Commensal Flora and Specimen Collection

Commensal Flora and Specimen Collection

Lecture Aims

• At the end of this lecture, you should be able to:

  • Describe the major microbial commensal flora at several body sites.

  • Explain the importance of commensal flora.

  • Describe how specimens are collected from different body sites.

  • Describe several different specimen types.

  • Explain why particular specimen types are best for diagnosing specific diseases.

  • Describe factors affecting pathogen survival within a specimen.

Normal Microbial Flora

• Commensal flora:

  • Population of microorganisms that inhabit the skin and mucous membranes of healthy individuals.

    • Bacteria, archaea, fungi, viruses, mites.

  • Also referred to as symbionts or microbiome.

  • Acquired rapidly after birth.

    • Composition shifts over a lifetime.

  • Do not cause disease in healthy people (most of the time).

  • Contrast with transient flora:

    • Colonise for hours to weeks, but no longer.

Commensal Flora - Body Sites

• Oral cavity:

  • Many anaerobic bacteria.

• Skin flora:

  • Largely Gram-positive bacteria.

  • Coagulase-negative staphylococci.

• Small intestine (upper bowel):

  • Enterobacteriaceae.

• Large intestine (colon or lower bowel):

  • Anaerobic bacteria (G- and G+).

• Vagina:

  • Lactobacilli, anaerobic bacteria.

  • Candida (low numbers).

Benefits of Commensal Flora

• Limit growth of pathogens

  • Compete for space and nutrients.

  • Colonisation resistance: form a colonisation barrier.

  • May produce bacteriocins which inhibit other organisms.

  • Alter environment to make it unfavourable (e.g., pH).

• Produce vitamins K, B12

  • Used by humans (e.g., intestinal flora).

• Aid in food absorption

  • Breakdown large molecules.

• Degrade harmful compounds

  • Cholesterol, toxins, drugs.

Commensal Flora & Specimen Collection

• Commensal flora can easily contaminate clinical specimens.

• Impact of commensal flora must be minimised during:

  1. Specimen collection

  2. Specimen transport and storage

  3. Specimen processing

Collection

• Contamination may be completely unavoidable

  • E.g., sputum or faecal samples: contain both commensal and pathogen.

• Contamination can sometimes be minimised

  • On skin by antisepsis

  • E.g., swab site with 70% alcohol or povidone iodine before collecting blood.

Transport or Storage

• Issue:

  • Commensal organisms in specimen can overgrow the pathogen

  • Contaminants may interfere with the growth of pathogen

• Minimise changes by

  • Transporting rapidly

  • Using transport medium if transport will be delayed

Specimen Processing

• Issue:

  • Must be able to distinguish between pathogen and non-pathogen

• Processing must minimise the risk of missing the pathogen

  • E.g., use selective media to inhibit commensals/encourage pathogen

Sampling Sites and Normal Microbiome

• Body sites that have normal commensal organisms:

  • Mouth

  • Nose

  • Upper respiratory tract

  • Skin

  • Gastrointestinal tract

  • Female genital tract

  • Urethra

• Body sites that are normally sterile:

  • Blood and bone marrow

  • Cerebrospinal fluid

  • Serous fluids

  • Tissues

  • Lower respiratory tract* (not completely sterile but typically pathogen-free)

  • Bladder* (not completely sterile but typically pathogen-free)

Types of Specimens - Overview

Ordering Specimens

• When are specimens ordered?

  • If antibiotic-resistant organisms are suspected

    • E.g., MRSA, VRE, ESBL

  • If specific pathogens or notifiable diseases are suspected

    • E.g., Legionella, Listeria, measles, chlamydia

  • Disease is chronic or unresolving

• When are they not?

  • If diagnosis has already been made based on symptoms and clinical picture

  • If empirical therapy has been started and is working

    • E.g., bacterial pneumonia

  • If the results are unlikely to be meaningful

    • E.g., too much commensal flora/ will not contribute to patient care

• Process:

  • Clinical examination -> Presumptive diagnosis -> Appropriate specimens taken -> Appropriate tests requested -> Lab results reported back.

