
beta 1-4GlcNAc. Infect Immun 1986; 53 : 573-81.
77. Calabi E, Calabi F, Phillips AD. Fairweather NF. Binding of
Clostridium difficile surface layer proteins to gastrointestinal
tissues. Infect Immun 2002; 70 : 5770-8.
78. Pechine S, Gleizes A, Janoir C, Gorges-Kergot R, Bare M-C,
Delmee M, et al. Immunological properties of surface proteins
of Clostridium difficile. J Med Microbiol 2005; 54 : 193-6.
79. Janoir C, Pechine S, Grosdidier C, Collignon A. Cwp 84, a
surface associated protein of Clostridium difficile is a cysteine
protease with degrading activity on extracellular matrix
proteins. J Bacteriol 2007; 159 : 7174-80.
80. Pothoulakis C. Pathogenesis of Clostridium difficile-associated
diarrhea. Eur J Gastroenterol Hepatol 1996; 8 : 1041-7.
81. Rupnik M, Brazier JS, Duerden BI, Grabnar M, Stubbs, SL.
Comparison of toxiotyping of Clostridium difficile strains and
description of novel toxinotypes. Microbiol 2001; 147 : 439-47.
82. Stubbs SL, Rupnik M, Gilbert M, Brazier JS, Duerden
BI, Popoff MR. Production of actin-specific ADPribosyltransferase (binary toxin) by strains of Clostridium
difficile. FEMS Microbiol Lett 2000; 186 : 307-12.
83. Just I, Selzr J, Wilm M, von Eichel-Streiber C, Mann M,
Akatories K. Glucosylation of Rho proteins by Clostridium
difficile toxin B. Nature 1995; 375 : 500-3.
84. Just I, Wilm M, Selzr J, Rex G, von Eichel-Streiber C, Mann
M, et al. The enterotoxin from Clostridium difficile (ToxA)
monoglucosylates the Rho proteins. J Biol Chem 1995;
270 : 13932-6.
85. Linevsky JK, Pothoulakis C, Keates S, Warny M, Keates, AC,
LaMont JT, et al. IL-8 release and neutrophil activation by
Clostridium difficile toxin-exposed human monocytes. Am J
Physiol Gastrointest Liver Physiol 1997; 273 : G1333-40.
86. Lima AA, Lyerly DM, Wilkins TD, Innes DJ, Guerrant RL.
Effects of Clostridium difficile toxins A and B in rabbit small
and large intestine in vivo and on cultured cells in vitro. Infect
Immun 1988; 56 : 582-8.
integrated response of the intestine to Clostridium difficile
toxins. Am J Physiol Gastrointest Liver Physiol 2001; 280 :
G178-83.
92. Brito GA, Fujji J, Carneiro-Filho BA, Lima AA, Obrig
T, Guerrant RL. Mechanism of Clostridium difficile toxin
A-induced apoptosis in T84 cells. J Infect Dis 2002; 186 :
1438-47.
93. Altaie SS, Meyer P, Dryja D. Comparison of two commercially
available enzyme immunoassays for detection of Clostridium
difficile in stool specimens. J Clin Microbiol 1994; 32 : 51-3.
94. Lyerly DM, Saum KE, MacDonald DK, Wilkins TD. Effect of
Clostridium difficile toxins given intragastrically to animals.
Infect Immun 1985; 35 : 1147-50.
95. Fiorentini C, Thelestam M. Clostridium difficile toxin A and
its effects on cells. Toxicon 1991; 29 : 543-67.
96. Siffert JC, Baldacini O, Kuhry JG, Wachsmann D,
Benabdelmoumene S, Faradji A, et al. Effects of Clostridium
difficile toxin B on human monocytes and macrophages:
possible relationship with cytoskeletal rearrangement. Infect
Immun 1993; 61 : 1082-90.
97. Just I, Fritz G, Giry M, Popoff MR, Boquet P, Hegenbarth S,
et al. Clostridium difficile toxin B acts on the GTP-binding
protein Rho. J Biol Chem 1994; 269 : 10706-12.
