Lec 3: Laboratory Diagnosis of Bacterial Disease
Responsibility of physician and laboratory for good cultures
Appropriate specimen collected
Delivered in a timely manner
Processed to maximize detection of pathogens
Lab provides information to physician for proper specimen for suspected diagnosis
Physician provides diagnosis information to assure proper test selection
Specimen Collection, Transport and Processing
Blood cultures are one of the most important procedures in the microbiology laboratory
Success of culture related to the collection process - timing with fever, symptoms
The most important factor is:
the volume of blood in the culture
Proper cleaning of skin to reduce contamination
Blood Cultures
50% of septic patients have <1 organism per mL of blood
40% more cultures are positive if 20 mL rather than 10 mL of blood are cultured
Skin cleaning is very important to prevent false positive cultures from skin flora entering bloodstream
Blood Cultures – how they are processed
Blood inoculated into bottles of nutrient broth at bedside
Bottles brought to the laboratory for incubation and monitoring for growth
No value in performing Gram stain directly from blood before incubation
Blood Cultures – detection of positivity
Microorganisms metabolize nutrients in the culture vial and release CO2 into the medium
Automated detection of metabolic activity is integrated in the bottle with a sensor read by an instrument at frequent intervals
The sensor detects the CO2 driven signal reacting with fluorescence to indicate growth (presumptive presence of organisms)
Blood Cultures – confirmation and isolation
When growth is detected, a sample of the broth is Gram stained
Inoculated on agar media to isolate the organism for identification & susceptibility testing
Blood Cultures – timing and reporting
Most clinically significant infections are detected as positive in the first 24–48 hours
Many are positive in 8–10 hours
Cultures incubated for 5–7 days
Positive results are critical values reported verbally immediately to the physician
Cerebrospinal Fluid (CSF)
Bacterial meningitis carries high morbidity and mortality if diagnosis is delayed
Specimens delivered to lab immediately and processed without delay
Common causes: Neisseria meningitidis (Gram-negative) and Streptococcus pneumoniae (Gram-positive)
Specimen is concentrated by centrifugation;
sediment inoculated onto culture media and Gram stain prepared for interpretation
Physician notified immediately if organisms are observed in Gram stain or when grown in culture
Media incubated and plates examined for growth after 18–24 hours
Normally sterile fluids
Fluids include:
peritoneal
pleural
synovial
pericardial (body fluids)
urine
spinal
Specimen is concentrated by centrifugation for smear and culture
If large volumes are submitted, can be inoculated into blood culture bottles to enhance sensitivity of culture
Swab samples: what to avoid
The slide lists the following as aspects to consider for swabs and specimens sent for microbiological studies:
Picks up extraneous microbes
Holds extremely small volume of specimen
Hard to get bacteria or fungi away from fibers and onto culture media
Inoculum not uniform across several different agar plates
Should vortex in 0.5 mL liquid before inoculating media
Upper Respiratory Tract (URT)
Swab used to collect pharyngeal specimens (dacron)
Target areas: tonsillar area, posterior pharynx, exudate or ulcerative areas
Avoid contamination with saliva
Group A Streptococcus (S. pyogenes) is a common pathogen to detect
For sinusitis: direct aspiration of the sinus; culturing nasopharynx is not useful for diagnosis due to high anaerobe potential
Rapid transport to laboratory
Lower Respiratory Tract (LRT)
Expectorated sputum
specimen quality assessed by Gram stain prior to culture
Specimens with many squamous epithelial cells and no neutrophils should not be processed for culture (reject)
Bronchoscopy, more invasive, may be necessary if expectorated sputum is inadequate or patient cannot cough up sputum
Eye and Ear Specimens
Tympanocentesis is often required to truly diagnose middle ear infection
Most ear infections are treated empirically without culture (common pathogens: Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, viruses)
Eye surface yields low organisms;
corneal scrapings provide media; intraocular aspiration provides media
Wounds, Abscesses, Tissues
Important to collect specimen from deep in the wound after surface skin has been cleaned
Avoid using swabs if an aspirate can be collected
Place tissues in a sterile container and add sterile saline to prevent drying
Deliver to the lab as soon as possible
Tissues
Tissue collection is often invasive or surgical
Collect enough tissue for pathology (histology) and culture
Do not place all tissue in a formalin container
formalin fixation renders tissue nonviable for culture
Communicate with laboratory regarding differential diagnosis if tissue is small
Urine
Most frequently submitted specimen
Urine cultures are a quantitative culture
Bacteria can continue to grow in urine after collection; important to avoid delay to lab for accurate culture
Refrigerate specimen if transport time to lab will exceed 2 hours
Escherichia coli is the most common pathogen
Genital specimens
Genital testing: Neisseria gonorrhoeae and Chlamydia trachomatis are most commonly detected by nucleic acid amplification tests (NAAT)
Submit genital swabs using specific collection devices provided by the laboratory
Syphilis (Treponema pallidum) is non-cultivable; best diagnosed by blood serology
Fecal specimens
Diarrheal specimens should be collected in a clean pan and transferred to a leak-proof container
Deliver to the lab promptly; floral bacteria will continue to metabolize and interfere with pathogen detection
Transport media available, but fresh is best
Several specialized media inoculated to recover a variety of pathogens with different growth requirements
Toxin testing requires specific orders as they are not part of routine culture
Final results for pathogenic bacteria may take 2–4 days due to mixing with normal fecal flora and isolation requirements
Bacterial Detection & Identification
Detection methods include:
Microscopy
Direct antigen detection
Nucleic acid detection
Culture
Serology is used to detect antibody responses
Summary: Refer back to the detection methods table and organism-specific study in later weeks
Antigen detection is often performed directly from specimen
Culture allows identification meeting 2–3 criteria by growth characteristics and rapid spot testing
Analysis of preformed enzymes, small-scale fermentation to identify organisms (same day as growth)
Nucleic acid-based tests
Antibody detection
Most common pathogenic organisms:
S. aureus, E. Coli, Pseudomonas aeruginosa, Klebsiella pneumoniae
identified on the same day as growth through:
Growth characteristics on culture media
Gram stain and other rapid tests
Prompt reporting to the clinician guides therapy choice
Antimicrobial Susceptibility Testing (AST)
Question asked: "What is it sensitive to?" (i.e., what antibiotics is the organism susceptible to?)
