DETECTION & ID OF MICROORGANISMS Flashcards
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
BLOOD
1ST step in detection
- Designed to avoid contamination from environment that may yield
false positives
- Equipment & reagents are utilized
SPECIMEN COLLECTION
- positive: blue/purple
- negative: pink/red
GRAM STAINING
- 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
SAMPLE PREPARATION
- Ex: mycobacterium tuberculosis
- Characteristic: have mycolic acid
- To differentiate the acid-fast and
non-acid fast bacteria
ACID-FAST STAINING
- Removal of acidic polysaccharides (inhibits DNA pol)
SPUTUM
- Collect midstream urine
- Centrifugation
- Inhibitors of DNA pol:
URINE
- For respiratory tract infections (RTI)
- Visually inspected for consistency and presence of saliva/other
contaminants - Removal of acidic polysaccharides (inhibits DNA pol)
SPUTUM
Organism is present in the sample
POSITIVE RESULT
Organism is absent in the sample
NEGATIVE RESULT
- Carry over of samples
- Antimicrobial therapy: to avoid, test 3-6 weeks after therapy
FALSE POSITVE
NA degradation: inhibition of amplification procedures (Hgb, lactoferrin, heparin, sodium polyanethol sulfonate, polyamines)
FALSE NEGATIVES
- 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)
BORDETELLA PERTUSSIS
- 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
CHLAMYDOPHILIA PNEUMONIAE
PROVIDE GUIDELINES FOR MOLECULAR METHODS IN THE LABORATORY:
- Clinical and laboratory standards institute
- Association for molecular pathology
- Food and Drug Administration
DETECTION METHODS:
- Agarose gel electrophoresis
- Amplification methods: PCR, TMA, LAMP
- Sequencing
- Immunoassays
- Western blots
- Mass spectrometry
SPECIMEN SOURCE OF Mycoplasma pneumoniae
BRONCHOALVEOLAR LAVAGE
- 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
LEGIONELLA PNEUMOPHILIA
- 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
MYCOPLASMA PNEUMONIAE
- 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
MYCOPLASMA PNEUMONIAE
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)
MYCOBACTERIUM TUBERCULOSIS
- 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
Streptococcus pneumoniae
- 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
Streptococcus pneumoniae
- 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
Streptococcus pneumoniae
- 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)
Neisseria gonorrhea
- 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)
Chlamydia trachomatis
- Most common STD
- Traditional diagnostic method:
o Culture
o EIA
o Direct fluorescent Antibody (DFA)
Chlamydia trachomatis
SPECIMEN SOURCE OF CHLAMYDIA TRACHOMATIS
o Urine
o Urethral
o Cervical
o Thin preparation vials/transport vials for PAP smears,
conjunctiva
- Spirochete, agent of syphilis, cannot be grown in vitro
Treponema pallidum SPECIES
- 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)
Treponema Pallidum
- 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
Mycoplasma genitalium
- Specimen source:
o Genital tract
o Amniotic fluid - Detection:
o Gene target (16S rRNA)
Mycoplasma hominis
- Specimen source:
o Genital tract
o Amniotic fluid - Detection:
o Gene targets (16S rRNA and urease gene)
Ureaplasma urealyticum
- 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
Haemophilus ducreyi
