Specimen Collection & Laboratory Diagnosis

Key principles of collection

1. Verify you are collecting the specimen from the correct patient

2. Get your sample collection container/swabs ready

• Check that you have the right collection container/swab

• Check the expiry date

• Ensure you have the specimen labels

3. Target site of infection

4. Avoid contamination

5. Timing:

• Time of day? Eg/ early morning sputum for AFB

• Before antibiotics

6. Volume of sample

7. Use appropriate PPE

8. Ensure lids are tight to prevent leakage

If there is a delay in transport, what do you do?

if delay in transport, store in appropriate conditions (RT, 2-8 degrees, frozen)

Key principles of the laboratory

1. Ensure test and specimen is appropriate for the test selected

2. Evaluate if the specimen is appropriately collected

3. Optimize methods to detect pathogens (direct examination, culture, molecular methods)

4. Determine susceptibility to antibiotics

5. Report results (into the EMR, critical calls to MD/RN, notification of IPAC)

6. Communicate test results of public health significance (communicable disease reporting to the public health unit)

What is the pre-analytical testing phase?

Patient evaluation → test order on ward → regulation paper on ward → specimen collection on ward → transport → specimen receipt in lab → determining accessibility in lab

When do most errors occur?

Most errors occur in the early “pre-analytical phase” :

• Wrong test, order entry, patient-specimen misidentification, quality of sample collection poor, wrong container, inappropriate storage and transport

What is the overall error rate? Does this have to do with test performance?

overall error rate in healthcare in the US is 31-69%

The error rate in the performance of the test is very low

What are the biggest reasons for cancelling or redrawing lab specimens/tests?

• quantity not sufficient

• other

• specimen clotted

• specimen not labeled

What are the consequences of improper collection?

• Affected quality reporting requirements and metrics

• Affected ability to detection infection

• Inadequate treatment

• Duplicated specimen collections and re-testing (unnecessary repeat)

• Decreased ability to provide up-to-date, proper patient care

• Exposure to bloodborne pathogens for nurses

• Unnecessary repeated specimen collection and patient dissatisfaction

What is the essential test information put on lab reqs?

• Unique identification of the patient

• Gender, age, DOB

• Test Ordered

• Date and Time of collection

• Who requested the test → most responsible physician

When ordering a test, what information should be considered?

• allergies to antibiotics (so the lab can do additional testing)

• specimen type

• anatomic site (where the tissue is from)

• clinical information (travel history, pregnancy, etc.)

What is one of the most important processes in specimen collection?

• Patient identification

  • Adverse outcomes related to errors related to this: 50% of deaths from transfusion related to errors in identification

What organization prioritizes accuracy of patient identification?

Joint Commission International (healthcare accreditation organization)

What is a patient identifier? How many are required?

Information directly associated with an individual that reliably identifies the individual as the person for whom the service or treatment is intended

Two are required:

• Full name

• Assigned identification number example medical record number, OHIP number

• Telephone number

• Date of Birth

• Electronic identification method such as RFID that includes two or more identifiers eg/ arm band (must be on the patient)

What is the only exception to pre-labelling the container (not labelling at the bedside)?

patient collection (i.e. urine culture)

Four best practices to labelling specimens

1. Take labels to the patient

2. Take a moment to check patient identifiers

3. Write time of collection and your initials on the label

4. Label immediately after collection

Common causes of specimen labelling errors?

• Specimen unlabelled

• Specimen labelling error

• Label illegible

• Specimen covered up

• Specimen label sideways

T/F: all transport media is the same for all specimens

false.

• there are different transport media for bacteria vs viruses vs parasites and for the type of test (culture vs molecular detection)

• Some transport media (example in blood culture bottles) have resins to bind to antibiotics

What specimens would get stored at room temperature if there’s a delay in transport?

• Anaerobic organisms

• Blood culture bottles

• Spinal fluids for bacterial culture

What specimens would get stored at 2-8 degrees celcius if there’s a delay in transport?

• Urine

• Sputum

• Bronchoscopy

• Swabs for respiratory virus

How long do you have until a specimen should be transported to the lab?

• All specimens should be transported within 2 hours

• Spinal fluids and STAT specimens = 1 hour

More than a 24 hour delay is not recommended for bacterial cultures. What specimens may be impacted if there is a delay greater than 12-24 hrs?

• Throat swabs for Group A streptococcus

• Cervical and urethral culture for N. gonorrhoeae (cervical culture)

• N. meningitidis (CSF, Blood culture)

• Anaerobes

What is stable for 2-3 days, given that they are stored properly?

