The document discusses laboratory diagnosis of urinary tract infections, including specimen collection and transport, microscopic examination of urine to detect white blood cells, bacteria, casts, crystals and parasites, and culture of urine samples to identify causative organisms and antibiotic susceptibility testing. Appearance of urine and findings on microscopic examination can provide clues to possible urinary tract infections or other underlying conditions. Proper collection and transport of urine samples is important for accurate laboratory diagnosis of UTIs.

1. LABORATORY DIAGNOSIS OF
URINARY TRACT INFECTIONS
PRESENTED BY DR.GARGI TIGNATH
GUIDED BY DR.PURTI AGRAWAL

2. Introduction:
The urinary tract ,from the calyces of kidneys to the
Urethra,is lined with a sheet of epithelium that is
Continuous with that of skin.
Protective factors:
1. Flow of Urine
2. sloughing of these epithelial cells serve to protect the
urinary tract from infection.
Micro-organisms,particularly bacteria,may enter the urinary
tract through the potential pathways of the epithelial
surface to cause infections.

3. 3
Only lower part of urethra has a
resident bacterial flora
Rest of the urinary tract is
normally sterile
Flushing effect of urine flow
Local phagocytic activity
Mucosal IgA and secretions
from prostatic and urethral
glands
Urinary Tract

4. Types of UTI:
 Upper UTI-Involves kidney or ureter
 Acute pyelitis: Infection of the pelvis of the
kidney
 Acute pyelonephritis – infection of the
kidney parenchyma.
 Lower UTI- Infection from the urinary
bladder downwards
 Cystitis-infection of the urinary bladder.
 Prostatitis-infection of prostate.
 Urethritis-infection of urethra.

5. o Uncomplicated UTI:
o Complicated UTI: Infection involving structurally
and functionally abnormal urinary tract
Infection involving structurally and
functionally normal urinary tract
(simple UTI)

6. EPIDEMIOLOGY AND RISK FACTORS
 50–80% of women in the general population- acquire at least
one UTI during their lifetime—uncomplicated cystitis in most
cases.
 Recent use of a diaphragm with spermicide, frequent sexual
intercourse, and a history of UTI are independent risk factors
for acute cystitis.
 Cystitis is temporally related to recent sexual intercourse, with
a sixtyfold increase in the relative odds of acute cystitis in the
48 h after intercourse.

7. In healthy postmenopausal women, sexual activity, diabetes mellitus, and
incontinence are risk factors for UTI.
Many factors predisposing women to cystitis also increase the risk of
pyelonephritis.
Factors independently associated with pyelonephritis in young healthy
women include frequent sexual intercourse, a new sexual partner, a UTI in
the previous 12 months, a maternal history of UTI, diabetes, and
incontinence.
The common risk factors for cystitis and pyelonephritis :
pyelonephritis typically arises through the ascent of bacteria from the
bladder to the upper urinary tract. However, pyelonephritis can occur without
clear antecedent cystitis.

8. PREDISPOSING FACTORS:
 Age-Incidence increases with age.
 Except among infants and the elderly, UTI occurs far more
commonly in females than in males.
 During the neonatal period, the incidence of UTI is slightly
higher among males than females because male infants more
commonly have congenital urinary tract anomalies.
 After 50 years of age, obstruction from prostatic hypertrophy
becomes common in men, and the incidence of UTI is almost
as high among men as among women.
 Between 1 year and ~50 years of age, UTI and recurrent UTI
are predominantly diseases of females.
 The prevalence of ABU is ~5% among women between ages
20 and 40 and may be as high as 40–50% among elderly
women and men.

9.  Sex-sexually active women are prone to UTI due to
-short urethra
-proximity to the anus
-urethral trauma during intercourse
 Pregnancy-
-Dialation of ureters and renal pelvis
-Stasis
-Incompetence of vesicourethral valves
-Hormonal changes.

