The document outlines the laboratory diagnosis of urinary tract infections, detailing methods for urine collection, processing, and interpretation of results. It includes instructions for various collection techniques, the significance of urine culture media, and guidelines for antibiotic susceptibility testing. The document also discusses how to interpret different laboratory and clinical scenarios related to UTIs.

1. Laboratory Diagnosis of Urinary
Tract Infections
Medical Microbiology & Immunology Department
Faculty of Medicine – Zagazig University
2023-2024

2. Intended Learning Outcomes
• Recognize the different methods of urine
sample collection, preservation and
transport.
• Describe urine sample screening and
processing.
• Interpret various laboratory and clinical
aspects of UTIs.

3. Collection Methods
• Clean-catch mid stream urine in males & females.
• Bag urine (babies and young children).
• Suprapubic aspiration.
• Sampling of Catheterized patients:
1. In and out catheter.
2. Sampling from Indwelling catheters.
Precautions:
 Always in a sterile container.
 If possible, before starting antimicrobial therapy.
 Preferably, First morning urine . Otherwise when at least 3 hours have
passed since the last urination.

4. Clean Catch Midstream Urine
Collection

5. Collection in Females
• Instructions:
 Wash hands with soap and water.
 With one hand , spread the labia apart.
 Wipe the urethral meatus from front to back with soap and water.
 Allow the first portion of the urine to pass in the toilet.
 Midstream urine specimen is passed into the sterile container.

6. Collection in Males
• Instructions:
 Wash hands with soap and water.
 Clean the head of the penis with soap and water.
 Retract foreskin (if uncircumcised).
 Allow the first portion of the urine to pass in the toilet.
 Midstream urine specimen is passed into the sterile container.

7. Collection in neonates and Infants
• Pediatric collection bags:
 Bags with hypoallergenic
skin adhesives attached over
the labia in girls or penis in
boys to collect specimens.
• Suprapubic aspiration:
 Collected by aspirating urine
from the distended bladder
through the abdominal wall,
using sterile technique.
 It may be used also to
collect urine from pregnant
ladies.
 It is the only urine specimen
suitable for anaerobic
culture of urine.

8. Sampling of Catheterized Patients
In and out catheter
• by placing a catheter through the urethra
into the bladder. It is only used in patients
who can`t evacuate the bladder by
themselves or in infants when we need to
take a sterile urine sample.
Indewelling Catheter:
• Clamp the catheter tube to allow collection
of freshly voided urine.
• Clean the catheter above the clamp with
70% ethanol.
• Use a sterile needle and syringe to puncture
the catheter tubing.
• Aspirate the urine directly from the tubing.
• Transfer the urine to a sterile container.
• Collection bag urine is NOT acceptable for
culture.

11. Transport to Laboratory
 Transfer to laboratory as soon as possible.
 Culture within 2 hours of collection.
WHY??
OTHERWISE
To keep bacteria viable but not multiplying so that you avoid
reporting of false high viable bacterial count.
Most pathogenic bacteria double in count every 20 minutes
 Refrigerate for 24 hrs. at 4°C. OR
 Add 1-2% of boric acid. Extends time up to 72hrs.
N.B.: Boric acid may inhibit growth of enterococci.

12. Rejection Criteria
(Specimens are unsuitable for culture)
• Urine from bedpan, diaper, catheter bag or tip.
• Delay more than 2 hrs. without refrigeration.
• Specimen received in a non sterile container.
• Specimen that have leaked out the container.
• Specimens for anaerobic culture except those collected by
suprapubic aspiration.

13. Processing of Urine Culture
• Inoculation & incubation of media plates.
• Macroscopic examination for PH, pyuria &
bacteriuria.
• Microscopic examination for pus cells, red cells &
epithelial cells.
• Identification of isolated uropathogen.
• Colony count of isolated uropathogen.
• AST of the isolated uropathogen
• Reporting of culture results.

14. Urine Dipstick Tests
A dipstick is a paper strip impregnated with chemicals that undergo a color
change when certain constituents of urine are present in a certain
concentration. The strip is vertically dipped into the urine sample, and after
the appropriate number of seconds mentioned on the strip container, the
color change is compared to a standard chart to determine the findings.

15. Screening for Bacteriuria (Nitrate Reductase)
• Nitrates in urine are derived from patients diet (e.g. vegetables).
• Gram negative bacilli such as Proteus & E.coli produce the enzyme
nitrate reductase which reduces nitrates to nitrites.
• Urine dipstick strips can detect the production of nitrites by a
colour change.
• A positive is highly specific for UT caused by splitting organisms,
such as Proteus species and E coli.
• However, the test is dependent on patient's diet and sufficient
bladder incubation time.
• Gram-positive uropathogens do not produce nitrite reductase and
therefore the dipstick test will be negative.

16. Screening for pyuria (Leucocyte esterase test)
• Leucocyte esterase is an enzyme produced by intact and damaged
neutrophils.
• A positive LE test indicates presence of pus cells in urine.
A Combination of nitrite and leukocyte esterase tests is rapid, low
cost and of good sensitivity and specificity.

