urine culture manual mt.sinai.pdf

MSH/TML Shared Microbiology Service Policy & Procedure Manual

Policy # MI\UR\v05 of 13

Section: Urine Culture Manual Subject Title: Table of Contents Issued by: LABORATORY MANAGER Original Date: February 2, 2000 Approved by: Laboratory Director Revision Date: October 10, 2003

Review Date: May 26, 2004

URINE CULTURE MANUAL

TABLE OF CONTENTS

INTRODUCTION ...........................................................................................................................3

SPECIMEN COLLECTION AND TRANSPORT..........................................................................4

A. VOIDED URINES Midstream urine ..............................................................................................................4 Neonatal bagged urine.....................................................................................................4 Indwelling catheter (Foley catheter) urine ......................................................................4 Ileal conduit urine............................................................................................................4

B. IN AND OUT CATHETER / CATHETER INSERTION URINE Segmented urines ............................................................................................................4 C. ASEPTICALLY COLLECTED AND OTHER URINES Nephrostomy urine ..........................................................................................................5 Suprapubic urine aspirate ................................................................................................5 Bladder / cystoscopy urine ..............................................................................................5

SPECIMEN REJECTION CRITERIA ............................................................................................5

SPECIAL REQUESTS Eosinophil stain ...............................................................................................................6 Bacterial latex agglutination............................................................................................6 Anaerobes........................................................................................................................6 Chlamydia detection........................................................................................................6 Legionella antigen detection ...........................................................................................6 Leptospira culture............................................................................................................6 Cryptococcus/Systemic Fungi.........................................................................................6 TB culture........................................................................................................................6 Viral culture.....................................................................................................................6 Parasitology .....................................................................................................................6

REAGENTS / MATERIALS / MEDIA ..........................................................................................6

PROCEDURE..................................................................................................................................7

PROCEDURE MANUAL MOUNT SINAI HOSPITAL/TORONTO MEDICAL LABORATORIES SHARED MICROBIOLOGY SERVICE

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Policy # MI\UR\v05 of 13

Section: Urine Culture Manual Subject Title: Table of Contents Issued by: LABORATORY MANAGER Original Date: February 2, 2000 Approved by: Laboratory Director Revision Date: October 10, 2003

Review Date: May 26, 2004

REPORTING RESULTS...............................................................................................................12

REFERENCES ..............................................................................................................................12

APPENDIX I (Reagents / Materials / Media) ...............................................................................13


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Policy # MI\UR\01\v04 of 13

Section: Urine Culture Manual Subject Title: Urines Issued by: LABORATORY MANAGER Original Date: February 2, 2000 Approved by: Laboratory Director Revision Date: November 24, 2000

I. INTRODUCTION

Urinary tract infections (UTI) are one of the most commonly encountered acute infectious diseases. Most UTIs occur as a result of bacteria ascending the urethra and entering the urinary bladder. Urine specimens for culture are collected when the following syndromes are suspected: cystitis, pyelonephritis, asymptomatic bacteriuria, and less commonly acute prostatitis, pyelonephric abscess, and urosepsis. Among the bacteria most commonly isolated from patients with acute uncomplicated cystitis are Escherichia coli, Klebsiella species, other Enterobacteriaceae and Staphylococcus saprophyticus. Hospitalized patients and patients with complicated urinary tract infections are commonly infected with E. coli, Klebsiella species, Proteus mirabilis, other Enterobacteriaceae, Pseudomonas aeruginosa and enterococci. Urine specimens can be divided into two categories based on clinical criteria, the possibility of urethral contamination, and the extent of microbiological work-up. A) Voided urines (non-sterile): Midstream urine (MSU)

Neonatal bagged urine Indwelling catheter (Foley catheter) urine Ileal conduit urine Suprapubic catheter urine

B) In and out catheter / catheter insertion urine

C) Aseptically collected and other urines: Nephrostomy urine Suprapubic urine aspirate Bladder / cystoscopy urine Segmented urines

Quantitative cultures of urine specimens are critical for diagnosis of UTI. The criteria to be used for distinguishing significant from non-significant growth may vary depending on the type of urine specimen received for culture.


