This document discusses diarrhea diseases and food poisoning. It begins with an introduction defining different types of diarrhea and the global burden. It then covers topics like the normal host defenses in the GI tract, diagnosing the cause of diarrhea through clinical history, physical exam and laboratory analysis of stool samples. The document outlines approaches to laboratory diagnosis of pathogens in stool through microscopy, stains, antigen detection and culture. It describes selective media used to recover different diarrheal agents. Treatment focuses on rehydration and appropriate use of antibiotics.

1. DIARRHEA DISEASES AND FOOD
POISONING
Presenter: Angela Charles
Facilitator: Dr. J. Manyahi
Date: November,2023

2. OUTLINE
Introduction.
Anatomic consideration.
Approach to diagnosis of the patient with diarrhea.
Laboratory analysis of a stool specimen.
Treatment and Prevention of Diarrhea.
CPL diarrhea diseases diagnosis, results, challenges
and recommendation

3. OBJECTIVES
1. To describe the normal host defenses at each level of the
gastrointestinal tract in preventing infection.
2. To associate the onset of symptoms, food ingested, travel
history, and clinical manifestations with the possible cause of
diarrheal illness.
3. To determine the factors that place individuals at risk of
gastrointestinal infection.
4. To describe diagnosis of Gastrointestinal infections and food
poisoning, prevention and treatments.

4. INTRODUCTION
 Diarrhea is defined as an “alteration in a normal bowel
movement characterized by an increase in the water content,
volume, or frequency of stools.
Types of Diarrhea
i. Acute diarrhea (persist for fewer than 14 days)
ii. Persistence diarrhea (persist for longer than 14 days)
iii. Chronic diarrhea (persist for more than 30 days)

5. Cont.
 Worldwide, the burden of diarrheal illnesses is responsible for
the deaths of 2195 children per day
 Estimates show that 1.8 million children die each year (more
than 6000/day) in developing countries of Asia, Africa, and
Latin America.
 This is more than human immunodeficiency virus (HIV)/
acquired immunodeficiency syndrome (AIDS), malaria, and
measles combined

6. Cont.
A foodborne outbreak is defined as the occurrence of GI or
neurologic symptoms within 72 hours of ingesting a
contaminated meal.
 The most commonly identified bacterial agents involved in
foodborne outbreaks are Salmonella sp., C. jejuni, S. aureus,
Clostridium botulinum, and C. perfringens. G. lamblia,
Crypto-sporidium, and Cyclospora .
 Chemical intoxications are often associated with fish
consumption

7. ANATOMIC CONSIDERATION
 Although there are exceptions, diarrheal pathogens are usually
acquired by ingesting a contaminated food or beverage.
 There are host defenses against infection such as
i. Gastric juice PH 1.6 - kills 99.9% of coliform bacteria except
cyst phase and spores
ii. Peristalsis – prevent adhesion of pathogens in small intestine
iii. Microbiota - 10^6/g of fecal, mostly anaerobic, produce toxin
Factors for GIs: median ID, Inadequate stomach acidic,
Antimicrobial exposure.

8. APPROACH TO DIAGNOSIS OF THE PATIENT
WITH DIARRHEA
 The cause of diarrhea can usually be determined by :
i. clinical history
ii. physical examination,
iii. laboratory analysis of a stool specimen.

9. CLINICAL HISTORY
 Recent dietary history going back 3 days before the onset of
symptoms is helpful.
 Travel history to countries with less effective sewage facilities
 Recreational activities such as haking,backpacking,swimming.
 Exposure to other sick individuals
 The duration of illness can also be helpful in narrowing the
differential diagnosis
 If the patient is immunosuppressed from AIDS, chemotherapy, or
organ transplantation, or because of other medical reasons,
opportunistic pathogens should be considered

10. PHYSICAL EXAMINATION
 The first step in the physical examination of a patient with a
diarrheal illness is to determine the patient’s state of hydration.
There are several signs suggestive of dehydration; for
example, a sunken appearance to the eyes, dry oral
membranes, skin tenting.
 Patients may also have fever, a decrease in blood pressure or
an increase in heart rate (orthostatic changes).
 Examination of the abdomen may also help lead to a diagnosis.

