Laboratory Diagnosis of Urinary Tract Infection (UTI)

A. Clinical Aspects:
1. Common clinical manifestations: Dysuria, frequency and urgency of
micturition
2. Fever with rigor and loin pain may be associated, particularly in cases
of pyelonephritis. Suprapubic tenderness may be present in cases of
cystitis.
3. Asymptomatic bacteriuria: Sometimes patients do not have local or
systemic symptoms referable to the urinary tract but microbiologically
urine samples show significant bacteriuria.
4. Urethral syndrome: Urethral syndrome is a term coined for the
presence of classic symptoms of urinary tract infection, viz. dysuria,
urgency,

and

frequency,

without the

presence

of

significant

bacteriuria. It is usually observed in young sexually active females

and is associated with bacterial counts of > 102 cfu/ ml.
Sometimes UTIs are classified as
I. Uncomplicated: Uncomplicated infections occur primarily in otherwise
healthy females and occasionally in male infants and adolescent and
adult males. Most uncomplicated infections respond readily to antibiotic
agents to which the etiologic agent is susceptible.
II. Complicated: Infection in a urinary tract with functional or structural
abnormalities, including indwelling catheters and calculi. It may occur in
both sexes.
Concept of significant bacteriuria: A means of differentiating between
contamination in the voided specimen and true urinary infection. It
describe the number of bacteria in voided urine that usually exceed the
numbers caused by contamination from commensal organisms present
in the anterior urethra.It usually refers to the laboratory finding of
>105 colony-forming units (CFU) of bacteria per ml of urine.
However, in the following conditions lower bacterial counts are also
considered significant:
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Patient on diuretics/ antibiotics

Suprapubic aspirate specimen

Pregnant women

Growth of gram positive cocci

Acute urethral syndrome

Growth of yeast/ fastidious

Catheterized specimen

organisms.

B. Pathogens commonly associated with UTI

Bacterial pathogens of urinary tract

Fungus

Gram Positive

Gram Negative

Candida species

S. aureus

E.coli

Parasites

S. saprophyticus

Klebsiella species

Schistosoma
haematobium

Streptococcus

Proteus species

Trichomonas vaginalis

Pseudomonas aeruginosa

Enterobius vermicularis*

Enterobacter species

Onchocerca volvulus*

Other Enterobacteriaceae

Wuchereria bancrofti*

species
Enterococcus
species

members
Salmonella species*
Neisseria gonorrhoeae*
* These are not primary pathogen of urinary tract infection
C. Sample collection
Acceptable methods for urine collection include:
A. Midstream clean catch voided sample
B. Catheter Collection
C. Suprapubic aspiration
I. Midstream clean catch voided sample: Sample of choice.
Procedure (in females)
The labia are held apart with the aid of a pair of sponges.
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The periurethral area, perineum and vulva are washed thoroughly from
front to back with two successive cotton pledgets or sponges soaked in
shop.
This is followed by a rinse with sterile saline or distilled water.
The first few milliliters of urine are not collected to flush out bacteria
from the urethra.
Then midstream portion of urine is collected in a sterile, wide mouthed
screw- caped container.
The soapy water preparation is not required for men; rather, simple
cleansing of the urethral meatus immediately before voiding and then
collection of midstream urine sample is usually sufficient.
II. Catheter collection:
Procedure Catheter is clamped proximal to the port so that freshly voided urine is
collected.
The catheter tube proximal to the clamp is disinfected with 70% ethanol.
Needle puncture is made using a needle and a syringe and urine is
aspirated.
Urine samples should not be obtained from catheter bags.
III.

Supra pubic bladder aspiration:

Reserved almost exclusively for neonates and small children.

Bladder should be full and palpable above pubis symphysis before
aspiration.

D. Sample Transportation and Preservation

Specimens are transported immediately to laboratory (within 2 hours)
after collection, to achieve accurate colony counts.
Urine is refrigerated for up to 24 hour at 40C if it cannot be delivered to
laboratory within 2 hours of collection. (Bacterial counts remain constant
at 40C).

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If refrigeration is not possible and specimen are delayed in transport,

urine is collected in transport tubes containing boric acid [concentration
of 10g/l (1% w/v)], glycerol and sodium formate.
Boric acid has been shown to be inhibitory to some Enterococci and
Pseudomonas strains so refrigeration (at 40C) is preferred over Boric acid
preservation.

E. Rapid Screening procedures

I.

Wet mount preparation:

Fresh urine specimen is required.
Procedure
A drop of uncentrifuged urine is placed onto slide.
Cover slip is applied and examined under high dry objective (40X).
Observation
WBCs greater than 5/high power field is considered positive for pyuria.
It may also show urinary parasite (trichomonads), fungus, cast and
crystals.

II.

Gram stain Can be useful in an emergency to initiate informed empirical

therapy.
Procedure
A drop of well mixed uncentrifuged urine is placed onto the slide.
It is allowed to air dry without spreading.
Gram stain is performed and examined under oil immersion to
determine bacteria.
Observation
The presence of at least one bacterium per oil immersion field
correlates with 105 bacteria or more per milliliter of urine.

