FINAL: DETECTION & ID OF MICROORGANISMS Flashcards
Routine
- colony morphology
- enzyme/pigment production
- carbohydrate fermentation patterns
Phenotypic
- genome, transcriptome, proteomes
Molecular
- Designed to avoid contamination from environments that may yield FALSE POSITIVES
- equipment and reagents are utilized
Specimen collection
Complete set of RNA molecules
Transcriptome
Complete set of proteins
Proteome
SAMPLE PREP
- dependent to microorganisms
Lysis procedure
Difficult to lyse due to THICK cell wall
Mycobacteria and fungi
Gram + bacteria has _____ cell wall compared to gram -
THICKER
This microorganisms has NO PLASMA
Mycoplasma
Concentration of microorganisms is done through
Centrifugation
SAMPLE PREP
- removal and inactivation should be included
Removal of RNAses for RNA analysis
Presence of inhibitors
SPECIMEN: BLOOD
- Removal of Hemoglobin in blood
samples
because Hgb inhibits ____ which causes _______
DNA pol
- causes FALSE NEGATIVE
SPECIMEN: BLOOD
WBC separation by?
Ficoll- Hypaque
SPECIMEN: BLOOD
______ & __________ can also be sources of microorganisms
SERUM (no clotting factors) & PLASMA (contains clotting factors & proteins)
SPECIMEN:
For Respiratory Tract infections(RTI)
SPUTUM
SPECIMEN: SPUTUM
Removal of ________ as it inhibits DNA pol
Acidic polysaccharides
SPECIMEN: URINE
- Centrifugation
- Inhibitors of DNA Pol:
o nitrate, crystals, hemoglobin,
o beta-human chorionic
gonadotropin
- Ensures accuracy
- integrity of specimens are critical
- Ensure the absence of inhibitors in
the sample
Quality control
CONTROLS:
- Ensures that assay system is working properly
- 2 Positive controls:
o LOWER and UPPER limit
Positive controls
CONTROLS:
- Should always yield NEGATIVE results
- All reagents except the target
Reagent Blank/contamination
controls
CONTROLS:
- For studies with NONTARGET organisms
- identify the presence of UNDESIRED TARGETS while avoiding any reaction with the desired target.
Negative template control
CONTROLS:
- Rule out amplification failure
- To confirm TRUE NEGATIVE result
- Ex. Housekeeping genes
o groEL, rpoB, recA, gyrB, ẞ -
actin, interferon-y, human
mitochondrial DNA
Amplification control
CONTROLS:
- Amplification controls that monitors particular step of amplification
method
- Can be:
o homologous extrinsic
o heterologous extrinsic
o heterologous intrinsic
Internal controls
- Target derive with a non target derived sequence
o homologous extrinsic
o heterologous extrinsic
o heterologous intrinsic
Homologous extrinsic
- Non-target derived
o homologous extrinsic
o heterologous extrinsic
o heterologous intrinsic
Heterologous extrinsic
- Non-target that are present in sample
o homologous extrinsic
o heterologous extrinsic
o heterologous intrinsic
Heterologous intrinsic
- Carry over of samples
- Antimicrobial therapy
o To avoid: test 3-6 weeks after therapy
False positives
HOW TO AVOID FALSE POSITIVES
test 3-6 weeks after therapy
- Nucleic acid degradation
- Inhibition of amplification procedures
- Inhibitors in Amplification
False negatives
- Inhibitors in Amplification
o Hemoglobin
o Lactoferrin
o Heparin
o Sodium polyanethol sulfonate
o Polyamines
SELECTION OF SEQUENCE TARGETS
Microorganisms share similar sequences
TRUE OR FALSE?
TRUE
MOLECULAR DETECTION OF
MICROORGANISMS
- AGAROSE GEL
ELECTROPHORESIS - AMPLIFICATION METHODS
- SEQUENCING
- IMMUNOASSAYS
- WESTERN BLOTS
- MASS SPECTROMETRY
- Non-molecular methods: lack
sensitivity and time-consuming - Bordetella, Legionella
- Mycobacteria, Chlamydia, and
Streptococcus spp.
