X : Serological & Molecular Detection of Bacterial Infections

d. release of lipid A from the bacterial cell.

All of the following are protective mechanisms against bacteria except
a. production of antimicrobial defense peptides.
b. phagocytosis.
c. activation of complement.
d. release of lipid A from the bacterial cell.

c. antibodies to one serotype protect against other serotypes.

All of the following are characteristics of streptococcal M proteins except
a. it is the chief virulence factor of Group A streptococci.
b. it provokes an immune response.
c. antibodies to one serotype protect against other serotypes.
d. it limits phagocytosis of the organism.

b. The patient has an antibody to a streptococcal exoenzyme other than streptolysin O.

An ASO titer and a streptozyme test are performed on a patient's serum. The ASO titer is negative, the streptozyme test is positive, and both the positive and negative controls react appropriately. What can you conclude from these test results?
a. The ASO is falsely negative.
b. The patient has an antibody to a streptococcal exoenzyme other than streptolysin O.
c. The patient has not had a previous streptococcal
infection.
d. The patient has scarlet fever.

b. Antibodies to Group A streptococci are believed to cross-react with heart tissue.

Which of the following applies to acute rheumatic fever?
a. Symptoms begin after S. pyogenes infection of the throat or the skin.
b. Antibodies to Group A streptococci are believed to cross-react with heart tissue.
c. Diagnosis is usually made by culture of the organism.
d. All patients suffer permanent disability.

a. Reduction of methyl green from green to colorless

Which of the following indicates the presence of anti- DNase B activity in serum?
a. Reduction of methyl green from green to colorless
b. Clot formation when acetic acid is added
c. Inhibition of red blood cell hemolysis
d. Lack of change in the color indicator

a. Acute rheumatic fever

Which of the following is considered to be a nonsuppurative complication of streptococcal infection?
a. Acute rheumatic fever
b. Scarlet fever
c. Impetigo
d. Pharyngitis

b. stimulation of chemotaxis.

All of the following are ways that bacteria can evade host defenses except
a. presence of a capsule.
b. stimulation of chemotaxis.
c. production of toxins.
d. lack of adhesion to phagocytic cells.

d. Antibody production takes at least a week before detection.

Antibody testing for Rocky Mountain spotted fever may not be helpful for which reason?
a. It is not specific.
b. It is too complicated to perform.
c. It is difficult to obtain a blood specimen.
d. Antibody production takes at least a week before detection.

c. Urease

Which of the following enzymes is used to detect the
presence of H pylori infections?
a. DNase
b. Hyaluronidase
c. Urease
d. Peptidase

c. Antibodies remain after initial treatment.

Which of the following reasons make serological identification of a current infection with Helicobacter pylori difficult?
a. No antibodies appear in the blood.
b. Only IgM is produced.
c. Antibodies remain after initial treatment.
d. No ELISA tests have been developed.

a. Cold agglutinins

M pneumoniae infections are associated with the production of which antibodies?
a. Cold agglutinins
b. Antibodies to ATPase
c. Antibodies to DNase
d. Antibodies to Proteus bacteria

b. Specific antibodies in the serum sample attach to the antigens fixed to a microscope slide. In a second step, the attached antibodies are stained with fluorescein-labeled anti-human immunoglobulin and visualized with the fluorescence microscope.

Which of the following best describes the principle of the IFA test for detection of antibodies produced in Rocky Mountain spotted fever?

a. Patient serum is applied to a microtiter plate coated with a monoclonal antibody directed against the target antigen. A detection antibody labeled with biotin and directed against the target antigen is added. After addition of a substrate, a color reaction develops indicating presence of the antigen.

b. Specific antibodies in the serum sample attach to the antigens fixed to a microscope slide. In a second step, the attached antibodies are stained with fluorescein-labeled anti-human immunoglobulin and visualized with the fluorescence microscope.

c. The serum sample is treated chemically to link the target antibodies to a fluorophore. The labeled sample is applied to a microscope slide to which the antigen has been attached. Following a wash step, the slide is examined for fluorescence.

d. Patient serum is applied to a slide to which a specific antigen is bound. Following a wash step, a chromogenic dye is applied that binds to the Fc region of IgG and IgM antibodies. After a second wash step, the slide is examined for fluorescence.

c. Endotoxin is released from the cell wall of dead bacteria, whereas exotoxin is released from live bacteria.

