WEEK 1: Introduction to culture and diagnosis of UTI

Question 1

Who are UTIs mostly common in?

Why?

Answer 1

UTIs common especially in adult women

Here are some key reasons why UTIs are more prevalent in females:

1. Shorter Urethra:
   Females have a shorter urethra compared to males. The urethra is the tube that connects the bladder to the external environment for urine elimination. A shorter urethra makes it easier for bacteria to travel from the outside into the bladder, increasing the risk of infection.
2. Proximity to Anus:
   The female urethra is located closer to the anus than in males. This proximity increases the likelihood of bacteria from the gastrointestinal tract, particularly E. coli, entering the urethra and ascending into the bladder.
3. Urethral Opening Position:
   The opening of the female urethra is close to the vagina, which can be a source of bacteria. Sexual activity, especially if not practicing proper hygiene, may introduce bacteria into the urethra, leading to an increased risk of infection.
4. Hormonal Factors:
   Hormonal changes, such as those occurring during pregnancy, menstruation, and menopause, can affect the pH of the vagina and the urethra. These changes may create an environment more conducive to bacterial growth and colonization.
5. Pregnancy:
   During pregnancy, hormonal changes and the pressure of the growing uterus can contribute to a higher risk of urinary stasis (incomplete bladder emptying) and, subsequently, an increased susceptibility to UTIs.
6. Menstrual Hygiene Products:
   Certain menstrual hygiene practices, such as using tampons and diaphragms, may introduce bacteria into the urethra, increasing the risk of infection.
7. Postmenopausal Changes:
   After menopause, decreased estrogen levels can lead to changes in the vaginal and urethral tissues, making them more susceptible to infection.
8. Voiding Habits:
   Some behavioral factors, such as delaying urination or not fully emptying the bladder during voiding, can contribute to the persistence of bacteria in the urinary tract.

Question 2

Describe the pathogenesis of UTI.

Answer 2

`1. Entry of Bacteria into the Urethra:
The most common route of entry for bacteria is through the urethra. Bacteria from the perineum, anus, or external genitalia can migrate into the urethra, especially in females due to the shorter length of the urethra.

2. Ascension to the Bladder:
   Once bacteria enter the urethra, they can ascend through the urinary tract to the bladder. The female anatomy, with its shorter urethra and proximity to the anus, facilitates this ascension. Sexual activity can also introduce bacteria into the urethra.
3. Adherence to Uroepithelial Cells:
   Bacteria that reach the bladder must adhere to the uroepithelial cells lining the urinary tract to establish colonization.
   Adherence is facilitated by specific bacterial adhesins interacting with receptors on the surface of uroepithelial cells.
4. Avoidance of Host Defenses:
   Successful pathogens must evade or resist the host’s immune defenses. Bacteria may possess mechanisms to resist being flushed out during urination, such as producing biofilms or adhesins that prevent detachment.
5. Colonization of the Bladder:
   Once bacteria adhere and avoid host defenses, they can proliferate and establish colonization within the bladder.
   The presence of a large bacterial population in the bladder contributes to the development of symptoms associated with UTIs, such as dysuria (painful urination), frequency, urgency, and lower abdominal discomfort.
6. Further Ascension to the Upper Urinary Tract:
   In some cases, bacteria can ascend further to the upper urinary tract, reaching the ureters and kidneys. This may result in more severe symptoms and complications, such as pyelonephritis (kidney infection).
7. Immune Response and Inflammation:
   The host’s immune system responds to the bacterial invasion by initiating an inflammatory response. Neutrophils are recruited to the site of infection, and cytokines are released to combat the infection.

Symptoms and Clinical Manifestations:

The inflammatory response and bacterial presence lead to the classic symptoms of a UTI, including pain or burning during urination, increased frequency of urination, urgency, cloudy or foul-smelling urine, and in some cases, hematuria (blood in the urine).

Question 3

State the causes of UTI.