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Urine Culture Manual Urine specimens arriving in the laboratory must be accessioned and processed as outlined below as soon as possible. All reagents, kits, media and the Autostreaker MUST be quality controlled before use. All tests must include appropriate controls. (Refer to Quality Control Manual).

II. SPECIMEN COLLECTION AND TRANSPORT Urine is normally a sterile body fluid. However, unless it is collected properly, it may become contaminated with normal flora from the urethra, vagina, prostate or perinium. A urine specimen should be collected into a clean, sterile containers and transported to the Microbiology Laboratory as quickly as possible. Processing of specimens within 2 hours of collection is recommended. If a delay in transport or processing is anticipated, the specimen should be placed in the refrigerator (40C to 80C) until processed. A. VOIDED URINES

Midstream urine (MSU) A midstream urine specimen is collected after careful cleansing of the urethral meatus. The first 10-20 mls of urine is voided and discarded in order to clear the urethra. The subsequent urine is collected into a clean, sterile container.

Neonatal bagged urine

A collection bag is placed over the external genitalia. Urine from the bag is transferred to a clean, sterile container.

Indwelling catheter (Foley catheter) urine

The specimen is obtained by aseptic puncture of the catheter tubing and transferred to a clean, sterile container.

Ileal conduit urine

After cleansing the stomal opening with alcohol, a sterile catheter is inserted and the urine is collected into a clean, sterile container.

B. IN AND OUT CATHETER / CATHETER INSERTION URINE

Urine is collected into a clean, sterile container immediately following the initial insertion of an indwelling catheter into the bladder.


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Urine Culture Manual Segmented Urines

These specimens are collected for the diagnosis of chronic bacterial prostatitis. Three urines plus prostatic secretions are collected and designated as follows:

VB1 = first voided urine representing the urethra VB2 = midstream urine representing the bladder VB3 = first voided urine after prostatic massage representing the prostrate EPS = expressed prostatic secretions C. ASEPTICALLY COLLECTED AND OTHER URINES Nephrostomy urine

Urine draining from a nephrostomy tube placed in the renal pelvis is collected into a clean, sterile container.

Suprapubic urine aspirate

Urine is aspirated through the bladder using a sterile needle and syringe. The urine is then transferred to a clean, sterile container.

Bladder / Cystoscopy / In and out catheter urine

Urine is collected into a clean, sterile container following temporary insertion of a sterile catheter or cystoscope into the bladder.

III. SPECIMEN REJECTION CRITERIA

Rejection criteria (Do not apply to pre-plated specimens)

Reporting procedure

Unsuitable specimens: - Condom catheter - Foley catheter tips and bags - Leaking specimens - Inappropriate / non-sterile container - >24 hr delay before specimen received - Unlabelled specimens - Mislabelled specimens - Duplicate specimens (more than one processed urine within 24 hrs.)

1. Document in LIS order entry 2. Enter resulting phrase and qualifier “Specimen unsuitable for culture…. because added qualifier". 3. Status "Final". 4. Phone ward / physician and document phone call in LIS.


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Urine Culture Manual IV. SPECIAL REQUESTS Eosinophil Stain –

Prepare smear by cytospin method and fix in absolute alcohol. Stain slide and examine for presence of eosinophils due to inter-tissue nephritis.

Bacterial Latex Agglutination – Bacteria

l antigen test is not done due to poor sensitivity and specificity.

Anaerobes – Collect urine into clean sterile container. Specimen must be transported immediately to the laboratory and cultured using pre-reduced Fastidious Anaerobic Agar (BRUC). Appropriate specimens are cytoscopy, bladder or suprapubic aspirate.

Chlamydia Detection –

Collect first voided urine into a clean, sterile container. Specimen processed in Virology.