98. Hecht G, Koutsouris A, Pothoulakis C, LaMont JT, Madara
JL. Clostridium difficile toxin B disrupts barrier function of
T84 monolayers. Gastroenterology 1992; 102 : 416-23.
99. Popoff MR, Rubin EJ, Gill DM, Boquet P. Actin-specific
ADP-ribosyltransferase produced by a Clostridium difficile
strain. Infect Immun 1988; 56 : 2299-306.
100. Rupnik M. How to detect Clostridium difficile variant strains in
a routine laboratory. Clin Microb Infect Dis 2001; 7 : 417-20.
101.Geric B, Johnson S, Gerding DN, Grabnar M, Rupnik M.
Frequency of binary toxin genes among Clostridium difficile
strains that do not produce large clostridial toxins. J Clin
Microbiol 2003; 41 : 5227-32.
102.Bartlett JG, Cang TW, Gurwith M, Gorbach SL, Onderdonk
AB. Antibiotic associated pseudomembranous colitis due to
toxin producing clostridia. N Engl J Med 1978; 298 : 531-4.
103.Wilkins TD, Lyerly DM. Clostridium difficile testing: after 20
years, still challenging. J Clin Microbiol 2003; 41 : 531-4.
87. Triadafilopoulos G, Pothoulakis C, Weiss R, Giampaolo C,
LaMont JT. Comparative study of Clostridium difficile toxin
A and cholera toxin in rabbit ileum. Role of prostaglandins
and leukotrienes. Gastroenterology 1987; 92 : 1174-80.
104.Kato H, Yokoyama T, Kato H, Arakawa Y. Rapid and simple
method for detecting the toxin B gene of Clostridum difficile
in stool specimens by loop mediated isothermal amplification.
J Clin Microbiol 2005; 43 : 6108-12.
88. Katyal R, Vaishnavi C, Singh K. Faecal excretion of brush
border membrane enzymes in patients with Clostridium
difficile diarrhea. Indian J Med Microbiol 2002; 20 : 178-82.
105.Teasley PG, Gerding DN, Olson MM, Peterson LR, Gebhard
RL, Schwartz MJ, et al. Prospective randomized trial of
metronidazole versus vancomycin for Clostridium difficileassociated diarrhea and colitis. Lancet 1983; 2 : 1043-6.
89. Hecht G, Pothoulakis C, LaMont JT, Madara JL. Clostridium
difficile toxin A perturbs cytoskeletal structure and tight
junction permeability of cultured human intestinal epithelial
monolayers. J Clin Invest 1988; 82 : 1516-24.
90. Castagliuolo I, Keats AC, Wang CC, Pasha A, Valenick
L, Pothoulakis C, et al. Substance P receptor expression in
intestinal epithelium in Clostridium difficile toxin A enteritis
in rats. Am J Physiol 1998; 275 : G68-75.
91. Pothoulakis C, LaMont JT. Microbes and microbial toxins:
paradigms for microbial-mucosal interactions II. The
106.Delmee M, Buts J-P. Clostridium difficile-associated diarrhoea
in children. In: Buts JP, Sokal EM, editors. Management of
digestive and liver disorders in infants and children. Elsevier
Science Publishers; 1993. p. 371-9.
107.Barbut F, Petit J-C. Epidemiology of Clostridium difficile
associated infections. Clin Microbiol Infect 2001; 7 : 405-10.
108.Nelson R. Antibiotic treatment for Clostridium difficileassociated diarrhea in adults. Cochrane Database Syst Rev
2005; CD004610.
Vaishnari: C. difficile pathogenesis
109.Aas J, Gessert CE, Bakken JS. Recurrent Clostridium difficile
colitis; case series involving 18 patients treated with donor
stool administered via a nasogastric tube. Clin Infect Dis
2003; 36 : 580-5.
110. Dutta P, Niyogi SK, Mitra U, Rasardy R, Bhattacharya MK,
Chakraborty S. Clostridium difficile in antibiotic associated
pediatric diarrhea. Indian Pediatr 1994; 31 : 121-6.