Answer 3

Etiology:

Commonly - Bacteria
Gram negative bacteria i.e. Enterobacteriaceae: *E. coli

Other Gram –ve bacteria:
Pseudomonas aeruginosa
Chlamydia trachomatis

Also:
Gram positive bacteria
*Staphylococcus saprophyticus
Enterococcus spp.
Streptococcus agalactiae- Group B streptococcus

Other pathogens
Fungi: Candida albicans

Parasites e.g. Trichomonas vaginalis.

Question 4

Presence of bacteria in urine not always indicative of a UTI, because peri-urethral & skin commensal bacteria present.

So,
For lab diagnosis: identification & counting of microbial pathogen using essential.

State the cutoff level.

Answer 4

For lab diagnosis: identification & counting of microbial pathogen using a “104 CFU/ml cut-off level” essential.

Question 5

Urine easily contaminated with commensal microflora from distal urethra, perineum or vagina (or fecal microflora in babies)

Describe the Optimal - morning & midstream urine method of specimen collection.

Answer 5

Urine voided first in the morning

Cleaning of periurethral area (with water not antiseptics)

Initial urine passed, washes off microflora from distal urethra - discarded.

Midstream urine should be collected into a sterile container.

Question 6

State the 3 current methods of urine collection.

Answer 6

i) Mid-stream-clean-catch (MSCC): patient instructed to clean labia before voiding into a sterile transport container

ii) **Suprapubic puncture: paediatric patients

iii) Urine catheter sample: hospitalized patients

Question 7

What is the main concern in specimen collection?

Answer 7

Main concern: lack of asceptic technique during collection increases risk of urine sample contamination.

**Normally, if patient has no UTI, the urine in the bladder is ‘sterile’. So suprapubic sample expected to be uncontaminated with commensal bacteria vs. MSCC which is prone to contamination from commensal microorganisms.

Question 8

State the duration and refrigeration requirements for urine sample to be valid.

Answer 8

Bacterial counts important in laboratory urine processing

Delays in transportation & processing of urine allow bacteria replication, affecting counts

Refrigeration at 4C
Ideally urine must be processed within 2 hours or refrigerated at 2-8C & processed within 24hr

Question 9

Why is How &/or type urine sample collected important?

Answer 9

May affect “accuracy” of results
Required as part of assessment of results

Question 10

State reasons why some urine samples can be rejected.

Answer 10

Urine samples that are rejected e.g.:
>2hr without refrigeration

Urine from urine bag of catheterized patient or bedpan or urinal

Leaky containers or contaminated with stool/ fecal material or menstrual blood.

Collected from patients on antibiotics.

Question 11

Visual analysis of urine.

State the normal colour of urine.

State some causes of Turbidity or cloudiness in urine sample.

Answer 11

Normal, fresh urine is clear & pale to dark yellow or amber in colour

Abnormal colour i.e. red/ red-brown color may be due to: blood or certain foods e.g. beetroot, certain drugs.

Turbidity or cloudiness may be caused by excessive cellular material or protein:
Leukocytes
Erythrocytes
Bacteria
Crystals
Fat

Question 12

Screening with Urinalysis dipstick

Describe urinalysis dipstick.

Answer 12

Typically, dipsticks

Reagents on test pads evaluate different properties of urine.

Question 13

Describe Microscopic process.

What can we look for?

Answer 13

Well-mixed urine spun in centrifuge & supernatant decanted

A drop of re-suspended sediment placed on slide & coverslip placed on top

Viewed with light microscope.

Wet mount performed for examination of:
White blood cells (wbc)
Red blood cells (rbc)
Bacteria
Fungi
Crystals

Question 14

What is pyuria?

What does it indicate?

Answer 14

Presence of abnormal no’s of wbcs in urine

May be due to an infection in lower or upper urinary tract.

Question 15

What is hematuria?

State possible causes of hematuria.

Answer 15

Normally no rbcs present

rbc under high power field = abnormal
NB. if contamination e.g., menstruation ruled out)

Possible causes:
Glomerular damage, nephritis, tumors eroding urinary tract, kidney trauma, urinary tract stones,

Rbc’s may be due to trauma during bladder cauterization.

Question 16

Epithelial cells appear larger than granulocytes & contain a large round or oval nucleus.

May be present into urine of healthy individuals in low no.s

What can cause them to be in high numbers?

