urinary tract infections-lab diagnosis (e.coli, klebsilla, proteus, pseudomonas)

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Laboratory Diagnosis

URINARY TRACT INFECTION

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Introduction• It is defined as a disease caused by microbial invasion of the genitourinary tract that extends from the renal cortex of the kidney to the urethral meatus.

•Route of infection to reach the urinary tract can be either- Ascending route Haematogenous route

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Causative organisms

• E.coli• Klebsiella• Pseudomonas• Proteus

Gram –ve Bacilli

• Staphylococcus• Enterococci

Gram +ve Bacilli

• M.tuberculosis• Citrobacter• Salmonella

Miscellaneous

• Candida albicans

Fungus

E.coli

c.albicans

Citrobacter

Staphylococcus

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UTI causing organisms

E.coli (63%)Klebssiella(15%)Pseudomonas(5%)Proteus(5%)Other Gram -ve bac-teriaStaphylococcusEnterococcus

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Sterile pyuria•Sterile pyuria is urine which contains white blood cells while appearing sterile by standard culturing techniques.

• Sterile pyuria is listed as a side effect from some medications such as paracetamol (acetaminophen).

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Community acquired UTI

•An infection contracted outside of a health care setting or an infection present on admission.•E.Coli is the most common cause followed by stapylococcus saprophyticus

s.saprophyticus

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Hospital acquired UTI•Acquired by a patient during a hospital visit.•Most common organism causing it- Klebsiella

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Patients with urinary catheters have a higher chance of getting UTI’s. This is because germs can travel along the tubing of the catheter and cause infection in the urinary system. This is then referred to as a “catheter associated” UTI.

Complications –•Cystitis• pyelonephritis• bacteremia• prostatitis•Rarely, endocarditis, septic arthritis, and meningitis

Catheter acquiredUTI

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Poor aseptic insertion of catheter: periurethral flora inserted into bladder.

Migration of bacteria along outer surface of catheter.

Open drainage.

Breaks in the closed drainage system.

Causes

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SPECIMEN COLLECTION

TRANSPORT and STORAGE

LABORATORY METHODS

LABDIAGNOSIS

1. 2.

3.

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SPECIMEN COLLECTION

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MID STREAM SPECIMEN(MSU)•Collected PRIOR to antibiotics.

•WASH hands properly.•Specimen is collected in a STERILE container

BEFORE ANTIBIOTICS

STERILE CONTAINER

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Method-

First morning specimen should be taken.

FEMALES- MALES-

Clean the area with soap and water , hold the labia apart and discard few ml of urine and then collect the midstream sample directly into the sterile container.

Clean the glans with soap and water, retract the foreskin, discard few ml of urine and the collect the mid stream sample directly into the sterile container .

•Discard the rest of the urine.•The first portion of urine adequately flushes out the normal urethral flora.

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CATHETER SPECIMEN

•Urine should be collected directly from the catheter and not from the collection bag.

•The catheter should not touch the container.

•Catheterization to obtain urine is not done because of risk of introducing infection though it yields excellent results.

URINE SPECIMEN FROM INFANTS

A clean catch specimen after cleansing of genitalia. OR Supra pubic aspiration.

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TRANSPORTand

STORAGE

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THE SPECIMEN SHOULD BE TRANSPORTED IMMEDIATELY.

•Urine is a good medium for the growth of microorganisms, hence delay in processing will hamper the results.

•If delay is unavoidable, the specimen can be refrigerated for up to four hours at 4˚c.

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Urine transport tubes have shown to preserve bacteria without refrigeration.It consistes of-

-Boric acid -Glycerol

-Sodium formate

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LABORATORY

METHODSMICROSCOPYCULTUREIDENTIFICATIONOTHER METHODSANTIBIOTIC SENSTIVITY TEST

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MICROSCOPYUrine is centrifuged and deposit is examined under the microscope.

Following can be seen-•Pus cell( 0-5/hpf is the normal value for pus cells in the human body).