Ideal Specimen Characteristics

Ideally, specimens for detecting pathogens should be:

1. Appropriate for the disease process

   • E.g., CSF for suspected meningitis

   • Many guidelines for which specimens to collect for disease/pathogen

2. Obtained before antimicrobial agents have been administered

   • Or after an antibiotic-free period

3. Free from contamination with normal flora

4. Collected in an appropriate container or transport media

   • Leak-proof containers

   • Within a sealable bag with separate paperwork section

5. Properly labelled

6. Large enough to process correctly

   • Too little is not good

Specimen Rejection by Laboratory

Any of the following may result in specimen rejection:

1. Labelling issues

   • Specimen is not labeled / too little information

   • Information on label does not match the requisition form

     • E.g., patient’s name or source of specimen

2. Transport issues

   • Transported at the improper temperature

   • Transported in incorrect medium

   • Transport delays

     • Time exceeds 2 hours post-collection

     • If >2 hours the specimen is not preserved

3. Specimen issues

   • Quantity of specimen is insufficient for testing

   • Specimen is leaking

   • Specimen is dried

   • Specimen was received in a fixative: no viable organisms will be present

   • Specimen unlikely to yield meaningful results

     • E.g., specimen for anaerobic culture from a site with anaerobes as part of the normal microbiota

     • Processing specimen would produce information of questionable medical value

• Poor specimen quality / rejection contributes to:

  • Delayed diagnosis or misdiagnosis

  • Inappropriate antimicrobial therapy

  • Increased patient length of stay and hospital cost

Specimen Quality Example: UTI Misdiagnosis

• Quality Problem:

  • Misdiagnosis of UTIs from false positive/negative culture results due to poor specimen collection and handling leads to inappropriate treatment, patient harm and wasted resources.

• Preventability/Improvement:

  • Overall false positive rates: 15-42% (CAP)

• Interventions/Practices:

  • Urine collection teams

  • Clean catch vs. catheterized collection

  • Time limitations on plating

  • Chemical preservation

  • Temperature preservation

• Intermediate Outcomes:

  • Contamination rates

  • Number of ID's and ASTS performed

  • Sample transport time

• Harms:

  • Misdiagnosis from false positives/negatives

  • Urinary catheter placement/replace

• Healthcare Outcomes:

  • Incorrect diagnosis

  • Unnecessary antibiotic use

  • Repeat collections/cultures/office visits

  • Additional testing/follow-up

  • Increased hospital LOS and/or visits

  • Healthcare-acquired infections

• Costs associated with above.

Transport of Specimens

• Critical elements in specimen transport:

  • Time, temperature, medium

• Time

  • Should be delivered to lab within 2 hours

  • Then processed as quickly as possible

  • If not processed immediately must be stored under correct conditions: stored for no longer than 24 h before processing

  • Must process immediately if these organisms are suspected:

    • Shigella spp.: Sensitive to pH changes

    • N. gonorrhoeae, N. meningitidis: Sensitive to temperature changes

    • H. influenzae: Sensitive to temperature changes

    • S. pneumoniae: Sensitive to oxygen

    • Anaerobes: Sensitive to oxygen

Temperature

• Many specimens can be stored at 4°C to prevent microbial growth

• Some should NOT be

  • E.g., CSF: one of the few specimens that should NOT be refrigerated

    • Fastidious organisms may not survive temp. fluctuations

• Recommendations vary according to specimen type and suspected pathogen:

  • E.g., Influenza A:

    • Specimens should be kept at 4°C for no longer than 3 days

    • Specimens can alternatively be frozen at ≤-70°C

    • Avoid freezing and thawing specimens if possible

  • E.g., Meningitis:

    • Transport CSF specimens to laboratory ASAP

    • Specimens for culture should not be refrigerated or exposed to extreme cold, excessive heat, or sunlight

    • Transport at temperatures between 20°C and 35°C

    • For proper culture results, CSF specimens must be plated within 1 hour

Transport Media

• Purpose is to:

  • Enhance survivability of pathogens in clinical material

  • Maintain microbial population in specimen as close as possible to what it was at the time of collection

• Transport media achieve this by

  • Preventing specimen from drying out

  • Non-nutritive: organisms won't multiply nor die

  • Contain buffers to prevent pH change

• Liquid or semi-liquid consistency

  • Semi-liquid achieved by adding agar

• May be available with or without swab

  • Choice depends on specimen type/disease

Transport Media Examples

1. Cary Blair medium

   • Semi-solid, good for enteric bacteria

   • Lacks any fermentable carbohydrates (reduces risk of pH change)

2. Stuart transport medium

   • Semi-solid, good for fastidious organisms

   • E.g., gonococci, streptococci

3. Amies charcoal medium

   • Charcoal absorbs fatty acids that are toxic to N. gonorrhoeae

4. Viral transport medium

   • Contains antibiotics and antifungals

   • Prevents bacteria and fungi growing

5. Anaerobic pouch or GasPak (anaerobes)

   • Or anaerobe transport medium (similar to thioglycollate)