Which of the following is true regarding exotoxins and endotoxins?
a. Both endotoxin and exotoxins are highly immunogenic allowing for the development of protective antibodies and vaccines.
b. Endotoxin has targeted activity whereas exotoxins have systemic effects when released.
c. Endotoxin is released from the cell wall of dead bacteria, whereas exotoxin is released from live bacteria.
d. Both endotoxin and exotoxins bind to specific receptors on a bacterial cell leading to cell lysis.

c. It is an important mechanism for protecting a bacterium against ingestion by PMNs.

Characteristics of a bacterial capsule include which of the following?
a. It cannot be used for vaccine development.
b. It is composed of peptidoglycan.
c. It is an important mechanism for protecting a bacterium against ingestion by PMNs.
d. It is what causes bacteria to stain as gram-negative.

b. It is the cause of most cases of acute food poisoning in the United States.

Which of the following statements regarding Helicobacter pylori is not true?
a. It is associated with an increased risk of gastric carcinoma.
b. It is the cause of most cases of acute food poisoning in the United States.
c. It is a major cause of peptic ulcers in the United States.
d. It is positive for urease.

Symbiotic

- Host & microbes live together long term.

Symbiotic

- Indigenous microbiota.

Mutualistic

- Both host & microbe’s benefit.

Parasitic

- Microbes cause harm to the host.

Infectivity

- Organism’s ability to establish an infection.

Pathogenicity

- Ability of an organism to cause dx.

Virulence

Extent of pathology caused by an organism when it infects a host

1. Endotoxin

2. Pili

3. Flagella

4. Capsule

5. Exotoxins

Bacterial Virulence Factors

Endotoxin

- The lipid A portion of LPS in gram – cell walls

Endotoxin

- Powerful stimulator of cytokine release

Pili

- Adherence to host cells

- Resistance to phagocytosis

Pili

- Conjugation – process of Horizontal gene transfer

Flagella

- Adherence to host cells

- Motility

Capsule

Blocks phagocytosis

Capsule

antibody attachment

Exotoxins

- Potent toxic proteins released from living bacteria

Exotoxins

- Neurotoxins, cytotoxins, enterotoxins

Exotoxins

- G –& +

Innate Defenses

Immune Defenses Against Bacteria

Identify if Innate or Adaptive Defense:

• Intact skin & mucosal surfaces (barriers to entry)

Innate Defenses

Immune Defenses Against Bacteria

Identify if Innate or Adaptive Defense:

• Complement proteins, cytokines, acute-phase reactants

Innate Defenses

Immune Defenses Against Bacteria

Identify if Innate or Adaptive Defense:

• Recognition of PAMPs by PRRs such as TLRs

Adaptive Defenses

Immune Defenses Against Bacteria

Identify if Innate or Adaptive Defense:

• Antibody Production

• Binding of Complement components, opsonization, neutralization of bacteria toxins

Adaptive Defenses

Immune Defenses Against Bacteria

Identify if Innate or Adaptive Defense:

• Cell-mediated immunity

• CD4 T cells produce cytokines that induce inflammation; cytotoxic T lymphocytes attack host cells that contain intracellular bacteria.

Culture of the Causative Agent

Laboratory Detection of Bacterial Infections

• Growth on broth / solid media.

Culture of the Causative Agent

Laboratory Detection of Bacterial Infections

• Major means of diagnosis, but may take time or may not be possible.

Microscopic Examination

Laboratory Detection of Bacterial Infections

Gram / special stains

Detection of Bacterial Antigens

Laboratory Detection of Bacterial Infections

Rapid testing by ELISA, LFA, or LA.

Molecular Detection of Bacterial DNA / RNA

Laboratory Detection of Bacterial Infections

Can obtain results in a few hours with PCR.

