1. health and fitness
2. disease

Case Study
Pathogenic Bacteriology
2009
Case # 20
Luu, Helen
Chiu, Allen
Navarrete, Maria
Case Summary









A 2-year-old male child experienced upper respiratory infection.
Anorexia and lethargy was seen.
At the emergency room, he had a fever of 39.9°C.
Physical examination showed a clear chest, exudative pharyngitis,
and bilaterally enlarged cervical lymph nodes.
Throat culture was taken and indicated no group A streptococci.
Penicillin was given, but worsened and he became increasingly
lethargic.
He developed respiratory distress when admitted to the hospital.
Examination revealed patient was febrile to 38.9°C and had an
exudate in the posteriour pharynx that was yellowish, thick
membrane which bled when scraped and removed.
Patient’s medical history indicated no immunizations received.
Key Information Pointing to
Diagnosis
 Site of infection was the upper respiratory, pharynx.
 Symptoms were anorexia, drowsiness, mild fever, and
respiratory distress.
 Examination showed exudate in posterior pharynx which
bled when scraped.
 Figure 1 was a special isolation medium used to isolate the
throat culture specimen.
 Figure 2 showed a Gram stain of the throat swab.
 No immunizations received.
Diagnosis

Pathogen causing infection : Corynebacterium diphtheriae

C. diphtheriae is normal inhabitant of the nasopharynx that causes upper
respiratory tract illness.
Stages of infection in respiratory disease:




Figure 1 shows black colonies on tellurite agar.


Initial stage: sore throat, low-grade fever; followed by adherent pseudomembrane on
the pharynx and tonsils.
Later stage: localized damage, bleeding, difficulty in breathing, and mocarditis and
peripheral neuritis.
Tellurite selects and differentiate amongst Corynebacterium.
Figure 2 shows small Gram-positive bacilli that are pleomorphic.

Arrangement is palisade or Chinese letters.
Figure 1
Figure 2
Classification,Gram Stain Results,
and Microscopic Appearance of
Corynebacterium diphtheriae




Family: Corynebacteriaceae
Genus: Corynebacterium
Species: diphtheriae
Small Gram-positive bacilli that are club-shaped.
 Arrangement = palisade or Chinese letters
 Classification based on colony morphology.
 Subspecies:
 C. diptheriae mitischodis : black colonies with a gray periphery
 C. diptheriae intermedius: large, gray colonies
 C. diptheriae gravis; small, dull gray to black
 All produce an immunologically identical toxin with gravis being
associated with the most severe disease.
 Gravis grows faster and depletes the local iron supply which allows earlier and
greater toxin production.
Diagnosis/Isolation/Identification/
of Corynebacterium diphtheriae
 Gram stain showing gram-positive bacillus with arrangement of
Chinese letters
 Loeffler’s agar slant:
 An enrichment medium that contains serum and egg to enhance the
formation of metachromatic granules in C. diphtheriae.
 Stained with methylene blue for better visualization.
 Requires selective media containing tellurite to isolate the organism.
Tellurite selects for Corynebacterium and other Gram-positive
organisms and inhibits Gram-negative organisms
 Cystine tellurite: longer shelf life
 Tinsdale: differentiate amongst Corynebacterium; helps to distinguish
other Corynebacterium species (diptheroids) that normally inhabit the
nasopharynx.
 Colonies appear black or gray due to tellurite reduction.
 C. diphtheria would form brown halos around the colonies due to formation of
ferric sulfide on tinsdale agar.
Diagnosis/Isolation/Identification/
of Corynebacterium diphtheriae


To prove that it can cause diphtheria, toxigenicity of C. diphtheriae strains is
demonstrated.
Most common in vitro assay is the Elek immunodiffusion test.
-Based on double
diffusion of diphtheria
toxin and antitoxin in an
agar medium.
- Organism is streaked on
the plate.
Low concentration of iron is
required in the medium which
triggers toxin production.
- A filter paper strip with
diphtheria antitoxin is
placed perpendicular to
he streak of the organism.
- Formation of a toxinantitoxin precipitin band at
the zone of equivalence
indicates production of
diphtheria toxin.
Diseases and Pathogenesis of
Disease Caused by
Corynebacterium diphtheriae


Normally found in the throats of healthy carriers.
Organism can cause:
 Diphtheria which starts as a local infection of the mucous membrane causing a
membranous pharyngitis
 Cutaneous diphtheria which necrotic lesions
with occasional formation of a local
pseudomembrane occur.
 More common in tropical and subtropical areas.