Answer 16

Appear larger than granulocytes & contain a large round or oval nucleus.

May be present into urine of healthy individuals in low no.s
But
No.s higher in nephrotic syndrome & others causing tubular degeneration.

Question 17

Microscopy of urine specimens.
What are Casts?
What are casts indicative of?
Describe the process of how they are formed and the different types.

Answer 17

Cylindrical structures formed in kidney nephron (distal tubule)

Note: indicative of kidney disease

*With renal tubular damage, epithelial cells slough into tubule lumen

*Assimilate with Tamm-Horsfall protein matrix (hyaline cast) & form ‘cellular casts’

*Over time epithelial cells degenerate forming coarsely granular, then finely granular casts

*Finally forming waxy casts indicating chronic tubular disease

Question 18

State other elements that also assimilate with hyaline casts & form various other casts.

Answer 18

Hyaline casts

RBC casts

WBC casts

Bacterial casts

Epithelial casts

Granular casts

Waxy casts; fatty casts

Question 19

Which casts are seen in individuals with fever or sometimes after strenuous exercise?
Vs.
Which casts are derived from degenerating cellular casts, thus presence in urine indicative of underlying pathology?

Which casts are indicative of glomerular bleeding, typically diagnostic of glomerulonephritis?
Post Streptococcal Glomerulonephritis

Which casts indicate inflammation of renal parenchyma?
Typical for acute pyelonephritis but may also be present with glomerulonephritis.

Answer 19

Hyaline cast (Tamm Horsfall protein) - seen in individuals with fever or sometimes after strenuous exercise.
Vs.

NB: Granular casts - derived from degenerating cellular casts, thus presence in urine indicative of underlying pathology.

RBC casts
Indicative of glomerular bleeding, typically diagnostic of glomerulonephritis
Post Streptococcal Glomerulonephritis

WBC casts
Indicate inflammation of renal parenchyma.
Typical for acute pyelonephritis but may also be present with glomerulonephritis.

Question 20

Describe the Gram staining principle & procedure.

Answer 20

Step 1. Label slide & heat fix colony (~30secs)

Step 2. Addition of crystal violet (1 min, then rinse with H2O)
Cell wall retains crystal violet

Step 3. Addition of iodine (1 min, then rinse with H2O)
Complexes with crystal violet, preventing easy removal of the dye, known as “fixing the dye”. All bacteria remain purple

Step 4. Addition of decolouriser e.g. ethanol or acetone
(~30secs, then rinse with H2O)
Gram+ve cell walls dehydrate, closing pores thus crystal violet-to-iodine complexes retained
In contrast
In Gram-ve cell walls: with decolouriser primary stain leaches into the solvent, washing away the dye, thus unstained

Length of time of decolorization is critical, prolonged time results in removal of primary stain from the ‘Gram+ve’ cells too, leading to ‘false negatives.

Step 5. Addition of counterstain e.g. safranin or fuchsin
(1 min, then rinse with H2O)

To visualize unstained Gram-ve bacteria, a counter stain is added. Safranin stains bacteria red (or pink)

Finally, viewing under the microscope with the oil immersion lens

Gram positive or negative?
Morphology? (rod, coccus, spiral)
Occur singly, chains, pairs etc.?
Size?

Question 21

Non selective media - growth of fastidious & non-fastidious bacteria
Bacteria can be differentiated by ability to secrete hemolysins:

α-hemolysis - incomplete (green colouring around colonies)

β-hemolysis - complete (clearing around colonies)

γ-hemolysis - no hemolysis

Name the media.

Answer 21

Blood (sheep, horse) agar

Question 22

Non-selective media for isolation & differentiation of urinary tract organisms

Differential media: lactose fermenters vs. non-lactose fermenters bacteria:
Lactose fermenters – acid produced turns pH indicator (bromothymol blue) green to yellow
Non-lactose fermenters - blue colonies

Electrolyte deficiency - inhibits swarming fProteusspecies.

Name the media.

Answer 22

Cysteine lactose electrolyte deficient medium (CLED)

Question 23

Selective & differential

Lactose fermenters vs. non-fermenters

Neutral red pH indicator - red at acidic pH & yellow at alkaline pH

NB. Selective for Gram negative bacteria

Name the media.