10. oStructural and functional abnormality of the urinary
tract-
-Obstruction due to urethral stricture,calculas,prostatic
hypertrophy and tumour
-Neurogenic bladder
-Vesico-urethral reflux
-Genital prolapse
oMetabolic- Diabetes mellitus
oIntervention- Instrumentation including catheterization
and any surgical procedure
oBacterial virulence-pilli and adherence to
uroepithelium

11. Etiological Agents Of Urinary Tract Infections:
•Bacteria-
•Gram-negative bacilli-
•E-coli
•Proteus species
•Klebsiella
•Enterobacter
•Pseudomonas
•Gram-positive cocci-
•Staphylococcus aureus
•Staphylococcus epidermidis
•Staphylococcus saprophyticus
•Enterococcus species

12.  Gram negative cocci-
 Neisseria gonnorhoeae
 Others-
 Mycobacterium tuberculosis
 Salmonella species
 Gardnella Vaginallis
 VIRUSES-
 Adenovirus
 FUNGI-
 Candida albicans
 PARASITES-
 Tricomonas vaginalis
 Schistosomia hematobium
 Enterobius vermicularis

14. 14
Ability to adhere urinary
epithelial cells
Some strains of E coli
(uropathogens) possess pili
interacting with galactose
containing receptor sites on
epithelial cell surfaces
Proteus sp. possess
urease, which raises the pH
and cause precipitation of
phosphate crystals leading
to stone formations
Pathogenicity
Entry of
microorganisms
By ascending route
following colonization
or periurethral area by
enteric organisms
Rarely
hematogenous

15. CLINICAL PRESENTATION
 Asymtomatic bacteriuria-
 About 5-7% of pregnant women have been reported to
have urinary tract infections without symptoms.
 Undetected and untreated bacteriuria-symptomatic
infection later in pregnancy-pyelonephritis and
hypertension-prematurity-perinatal death.

16.  Symptomatic UTI:
 Urgency
 Frequency of micturation
 Associated with pain and dyscomfort.
 Pyelonephritis (upper UTI)-
-loin pain,tenderness,high grade fever and rigour.
Cystitis/ lower UTI-
-Dysuria,fever with chills and increased frequency.

17. COLLECTION AND TRANSPORT OF SPECIMEN
Specimen
collection urine
MSU
MALE
FEMALE
CSU
DURING
CYSTOSCOPY
SUPRAPUBIC
ASPIRATE
Children,infants,older
women
EMU TB of urinary tract
Initial flow
Urethritis,prostatitis

18. COLLECTION:
 Mid Stream Urine(MSU)-
 Appropriate for bacterial culture.

19. 19
Sterile specimen container
Female patients
Instruction for collection of mid stream urine
Spread labia,
using plain
soap or
antiseptic wipe
front to back,
dry with tissues
Retract prepuce,
using plain soap
or antiseptic
clean glans. Dry
with tissues.
Male patients
Begin passing urine
Stop flow in midstream
Pass several ml into pen container
without touching rim
Stop flow before it ends
Recap container
Pass remaining urine into lavatory
Send specimen to laboratory
immediately
(refrigerate if prolonged transport
time)

20. CATHETER SAMPLE URINE(CSU):
 If the patient has been catheterized,the sample is
collected as follows:
 Area over the catheter is first cleaned with
alcohol,after donning clean gloves.
 With the help of a sterile syringe and needle,the
urine sample is drawn and put into the universal
container.
 Urine should be never collected from the urobag or
by opening the draining tube.

21. SUPRAPUBIC ASPIRATE:
 Sample for culture.
 suprapubic aspirate is the gold standard for obtaining
urine specimens for culture.
 Any growth of pathogenic bacteria in an SPA specimen
is felt to be significant.
 It is a simple, safe, rapid and effective technique in
infants/pediatric age group.

22. Indications:
 Young unwell infants for whom there is a need to
obtain specimens as part of a septic work-up
where antibiotic administration should not be
delayed while awaiting a clean-catch urine.
 For a child (< 2 years) when it is deemed important
to confirm a UTI. Eg in a child with recurrent urinary
tract infections with positive cultures but minimal
cellular response.
 Children with previous UTIs with unusual or
resistant organisms
 Children on prophylactic antibiotics

23.  Contraindications:
 Bleeding diathesis
 Abdominal distension
 Complications:
 Microscopic haematuria is common.
 rare complications include: macroscopic
haematuria, bladder haematoma, bladder
haemorrhage, intestinal perforation.

24.  Equipment
 One assistant to hold the infant (not parent)
 Specimen jar for urine
 23G needle (25G for premature infants)
 3ml or 5 ml syringe
 Ultrasound/Bladder scanner and gel

25. SPA METHOD
 Assistant to hold infant supine with legs extended and together.
To prevent voiding in boys, the shaft of the penis should be
squeezed to occlude the urethra.
 Identify insertion point – midline, lower abdominal crease .Wipe
the overlying skin with an alcohol swab.
 Insert needle perpendicular to the skin in all directions. Do not
aim the tip of the needle down into the pelvic region. (The bladder
in a baby is predominantly an abdominal rather than pelvic
organ).
 The skin should be punctured quickly as if popping a balloon with
a needle. Insert needle to the hub and aspirate. If urine is not
immediately aspirated, continue aspirating as the needle is
withdrawn.