Legionella Antigen Detection –

Collect urine into clean sterile container. Send to Public Health Laboratory.

Leptospira Detection – Collect urine into clean sterile container. Notify microbiologist. Send to Public Health

Laboratory. Cryptococcus/Systemic Fungi – Urine is collected into a clean, sterile container. Process culture plates in Mycology.

ged specimen for stain and culture. Use centrifu TB Culture –

Collect first morning voided urine on three consecutive days into clean, sterile containers. Send to Public Health Laboratory.

Viral Culture –

Collect urine into a clean, sterile container. Process specimen in Virology.

Parasitology – Schistosomiasis – Collect mid-day urine into a clean, sterile container. Process specimen in Parasitology. V

. REAGENTS / MATERIALS / MEDIA

Refer to Appendix I.


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Urine Culture Manual VI. PROCEDURE

A. Processing of specimens:

a) Direct Examination: Gram stain: Not routinely performed. If specifically requested, perform Gram stain directly on unspun specimen.

Fungal stain: Not routinely performed. If dimorphic fungus or cryptococcus specifically requested, prepare 2 slides with spun urine (3500 rpm x 15 min.). Send unstained slides to mycology for staining and nterpretation. i

Eosinophial stain: Not routinely performed. Prepare 1 slide by cytospin. Stain slide as per Technical manual.

b) Culture:

The urine specimen is mixed and a sterile calibrated loop is dipped vertically into the sample. The loop is streaked down the centre of the plate and then cross-streaked at a 900 angle to the inoculum. (See diagram below). If using the Autostreaker, inoculate the plate with a small streak using a sterile calibrated loop (See diagram below). The amount of urine inoculated is dependent on the type of urine specimen that is processed. (See Table 1 below)

i) Autostreaker ii) Manual inoculation


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Urine Culture Manual If anaerobic culture request, use 0.001 mL loop on cystocopy or bladder urines. Add pre-reduced Fastidous Anaerobic Agar. TABLE 1. Specimen type inoculum, media and incubation conditions.

Media Incubation Voided urine: use 0.001 mL loop Blood Agar (BA) MacConkey Agar (MAC)

02 350C x 18 – 24 hours 02 350C x 18 – 24 hours

Aseptically collected urine: use 0.001 mL loop Blood Agar (BA) MacConkey Agar (MAC)

02 350C x 18 – 24 hours 02 350C x 18 – 24 hours

Suprapubic Urine Aspirate: use 0.001 mL loop Blood Agar (BA) MacConkey Agar (MAC) Pre-reduced Fastidious Anaerobic Agar (BRUC)

02 350C x 18 – 24 hours 02 350C x 18 – 24 hours

An02 350C x 48 hours Segmented urines: use both 0.01 mL and 0.001 mL loops (2 sets) Blood Agar (BA) MacConkey Agar (MAC)

02 350C x 18 – 24 hours 02 350C x 18 – 24 hours

Urine for yeast, candida or unspecified fungi: use 0.001 mL loop Blood Agar (BA) First day: Second day: MacConkey Agar (MAC)

02 350C x 18 – 24 hours Room temperature x 18-24 hours

02 35 C x 18 – 24 hours

0

Urine for dimorphic fungi: spin urine at 3500 rpm, for 15 mins. EBM FMA Inoculate 1 drop of sediment per plate. Forward plates to Mycology

02 30oC x 3 weeks. 02 30oC x 3 weeks.


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Urine Culture Manual Note: 1. Specimens processed after 1600 hours should be placed in a separate basket in the

incubator.