Viruses

Review of Key Collection Principles

Maximize ability to isolate pathogen

Minimize contamination with normal flora during collection

Make it safe for the patient, yourselves and the lab

Store specimens at the appropriate temperature

Transport to the lab as soon as possible

Best practices of pre-analytic factors regarding blood cultures: Ordering

Patient meets clinical criteria for collection eg/ sepsis

Best practices of pre-analytic factors regarding blood cultures: Timing

Prior to antimicrobial administration

Best practices of pre-analytic factors regarding blood cultures: Antisepsis

Wash hands, use gloves, disinfect skin, disinfect BC bottle top

Best practices of pre-analytic factors regarding blood cultures: Draw type and location

Peripheral vs line draw

Different sites for each set

Best practices of pre-analytic factors regarding blood cultures: Number of sets

At least 2 sets

Best practices of pre-analytic factors regarding blood cultures: Volume

Adults 8-10 ml per bottle; pediatrics based on weight

Best practices of pre-analytic factors regarding blood cultures: Labelling

2 patient identifiers, collection date and time, name of collector

Best practices of pre-analytic factors regarding blood cultures: Training/education

Collector should be trained and competency assessed

Dedicated phlebotomy team preferred

Feedback of quality metrics and stewardship

Collecting 2 sets - blood cultures. What should you not do?

Collecting two sets means 4 bottles

• Do not collect 2 sets from the same venipuncture site

• Do NOT collect only ONE set

  • Lower volume so decreases ability to growth the pathogen - blood cultures are 20% less sensitive

  • Difficult to determine if a contaminant for some bacteria such as coag negative staphylococcus

• Use chlorhexidine to disinfect patient skin!

Ratios regarding blood culture volume

• The yield is proportional to the amount of blood

• The ratio of the volume of blood to the volume of the broth in the blood culture bottle is important to allow adequate dilution of the blood to prevent inhibition of growth if the patient is on antibiotics

What can blood culture contamination lead to?

• Difficult for clinicians to know if the bacteria isolated is the cause of infection and needs treatment

• Increase in hospital length of stay

• Increase in costs for antibiotics, investigations and other costs

Recommended that hospitals keep their blood culture contamination to what?

hospitals keep their blood culture contamination ≤ 3%

How to decrease blood culture contamination?

1. Skin Preparation:

• Choose the right disinfectant: chlorhexidine, tincture of iodine

• Need contact time of the disinfectant on the skin

  • Proviodine requires 1.5-2 minutes

  • Tincture of iodine: 30 seconds

  • Chlorhexidine: 30 seconds

2. Blood Culture Bottle Preparation

• The rubber septum is not protected by the cap on the bottle

• Disinfecting the septum, usually with alcohol, reduces contamination

• Do not blow or wave air on the septum to dry

• In the summer or during construction, an increase in Bacillus spp contamination occurs as it is commonly in the environment and survives as a spore

Why do we not like drawing blood from BC catheters?

• Increase in false positive cultures with BC catheter draw

• Only take culture from a central line catheter or PICC if you suspect a line-related infection (CLABSI) or if the patient is a very difficult draw – may need to take from different lumens

• The second set should be a peripheral draw

What is one of the most common lab specimens received in the laboratory?

urine cultures

When should urine cultures be collected? What is the exception to the rule?

• Cultures should only be collected if the patient is symptomatic:

  • Dysuria, hematuria, urgency

  • Fever, rigors, back pain

• Asymptomatic patients should only have urine cultures if:

  • Pregnant – routine screening at 16-18 weeks

  • Invasive urologic procedure where bleeding is expected (eg TURP)

What are the non-invasive types of urine cultures?

• Clean-catch midstream specimens

• Catheter (indwelling or in and out)

• Ileal conduit

• Bagged specimens in pediatrics

What are the invasive types of urine cultures?

• Cystoscopic

• Ureteral

• Percutaneous nephrostomy

• Supra-pubic aspirate

Can we use a bag for collection of urine culture to diagnose UTIs?

no- high rate of false positive results due to contamination with perineal flora

Patient instructions for mid-stream urine cultures

• Men: Clean the glans

• Void the first part of urine into toilet and then without stopping collect midstream portion ➔ prevents urethral contaminant – VERY IMPORTANT FOR URINE CULTURES FROM MEN

How to collect a urine culture from a foley catheter?