26. If unsuccessful, withdraw the needle to just under
the skin, and advance at an angle with the needle
aimed further away from the pelvis. Do not repeat
this procedure more than once.
If urine is obtained, remove needle and place
urine into sterile urine jar.

28. Factors increasing the likelihood of a
successful SPA:
History of no voiding in the past 30
minutes, and the presence of a dry nappy
Prehydration
Bladder dull to percussion

29. EARLY MORNING SAMPLE(EMU)
 Indicated if renal tuberculosis is suspected.
 Three urine samples are collected on consecutive
days.
 Entire morning sample is collected

30. TRANSPORT:
 Sample must be transported at room temperatute
with in half an hour.
 OR Refrigerated at 4 degree Celcius upto 4 hrs
 Beyond 4hrs urine should not be processed for
bacterial culture.
 If the sample is from a patient who has no
immediate healthcare facility-a special container
with 1.8% boric acid is provided,and urine can be
kept for upto 24 hrs

31. APPROACH TO DIAGNOSIS OF UTI
UTI
MICROSCOPY
PUS
CELLS,RBCS,BACTERIA
GRAM STAIN
CULTURE
QUANTITATIVE
METHODS
SEMI-
QUANTITAVE
METHOD
ANTIBIOTIC SENSITIVITY

32. LABORATORY EXAMINATION OF URINE
 DAY-1
1.Describe the appearance of urine
 colour of specimen-
 whether it is Cloudy or clear

33. APPEARENCE POSSIBLE CAUSE
CLOUDY-(Possibly urine hasan
unpleasent smell and
containsWBCS)
• Bacterial urinary infection.
RED AND CLOUDY(Due to red
cells)
•Urinary schistosomiasis
•Bacterial infection
BROWN AND CLOUDY(due to
haemoglobin)
•Black water fever
•Other conditions-causing intra
vascular haemolysis
YELLOW BROWN/GREEN
BROWN(Due to bilirubin)
•Acute viral hepatitis
•Obstructive jaundice
YELLOW ORANGE( due to
urobilin-oxidized urobilinogen)
•Hemolysis
•Hepatocellular jaundice
MILKY WHITE(Due to chyle) •Bancroftian filariasis

34.  Points to remember:
 1.Colour of urine can be caused by the ingestion of
certain foods,herbs ,drugs especially vitamins.
 Freshly passed urine is clear and pale yellow to
yellow depending on concentration.
 When urine left to stand-cloudiness may develop-
1.Due to precipitation of urates in acidic urine(pink
orange colour).
2.Phosphates and carbonates in an alkaline urine.

35. 2.MICROSCOPIC EXAMINATION:
 Wet preparation : To detect-
 Significant pyuria,(WBCs in excess of 10 cells/µl of
urine.counting -1WBCs per low power field-
correspond to 3 cells per/µl ).
 Red casts
 Yeast cells
 T.vaginalis motile trophozoites
 S.haematobium eggs
 Bacteria(provided urine is freshly collected).

36. REPORTING OF WET PREPARATION:
1.Bacteria-seen as rods,but sometimes cocci or
streptococci
-bacteria is usually accompanied by pyuria(pus cells
in urine).
2.WBC-Round 10-12µm in diameter,cells that
contain granules .(in UTI- found in clumps).
-In urine sediments,WBCs/hpf40X)-
Few upto -10WBCs/hpf
Moderate-11-40 WBCs/hpf
Many-40 WBCs/hpf

38.  Significant pyuria->10 WBCs/µl
 Bacteriuria without pyuria-
 Diabetes
 Enteric fever
 Bacterial endocarditis
 Contaminated urine
 Pyuria with a sterile routine culture-
 Renal tuberculosis
 Gonnococcal urethritis
 C.Trachomatis
 Leptospirosis
 Patient of UTI treated with antimicrobials

39. Red cells:
 Smaller and more refractile than white cells,have
definate outline and contain no granules.
 Isotonic urine ringed appearance.
 Hypertonic urine-small and crenated.
 Glomerulonephritis-dysmorphic(vary shapes and sizes).
 Causes of hematuria -
 acute glomerulonephritis
 Bacterial infections
 Urinary schistosomiasis.
 Red cells in urine of women –may be due to
menstruation

41. Casts:
 Solidified protein and are cylindrical in shape.(formed in
kidney).
 Hyaline casts-colourless and empty,-damage to the
glomerular filter membrane
 Seen following strenous exercise or during fever.
 Waxy casts-hyaline casts remained in kidney for longer
time,appear thick ,dense ,indented or twisted,may be yellow in
colour.
 Cellular cast-
 WBCs-inflammation of kidney,pelvis or tubules
 Red cells casts-appear orange red –indicates hemmorhage
into the renal tubules or glomerular bleeding.