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III.

Other screening tests-

S.No

Test

Principle

Remarks

Nitrate

Nitrate-reducing enzymes that are

False negative with non- nitrate

reductase

produced by the most common

reducing organisms, e. g.

test

urinary tract pathogens reduce

Enterococcus faecalis,

nitrate to nitrite.

Pseudomonas species,
Staphylococcus species and
Fungus.

Leukocyte

It measures esterase enzyme

Neutropenic,

esterase

produced by inflammatory cells

immunocompromised patients

Test

(WBCs).

have very few WBCs in urine

which may give false negative

Catalase

It is based on the detection of catalase False negative results with

test

enzyme present in most bacterial

Streptococci and Enterococci.

species commonly causing urinary

tract infection
Note - None of the screening methods is as sensitive or reliable as a
culture. In general, screening methods are insensitive at levels below 105
CFU/ml. Therefore, they are not acceptable for urine specimens collected
by suprapubic aspiration or catheterization.

F. Urine culture
Gold standard method for establishing the diagnosis of UTI as:
It establishes the identity of organism
It enables their characterization, including antibiotic sensitivity testing
It allows the determination of bacterial count.
I.

Pour plate method, where a known volume of the urine sample is mixed
with a fixed volume of molten nutrient agar and plates are poured. The
number of bacteria per unit volume can be determined by multiplying the

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number of colonies with a dilution factor of the urine. Although this method
is most precise, it is cumbersome and time consuming.
II.

Semi-quantitative method of counting viable bacteria:

a) Standard Loop Method By using an inoculating loop of standard
dimension calibrated to hold a fixed volume of urine held in the form of
flat sided drop.
Procedure (using a calibrated loop delivering 0.01 ml of urine)
The calibrated wire inoculating loop is flamed and allowed to cool.
Loop is inserted vertically in well mixed urine to allow urine to adhere to
the loop.
Loopful of urine is touched over the surface of the agar plate at the
centre of the plate, from which the inoculum is spread in a line across
the diameter of the plate. Without flaming or reentering urine, the loop is
drawn across the entire plate, crossing the first inoculum streak
numerous times to produce isolated colonies.
Plates are incubated for at least 24 hours at 370C.
b) Filter paper methodA strip of filter paper is folded to obtain an L- shape.
Angulated end and foot of the filter paper is dipped in the uncentrifuged
sample of urine.
Filter paper charged with urine specimen is withdrawn from specimen
container
Filter paper is allowed to absorb the urine.
Foot is pressed on the culture medium (ensuring whole area of foot
makes contact with agar surface).
Strip is removed and discarded in disinfectant.
Plates are incubated at 370C for 24 hour.

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Colony Count for

Culture methods

Remarks

significant
bacteriuria

0.01 ml
Standard

Loop

Loop

0.005 ml

1000 colonies

500 colonies

Loop
Filter paper

25 colonies of bacilli

Porosity and absorptive

(4 mm X 4 mm dimension of and 30 colonies of

power of the paper obtained

the foot- end)

from different

cocci

manufacturers often very.

G. Bacterial identification tests depending on colony morphology

observed
1. Cystine lactose electrolyte deficient agar (CLED) Differentiates between
lactose

fermenter

colonies

(yellow)

from

non-

lactose

fermenter

(blue/gray/green) colonies and inhibits swarming of Proteus. It also

supports the growth of certain Staphylococci, Streptococci and Candida that
fail to grow on MacConkey.
Lactose fermenter bacteria Appears as yellow due to acid
production from lactose utilization, e.g. E.coli, Klebsiella,
Staphylococcus
Non- lactose fermenter bacteria - Appears as green, e.g. Proteus
2. Blood Agar It has the advantage of promoting the growth of nutritionally
exacting strains.

Colony Morphology

Identification Test

Gram

Lactose

E. coli: IMViC ++--, TSI Acid/Acid with gas, Motile, Sugar

negative

Fermenting fermentation (Glu, Lac, Suc, Mal, Mann, Man. and Xyl.)1 sugars

bacilli

(LF

except sucrose with acid and gas.

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Bacteria)

Klebsiella pneumoniae: IMViC --++, TSI Acid/Acid with gas, Non

Motile, Sugar fermentation (Glu, Lac, Suc, Mal, Mann, Man. and
Xyl.) 1with acid and gas.

Non-

Pseudomonas sp: Oxidase +ve , TSI Alkaline/Alkaline, Motile,

Lactose

Oxidation and fermentation test- Oxidative.

Fermenting Proteus sp: Oxidase ve , TSI Alkaline/Acid with H2S, Motile,

(NLF

Phenylalanine deaminase test +ve .

Bacteria)
Gram Positive Cocci

S. aureus: Catalase +ve , Coagulase +ve, Mannitol fermented,

S. saprophyticus: Catalase +ve , Coagulase -ve, Novobiocin Disc
Susceptibility - Resistant

1Glu.

Glucose, Lac. - Lactose, Suc. - Sucrose, Mal. - Maltose, Mann. Mannitol, Man. Mannose, Xyl. - Xylose

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