111. Dhawan B, Chaudhry R, Sharma N. Incidence of Clostridium
difficile infection: a prospective study in an Indian hospital.
J Hosp Infect 1999; 43 : 275-80.
112. Vaishnavi C, Kochhar R, Bhasin DK, Thapa BR, Singh K.
Detection of Clostridium difficile toxin by an indigenously
developed latex agglutination assay. Trop Gastroenterol 1999;
20 : 33-5.
113. Vaishnavi C, Bhasin DK, Singh K. Faecal lactoferrin assay
as a cost effective tool for intestinal inflammation. Am J
Gastroenterol 2000; 95 : 3002.
114. Vaishnavi C, Thapa BR, Thennarasu K, Singh K. Faecal
lactoferrin assay as an adjunct to Clostridium difficile diarrhea.
Indian J Pathol Microbiol 2002; 45 : 69-74.
115. Gupta U, Jadav RN. Clostridium difficile in hospital patients.
Indian J Med Res 1985; 82 : 398-401.
116. Niyogi SK, Dutta P, Dutta D, Mitra U, Sikdar S. Clostridium
difficile and its cytotoxin in hospitalized children with acute
diarrhea. Indian Paediatrics 1991; 28 : 1129-32.
117. Niyogi SK, Bhattacharya SK, Dutta P, Naik TN, De SP, Sen
D, et al. Prevalence of Clostridium difficile in hospitalised
patients with acute diarrhoea in Calcutta. J Diarrhoeal Dis
Res 1991; 9 : 16-9.
118. Bhattacharya MK, Niyogi SK, Rasaily R, Bhattacharya SK,
Dutta P, Nag A, et al. Clinical manifestation of Clostridium
499
difficile enteritis in Calcutta. J Assoc Physicians India 1991;
39 : 683-4.
119. Niyogi SK. Antimicrobial susceptibility of Clostridium
difficile strains isolated from hospitalised patients with acute
diarrhoea. J Diarrhoeal Dis Res 1992; 10 : 156-8.
120.Kochhar R, Ayyagari A, Goenka MK, Dhali GK, Aggarwal
R, Mehta SK. Role of infectious agents in exacerbation of
ulcerative colitis in India: A study of Clostridium difficile.
J Clin Gastroenterol 1993; 16 : 26-30.
121.Vaishnavi C, Bhasin DK, Kochhar R, Singh K. Clostridium
difficile toxin and faecal lactoferrin assays in adult patients.
Microbes Infect 2000; 2 : 1827-30.
122.Kang G, Srivastava A, Pulimood AB, Dennison D, Chandy M.
Etiology of diarrhea in patients undergoing allogeneic bone
marrow transplantation in South India. Transplantation 2002;
73 : 1247-51.
123.Gogate A, De A, Nanivadekar R, Mathur M, Saraswathi K, Jog
A, et al. Diagnostic role of stool culture and toxin detection in
antibiotic associated diarrhoea due to Clostridium difficile in
children. Indian J Med Res 2005; 122 : 518-24.
124.Vaishnavi C, Kaur S, Singh K. Clostridium perfringens type
A and antibiotic associated diarrhoea. Indian J Med Res 2005;
122 : 52-6.
125.Balamurugan R, Balaji V, Ramakrishna BS. Estimation
of faecal carriage of Clostridium difficile in patients with
ulcerative colitis using real time polymerase chain reaction.
Indian J Med Res 2008; 127 : 472-7.
126.Chaudhry R, Joshy L, Kumar L, Dhawan B. Changing pattern
of Clostridium difficile associated diarrhoea in a tertiary care
hospital: a 5 year retrospective study. Indian J Med Res 2008;
127 : 377-82.
Reprint requests:Dr C. Vaishnavi, Additional Professor (GE Microbiology), Department of Gastroenterology, Postgraduate Institute of
Medical Education & Research, Chandigarh 160 012, India
e-mail: [email protected], [email protected]