42.  Granular casts:
 Irregular sized granules originating from degenerate
cells and protein.
 Seen in renal damage.
Epithelial cells:
 Nucleated vary in size and shape.
 Reported as few,moderate and many.
 Normally few are seen in urine.
 Large no. indicates inflammation of urinary tract.
 Vaginal contamination of the specimen.

44.  Yeast cells:
 oval in shape and some show single budding
 Can be differentiated with RBCs-run a drop of
dil.acetic acid under cover slip-red cells will be
haemolysed but not the yeast cells.
 Seen in women with vaginal candidiasis
 In diabetic and immunocompromised patient.

46.  Trichomanas vaginalis:
 Littile larger than white cells.
 motile,move by flagella and an undulating
membrane.
 seen in Acute vaginitis
 Eggs of s.haematobium:
 Large size(145x55µm)
 Spine at one end.

47. EGGS OF SCHISTOSOMIA

48.  CRYSTALS:
 Characteristic refractile appearance.
 Indicator of urinary tract calculi.
Type of crystal Acidic urine
Calcium oxalate crystals Colourless,small,octahedral or
oval spheres or biconcave
disc(dumbell shape).

49. EXAMINATION OF GRAM STAINED SMEAR:
 Prepare and examine a Gram stained smear of the
urine when bacteria or WBCs seen in wet
preparation.
 Transfer a drop of the urine sediment to a slide and
spread it to make a thin smear, heat fix/methanol fix
and stain it by Gram technique.

50.  Look for bacterias associated with urinary infections –
gram negative rods.
 Occasionally,Gram positive cocci and streptococci may
be seen.
 Single type of organism-uncomplicated acute UTI.
 More than one type of organisms-chronic or reccurrent
infections
- In acute urethritis of male
 To make presumptive diagnosis of gonnorhea-gram
negative intracellular dipplococci in pus cells

51. 3.BIOCHEMICAL TESTS
 Protein
 Nitrate
 Leucocyte esterase

52. PROTEINS:
 Sulphosalicylic acid reagent test
 Protein reagent strip test
 Significance of the test:
 Proteinuria is found in most bacterial urinary tract
infections .
 Other causes include: glomerulonephritis,nephrotic
syndrome,ecclampsia,urinary
schistosomiasis,hypertension and severe febrile
illness.

53. GRIESS TEST OR NITRITE REAGENT STRIP TEST
 Principle:
 urinary pathogens:E coli(commonest cause of
UTI),proteus species and klebsiella species are able to
reduce the nitrite ,normally present in urine to nitrite.
 Used to screen UTI in pregnancy in antenatal clinics.
 Test is false negative :
 when infection is caused by pathogens that do not
reduce nitrite,
 e.g-enterococcusfaecalis,
pseudomonas,staphylococcus,candida organisms.
 Occasionally person is on diet lacking vegetables.
 Bacterias are very few in urine.

54. LEUCOCYTE ESTERASE TEST(LE)
 This is an enzyme specific for polymorphonuclear
neutrophil(pus cells).
 Detects the enzyme from active and lyzed WBCs
 An alternative method of detecting pyuria when it
is not possible to examine fresh urine
microscopically for white cells.
 when the urine is not fresh and likely to contain
mostly lyzed WBCs.
 LE-using a reagent strip test such as the BM-Test-
LN(Boehringer strip) –detects both nitrite and
leucocyte esterase.

55.  False negative strip tests:
 Urine contains boric acid.
 Excessive amount of protein (>500mg/100ml).
 Excessive amount of glucose(>2g/100ml).