Serology

Laboratory Detection of Bacterial Infections

Detects antibodies to bacterial antigens

1. Culture of the Causative Agent

2. Microscopic Examination

3. Detection of Bacterial Antigens

4. Molecular Detection of Bacterial DNA / RNA

5. Serology

Laboratory Detection of Bacterial Infections

T

Uses of Laboratory Detection of Bacterial Infections

• To detect & confirm infections for which other laboratory methods are not available.

(t/f)

F

• To diagnose infections for which clinical symptoms are nonspecific.

Uses of Laboratory Detection of Bacterial Infections

• To diagnose infections for which clinical symptoms are specific. (t/f)

F

• Current infection indicated by presence of IgM, a high IgG titer, or a fourfold rise in antibody titer between acute & convalescent samples

Uses of Laboratory Detection of Bacterial Infections

• Current infection indicated by presence of IgM, a low IgG titer, or a twofold rise in antibody titer between acute & convalescent samples (t/f)

T

Uses of Laboratory Detection of Bacterial Infections

• To determine a past exposure to & organism (IgM-. IgG+) (t/f)

T

Uses of Laboratory Detection of Bacterial Infections

• To assess reactivation of re-exposure. (t/f)

• Delay between start of infection & production of antibodies.

• Low antibody production by immunosuppressed patients.

Disadvantages of Laboratory Detection of Bacterial Infections

Group A Streptococci

- Gram + cocci arranged in pairs / chains.

Person-to-person transmission.

Group A Streptococci MOT

• Pharyngitis / Strep Throat

• Pyoderma / Impetigo

• Scarlet Fever

• Toxic Shock Syndrome (TSS)

• Necrotizing Fasciitis

• Treated with Antibiotics

• Enanthem – rashes in mucosal

• Exanthem – rashes in the skin

Clinical Manifestations of Acute Group A Streptococcal Infection

Pharyngitis / Strep Throat

– most common clinical manifestations of Group A Streptococcal infection. Symptoms include fever, chills, severe sore throat, headache, tonsillar exudates, petechial rash on the soft palate, & anterior cervical lymphadenopathy.

Pyoderma / Impetigo

– most common clinical manifestations. It is characterized by vesicular lesions on the extremities that become pustular & crusted which tend to occur in young children.

Enanthem

rashes in mucosal

Exanthem

rashes in the skin

Acute Rheumatic Fever / Arthritis

- Develops a sequelae 1–3 weeks after pharyngitis/tonsillitis in 2% – 3% of infected individuals.

Acute Rheumatic Fever / Arthritis

- Most likely caused by immune responses to streptococcal antigens that cross-react with human heart tissue.

• Fever

• Joint pain

• Inflammation of the heart

JIF

- Symptoms of Acute Rheumatic Fever / Arthritis

Poststreptococcal Glomerulonephritis

- May follow strep infection of the skin or pharynx

Poststreptococcal Glomerulonephritis

- Damages glomeruli in the kidney, producing hematuria, proteinuria, edema, hypertension, malaise, backache, abdominal discomfort, & impairment in renal function.

Poststreptococcal Glomerulonephritis

- Deposits of immune complexes containing streptococcal antigens in glomeruli.

• Culture on Sheep Blood Agar (SBA) – Small translucent colonies surrounded by clear zone of β hemolysis.

• Rapid assays to detect group A streptococcal antigens

• Lateral Flow Immunochromatographic Assays(LFA)

Laboratory Diagnosis of Acute Group A Streptococcal Infections

Small translucent colonies surrounded by clear zone of β hemolysis.

Laboratory Diagnosis of Acute Group A Streptococcal Infections

Culture on Sheep Blood Agar (SBA) shows

Lateral Flow Immunochromatographic Assays(LFA)

Laboratory Diagnosis of Acute Group A Streptococcal Infections

• Rapid assays to detect group A streptococcal antigens such as

Antistreptolysin O (ASO)

Serological Detection of Group A Streptococcal Sequelae

- Nephelometric methods currently used that measure light scatter produced by immune complexes containing streptolysin antigen.