2. Process specimens for anaerobic culture immediately. B. Interpretation of cultures: Examine plates after appropriate incubation time.

a) Cultures with no growth:

Discard negative routine cultures after 18-24 hrs incubation and report as “No growth”. Except when yeast or non-specified fungus is requested (re-incubate x 24 hrs at room temperature). Urine specimens processed after 1600 hrs should be incubated in a separate basket labelled, "After 4 p.m.", the previous day with no growth are re-incubated until 1400 hrs and re-examined. If cultures remain negative, then discard and report as “No growth”.

b) Cultures with growth:

Count the colonies and multiply by the appropriate dilution factor in SI units. Work-up cultures according to the criteria in Tables 2, 3, 4 and 5. NB: The following tables are meant to serve as a guide only. Example: 0.001 mL loop -1 colony = 1 x 106 CFU/L

0.01 mL loop -1 colony = 0.1 x 106 CFU/L TABLE 2: Criteria for the identification and sensitivity testing of organisms isolated from Voided Urines (MSU, neonatal bagged urine, indwelling catheter (Foley catheter urine, ileal conduit urine, and suprapubic catheter). See exception in Table 3. No. of Potential Uropathogen1, 2

No. of colonies of each type Colony count / L

Work up

1 > 10 ≥ 10 x 106 CFU/L ID+Sens 2 Both > 100 >100 x 106 CFU/L ID+Sens on both 2 One > 100

One < 100 >100 x 106 CFU/L < 100 x 106 CFU/L

ID+Sens Ignore

2 Both < 100 < 100 x106 CFU/L No work-up ≥ 3 All ≥ 10 ≥ 1 x106 CFU/L No work-up

ID = Identification; Sens = Sensitivity testing. Note: 1. Ignore any < 10 colonies when counting the types of organisms.

2. Ignore any number of colonies of non-uropathogens (Lactobacillus, diphtheroids, viridans Streptococci or Bacillus species).


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Urine Culture Manual TABLE 3. Criteria for identification and sensitivity testing of organisms isolated from In and Out catheter urines. No. of Potential Uropathogen1, 2

No. of colonies of each type Colony count/ L

Work-up

1 > 10

≥ 10 x 106 CFU/L ID+Sens

2 Both > 10

≥ 10 x 106 CFU/L ID+Sens

2 One >10

One<10 or factor of 10x less

≥ 10 x 106 /L

Use phrase - Light growth -on LIS TEST Comment

(do not enter as ISOLATE)

ID+Sens

Describe3

3 >10

Other(s) factor of 10x less

> 10 x 106 CFU/L

Use phrase - Light growth -on LIS TEST Comment

(do not enter as ISOLATE)

ID+Sens

Describe3

Any # All <10 < 10 x 106 CFU/L No work-up

≥ 4 All ≥ 10 -- No work-up ID = Identification; Sens = Sensitivity testing Note: 1. Ignore urethral/skin flora <10 colonies when counting the types of organisms.

2. Ignore any number of colonies of non-uropathogens (Lactobacillus, diphtheroids, viridans Streptococci or Bacillus species).

3. Describe as Gram positive cocci, Gram positive bacilli, Gram negative bacilli. TABLE 4. Criteria for the identification and sensitivity testing of organisms isolated from suprapubic urine aspirates, bladder/cystoscopy urine, nephrostomy urine and segmented urines).

Types of Organisms

# of colonies of each type Colony count/ L

Work-up

Any # of colonies

Identify any type of

organism

Quantitate using

appropriate dilution factor

Do sensitivity testing as

appropriate


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Urine Culture Manual TABLE 5. Identification and sensitivity testing methods for common urinary tract isolates. Suspect Organism Tests to be performed and expected result Identification /

Susceptibility E.coli (Lactose fermenter)

Oxidase: Negative MUG: Positive Indole: Positive

Vitek Sens

Enterobacteriaceae

Oxidase: Negative

Vitek ID + Sens

Pseudomonas aeruginosa

Oxidase: Positive Characteristic appearance Cetrimide plate

Vitek Sens

Non-fermenters Oxidase: Negative / Positive

Vitek ID + Sens or API NE + KB Sens if applicable

Yeast • If isolated from Bladder Urine: Wet mount Germ tube-positive : Report as C. albicans -negative : Identify to species • If isolated from Voided Urine: Wet mount Do not identify. Report as yeast.