WHAT TYPE OR BACTERIA?
RESPIRATORY
Respiratory BACTERIA
- Upper respiratory tract
- Whooping cough
- specimen source: nasopharyngeal
Bordetella pertussis
Respiratory BACTERIA
- Lower respiratory tract
- 3RD MOST COMMON CAUSE OF COMMUNITY ACQUIRED PNEUMONIA
- Legionnaires’ disease
- specimen source: deep respiratory secretions, serum, buffy coat, urine
Legionella pneumophila
Respiratory BACTERIA
- 10% of community-acquired pneumonias
- implicated in atherosclerosis &
coronary artery disease
- specimen source: respiratory, throat, atherosclerotic lesions
Chlamydophila pneumoniae
Respiratory BACTERIA
- TB: contagious infection disease primarily affecting the lungs
- Detection (Traditional)
o Smear and culture
o Kinyoun and Ziehl Neelsen
stains -
o Flurochromess
- qPCR
o Targets rRNA internal
transcribe spaces (ITS) - specimen source: sputum, bronchoalveolar lavage, bronchial washings, gastric aspirators
Mycobacterium tuberculosis
Respiratory BACTERIA
- damages living of respiratory system (throat, lungs, trachea)
- specimen source: bronchoalveolar lavage,
DETECTION:
- multilocus variable-number
tandem-repeat (VNTR) analysis
- multilocus sequence typing.
- matrix-assisted laser desorption
ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Mycoplasma pneumoniae
Respiratory BACTERIA
- Major/LEADING cause of community-acquired
pneumonias.
- significant cause of MENINGITIS
- can cause BACTEREMIA which can lead to SEPTICEMIA
- specimen source: bronchoalveolar lavage,
Streptococcus pneumoniae
- Organisms
o Neisseria gonorrhoeae
o Chlamydia trachomatis - Cervical swabs. Urine, transport
vials for PAP smears - Culture can be performed along
with molecular methods
BACTERIA in the Urogenital tract
BACTERIA in the Urogenital tract
- gonorrhea
- specimen source: urine, urethral, cervical, thin preparation vials/ transport vials for PAP smears
- Traditional diagnostic method: culture
- DETECTION: PCR
Neisseria gonorrhea
BACTERIA in the Urogenital tract
- MOST COMMON STI
- CHLAMYDIA
- specimen source: urine, urethral, cervical, thin preparation vials/ transport vials for PAP smears, conjunctiva
- Traditional diagnostic method: culture, EIA, direct fluorescent anti-body (DFH)
Chlamydia trachomatis
BACTERIA in the Urogenital tract
o Agent of syphilis
o A spirochete
o Cannot be grown in vitro
- Stages:
o PRIMARY- formation of hard
chancre in site
o SECONDARY- disseminated rash
o LATENT- asymptomatic
o TERTIARY- CNS and CV
- Laboratory diagnosis of Syphilis
o Serologic
- Hemaglutination
- EIAS (fluorescent treponemal
antibody absorption FTA-ABS)
- Detection of cardiolipin
Rapid Plasma Reagin
(RPR)
Venereal disease
laboratory (VDRL)
o PCR assays
- specimen source: GENITAL ULCERS, BLOOD, BRAIN TISSUE, CSF, amniotic fluid, placenta, umbilical cord, fetal tissue, serum
Treponema pallidum subsp. Pallidum
Agents of nongonococcal
urethritis
TRADITIONAL METHOD: CULTURE
Mycoplasma hominis, Mycoplasma
genitalium, Ureaplasma
urealyticum and Haemophilus ducreyi
Agents of nongonococcal
urethritis
- causes CHANCROID, STI characterized by painful genital ulcers
- specimen sources; samples collected from the ulcers
Haemophilus ducreyi