Obtain sample from the sampling port using aseptic technique

• never from the catheter bag (highly contaminated) or never from the tip of the foley catheter

If a specimen required refrigeration but is kept at RT for more than four hours, what happens?

Refrigeration at 2-8°C. If kept at room temperature ≥4 hours can get overgrowth of pathogens and contaminating organisms (from urethra, perineum)

Why should you not obtain swabs from superficial ulcers or surgical procedures

ulcers: they are prone to both false positive and false negative results

surgical procedures: fluid and/or tissue samples can be collected

What is the best specimen of collection for abscess/wound cultures?

fluid or tissue

• If closed abscess -→ aspirate fluid with a needle – done by MD

• Tissue from biopsy or debridement – done by MD

• Many specimens do not have enough fluid to aspirate so a swab is used

how to take a swab from a wound?

• open areas are usually contaminated

• clean the area with sterile water or nonbacteriostatic saline with a sponge or gauze

• Always sample the advancing margin

• Pus itself may not grow anything as the organism may be dead

Advantages of fluid or tissue

• Maximizes volume which improves detection of pathogens

• Laboratory provides testing of anaerobic organisms in addition to aerobic organisms

• Swabs are NOT usually inoculated on to media and incubated in anaerobic conditions

• Less likely to be contaminated by epithelial cells/skin commensals

What is Candida auris?

• first isolate Japan in 2009 and has spread rapidly worldwide to six continents and 45 countries

• first reported in the US in 2016

• mortality 30-60%

• causes invasive and non-invasive (wound, ear) infection

• in India and South Africa cause of 25-40% of candidemia cases

• most commonly colonized sites are axilla and groin

Virulence of candida auris

• heat and salt tolerant

• Capacity to form biofilms when exposed to human sweat

• Adhesin surface colonization factor Scf1

Candida auris in Canada

In Canada:

• 51 cases have been identified from 2012-Oct 2023 and 1/3 are MDR

• All four clades have been identified in Canada and there has been clonal spread

• Now up to 62 cases reported with 51% occurring in the last 3 years, 3 outbreaks

Screening for Candida auris based off the PHAC

• admitted to a hospital or LTC home outside of Canada (including in the US) within the prior 12 months

• transferred from a Canadian healthcare facility with an ongoing outbreak

screening sites include: bilateral axilla and groin, previously colonized sites; clinically relevant sites eg wounds or exit sites)

screening frequency: admission screening and contacts- if negative minimum two additional screens a week apart

Causes of infectious diarrhea

• Salmonella, Shigella, Campylobacter, Yersinia entercolitica, Shiga-toxin producing E. coli (includes E. coli 0157)

• Clostridium difficile (most common cause of diarrhea that develops in a hospitalized patient)

• Rare bacterial causes: Vibrio cholera Aeromonas, Plesiomonas Noravirus, adenovirus, astrovirus, enterovirus

• Giardia, Entamoeba histolytica/dispar, Dientamoeba fragilis

• Tapeworms

• Nematodes: ascaris, strongyloides

• Cryptosporium, Cyclospora

Laboratory Rejection Criteria for C diff

• Formed stool

• From a patient < 1 year

Laboratory Rejection Criteria for a hospitalized patient

Bacterial stool culture in a patient admitted for > 72 hours

Who collects sterile fluids?

physician as they are considered irretrievable specimens

• ex. Cerebrospinal fluid

  ◦ Pleural fluid

  ◦ Joint Aspirate

  ◦ Pericardial fluid

  ◦ Vitreous fluid

how are spinal fluids collected?

• Collected into sterile containers using aseptic technique

• For spinal fluids, often 4 tubes are collected:

  • The order of collection important

  • Tube 1, 4 sent for cell count (4 preferred if only 1 tube used for cell count)

  • Tube 2 for chemistry (glucose, protein)

  • Tube 3 for microbiology – need large volumes if fungal or TB cultures requested (10 ml)

timeline for spinal fluids

• must be sent to the on-site laboratory within one hour of collection

• Important for the cell count – get degradation, also clinically essential to determine if infectious vs non-infectious

• Both the cell count and gram stain will be reported by the laboratory ASAP

• The lab quality metric is that the gram stain is reported within one hour of receipt

Critical Call Notification: Role of Laboratory vs Role of RN/Physician

Life threatening results are called to the nurse/physician involved in the care of the patient by the laboratory

Laboratory:

◦ Indicates the laboratory result is a critical result

◦ Asks the full name of the person taking result

◦ Provides the lab result

◦ Asks the person to read back the result

RN or physician

◦ RN documents has taken the critical result

◦ Must call the MRP and provide the result

◦ If cannot contact the MRP must call the physician on call

◦ Someone must take the result

What are the most common bacterial pathogens that are inoculated?

