Laboratory diagnosis of Urinary tract infections

 

Learning objectives:

               Whenever a clinically suspected case of urinary tract infection is presented, for microbiological evaluation and management of patient - students must know

           1. Common aetiological agents of UTI

           2. Proper method and containers for sample collection; sample transportation.

       3. Sample processing: Culture media, culture method, incubation, result interpretation including identification of isolated bacteria and colony count.

               4. Clinical interpretation of result.

UTI: Disease caused by microbial invasion of urinary tract

Definition

·        Bacteriuria-Presence of bacteria in the urine

·        Pyuria-Presence of WBCs in the urine

·        Significant bacteriuria: Normal urine is sterile; it may get contaminated during voiding with normal urethral flora. A count of >10⁵ CFU count/ml of urine considered to be Significant infection in most cases (Kass concept)

·        Asymptomatic bacteriuria: Without symptoms, Common in pregnancy (5-7%)

·        Lower UTI:

§  Urethritis – Dysuria(Burning sensation or pain during micturition), frequent urination, urgency

§  Cystitis–Dysuria,frequent urination,Suprapubic pain, tenderness and occasionally hematuria,Urine: cloudy

·        Upper UTI

§ Acute Pyelonephritis-flanks pain, renal tenderness,hematuria, Fever, chills and vomiting(Systemic manifestation)

o + symptoms of lower UTI present

 

Etiology of UTI

·        E. Coli is commonest bacteria causing UTI without catheter and urological abnormalities or calculus

·        Other bacteria like Klebsiella, Proteus mirabilis, Enterobacter, Enterococcus spp., Staphylococcus aureus, Staphylococcus saprophyticus- (in sexually active female) and fungus Candida may be responsible for UTI

Note:

v  Bacteriuria without Pyuria:

·        Enteric fever, Bacterial endocarditic, diabetes

v  Pyuria without bacteriuria

·        Renal tuberculosis, Gonococcal Urethritis, Chlamydia trachomatis infection, Ureoplsma,   Leptospirosis, Trichomonas, Pt. treated with antimicrobials, Urological abnormality

 

Laboratory diagnosis

Specimen collection: Urine should be collected in wide mouth screw capped sterile container by

·        Clean Catch Midstream early morning Urine:

§  Female:

·        Clean area with soap and water hold labia apart and begin voiding in commode.

·        After several ml have passed collect mid-stream

§  Male:

·        clean glans penis with soap and water retract foreskin

·        After several ml have passed collect mid-stream

·        Suprapubic aspirate:

§  In neonates and small children when urine difficult to collect

§  Needle aspiration above symphysis pubis through abdomen into bladder.

·        In catheterise patients:

§  Urine aspirated from catheter tube after clamping distally and disinfecting through syringe and needle

§  Never collect from urine bag

Transportation

·        Label and transport as early as possible to laboratory

·        If not possible: if delay > 2 hours, refrigerate the urine at 4-6°C or add boric acid preservative (0.1gm/10 ml urine)

 

 

Laboratory procedures:

Day 1

 

 

 

Microscopic Examination (wet preparation - HPF)

·        Pus cells:> 10 pus cells/HPF  correlate with significant infection

·        RBCs: Stone, Acute glomerulonephritis

·        Crystals-Calcium oxalate: stone                                                                                                   

·        Epithelial cells- if in large no -inflammation or vaginal contamination of urine          

·        Yeast cells- Candida

·        Motile trophozoites:Trichomonasvaginalis

·        Gram stain:In gram stain presence of at least one bacterium in oil immersion field (examining 20 fields) uncentrifuged urine correlate with significant bacteriuria (>10⁵ /ml)

 

 

Culture: 

·        Nutrient agar, MacConkey agar

·        Semi Quantitative Method: Standard loop technique : Un-centrifuged urine(0.01 ml)with loop of 4 mm diameter

·        Plates incubated for 37 degree for 24 hours.

 

         Laboratory Examination of Urine- Day2

·        Colony counts in Nutrient agar by colony counter: number of colony X 100= total CFU/ml.

·        If colony count >10⁵CFU/ml is considered Significant bacteriuria

·        Identification of bacteria isolated on culture plates are done by Gram stain and Biochemical reactions

·        The isolated bacteria are further processed for Antibiotics Sensitivity Test –By disc diffusion method

§  Routinely following antibiotics for UTI are used

·        Quinolones(Norfloxacin, Ciprofloxacin, Levofloxacin),

·        Nitrofurantoin,

·        Ampicillin/Sulbactam, Amoxicillin/clavulanic acid,

·        Cotrimoxazole ,

·        cefuroxime,

·        Amino glycosides (Amikacin, Gentamicin)

Note:

·        For Proteus spp - Do not use Nitrofurantoin and tetracycline

·        Chloramphenicol must not be tested / given for UTI

 

·        Report of Urine CS includes:

§  Microscopic finding:  Pus cells, RBC, Epithelial cells

§  Culture:  No Organism Isolated / No significant Bacteriuria/ Significant Bacteriuria

§  Antibiotic sensitivity:      The tested antibiotic s are interpretated as Sensitive /Resistant/Intermediate sensitive depending on zone of inhibition (ZOI) as per CLSI criteria

Take Home massage:

Ø  Sample to be collected – Clean Catch Mid stream urine

Ø  Presence of >10 pus cells/hpf in urine microscopy correlate with significant infection

Ø  Culture - In mid-stream early morning urine sample: Colony count of >10⁵CFU/ml - Significant Bacteriuria

Ø  Most Common Pathogen responsible uncomplicated UTI is E. coli

Ø  Nitrofurantoin is oral drug only for UTI in both gram-positive cocci and gram-negative bacilli except Proteus spp.

Ø  Fluoroquinolons are commonly used for UTI.

 

              

 

Exercise:

A 25 years old female presented with high grade fever with rigor since 2 days & complains for burning micturition and suprapubic pain. She has a urine routine and microscopic results which is suggestive of pyuria. Urine culture examination is strongly recommended. Answer the following questions

Q. 1        Which container is used for sample collection?

Q. 2        What instructions should be given to patient for sample collection?

Q.3         Which culture media are used for culture?             

Q.4         What is the culture method for quantification of bacteria in urine?              

Q. 5        Which antibiotics are preferred for sensitivity testing for Escherichia coli isolated from urine culture?