Diarrheal Diseases in Underdeveloped Countries

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Diarrheal Diseases in Underdeveloped Countries

• Worldwide Problem
• More than 10,000 deaths per day
• On average, about 18 diarrheal episodes per year
• Primarily in children

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Diarrheal Diseases in Developed Countries

• Continues to be a problem
• 25 million enteric infections per year
• 10,000 deaths per year in the U.S.A
• Highest rate in children under 5

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Immunodiagnostics for Diarrheal Disease
Clostridium difficile Entamoeba
histolytica/dispar Giardia Cryptosporidium Clostri
dium perfringens Fecal Leukocytes Inflammatory
Bowel/Irritable Bowel
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Clostridium difficile
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• Clostridium difficile is a gram positive, spore
  forming bacillus
• Now known to be the cause of pseudomembranous
  colitis (PMC)and antibiotic associated
  diarrhea(AAD)
• Causes almost all cases of PMC but only 25 of
  AAD. The most common cause of nosocomial diarrhea

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• Organism first described in 1935 - isolated from
  feces of normal infants and named Bacillus
  difficilis because of difficulty in isolation
• Only recognized as a pathogen in late 1970s
• Diarrhea is efficient method of spreading spores

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In early 80s Dr Wilkins and his research team at
the Anaerobe Lab at Virginia Polytechnic
Institute (VPI) isolated and characterized toxin
A and toxin B of Clostridium difficile. Dr
Bartlett at Johns Hopkins University had
recognized that C.difficile produces two toxins
in 1980
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• Dr Wilkins research group also developed the
  first diagnostic test for the disease and
  developed and patented the antibodies
  subsequently used by all other companies for the
  development of their competitive EIAs.
• The names Dr Wilkins and/or Dr Lyerly (the
  founders and co-owners of TechLab Inc) are to be
  found in the reference list of ALL competitive
  companies kit inserts

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Clinical Terms

• Antibiotic Associated Diarrhea
• Antibiotic Associated Colitis
• Pseudomembranous Colitis

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Sequence of events leading to C.difficile
infection

• Alteration of normal gut flora
• Nosocomial infection by C.difficile
• Growth and production of toxins
• Tissue damage by toxin A, exacerbated by toxin B
• Diarrhea and colitis due to tissue damage and
  influx of fluid

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Biological Activities of Toxin A and B of
C.difficile
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Are there unusual strains that may be missed with
an ELISA that detects only toxin A?
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YES!
Multicenter Evaluation of the Clostridium
difficile TOX A/B TEST Journal of Clinical
Microbiology, Jan 1998, p. 184-190 An Outbreak of
Toxin A negative, Toxin B positive Clostridium
difficile Associated Diarrhea in a Canadian
Tertiary Care Hospital. Canada Communicable
Disease Report, April 1999 Characterization of a
Toxin A negative, Toxin B positive Strain of
Clostridium difficile Responsible for a
Nosocomial Outbreak of Clostridium difficile
Associated Diarrhea Journal of Clinical
Microbiology, July 2000, p. 2706-2714 Pseudomembra
nous Colitis Caused by a Toxin A- Toxin B strain
of Clostridium difficile Journal of Clinical
Microbiology, April 2000, p. 1696-1697
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• These strains have caused fatalities in Europe
  and North America
• Actual incidence (at this time) is unknown - we
  believe it is around 10, however, a study in
  Israel showed an incidence of more than 66 in
  one hospital setting

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• These strains do not lack toxin A - they only
  lack the region of the toxin A gene that codes
  for the antibody binding site -known as the
  receptor region.

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Analysis of A-B isolates by PCR

• 52 A-/B isolates tested
• All 52 were from clinical specimens that tested
  A-/B
• All 52 have the same large deletion in Toxin A

A/B
A-/B
Antibody binding site-(repeat region)
Toxin B
Toxin A
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All Broad Spectrum Antibiotics cause C.difficile
Infections

• Not due to resistance of C.difficile
• Due to susceptibility of normal flora
• Diarrhea sometimes can be stopped by stopping the
  inciting agent

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Epidemiology of C.difficile Disease

• lt3 normal carrier rate in healthy adults
• 50 or higher in infants
  usually asymptomatic
• Outbreaks occur in hospitals and medical centers

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Transmission of C.difficile

• Primarily a nosocomial pathogen - however can be
  present in the community (mainly a disease of the
  aged)
• Spore former - study showed that spores spread on
  floor of clean hospital room could be isolated
  and cultured months later
• Has been isolated from hands of health care
  workers, library books, medical equipment, cords
  used to summon nurses, carpet etc.

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Benefits of TechLab TOX A/B II TEST

• Has highest correlation with tissue culture (gold
  standard), highly sensitive and specific
• Detects both toxins
• Diluent stabilizes toxin for transport or storage

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Benefits of TechLab TOX A/B II TEST

• Can be performed in as little as 40 minutes
  (rapid format)
• Developed by the pioneers of C.difficile testing
  (Competitors use the TechLab patented monoclonal
  antibody)
• No indeterminates, no repeat testing
• Can be used with the TechLab stool preparation
  device to cut down on technician time and
  stabilize the toxin for transport

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Clostridium difficile Testing

• Culture
• Latex agglutination
• Tissue Culture
• ELISA

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Culture

• Culturing is not standardized
• Requires anaerobic techniques, special selective
  media and expertise not readily available in may
  labs
• A toxin test has to be performed on the cultured
  isolates to determine whether the organisms is
  toxigenic or nontoxigenic
• Organisms from asymptomatic patients can be
  isolated - these patients do not require
  antibiotic therapy
• Organisms present in very low numbers can also be
  cultured - these may or may not cause disease

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Latex Agglutination

• Detects glutamate dehydrogenase, an enzyme
  produced by both toxigenic and nontoxigenic
  C.difficile and some other Clostridia
• Very low sensitivity and specificity

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Tissue Culture (Cytotoxicity Test)

• Gold standard as it was the first test available
  and can detect picogram quantities of toxin B
  (remember, toxin B is the most potent cytotoxin
  known to man - toxin A is also cytotoxic but at
  the dilutions used in this test it has no
  cytotoxic effect).
• Test requires that sample be diluted,
  centrifuged, filter sterilized and dispensed into
  the wells of a microtiter plate containing a
  confluent sheet of mammalian cells
• Labor intensive and takes 24-48 hours for a
  result
• Requires considerable expertise and specialized
  equipment

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ELISA
Clostridium difficile Tests that are toxin A
specific

• TechLab TOX A TEST
• Meridian Premier Toxin A
• Bartels Prima (recently purchased by Trinity)
• Alexon ProspecT Toxin A
• Vidas CDA
• Becton Dickinson Culturette CD
• Biosite Triage
• Becton Dickinson ColorPac Toxin A

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EIA TESTS that detect both toxin A and toxin B

• TechLab TOX A/B TEST
• Meridian Premier AB