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MOLECULAR BIOLOGY | MIDTERMS > DETECTION & ID OF MICROORGANISMS > Flashcards

DETECTION & ID OF MICROORGANISMS Flashcards

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1
Q

Removal of hemoglobin in blood samples (Hgb inhibits DNA pol →
False negative)
- WBC separation by Ficoll-Hypaque
- Serum and plasma can also be sources of microorganism NA
- 4 layers (from bottom to top):
o Blood (RBC)
o Ficoll
o WBC
o Plasma

A

BLOOD

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2
Q

1ST step in detection
- Designed to avoid contamination from environment that may yield
false positives
- Equipment & reagents are utilized

A

SPECIMEN COLLECTION

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3
Q
  • positive: blue/purple
  • negative: pink/red
A

GRAM STAINING

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4
Q
  • Lysis procedures
    o Dependent to microorganisms
    o Mycobacteria and fungi
    ▪ Difficult to lyse due to thick cell walls
    o Gram positive bacteria
    ▪ Thicker cell wall than gram negative bacteria
    o Mycoplasma: no cell wall
    o Concentration of organisms: centrifugation
    o Presence of inhibitors:
    ▪ Removal ad inactivation should be included
    ▪ Removal of RNases for RNA analysis
A

SAMPLE PREPARATION

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4
Q
  • Ex: mycobacterium tuberculosis
  • Characteristic: have mycolic acid
  • To differentiate the acid-fast and
    non-acid fast bacteria
A

ACID-FAST STAINING

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5
Q
  • Removal of acidic polysaccharides (inhibits DNA pol)
A

SPUTUM

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6
Q
  • Collect midstream urine
  • Centrifugation
  • Inhibitors of DNA pol:
A

URINE

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6
Q
  • For respiratory tract infections (RTI)
  • Visually inspected for consistency and presence of saliva/other
    contaminants
  • Removal of acidic polysaccharides (inhibits DNA pol)
A

SPUTUM

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7
Q

Organism is present in the sample

A

POSITIVE RESULT

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8
Q

Organism is absent in the sample

A

NEGATIVE RESULT

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8
Q
  • Carry over of samples
  • Antimicrobial therapy: to avoid, test 3-6 weeks after therapy
A

FALSE POSITVE

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9
Q

NA degradation: inhibition of amplification procedures (Hgb, lactoferrin, heparin, sodium polyanethol sulfonate, polyamines)

A

FALSE NEGATIVES

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9
Q
  • Upper respiratory tract
  • Whooping cough
  • Traditional diagnostic methods:
    o Culture
    o Direct fluorescent antibody
  • Specimen source: Nasopharyngeal
  • Detection:
    o Primer/ probes ASR by qPCR targeting
    IS481 and IS1001, other gene
    targets/adenylate cyclase gene, porin
    gene, pertussis toxin promoter region)
A

BORDETELLA PERTUSSIS

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10
Q
  • Cough, sore throat, hoarseness, nasal congestion, and mild to moderate pneumonia
  • Traditional diagnostic methods:
    o Culture
    o Serology
  • Specimen sources:
    o Respiratory
    o Throat
    o Atherosclerotic lesions
  • Detection:
    o PCR targeting cloned Pst 1 fragment. 16S
    rRNA, MOMP
A

CHLAMYDOPHILIA PNEUMONIAE

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10
Q

PROVIDE GUIDELINES FOR MOLECULAR METHODS IN THE LABORATORY:

A
  • Clinical and laboratory standards institute
  • Association for molecular pathology
  • Food and Drug Administration
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11
Q

DETECTION METHODS:

A
  • Agarose gel electrophoresis
  • Amplification methods: PCR, TMA, LAMP
  • Sequencing
  • Immunoassays
  • Western blots
  • Mass spectrometry
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12
Q

SPECIMEN SOURCE OF Mycoplasma pneumoniae

A

BRONCHOALVEOLAR LAVAGE

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12
Q
  • Lower respiratory tract, 3rd most common cause of
    community-acquired pneumonias
  • Legionnaire’s disease
  • Traditional diagnostic methods:
    o Culture
    o Antigen detection
  • Specimen sources:
    o Deep respiratory secretions
    o Serum
    o Buffy coat
    o Urine
  • Detection:
    o PCR targeting 5S rRNA macrophage
    infectivity potentiator (mip) gene and 16s
    rRNA
A

LEGIONELLA PNEUMOPHILIA

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12
Q
  • Damages lining of respiratory system (throat, lungs,
    trachea)
  • Traditional diagnostic methods:
    o Culture
    o Serology
  • Specimen source:
    o Bronchoalveolar lavage
  • Detection:
    o Multilocus variable – number tandem repeat (VNTR) analysis
    o Multilocus sequence typing
    o Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALOITOF MS)
    o Gene targets: 16S rRNA, 16S rDNA, species-specific protein gene, PI adhesion
    gene
A

MYCOPLASMA PNEUMONIAE

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13
Q
  • Damages lining of respiratory system (throat, lungs,
    trachea)
  • Traditional diagnostic methods:
    o Culture
    o Serology
  • Specimen source:
    o Bronchoalveolar lavage
  • Detection:
    o Multilocus variable – number tandem
    repeat (VNTR) analysis
    o Multilocus sequence typing
    o Matrix-assisted laser desorption ionization
    time-of-flight mass spectrometry (MALOITOF MS)
    o Gene targets: 16S rRNA, 16S rDNA,
    species-specific protein gene, PI adhesion
    gene
A