Answer 23

MacConkey Agar

Question 24

State some tests for Gram negative rod bacteria.

Answer 24

Oxidase test
Urease test
Citrate agar test
Motility test
Indole test

Question 25

State Some tests for Gram positive cocci.

Answer 25

1. Catalase test
2. DNase plate
   Divide plates accordingly.
   Inoculate plate with organisms Incubate 37C / 24hr.
3. Tube Coagulase
   3 tubes with 0.5ml plasma
   & add
   0.5ml Positive control
   0.5ml Negative control
   0.5ml Gram+ve bacterium

Question 26

Describe way of Calculating Colony Forming Units/mL.

Answer 26

Initial calibration loop vol. i.e. 1µL
Count colonies.
Multiply no. by x1000.

e.g. 21 colonies x 1000 = 21 000
Report as Colony Forming Units CFU /mL

FOR
Initial calibration loop vol. i.e. 10µL
Count colonies.
Multiply no. by x100.

e.g. 21 x 100 = 2 100
Report as CFU /mL.

Infection threshold
i.e. Cut off ≥ 104

NOTE: In some distinct cases threshold may be lower or disregarded i.e. colonies of certain bacteria e.g. Group B Strep
i.e urine collected suprapubic aspiration

Question 27

Summary.

Name the common agar plates used in the lab promote growth of bacteria that commonly causing UTIs.

Other bacteria that cause UTIs less, may be fastidious, grow slowly or not at all on the commonly used media e.g. N. gonorrhoea, Mycobacterium spp-repeat with specific selective media and incubation conditions

Although less common, when a UTI is truly caused by unusual bacteria the likelihood of a urinary tract malformation is higher, so it is important to diagnose accurately

Even with a ‘Cut off of 104 CFU/ml’, there may be ‘true infections’ with lower CFU/mL . So it is important to consider patient symptoms, patient history & microorganism species identification

Answer 27

The common agar plates used in the lab i.e blood, MacConkey & CLED, promote growth of bacteria that commonly causing UTIs

Other bacteria that cause UTIs less, may be fastidious, grow slowly or not at all on the commonly used media e.g. N. gonorrhoea, Mycobacterium spp-repeat with specific selective media and incubation conditions

Although less common, when a UTI is truly caused by unusual bacteria the likelihood of a urinary tract malformation is higher, so it is important to diagnose accurately

Even with a ‘Cut off of 104 CFU/ml’, there may be ‘true infections’ with lower CFU/mL . So it is important to consider patient symptoms, patient history & microorganism species identification

Question 28

Urinary tract infections (UTIs) are one of the most common type of infections.
Can be categorized as____________and or ___________.

Affect _________more than _________.

Recognizing diagnostic UTI hallmarks facilitates rapid assessment & treatment (can also help in differentiating lower UTIs from more serious upper UTIs)
Colour, odour, leukocyte esterate & nitrites, WBCs, RBCs, RTEs, casts

Diagnosis based only on clinical symptoms not appropriate. Diagnostic precision is increased by:

Physical examination of the urine & microscopy of urine sediment

Detection of leukocyte esterase & nitrite (i.e. by urinalysis dipsticks) & other hallmarks of UTIs

Culture + qualitative & quantitative lab analysis of the bacterial culture

Answer 28

Urinary tract infections (UTIs) are one of the most common type of infections. Can be categorised as:
‘uncomplicated’ vs ‘complicated’ &/ OR ‘lower vs upper’

Affect women more than men

Recognising diagnostic UTI hallmarks facilitates rapid assessment & treatment (can also help in differentiating lower UTIs from more serious upper UTIs)
Colour, odour, leukocyte esterate & nitrites, WBCs, RBCs, RTEs, casts

Diagnosis based only on clinical symptoms not appropriate. Diagnostic precision is increased by:
Physical examination of the urine & microscopy of urine sediment
Detection of leukocyte esterase & nitrite (i.e. by urinalysis dipsticks) & other hallmarks of UTIs
Culture + qualitative & quantitative lab analysis of the bacterial culture