56. URINE CULTURE:
 cultures are indicated in the following situations:
 – Complicated UTI including pyelonephritis
 – UTI in past 3 weeks indicating possible relapse or the
presence of symptoms for > 7 days
 Recent hospitalization or catheterization indicating
possible nosocomial infection
 – Transplant patients
 – MS patients
 – Prostatitis patients
 – Pregnancy
 – Diabetes
Indications

58.  Conventional: 5 % sheep BAP and MAC
 – CLED: Cysteine lactose electrolyte deficient medium: inhibit
 Proteus swarming and accommodate common pathogens
 • Paddles or “Dip”type devices
 – SOLAR-CULT( Solar Biologicals, NY)
 – OnSite™(Trek Diagnostics, Cleveland, OHIO)
 – DipStreak(Novamed, Israel)
 – Other
 • Chromogenic media
 – BBL CHROMAgar
 – bioMerieuxCPS ID2
 – RemelSpectra UTI
 • CCF: BAP and MAC is usual

59.  Routine or non-invasive:
 Clean-Catch mid-void; indwelling catheter or pediatric “bag”
– 0.001 ml calibrated loop onto BAP and MAC, Streak down
center; spread out from there.
– > 16 hr incubation, 35 °in an O2 incubator before reading
plates initially; most laboratories discard as no growth at ~
24 hrs.

60.  Invasive collection methods:
 straight catheter, suprapubic aspirate, cystoscopy,
nephrostomy; “low colony count urine .
– 0.01ml calibrated loop onto BAP and MAC
– Streak down center and spread out from there
– Consider > 24 -48 hr* incubation, 35 °C in a O2incubator
– May want to include the 0.001 ml inoculums well for
easier CFU determination
 We need to know what type of urine it is!
 *incubation time may vary if specific organisms.

61.  Normal specimens may contain small no.
organisms,,usually less than10,000 per /ml of urine.
 Urine from aperson with untreated acute urinary
tract infection usualy contains 100,000 or more
bacteria/ml.
 Approximate no. of bacteria per ml of urine,can be
estimated by using acaliberated loop or a measured
piece of filter paper
 Single colony represents-1 organisms.
 E.g if an innoculum of 1/500 ml produces 20
colonies, the number of organisms represented in
1/500 ml of urine is 20 or10,000 in 1 ml.

62. CYSTEIN LACTOSE ELECTROLYTE-DEFICIENT
AGAR
 Mix the urine(freshly collected clean-catch
specimen) by rotating the container.
 Using a sterile caliberated wire loop e.g one that
holds 1/500 ml,inoculate a loopful of urine on a
quarter plate of CLED agar,if microscopy shows
many bacteria,use a half plate of medium.
 Incubate the plate aerobically at 35-37 degree
overnight

63.  Growth of both Gram positive and Gram negative
pathogens.
 Indicator in CLED agar-bromothymol blue-therefore
lactose fermenting colonies appear yellow.
 The medium is electrolyte deficient to prevent
swarming of proteus species.

68. E. COLI COLONIES ON MAC CONKEY AGAR

69. DAY-2
EXAMINE AND REPORT THE CULTURE:
 Negative urines (no growth)
– 0.01 ml inoculum
• Sterile or < 100 CFU/ml OR
• No growth of >100 CFU/ml
– 0.001 ml inoculum
• Sterile or < 1000 CFU/ml OR
• No growth of >1000 CFU/ml
 Positive cultures: colony count reported along with
ID (with or without AST)
• Mixed cultures: reported as such with note about
calling for consultation and/or further work-up
• Unusual pathogens/isolates
– Bring it to attention of supervisors
– Call clinician or other health care provide

70. REPORTING OF URINE CULTURE
 Count approximate no. of colonies=Estimate the
number of bacteria,that is colony-forming units per
ml of urine.
 <10,000 organisms/ml,not significant.
 10,000-100,000,doubtful significance.
 >100,000/ml,significant bacteuria.

71. MOST COMMONLY USED
QUANTITATIVE CRITERIA FOR UTI
 • Symptomatic women
 – >103 CFU bacteria/ml = most likely cause of the UTI•
IDSA: 1000 CFU/ml: 80% sensitivity and 90%
specificity– >10 2 CFU/ml= 95% sensitivity and 85%
specificity for cystitis in women
 • Symptomatic men
– >103CFU bacteria/mL
 • Catheterized patients
>103CFU bacteria/mL
 • Asymptomatic individuals
– >105CFU bacteria/mL(IDSA recommends 2 urine
samples)
 • Usually any growth of a pathogen in a suprapubic
aspirate or intraoperatively obtained sample is
considered significant

72. ANTIMICROBIAL SENSITIVITY TESTING
 Indications-
 Urine with significant bacteriuria,particularly from
patient with reccurent UTI.
 Complicated UTI.