Standards and rationale
Approved standards used: CLSI (Clinical Laboratory Standards Institute)
Based on achievable blood or urinary tract levels of drugs
Aims to predict clinical efficacy of therapy
General methods used: two main families
Broth dilution tests
18–24 hour old culture colonies are used as inoculum (peak growth)
McFarland inoculum standard used to ensure a consistent density
Suspension is inoculated into tubes, microtiter trays, or cards containing growth media and known 2-fold dilutions of antibiotics
Incubated at 35°C for 16–24 hours
Outcome: determines the Minimum Inhibitory Concentration (MIC)
MIC is reported as Susceptible, Intermediate, or Resistant based on CLSI standards
ug/mL of an antimicrobial agent required to inhibit or kill a microorganism
The lowest concentration that inhibits visible growth is the MIC
Visual description (tube/dilution):
Serial dilutions of an antimicrobial agent are prepared
Inoculum is added and tubes are incubated
Growth occurs in tubes with antibiotic concentrations below the MIC
ORGANISM GROWS IN 4UG/ML BUT NOT AT 8UG/ML
This indicates that the minimum inhibitory concentration (MIC) for this organism is between 4 µg/mL and 8 µg/mL
suggesting resistance to doses equal to or above 8 µg/mL.
Agar diffusion tests (KB/Kirby Bauer)
Standardized inoculum spread over agar surface
Disks or strips impregnated with antibiotics placed on agar surface
as the distance from the disk increases, the concentration decreases logarithmically, creating drug gradient
Zone of inhibition around each disk is measured and compared to breakpoints to determine susceptibility
Drug diffuses in agar creating a concentration gradient; bacteria grow where concentration is insufficient to inhibit, forming a lawn with a clear zone around the disk
Procedure:
Standard seeding, agar, and disks
Incubation is typically 24 hours (some conditions may require longer)
Advantages
Technically simple
Very reproducible
No special equipment required
Provides easily understood susceptibility categories for clinicians
Flexible selection of antibiotic agents
Disadvantages
Limited spectrum of organisms approved for testing
Inadequate for detection of vancomycin-intermediate S. aureus (VISA) and some resistance mechanisms
May not detect daptomycin resistance in staphylococci/enterococci or colistin resistance in Gram-negative rods
Provides only qualitative results rather than precise MIC values
Gradient diffusion testing (E test) combines features of both
Quantitative AST using a preformed antimicrobial gradient on a plastic strip placed on an inoculated agar plate
Combines simplicity of disk diffusion with ability to determine MICs
Procedure:
Plate inoculated with standard suspension of organism
E Test strip placed on agar; incubation proceeds as with disk diffusion
Read MIC directly from the scale on the strip at the point where the ellipse of inhibition intersects the strip
Advantages
MICs that align with broth dilution methods
Flexible and relatively simple; allows intermediate MICs to be read directly
Can test up to 5 strips on a single large Mueller-Hinton plate
Disadvantages / considerations
Strips are more expensive than standard disks
Requires careful interpretation and proper QC
Common Sources of Error in AST
Inoculum preparation
Must be pure and at the correct density
Incubation conditions
Temperature, atmosphere, and duration must be appropriate
Endpoint interpretation
Time to read:
dilution method typically 16–20 h;
disk diffusion 16–18 h;
some drug–bug combinations may require up to 24 h
Quality control (QC)
Daily QC for first 30 days, weekly after establishing performance
Deviations may prompt returning to daily QC
Summary
The laboratory strives to provide clinically relevant, accurate information as soon as possible
Specimen quality is crucial - "garbage in, garbage out"