Toxigenic strains cause severe disease.
 Secretes a potent heat-labile
polypeptide exotoxin that cause diphtheria.
 Exotoxin causes diphtheria, not the organism.

In order for toxin production to occur:
 Bacterium must be lysogenized by a
bacteriophage that carries the tox gene.
 Aerobic conditions, alkaline pH of 7.8-8.0,
and a low iron level.
Diseases and Pathogenesis of
Disease Caused by
Corynebacterium diphtheriae

C. diphtheria toxin:




Toxin binds to its cell surface receptor and enters through receptor mediated endocytosis.
Acidification of endocytic vesicle allows A to dissociate from B.
A enters the cytoplasm and catalyzes the transfer of ADP-ribose from NAD to the eukaryotic
elongation factor-2.
This results in inhibition of protein synthesis and cell death.
Therapy, Prevention and
Prognosis of Patient Infected
with diphtheria

For a patient infected with C. diphtheria, therapy treatment with antitoxin
and antibiotics.
 Goal for both is to kill the organism and stop toxin production.
 Antitoxin is only effective before toxin enters the cell.
 The antitoxin neutralizes circulating (unbound) toxin that prevents progression of the
disease.
 Sensitivity testing needs to be done prior to using antitoxin.
 Antibiotics such as penicillin, erythromycin to eliminate the organism.
 Usually given for 14 days.


Individuals that recover from diphtheria may become asymptomatic carriers
of the organism.
Transmission is from person-to-person via
respiratory droplets.


Disease can keep spreading and become epidemic.


Rarely from skin to skin contact.
Historically, diphtheria has been a deadly disease mainly for children.
Patients infected with diphtheria should be isolated for a couple of days to
prevent other individuals catching this contagious disease.
Therapy, Prevention and
Prognosis of Patient Infected
with diphtheria

Prognosis depends on the size and location of the membrane.
 Early detection and treatment will eliminate the organism and toxin.
 The longer the delay, the higher the death rate.
 If untreated, it is fatal due to airway obstruction, kidney and heart problems.

Vaccination
 Childhood vaccination with diphtheria/pertussis/tetanus (DPT).
 Primary 3 doses given within half a year with another
dose given later more than a year after that.
 Tetanus-diphtheria toxoid (Td) is also available and recommended as a booster
every 10 years.
 Due to waning antitoxin titers, most individuals have antitoxin levels below the optimal
level 10 years after the last dose.

Schick SkinTest:
 Used to test for an individual’s immunity to diphtheria toxin.
 Done by injecting a small amount of diphtheria toxin intradermally
 Observance for a local erythematous and necrotic reaction.
 An erythematous reaction with or without necrosis indicates that there is no antitoxin
antiboides to neutralize the toxin and therefore, the individual is susceptible to
diphtheria.
Primary Research Article

Van Damme, P., Burgess, M., 2004. “Immunogenicity of a combined diphtheriatetanus-acellular pertussis vaccine in adults.” Vaccine. 22. 305-308

Two clinical studies were set-up (1 in Belgium and 1 in Australia) to evaluate the
immunogenicity of a booster vaccination, using either a dTpa vacine (Boostrix)
or a licensed bivalent tetanus and diphtheria (Td) vaccine.

METHODS:
Randomly, chose 824 people that are over 18 years of age to receive either one
dose of Boostrix or a licensed bivalent Td vaccine.




No history of diphtheria or tetanus vaccination.
No previous exposure to disease, for at least 5 years.
Two blood samples, 1 prior to the vaccination and 1 one month after
vaccination, were taken to evaluate the serostatus and imunnogenicity of the
adults.
Used ELISA test to measure antibody concentrations for diptheria, tetanus, and
pertussis.
 Seroprotection for each antibody concentration were as followed:
 Anti-diphtheria antibody concentrations > 0.1 IU/mL
 Anti-tetanus antibody concentrations > 0.1 IU/mL
 Cutt off for pertussis was 5 E1.u/mL
Primary Research Article


RESULTS:
From the blood sample collected prior to vaccination, about a quarter or more of the
test subjects were seronegative for antibodies against diphtheria, tetanus, and
pertussis.
From the blood sample collected 1 month post-vaccination, almost all individuals had
seroprotection for both diphtheria and tetanus.