• S. pneumoniae

• N. meningitidis

• Haemophilus influenzae

• Listeria monocytogenes

• Group B streptococcus

• E. coli

Incubation of media for CSF: Atmospheres

• 5% CO2 - Neisseria gonorrheae, N. meningitides, S. pneumoniae

• Ambient air – Pseudomonas aeruginosa

How is Acute pharyngitis diagnosed?

Detection of S. pyogenes (Group A Streptococcus)

• Culture based still the most common

• ED and offices may use antigen detection methods

• Molecular methods (PCR)

Advantages of using molecular methods (nucleic acid is extracted from the specimen or isolate) to diagnose

• Increased sensitivity compared to culture based method

• Can detect organisms where specimen may be delayed in transit

• Detect organisms that cannot be cultured eg/ Whipple’s disease

• Detect new organisms more rapidly then traditional methods eg/ SARS virus

• Provide quantitative result

Disadvantages of using molecular methods (nucleic acid is extracted from the specimen or isolate) to diagnose

• Contamination can occur in the laboratory

• If the target site of the organism mutates, the assay will not detect organism = false negative

• Susceptibility testing cannot be performed

• Can be extremely sensitive so picks up “colonization” not necessarily disease

• Result can remain positive for a long period of time – can reflect dead organism

TB specimens

• Sputum – first morning x 3 days; do not rinse with tap water as environmental mycobacteria can lead to false positive stains

• Bronchoscopy

• Spinal fluid

• Blood culture – require special tube

• Tissue/aspirate

• SWABS are NOT recommended as the hydrophobic nature of the cell wall prevents its release from the swab

WHO Priority List of Fungi: Critical Group

• cryptococcus neoformans

• candida auris

• aspergillus fumigatus

• candida albicans

WHO Priority List of Fungi: High Group

• nakaseomyces (candida) glabrata

• histoplasma spp

• eumycetoma causative agents

• mucorales

• fusarium spp

• candida tropicalis

• candida parapsilosis

WHO Priority List of Fungi: Medium Group

• scedisporium spp

• lomentospora prolificans

• coccidiodes spp

• pichia (candida) kudriavzeveil

• cyptococcus gattil

• talaromyces marneffei

• pneumocytsis jirovecii

• paracoccidiodes spp

criterion for who priority list of fungi

• deaths

• annual incidence

• current global distribution

• trends in last 10 years

• inpatient care

• complications and sequelae

• antifungal resistance

• preventability

• access to diagnostic tests

• evidence based treatments

Host factors of Cryptococcus neoformans/gattii

• HIV, organ transplantation, diabetes, older age, renal and liver disease

• corticosteroids, nastozole (aromatase inhibitor), ibrutinib, JAK inhibitors, alemtuzumab, fingolimod, eculizumab

• idiopathic CD4 lymphopenia

  • HIV decreasing role with only 10% of cases in Australia/NZ

• C. gattii vs C. neoformans is more frequently found in normal host

Diagnostic methods of Cryptococcus neoformans/gattii

Direct microscopy: spherical to oval, 5-10 µm (wide range 2-20), narrow budding

Detection of Cryptococcus antigen (latex agglutination, lateral flow, EIA)

Test methods to diagnosis invasive aspergillus infections

1. Tissue pathology

2. Fungal direct microscopy and culture

3. Galactomannan – done on blood and bronchoscopy specimens

4. Aspergillus PCR done on bronchoscopy specimens

Malaria: What is it, specimens, clinical information, and tests

• In Canada, malaria occurs in travellers and immigrants from endemic countries

• Life-threatening with significant morbidity and mortality

Specimens:

• Blood by venipuncture

• Blood by capillary puncture - higher sensitivity when take blood from fingerprick capillary sample

Clinical information:

• Country of travel, symptoms

Tests:

• Antigen detection, Blood smear and microscopy, PCR

Enzyme immunoassay (EIA)

• Most common method

• Uses an enzyme label to detect that the patient’s sample reacted with the antibody or antigen

• Eg/ HIV antibody detection

Serology

Blood sample used to diagnosis acute or chronic infection

• IgM or seroconversion of IgG to diagnose acute infection

• Hepatitis B chronic infection – persistence of Hep B sAg

• Syphilis

Immune status is varicella, mumps, measles, rubella, hepatitis A, B