Laboratory Diagnosis

ANATOMY OF THE URINARY TRACT

FEMALE

MALE

Types of Specimens
Mid-stream urine (MSU)
Catheter specimen of urine (CSU) Suprapubic Aspirate (SPA)
Note!
This is an invasive procedure and should be used only when highly necessary!

Specimen Collection
Note! Sterility is important! Use a sterile container for collection of urine

Ensure that container is labelled properly

Name of patient-------Type of specimen-----Date and time of collection----------------

Specimen Collection

Mid stream specimen Urine from indwelling catheter

Specimen Collection

Catheter specimen of urine (CSU)

Laboratory Procedures
Macroscopic appearance
Microscopy Culture

Antimicrobial Susceptibility testing of isolates

Macroscopic appearance
Cloudy, murky, or turbid (muddy) urine is characteristic of a urinary tract infection (UTI)

Clear urine! May suggest the absence of UTI. This is not always so

red or smokey brown urine may suggest the presence of red blood cells Foul or strong urine odor may suggest infection

Microscopy
Methods employed: Wet preparation

Gram stain
Ziehl Neelsen (if indicated in clinical diagnosis) Other stains

Microscopy
Slide is examined under the light microscope

Wet preparation

bacteria White blood cells(WBCs)

Possible Gram stain results

Gram stain from a cloudy urine Pus cells and gram negative bacilli

Gram negative bacilli (GNB) Pus cells

Possible Gram stain results

cloudy urine Pus cells and gram negative bacilli
Gram stain from a

Gram negative bacilli (GNB) Pus cells

Possible Gram stain results

Gram stain from a cloudy urine No pus cells, gram positive cocci in chains

gram positive cocci in chains

Possible Gram stain results

Gram stain from a cloudy urine Many pus cells and gram negative diplococci seen intracellularly

Pus cells

Gram negative diplococci within pus cells (intracellular)

Possible Gram stain results

Squamous epithelial cell Budding yeast cells Gram negative bacilli

Gram stain from a cloudy urine No pus cells, gram positive ovoid bodies (budding yeast cells)

Pseudo hyphae of yeast

Possible Gram stain results

Gram stain from a bloody urine Many red blood cells, few white blood cells, no organisms seen

Culture of urine
What is the organism in the patients urine? Qualitative analysis Is the organism(s) found of numerical significance?

Semi- quantitative analysis Antimicrobial susceptibility of isolates What are the treatment options based on in vitro susceptibility testing?

Semi- quantitative analysis

We assume: Significant plate count 100,000 bacteria/ml Doubtful significance >10,000 but <100,000 Insignificant plate count 10,000

Semi- quantitative analysis

Assuming we use a calibrated loop to deliver 2ul of urine and significant plate count is 105 bacteria/ ml.(colonies) Then: 1ml.(1,000 ul) 100,000 bacteria (colonies)

.02 ml.(2ul)
significant plate count
And Insignificant plate count

100,000 x 2 1,000 = 200 Bacteria (colonies) = 200 Bacteria (colonies) using a calibrated loop of 2ul = 20 Bacteria (colonies)

Note! We assume that one colony seen on the culture medium is a progeny of one bacterium seeded onto the agar

Qualitative analysis:
Choice of Media:

Blood Agar - will support the growth of most bacteria implicated in UTI

MacConkeys Agar -- will identify members of the Enterobacteriaceae

Other Media: e.g. Sabourauds Dextose Agar - for yeast (if indicated)

INOCULATION OF CULTURE MEDIA

Loop to deliver 2ul urine A loop full of urine is taken and inoculated onto culture media . Plates are incubated at 370 c overnight

Interpretation of Culture
Conclusion: Significant growth of a lactose fermenter

>200 colonies (plate count>105cfu/cc) significant plate count on blood agar

Lactose fermenter on MacConkeys agar

Identification of isolates
Gram stain Biochemical Reactions etc.

E.g. organism seen on previous slide showed a significant plate count of a lactose fermenter

I.D. Gram stain gram negative bacilli Biochemical Reactions - Lactose Acid - Glucose Acid with gas production - Urease negative - Citrate - positive - Motile - Indole positive Therefore isolate identified as Escherichia coli or E. coli

Kligler A/AG

Identification of isolates
e.g. if Gram stain gram positive cocci catalase positive coagulase negative Novobiocin resistant Then the organism is identified as Staphylococcus saprophyticus

Susceptibility testingdisc diffusion technique

Susceptible

Antimicrobials commonly used include: Naladixic acid (NA) Nitrofuradantoin (F) Sulphamethoxazole trimetroprim (SXT)
Gentamicin (GM) Ampicillin (AM) Augmentin (AMC) Norfloxacin (NOR) Others when there is multiple resistance

Resistant

Reporting:
e.g. Direct Microscopy: Many pus cells, few epithelial cells, many gram negative bacilli.
Culture: Significant growth of E. coli plate count > 105 bacteria/cc. Susceptible to/ etc. Resistant to/ etc.

Common Isolates
CYSTITIS
Bacterial : E. coli Klebsiella sp. Proteus sp. Pseudomonas sp Mycotic: Candida albicans Parasitic: Schistosoma haematobium

RENAL ABSCESS:
Staphylococcus aureus Mycobacterium tuberculosis E. Coli Proteus sp. Pseudomonas aeruginosa Klebsiella pneumoniae

Common Isolates
PYELONEPHRITIS/PYE LITIS
E. coli

IV CATHETER:
Staphylococcus aureus Yeast Klebsiella pneumoniae E. Coli

Klebsiella sp. Proteus sp. Pseudomonas sp Enterococcus faecalis

URINARY CATHETER:
E. coli Klebsiella pnemoniae Proteus sp. Pseudomonas sp.

Identification of common isolates

Escherichia coli: Colonial morphology: flat dry LF Gram reaction: Gram negative bacilli Oxidase: negative Kliglers: Acid/Acid Gas Urea: negative Citrate: negative Motility: Motile Indole: Positive Lysine: Variable
flat dry lactose fermenter (LF)

Identification of common isolates

Klebsiella pneumoniae
Colonial morphology: mucoid raised LF Gram reaction: Gram negative bacilli Oxidase: negative Kliglers: Acid/Acid Gas Urea: Positive Citrate: Positive Motility: Non-Motile Indole: Negative Lysine: Positive

mucoid raised lactose fermenter (LF)

Identification of common isolates

Proteus mirabilis
Colonial morphology: swarming growth Gram reaction: Gram negative bacilli Oxidase: Negative Kliglers: Akaline /Acid + H2S+Gas(co2, Urea: Positive Citrate: Positive Motility: Motile Indole: Negative Lysine: Negative PPA Positive

Identification of common isolates

Pseudomonas aeruginosa
Colonial morphology: green pigment Gram reaction: Gram negative bacilli Oxidase: Positive O/F

Identification of common isolates

Staphylococcus spp.
Colonial morphology: white or golden Gram reaction: Gram positive cocci catalase: positive Coagulase positive (aureus) Novobiocin Resistant (saprophyticus)

Identification of common isolates

Streptococcus spp.
Colonial morphology: Note type of haemolysis! Gram reaction: Catalase: Aesculin hydrolysis: CAMP test Bacitracin Gram positive cocci Negative Positive (Group D Strep.) Positive (Group B Strep.) Sensitive (Group A Strep.)

For Group D strep. do further tests to exclude Enterococcus faecalis

Identification of common isolates

Candida albicans
Colonial morphology: white or creamy Gram reaction: large Gram positive ovoid bodies

Germ tube

Positive