17. pH of urine specimen:
Normal urine pH is slightly acidic, with usual values of 6.0 to
7.5, but the normal range is 4.5 to 8.0.
•A urine pH of 8.5 or 9.0 is indicative of a urea-splitting
organism, such as Proteus, Klebsiella, or Ureaplasma
urealyticum.

18. Microscopic Examination
1- Pus cells/HPF.
2- Red cells /HPF.
3- Squamous epithelial cells.

19. Microscopic Examination
Pus Cells Red Cells
Significant pyuria= > 10 pus
cells/HPF of urinary sediment
• In some UTIs (e.g. honeymoon
cystitis & prostatitis)
• In some autoimmune disorders
(e.g. post streptococcal
glomerulonephritis)

20. Squamous Epithelial Cells
Usually few in properly collected urine specimen Many
epithelial cells in urine may indicate specimen
contamination by vaginal secretions since this is the
epithelial lining of vagina and distal urethra. What is
the epithelial lining of urinary tract?

21. Urine Culture Media
• MacConkey Agar
PLUS
• Blood Agar
OR
• CLED Agar
(Cystine–lactose–electrolyte-deficient agar)
A differential medium used for isolation and enumeration
of bacteria from urine. The medium supports the growth
of all urinary pathogens and provides a distinct
colony morphology and colour.

22. Growth & Identification
• CLED
• Blood/ MacConkey

23. Viable Bacterial Count
Calculation:
• The number of bacteria in a urine sample is expressed as colony forming
units per ml (CFU/ml).
Reporting & interpretation :
 Less than 10 000 CFU/ml (104
CFU /ml) = Contamination (non
significant).
 10 000–100 000 CFU/ml (104 –
105
CFU/ml) = Doubtful significance
(suggest repeat specimen)
 More than 100 000 CFU/ml (105
CFU /ml) = Significant bacteriuria.
Many guidelines and studies consider lower counts than 100 000/ml. as
indicative of UTI

24. Antibiotic Susceptibility Test (AST)
• Disk diffusion Method is usually used in clinical laboratories.
• Kirby - Bauer method on Muller Hinton agar is the standard method.
• After over night incubation, inhibition zones are measured and reported as
sensitive, intermediate and resistant based on guidelines of CLSI (clinical
laboratory standard institute).
• Antibiotic choice for AST is based on:
 Type of isolated organism.
 Patient age.
 Physiological state of the patient (e.g. pregnancy, lactation).
 Clinical condition (e.g. renal, hepatic).
 Site of infection (e.g. prostate & meninges ).

26. How would you interpret and take decisions?
Based on the knowledge that you have
acquired in this module you are expected to
correctly interpret & take decision in
various laboratory and clinical aspects of
UTIs.
NOW, LET US SEE

27. How would you interpret and take a decision
1. A positive urine culture without pyuria. Explain.
2. A negative urine culture with pyuria. Explain.
3. A patient with recurrent symptoms of UTI, but with normal pus cells count
in urine. Should you request a urine culture or not?
4. A 25 years old female with symptoms of UTI & pyuria. Should you
prescribe antibiotic treatment or send her first for culture and sensitivity
test?.
5. A 25 years old male with symptoms of UTI & pyuria. What is your next
move?. A request for culture or a prescription of antibiotic treatment?.
6. A catheterized patient without complaint but with high pus cells in urine
and a positive urine culture. What is your diagnosis & treatment decision?
7. In hospitals where epidemiological & statistical data about common
uropathogens and their antibiotic susceptibility is available. Should you
primarily prescribe antibiotic treatment without culture and sensitivity?.
8. A 28 years old female with symptoms of UTI, urine culture reported
isolation of Staph. saprophyticus in a colony count of 8 000 CFU/ml. Would
you consider this finding diagnostic for UTI?.

28. Answers
1. Pyuria is not a mandatory finding for diagnosis of UTI. Estimation of pus cells
/HPF in urine is variable and is affected by many factors.
2. A case of sterile pyuria (Revise possible causes).
3. Yes you should. Culture request is indicated by patient`s clinical picture and
whether the case is a complicated or uncomplicated UTI and the absence of pus
cells in urine doesn`t rule out UTI. (Revise number 1 answer).
4. A typical case of uncomplicated UTI for empirical treatment without culture
and sensitivity using first line antibiotics (Revise).
5. Male UTI below the age of 50 y is among the definition of complicated UTI
where culture and sensitivity is the correct procedure for diagnosis and
treatment.
6. This is NOT CAUTI since there is no complaint. No treatment is needed. The
presence of pus cells is due to the inflammation caused by the mere presence
of the catheter & bacteriuria is a result of biofilm formation on the catheter.
7. Yes you should use the data for guided empirical treatment based on antibiotic
stewardship of the hospital.
8. Yes. Staph. saprophyticus is a common uropathogen in females suffering from
cystitis in the sexually active period (Acute urethral syndrome) and it is usually
of low bacterial count in urine culture.