MYCOPLASMA PNEUMONIAE

13
Q

Tuberculosis (TB): contagious infectious disease primarily affecting the lungs
* Traditional diagnostic methods:
o Smear (104 organisms/mL) and culture
(101-102 organisms/mL)
o Kinyoun and Ziehl-Neelsen stains
o Fluorochromes
* Specimen sources:
o Sputum, bronchoalveolar lavage,
bronchial washings, gastric aspirates
* Detection:
o PCR targeting IS 6110 and 16S rRNA; qPCR
targets rRNA internal transcribe spaces
(ITS)

A

MYCOBACTERIUM TUBERCULOSIS

13
Q
  • Leading cause of community-acquired pneumonia, particularly in children, the elderly, and individuals with weakened immune system
  • Significant cause of meningitis
  • Can cause bacteremia, which can lead to septicemia
  • Traditional diagnostic method: culture
  • Specimen source:
    o Blood
    o CSF
    o Serum
    o Sputum
A

Streptococcus pneumoniae

14
Q
  • Leading cause of community-acquired pneumonia, particularly in children, the elderly, and individuals with weakened immune system
  • Significant cause of meningitis
  • Can cause bacteremia, which can lead to septicemia
A

Streptococcus pneumoniae

15
Q
  • Significant cause of meningitis
  • Can cause bacteremia, which can lead to
    septicemia
  • Traditional diagnostic method: culture
  • Specimen source:
    o Blood
    o CSF
    o Serum
    o Sputum
A

Streptococcus pneumoniae

15
Q
  • Gonorrhea
  • Traditional diagnostic method: culture
  • Specimen source:
    o Urine
    o Urethral
    o Cervical
    o Thin preparation vials/ transport vials for PAP smear
  • Detection:
    o Gene targets (Omp III gene, Opa gene, cytosine DNA
    methyltransferase gene, CPPB gene, site-specific
    recombinase gene)
A

Neisseria gonorrhea

15
Q
  • Chlamydia; asymptomatic
  • Most common STD
  • Traditional diagnostic method:
    o Culture
    o EIA
    o Direct fluorescent Antibody (DFA)
  • Specimen sources:
    o Urine
    o Urethral
    o Cervical
    o Thin preparation vials/transport vials for PAP smears,
    conjunctiva
  • Detection:
    o Gene targets (MOMP, 16S rRNA)
A

Chlamydia trachomatis

15
Q
  • Most common STD
  • Traditional diagnostic method:
    o Culture
    o EIA
    o Direct fluorescent Antibody (DFA)
A

Chlamydia trachomatis

15
Q

SPECIMEN SOURCE OF CHLAMYDIA TRACHOMATIS

A

o Urine
o Urethral
o Cervical
o Thin preparation vials/transport vials for PAP smears,
conjunctiva

15
Q
  • Spirochete, agent of syphilis, cannot be grown in vitro
A

Treponema pallidum SPECIES

16
Q
  • Spirochete, agent of syphilis, cannot be grown in vitro
  • Stages:
    o Primary
    ▪ Formation of hard chancre in site
    ▪ 30-90 days after exposure
    o Secondary
    ▪ Disseminated rash
    ▪ 4-10 weeks after initial infection
    o Latent
    ▪ Latent
    o Tertiary
    ▪ CNS & CV manifestations
    ▪ 3-15 years after initial infection
  • Traditional diagnostic methods:
    o Serologic
    ▪ Presence of an antibodies against cardiolipin
    – Rapid plasma (RPR), venereal disease
    research laboratory (VDRL)
    o Direct antigen detection (Dark field, DFA)
    o Hemagglutination assays
  • Specimen sources:
    o Genital ulcers
    o Blood
    o Brain tissue
    o CSF
    o Amniotic fluid
    o Placenta
    o Umbilical cord
    o Fetal tissue
    o Serum
  • Detections:
    o Gene targets (TpN44.5a, TpNI9, TpN39, p0IA, TpN97,
    16S rRNA, polA)
A

Treponema Pallidum

17
Q
  • Specimen sources:
    o Urine
    o Urethral vaginal
    o Cervical
  • Detection:
    o Gene targets - MgPa (adhesion gene), rDNA gene
  • Smallest genome and 1st organism genome sequenced
A

Mycoplasma genitalium

17
Q
  • Specimen source:
    o Genital tract
    o Amniotic fluid
  • Detection:
    o Gene target (16S rRNA)
A

Mycoplasma hominis

18
Q
  • Specimen source:
    o Genital tract
    o Amniotic fluid
  • Detection:
    o Gene targets (16S rRNA and urease gene)
A

Ureaplasma urealyticum

19
Q
  • Causes:
    o Chancroid
    o STI characterized by painful genital ulcers
  • Traditional diagnostic methods:
    o Gram stain
    o Culture
    o Serological
  • Specimen source:
    o Samples collected from the ulcer
  • Detection:
    o Gene targets (1.1 Kb target, groEL gene, intergenic
    spaces between 16S & 23S rDNA, p27, 16S rDNA gene
A

Haemophilus ducreyi

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MOLECULAR BIOLOGY | MIDTERMS (2 decks)

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