11. LABORATORY ANALYSIS OF A STOOL
SPECIMEN.
 Evaluation of the patient’s peripheral blood cell count/ fecal
leukocytes in the stool may reveal a leukocytosis in invasive
infections.
 Anemia may be present in cases of severe GI blood loss or
hemolytic infection.
 Thrombocytopenia (low platelet count) may be present in
some infections.
 Evaluation of the patient’s blood chemistry test results can
show electrolyte abnormalities.

12. Approach to Diagnosis of the Patient with Diarrhea
• Common Pathogens Involved In Diarrhea

13. Laboratory Diagnosis of Gastrointestinal Pathogens
 Specimen Collection and Handling
 Specimens delivered to the laboratory within 30 minutes may be
collected in a clean plastic container.
 Volume of a liquid stool at least equal to 1 teaspoon (5 mL) or a
pea-sized piece of formed stool is necessary for most procedures
 Preservatives must be avoided if bacterial cultures are ordered
 Rectal swabs are to be processed if stool is unavailable, Cary-
Blair or a similar transport medium should be used.

14. Cont.
 Stool Specimens for Bacterial Culture
 If a delay longer than 2 hours is anticipated for stools for bacterial culture, the
specimen should be placed in transport medium.
 The Cary-Blair transport medium preserves the viability of intestinal bacterial
pathogens, including Campylobacter and Vibrio spp. However, the media
produced by different manufacturers can vary.
 Because Shigella spp. are sensitive to environmental factors, a transport medium
of equal parts of glycerol and 0.033 M phosphate buffer (pH 7.0) increases the
viability of Shigella in comparison to Cary-Blair.

15. Cont.
Stool Specimens for Viruses
 Stools for virus culture must be refrigerated if they are not inoculated
into cell cultures within 2 hours.
 A rectal swab, transported in modified Stuart’s transport medium or
another viral transport medium, is adequate for recovery of most
viruses from feces.

16. Direct Detection Of Agents Of Diarrhoea In Feces
Direct Microscopic Examination
 Microscopic examination of the stool may reveal white blood
cells in cases of inflammatory diarrhea (e.g. Salmonella,
Shigella, Yersinia, Campylobacter, EIEC, and various Vibrio
spp.).
 Red blood cells may be present because of intestinal wall
bleeding
 Characteristic darting motility may be seen. E. histolytica,
Vibrio cholera
 However, for practical reasons most laboratories do not use a
wet mount.

17. Direct Detection Of Agents Of Diarrhea In Feces
Stains
 Feces may be Gram stained for detection of certain etiologic agents.
 For example, many thin, comma-shaped, gram-negative bacilli may
indicate Campylobacter infection (if vibrios have been ruled out).
 In addition, polymorphonuclear cells may also be detected.
 An acid-fast stain can be used to detect Cryptosporidium spp.,
Mycobacteria, and Isospora spp.

18. Direct Detection Of Agents Of Diarrhea In Feces
Antigen Detection
 Enzyme immunoassays (EIAs) can detect numerous microorganisms capable
of causing GI tract infections.
 For example, EIAs are commercially available to detect E. coli O157:H7
and Campylobacter spp., the presence of the Shiga toxins produced by
EHEC, or the presence of C. difficile toxins A or A and B.
 In addition, rotavirus is detected using a solid-phase EIA procedure.
 EIA methods have also been evaluated for detection of certain bacterial
pathogens. The laboratory diagnosis of C. difficile has been inadequate when
using traditional EIAs.

19. Direct Detection Of Agents Of Diarrhea In Feces
Antigen Detection
 Newer kits are coupling glutamate dehydrogenase (GDH) and A/B
toxin in a combination assay.
 However, the combination kits do not seem to be more specific than a
GDH assay alone.
 Laboratories have demonstrated excellent sensitivity and specificity
using the GDH assay followed with PCR for definitive confirmation.

20. Direct Detection Of Agents Of Diarrhea In Feces
Molecular assays
• Development of amplification techniques has led to numerous
publications for the direct detection of many enteric pathogens.
• A disadvantage with probe technology is that the organism itself is not
available for susceptibility testing, which is important for certain
bacterial pathogens (e.g., Shigella) for which susceptibility patterns
vary.