Swabs Used to Collect Specimens: Tips

• Swabs have several components can affect pathogen viability

  1. Swab tip

  2. Swab shaft type

  3. Transport medium

• Most swabs go into transport medium; not dry

• Guidelines recommend specific swabs for each pathogen

A. Cotton-Tipped Swabs

• Cotton contains fatty acids that can inhibit bacterial growth

  • Not recommended for Chlamydia, Neisseria gonorrhoea, Bordetella

  • Not recommended for viruses

• Suitable for non-fastidious bacteria

B. Calcium Alginate-Tipped Swabs

• Calcium alginate extracted from seaweed

• No fatty acids

  • But may be toxic for lipid-enveloped viruses eg. HSV

• Calcium alginate may inhibit PCR

  • Not recommended for PCR-based detection of B. pertussis

  • But is fine for culture-based detection

  • Not recommended for COVID19 specimens

C. Dacron and Rayon-Tipped

• Both are synthetic fibres

• Wide range of uses

• Dacron are the least toxic of swabs: recommended for

  • Detection of viruses

  • Bordetella, N. gonorrhoea, Mycoplasma

• Suitable for both PCR and culture-based detection

Swabs Used to Collect Specimens: Shafts

Shafts are typically made of wood, plastic, or wire

A. Wooden Shafts:

• Not recommended for viruses

• Can be toxic to Ureaplasma, Neisseria spp.

• Can introduce dust/debris

B. Flexible Wire Shafts and Small Tips

• Aluminium or stainless steel

• Used mostly for nasopharyngeal specimens (Bordetella pertussis)

  • Aluminium shafts shown to inhibit PCR for B. pertussis

• Used for male urethral specimens for diagnosis of gonorrhoea

C. Plastic

• Inert, no dust

Coronavirus (COVID-19)

• Swab Sample Collection Guidelines for sample preparation

• According to WHO guidelines:

  • Respiratory material* (nasopharyngeal and oropharyngeal swab in ambulatory patients and sputum (if produced) and/or endotracheal aspirate or bronchoalveolar lavage in patients with more severe respiratory disease)

  • Serum for serological testing, acute sample, and convalescent sample (this is additional to respiratory materials and can support the identification of the true agent, once serologic assay is available)

    • *Modifiable with information on whether upper or lower respiratory material is better for coronavirus detection.

• For Oral Coronavirus testing buccal swabs are commonly used. The guidelines from the FDA and US Centers for Disease Control and Prevention (CDC) specify the type of swab that should be used in the procedure:

  • "Swab specimens should be collected using only swabs with a synthetic tip, such as nylon or DacronⓇ, and an aluminium or plastic shaft. Calcium alginate swabs are unacceptable and cotton swabs with wooden shafts are not recommended."

Body Fluids: Blood

• Specimen type: blood

• Culture is the most sensitive method available for detecting organisms in blood

  • Bacteria or fungi

  • Obtained by venepuncture or intravascular catheter

• Multiple blood cultures are required to detect bacteraemia

  • At least two separate specimens

  • Reduces possibility of detecting a specimen contaminant

• Blood culture bottles contain growth medium and

  • Anticoagulant sodium polyanethanolsulfonate (SPS)

    • Can inhibit several pathogens

    • May also have compounds to neutralise antibiotics

• Different types of bottles are available

  • E.g., aerobes, anaerobes, paediatric bottles

Blood Culture Methods

Positive cultures may be detected manually or automatically

• Manual systems used to incubate blood culture bottles

  • Septi-Chek, Isolator and Signal

  • Bacterial growth is detected by direct observation

• Instrumental or automatic systems:

  • BacT/Alert/BACTEC systems

  • CO_2 sensors attached to individual bottles detect bacterial growth

Body Fluids: Other

Cerebral Spinal Fluid (CSF)

• CSF required for diagnosis of meningitis

  • Specimen obtained by lumbar spinal puncture

  • Large volumes are preferred

  • Transport immediately to lab at room temperature

Other Body Fluids

• E.g., pericardial, pleural, peritoneal, peritoneal dialysis, synovial fluids, serous fluids

• 1 to 5 ml usually adequate

• Tests:

  • Various: chemistry, microscopy, culture

Laboratory Diagnosis of Meningitis

Gastrointestinal Tract Specimens

• Specimen types: faeces and rectal swabs

  • To diagnose infectious diarrhoea or food poisoning

• Faecal specimens must be

  • In a clean container with a tight lid

  • Free from contamination by urine, barium or toilet paper

  • Minimum volume ~5 ml

• Considerations

  • Normal gut flora can acidify the specimen

    • Due to fermentation of carbohydrates

  • If specimen cannot be processed within 2 h:

    • Place into Cary-Blair transport medium soon after collection

  • Rectal swabs should also go into Cary Blair transport medium

Urinary Tract Specimens

• Specimen: Urine

• Very common specimen in a clinical laboratory

• Used to detect UTI or cystitis: bladder infection

  • Pyelonephritis: infection of renal parenchyma

• Specimen obtained by

  • Midstream specimen of urine (msu)

  • Catheter specimen of urine (csu)