T

Serological Detection of Group A Streptococcal Sequelae (t/f)

Antistreptolysin O (ASO)

• Titer elevated in 85% of patients with acute rheumatic fever

Antistreptolysin O (ASO)

Serological Detection of Group A Streptococcal Sequelae

- Does not increase in patients with skin infection.

Anti-DNase B

Serological Detection of Group A Streptococcal Sequelae

- Produced by both rheumatic fever & impetigo px.

EIA & nephelometric methods.

Serological Detection of Group A Streptococcal Sequelae

Anti-DNase B is tested by

Streptozyme Test

Serological Detection of Group A Streptococcal Sequelae

- Is a slide agglutination screening test for the detection of antibodies against streptococcal antigens.

• Antistreptolysin O (ASO)

• Anti-hyaluronidase (AHase)

• Hyaluronidase

• Anti-streptokinase (ASKase)

• Anti-nicotinamide adenine dinucleotide (anti-NAD)

• Anti-DNase B – enzyme that degrades DNA

Serological Detection of Group A Streptococcal Sequelae

Streptozyme Test detect antibodies to 5 streptococcal products:

Antistreptolysin O (ASO)

Serological Detection of Group A Streptococcal Sequelae

– detect antibodies to the streptolysin O enzyme produced by Group A streptococcus.

Hyaluronidase

Serological Detection of Group A Streptococcal Sequelae

enzyme produced by gas that breaks hyaluronic acid

Anti-streptokinase (ASKase)

Serological Detection of Group A Streptococcal Sequelae

can dissolve blood clots

Anti-nicotinamide adenine dinucleotide (anti-NAD)

Serological Detection of Group A Streptococcal Sequelae

– break down

Anti-DNase B

Serological Detection of Group A Streptococcal Sequelae

enzyme that degrades DNA

F

Gram – microaerophilic spiral bacterium

Helicobacter pylori is a gram + microaerophilic rods bacterium (t/f)

fecal-oral route

Helicobacter pylori is transmitted by

gastric & duodenal ulcers

Helicobacter pylori’s major cause is

urease

Helicobacter pylori can survive in acid environment of stomach because of production of -, which provides a buffering zone around the bacteria.

Urease

provides a buffering zone around the bacteria.

antibiotics & anti-ulcer medications

Treatment of Helicobacter pylori

gastric carcinoma or mucosa-associated lymphoid tumors

If Helicobacter pylori is left untreated, it can lead to

• Detect urease in stomach biopsy (CLOtest)

• Urea Breath Test

• H pylori antigen

• H pylori Antibodies

• ELISA is method of choice.

• IgG in serum indicates an active infection.

• Titers decrease AFTER successful treatment.

Detection of Helicobacter pylori Infection

T

Helicobacter pylori (t/f)

Titers decrease AFTER successful treatment

ELISA

method of choice for the detection of Helicobacter pylori Infection

IgG

what indicates in a serum an active infection

- Tiny bacteria that lack a cell wall

Mycoplasma pneumonia

- Leading cause of respiratory infections

Stevens-Johnson syndrome

Mycoplasma pneumonia causes what dx in minority of cases which is a condition in which the top layer of the skin dies & sheds

Fever, headache, malaise, & cough

Symptoms of Mycoplasma pneumonia

“Walking pneumonia”

Mycoplasma pneumonia is commonly known as

Raynaud Syndrome

transient vasospasm of the digits in which the fingers turn white when exposed to the cold.

1. Culture

2. Antibodies to M. pnuemoniae

3. Cold Agglutinins

4. Molecular Methods

Laboratory Diagnosis of M. pnuemoniae Infection

- Produces mulberry colonies with a “fried egg” appearance on specialized media

Culture of M. pnuemoniae Infection produces what colonies and appearance on specialized media

Culture

- Is gold standard for detection of M. pnuemoniae but rarely performed in clinical laboratories because organism is difficult to grow.

Antibodies to M. pnuemoniae

Most useful diagnostic assay of M. pnuemoniae