21. Culture Of Fecal Material For Isolation Of Etiologic Agents
Bacteria
 Fecal specimens for culture should be inoculated to several media for
maximal yield, including solid agar and broth.
 The choice of media is arbitrary and based on the particular
requirements of the clinician and the laboratory.
 Selective and differential culture media are commonly used to attempt
to identify bacterial pathogens in stool.

22. Culture Of Fecal Material For Isolation Of Etiologic Agents
Organisms for Routine Culture
 Stools received for routine culture should be examined for the
presence of Campylobacter, Salmonella, and Shigella spp. under all
circumstances.
 Protocols for culture of enterohemorrhagic E. coli (e.g., E. coli
O157:H7) vary greatly;
based on incidence of disease, laboratories routinely culture for
this organism when cases of severe diarrhea are implicated.

23. Culture Of Fecal Material For Isolation Of Etiologic Agents
Routine Culture Methods
 Specimens received for detection of the most frequently isolated
Enterobacteriaceae and Salmonella and Shigella spp.
should be plated to a supportive medium and selective and
differential medium.
 Blood agar is an excellent general supportive medium.
The absence of normal gram-negative fecal flora or the presence of
significant quantities of organisms such as S. aureus, yeasts, and P.
aeruginosa can be evaluated.

24. Culture Of Fecal Material For Isolation Of Etiologic Agents
Routine Culture Methods.
 The selective and differential agar should support growth of most
Enterobacteriaceae, vibrios, and other possible pathogens;
MacConkey agar works well.
 All lactose-negative colonies should be tested further, ensuring
adequate detection of most vibrios and most pathogenic
Enterobacteriaceae.

25. CULTURE OF FECAL MATERIAL FOR ISOLATION OF
ETIOLOGIC AGENTS
Routine Culture Methods
 Salmonella/Shigella.
 The specimen should also be inoculated to a moderately selective
agar such as Hektoen enteric (HE) or xylose-lysine desoxycholate
(XLD) media.
 These media inhibit growth of most Enterobacteriaceae, allowing
Salmonella and Shigella spp. to be detected.
 All these media are incubated at 35° to 37° C in ambient air and
examined at 24 and 48 hours for suspicious colonies.

26. Culture Of Fecal Material For Isolation Of Etiologic Agents
Routine Culture Methods
 Campylobacter.
 Cultures for isolation of C. jejuni and C. coli should be inoculated to a
selective agar containing antimicrobial agents that suppress the growth of
normal flora.
 Commercially produced agar plates for isolation of campylobacters are
available from several manufacturers.
 These plates are incubated in a microaerophilic atmosphere at 42° C and
examined at 24 and 48 hours for suspicious colonies.

27. CULTURE OF FECAL MATERIAL FOR ISOLATION OF
ETIOLOGIC AGENTS
Routine Culture Methods
 Enrichment broths
 Enrichment broths are sometimes used for enhanced recovery of
Salmonella, Shigella, Campylobacter, and Y. enterocolitica, although
Shigella usually does not survive enrichment.
 selenite F broth yields good recovery.
 Enrichment broths for Enterobacteriaceae should be incubated in air at
35° C for 6 to 8 hours and then several drops should be subcultured to
at least two selective media.

28. Laboratory Diagnosis Of Clostridium Difficile–associated Diarrhea
• The definitive diagnosis of C. difficile–associated diarrhea is based on
clinical criteria combined with laboratory testing.
• Visualization of a characteristic pseudomembrane or plaque on
endoscopy is diagnostic for pseudomembranous colitis
• Since, patients may be colonized with non–toxin producing strains.
Instead, stool specimens are often examined for the presence of the C.
difficile toxins.
• No single laboratory test will establish the diagnosis unequivocally.

29. Laboratory Diagnosis Of Clostridium Difficile–associated Diarrhea
 Two tests are available for routine use: culture, detection of cytotoxin by
tissue culture, and antigen detection assays (e.g., enzyme immunoassay,
latex agglutination) for C. difficile toxin.
 In addition, many laboratories are now using polymerase chain reaction.
 Commercially available PCR assays include BD Gene Ohm (BD
Diagnostics, La Jolla, CA), Cepheid Xpert (Cepheid, Sunnyvale, CA),
etc.
 According to a recent study, PCR demonstrates high sensitivity and
specificity for the diagnosis of C. difficile-associated diarrhea.