Refer to Mycology

Group B streptococcus

Strep. Latex Aggl’n: Postive Bile esculin: Negative

Vitek Sens

Staphylococcus species: aureus

Staph. Latex Aggl’n: Positive

Vitek Sens Oxacillin screen

saprophyticus

CNST

Staph. Latex Aggl’n: Negative Novobiocin: Resistant Patient: < 60 yrs and females only Novobiocin: Sensitive Staph. Latex Aggl’n: Negative

Vitek Sens Vitek Sens

Enterococcus species Bile esculin: Positive Vitek Sens Vancomycin screen


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Urine Culture Manual Notes:

1. Lactobacillus, diphtheroids, viridans streptococci and Bacillus species do not require sensitivity testing. Except for bladder urines, report these organisms as, "No significant growth" regardless of colony count even if grown in pure culture.

2. Identification and sensitivity testing results can be referred to previous specimen if received and processed within 7 days.

3. The above table serves as a guide only. Refer to the appropriate section(s) as required. 4. Save positive yeast cultures for 7 days at room temperature in case further work-up is

required. D. Susceptibility Testing: Refer to Susceptibility Testing Manual VII. REPORTING RESULTS Eosinophil Stain: Negative report: “No Eosinophils seen”

Positive report: “Eosinophils present” Culture:

Negative report: “No Growth”

<106 CFU/L: “No significant growth”

Mixed cultures: “No significant growth”

Preliminary Positive report: Morphologic description of organism with corresponding colony

count/L. “Identification and susceptibility to follow”. Final positive report: Organism name with corresponding colony count/L and sensitivity

testing results. VIII. REFERENCES 1. Clarridge, J.E., Johnson, J.R., Pezzlo, M.T. 1998. Cumitech 2B, Laboratory Diagnosis of

Urinary Tract Infections, Coordinating ed., A. S. Weissfield. ASM, Washington, D.C. 2. Murray, P., Baron, E.J., Pfaller, M.A., Tenover, F.C., Yolken, R.H. 1999. Manual of

Clinical Microbiology, 7th Edition. The ASM Press, Washington, D.C.


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Policy # MI\UR\01\v04 Page 13 of 13

Urine Culture Manual

APPENDIX I Reagents / Materials / Media: .01 ml sterile calibrated loop .001 ml sterile calibrated loop autostreaker Eosinophil stain – Hansel Gram stains refer to Media section for preparation Blood Agar (BA) - PML MacConkey Agar (MAC) - PML Fastidious Anaerobic Agar (BRUC) – Medprep Blood Egg Albumin (BEAA) – Medprep Esculin Base Medium (EBM) – Inhibitory Mold Agar (IMA) – Novobiocin disc (NB) – Deoxyribonucleic Acid Agar (DNA) Staph. Latex Agglutination (Sanofi) Bile Esculin Prolex Strep Grouping – ProLab Vitek susceptibility cards – BioMerieux Vitek identification cards - BioMerieux Vancomycin Screen plate – refer to Media section for preparation Oxidase reagent – refer to Media section for preparation Beta-glucoronidase (MUG) – Spot Indole reagent – refer to Media section for preparation.



Section: Urine Culture Manual Subject Title: Appendix II – CHROMAgar Colonies

Issued by: LABORATORY MANAGER Original Date: November 8, 2004 Approved by: Laboratory Director Revision Date:

Review Date:


Escherichia coli

Staphylococcus saprophyticus

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T:\Microbiology\New Manual\Live Manual\Urine Appendix II Chromagar colonies.doc



Section: Urine Culture Manual Subject Title: Appendix II – CHROMAgar Colonies

Issued by: LABORATORY MANAGER Original Date: November 8, 2004 Approved by: Laboratory Director Revision Date:

Review Date:


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T:\Microbiology\New Manual\Live Manual\Urine Appendix II Chromagar colonies.doc

Enterococcus mixed with Escherichia coli (pink colonies)

Streptococcus agalactiae

urine culture manual mt.sinai.pdf

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