Individuals vaccinated with Boostrix were also seropositive for the pertussis antigens PT,
FHA, and PRN.
Primary Research Article



CONCLUSIONS:
Even with childhood vaccination programs available in these countries, this
shows that a portion of the adults in both Belgium and Australia is seronegative
for antibodies against diphtheria and tetanus antitoxin.
After one vaccination, individuals had antibody concentrations for the diseases
that were above seroprotection limits.
Despite the recommendation of getting vaccinated for the infectious diseases,
this study comes to show that many individuals are either not up-to-date with
getting a booster shot or have never been vaccinated before.


Individuals are susceptible to the diseases and can become an epidemic problem
once again.
Vaccination for DPT wears off over time
and in order to prevent the spread of this
infection or get infected, booster shots
must be taken at least every 10 years.
Take Home Messages

Disease includes diphtheria that starts as a local infection of the mucous membrane
involving an exotoxin.

High yields of toxin are synthesized only by lysogenic bacteria under iron deficiency
conditions.


Cutaneous diphtheria also seen but mainly in tropical areas.
Typical symptoms are symptom low-grade fever,
malaise, inflammation, edema, and exudate
pharyngitis.

Pathogen is the diphtheria toxin that the organism produces which can disseminate in
the bloodstream and lymph nodes.
Diagnostics include looking at site of infection, followed with culture swab to perform
Gram stain, isolation on selective agar containing tellurite, and followed with in vitro or
in vivo tests (ie. ELEK plate) to show presence of toxigenicity.
Therapy is based on sensitivity testings followed by treatment with antitoxin and
antibiotics.
Prognosis depends on the size and location of the membrane.








Early detection and treatment is crucial.
Delayed treatment or no treatment can lead to more severe, fatal infections and even death.
Prevention is DPT vaccination followed with a booster Td vaccine every 10 years.
Transmission is spread by respiratory droplets from person-to-person.
Threat is severe in children, unimmunized individuals, low levels of immunity
individuals and frequent travelers.
References










"Diphtheria (Corynebacterium diphtheriae)." Menopause and all about Health and Beauty. 01 Mar.
2009 <http://www.drlera.com/bacterial_diseases/diphtheria_.htm>.
"Disease Listing, Diphtheria, Technical Information | CDC Bacterial, Mycotic Diseases." Centers
for Disease Control and Prevention. 06 Oct. 2005. 09 Mar. 2009
<http://www.cdc.gov/NCIDOD/DBMD/DISEASEINFO/diptheria_t.htm>.
Efstratiou, Androulla, et.al. "Current Approaches to the Laboratory Diagnosis of Diphtheria." The
Journal of Infectious Diseases 181 (2000): S138-145.
Frassetto, Lynda A. "Corynebacterium Infections: Overview - eMedicine Infectious Diseases."
EMedicine The Continually Updated Clinical Reference. 30 June 2008. 09 Mar. 2009
<http://emedicine.medscape.com/article/215100-overview>.
Leboffe, Michael J., and Burton E. Pierce. A Photographic Atlas for the Microbiology Laboratory.
3rd ed. Englewood: Morton Company, 2004.
Mahon, Connie R., George Manuselis, and Donald C. Lehman. Textbook of Diagnostic
Microbiology. 3rd ed. Philadelphia: Saunders, 2006.
Mattos-Guaraldi, Ana Luiza, et. al. "Diphtheria Remains a Threat to Health in the Developing
World - an Overview." Mems Inst Oswalda Cruz 98 (2003): 987-93.
McQueen, Nancy. “Corynebacterium,” 2009.
Todar, Kenneth. "Corynebacterium diphtheriae and Diphtheria." Online Textbook of Bacteriology.
2008. 09 Mar. 2009 <http://www.textbookofbacteriology.net/diphtheria.html>.
Van Damme, Pierre, and Margaret Burgess. "Immunogenicity of a combined diphtheria-tetanusacellular pertussis vaccine in adults." Vaccine 22 (2004): 205-308.