  • Cystoscopy collection or suprapubic aspiration

• First-voided morning urine is optimal

  • Pathogens are ‘concentrated’

  • Mid-stream to prevent contamination with urethral colonisers

  • Transport immediately to lab

  • Process within 2 hours of collection

  • Store at 4°C for up 24 h if not processed immediately

• If cannot be processed within 2 h specimen must be preserved

  • Urine transferred into transport tubes

  • Contain buffered boric acid, glycerol, formate and Na

  • Preserve bacteria without refrigeration for up to 48 h at ambient temp

Genital Tract Specimens

• Specimen types: typically vaginal or urethral swabs

• Used to diagnose a range of syndromes / infections

• Many specimens will be contaminated with normal flora of the skin or urogenital tract

• Some specimen types:

  • High vaginal swab: used to detect

    • Yeast spp.

    • Group B streptococci (pregnancy)

    • Trichomonas vaginalis (wet prep)

  • Cervical/urethral swabs: used to detect

    • N. gonorrhoeae, Chlamydia

  • Gram-stained smear: used to detect

    • Bacterial vaginosis

    • Look for ”clue cells”

    • Epithelial cells covered in bacteria

Wounds, Abscesses, Skin & Soft Tissue Specimens

• Specimen types:

  • Tissue

    • These are the best / most ideal specimens

    • Should be kept moist during transport

  • Aspirates: from abscess

  • Swabs

    • Swabs generally inferior to aspirates or biopsies

    • Contain less material

    • More likely to be contaminated with normal or transient flora

    • Not amenable to optimal anaerobic transport

  • Important to also culture for anaerobes

    • In all except skin specimens

Upper Respiratory Tract Specimens

• Many different sites:

  • External nares, nasopharynx, throat, oral ulcerations and inflammatory material from the nasal sinuses

• Typical specimen types:

  • Throat swabs: used to detect

    • Pharyngitis caused by Streptococcus pyogenes

    • Infection with Neisseria gonorrhoeae

    • Carriers of Staphylococcus aureus

  • Nasopharyngeal secretions

  • Aspirate (mostly in young children)

• Deliver specimens to the laboratory promptly

  • Process without delay (within 1 – 2 h of collection)

Nasopharyngeal Secretions

Secretions may be used to detect

• Carriers of Bordetella spp., N. meningitidis

• Respiratory viruses

  • E.g. influenza, respiratory syncytial virus, COVID

• Vincent’s angina

  • Oral infection diagnosed by direct examination of a Gram-stained smear

• If collected by endoscopy

  • Avoids contamination with commensal flora

Lower Respiratory Tract Specimens

• Required for diagnosis of bronchitis, pneumonia, lung abscess and empyema

• Specimen types:

  • Expectorated/induced sputum

  • Endotracheal tube aspirations (intubated patients)

  • Bronchoscopy specimens

    • Bronchoalveolar lavage (BAL), bronchial brushings, washes

  • Pleural fluid

    • Pleural effusion may be due to pneumonia

Ear Specimens

• Specimen types:

  • Swab

  • Aspirate

• Collected for the diagnosis of:

  • Otitis externa

  • Otitis media

Otitis Externa:

• Specimen typically a swab

• Will be contaminated with normal flora

Otitis Media

• Specimen is middle ear fluid

• Rarely examined: infection is diagnosed on clinical symptoms alone

• Anaerobic bacteria may be involved

• Contamination with normal flora less likely than externa

• Potential pathogens differ according to disease

  • Acute otitis media

    • S. pneumoniae, H. influenzae, M. catarrhalis

  • Chronic otitis media with effusion

    • P. aeruginosa, S. aureus

Eye Specimens

To detect pathogens for the following conditions:

• Conjunctivitis:

  • Conjunctival scrapings collected with swab or sterile spatula

  • E.g. detect adenovirus, HSV, varicella-zoster virus

• Keratitis:

  • Corneal scrapings collected with a sterile spatula

  • E.g. N. gonorrhoea, HSV, varicella-zoster virus, acanthamoeba

• Endophthalmitis:

  • Vitreous fluid collected by aspiration

  • Range of pathogens due to diverse reasons for infection

    • Endogenous, traumatic (B. cereus), post-surgery (coag-neg Staphs)

• Periorbital cellulitis:

  • Fluid collected by aspiration or tissue biopsy

  • Bacteria: S. aureus, S. pneumoniae, S. pyogenes

Resources

• Chapter 32 Diagnosis of infection and assessment of host defence mechanisms – Mims' Medical Microbiology and Immunology, 6th Edition. Elsevier

• Chapter 5 Specimen Management – Bailey & Scott’s Diagnostic Microbiology

• What’s new in the world of Swabs? (January 2008) – http://www.americanmedtech.org/files/steponlinearticles/332.pdf