30. Selective Media Commonly Used to Recover Diarrheal Agents
Characteristic Morphology
Culture Medium Purpose Pathogens Colon Flora
MacConkey agar Recover Enterobacteriaceae and
other nonfastidious, GNR;
Salmonella, Shigella (with few
exceptions) organisms;
Edwardsiella appear clear and
colorless
Lactose fermenters, such as E. coli,
Klebsiella spp., etc appear dark pink
to red.
Hektoen enteric (HE)
agar
Recover primarily Salmonella and
Shigella spp.;
contains indicators to detect
hydrogen sulfide (H2S)
production
Salmonella spp. appear green
to blue-green with black
centers ;
Shigella spp. appear green
without black centers
Lactose fermenters, such as E. coli,
are slightly inhibited and appear
orange to salmon pink;
Proteus spp. are slightly inhibited;
small, clear colonies with black
centers may appear.
Thiosulfate-citrate-
bile salts-sucrose
agar (TCBS)
Recover Vibrio spp., including
Vibrio cholerae, from stool and
food;
Aeromonas spp. May be recovered
from this medium
Vibrio spp. such as V. cholerae
and V. alginolyticus produce
yellow colonies;
V. parahaemolyticus and V.
vulnificus produce blue-green
colonies
Inhibitory to most colon flora, except
for occasional
Pseudomonas isolates, which may
also appear blue-green

31. Selective Media Commonly Used to Recover Diarrheal Agents
Characteristic Morphology
Culture Medium Purpose Pathogens Colon Flora
Sorbitol-
MacConkey
(SMAC) agar
Detects sorbitol negative E coli; E. coli O157:H7 appears
colorless; does not ferment
sorbitol
Most appear pink.
Campylobacter
blood agar
(CAMPY-BA)
Enrichment-selective medium
primarily to isolate and cultivate
Campylobacter spp. from stool
Campylobacter jejuni appears
pinkish gray, moist, and runny
when incubated at 42° C
Cefsulodin-irgasan-
novobiocin (CIN)
Isolate and recover Yersinia
enterocolitica and Aeromonas spp.;
Plesiomonas shigelloides may also
be recovered;
Y enterocolitica produce
colonies that look like bull’s
eyes; center is red and periphery
appears colorless;
Except for P. aeruginosa,
Citrobacter, and Serratia, most
colon flora are inhibited.
Xylose-lysine-
deoxycholate(XLD)
agar
Isolate Salmonella and Shigella spp.
from stool
Salmonella spp. appears red with
black centers ,
Shigella spp. appear red or clear
Other intestinal flora that may
grow ferment one or all of the
carbohydrates in this medium,
resulting in yellow colonies.

32. Treatment and Prevention of Diarrhea
1. Rehydration - The best hydration solution contains both
glucose and sodium (oral rehydration/IV infusion ).
2. Antimicrobial therapy is ineffective against the viral causes
of gastroenteritis.
3. Vaccination eg: rotavirus and Salmonella typhi
4. Antibiotics may be used against some bacterial pathogens
such as Salmonella.
5. Travelers to high-risk areas should be advised to drink only
bottled beverages and avoid consuming undercooked food
and ice.
6. Ideally, fruits and vegetables would be peeled before use.

33. CPL LABORATORY DIAGNOSIS OF DIARRHEA
DISEASE

34. CPL RESULTS
STOOL CULTURE

35. Cont.
WIDAL TEST

36. CHALLENGES AT CPL
Absence of enrichment media for stool specimen such
as Selenite F broth, Cary Blair
Absence of sorbial MCA and serological method for
tubtying of 0150 E.coli
The use of slide widal test only

37. RECOMMENDATION
 Availability of enrichment media to enhance isolation of
enteric pathogens
 Availability of sorbital MCA and serological test for isolation
and typing of o150 E.coli respectively.
 We recommend the use of at least two tests to diagnose
typhoid fever (Blood culture/Stool Culture).
 If Widal test must be performed, a positive slide test should
always be confirmed by the tube test.

38. References
1. Textbook Of Diagnostic Microbiology By Connie Mahon
And Donald Lehman 7th ed,2018.
2. District Laboratory Practice in Tropical Countries Part 2
Second Edition Monica Cheesbrough 2006.
3. CPL data collection books