•RBC

•Epithelial cells

•Bacteria

Bacteria

Pus cell

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CULTUREMost lab use a semi quantitative method (STANDARD LOOP TECHNIQUE) for culture.

This technique involves a standard calibrated loop which transfers a fixed and small volume of urine.

STANDARD CALIBRATED LOOP

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Fixed and small amount of uncentrifuged urine is transferred to BLOOD and MacCONKEY’S AGAR.

Next day, the number of colonies grown is counted and total count per ml is calculated.

Standard loop technique

•BLOOD AGAR- gives a quantitative measurement of bacteriuria.•Mac CONKEY’S AGAR- gives presumptive diagnosis of the bacteria.

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INTERPRETATION OF RESULTS

•Bacteriological diagnosis of UTI is carried out by ‘SIGNIFICANT BACTERIURIA’ using quantitative cultures developed by KASS(1956).

•This is based on the fact that normal urine is sterile but during voiding may become contaminated with genital commensals.

SIGNIFICANT BACTERIURIA

DOUBTFUL SIGNIFICANCE

NO SIGNIFICANT GROWTH

•When bacterial count is more than 105/ml of a single species.

•It indicates ACTIVE UTI.

•Between 104 to 105 bacteria per ml.

•Specimen should be repeated for culture.

• <103 bacteria per ml are regarded as contaminated.

• Contamination is also considered when 3 or more bacteria are isolated.

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IDENTIFICATION

The organisms are identified by-

colony characters gram staining motility biochemical test slide agglutination test serological test

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SIZE 1-2 mm

COLONY CHARACTER Blood agar

Mac Conkey agar

Grey-white, moist, smooth, opaque, non mucoid Pink colony( due to lactose fermentation)

GRAM STAINING Gram –ve bacteria

BIOCHEMICAL TEST Indole Methyl red Voges-Proskauer(VP) Citrate

++--

MOTILITY Motile

E.coli

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BLOOD AGAR MacCONKEY’S AGAR

GRAM STAINING

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SIZE 2-4 mm

COLONY CHARACTER Blood agar

Mac Conkey agar

Grey-white, moist, smooth, opaque, mucoid Pink colony( due to lactose fermentation)

GRAM STAINING Gram –ve bacteria

BIOCHEMICAL TEST Indole Methyl red Voges-Proskauer(VP) Citrate

--++

MOTILITY Non-Motile

Klebsiella

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BLOOD AGAR

GRAM STAINING

MacCONKEY’S AGAR

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ProteusCOLONY CHARACTER Blood agar Nutrient agar Mac Conkey agar

Exhibits SWARMING(fishy odour)Exhibits SWARMINGSmooth pale colonies

GRAM STAINING Gram –ve bacteria

BIOCHEMICAL TEST Indole TSI PPA Urease Oxidase

-(P.mirabilis)+(P.vulgaris)H2S production++-

MOTILITY Motile

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BLOOD AGAR

GRAM STAINING

NUTRIENT AGAR

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PseudomonasSIZE 2-4mm

COLONY CHARACTER Blood agar Nutrient agar Mac Conkey agar

Smooth, large, convex, translucent(sweetish aromatic odour)Same as on blood agarSmooth pale colonies

GRAM STAINING Gram –ve bacteria

BIOCHEMICAL TEST Indole TSI PPA Urease OxidaseCitrate

-K/ no change (without H2S production)-+/-+-

MOTILITY Motile

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BLOOD AGAR

GRAM STAINING

NUTRIENT AGAR

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OTHER METHODS-

Dip slides

Pour plate method

Triphenyltetrazolium

chloride (TTC) test

Griess nitrite test

Glucose test paper

Polymorphonuclear

neutrophils (PMNs)

Catalase test

Leucocyte esterase

Detection of lipopolysaccha

ride (endotoxin)

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DIP SLIDES

In this test commercially available plastic slides coated with CLED agar (cysteine lactose electrolyte deficient) on one side and MacConkey’s agar on the other is dipped in urine then placed in a sterile container is incubated at 37˚c.

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POUR PLATE METHOD•It is a quantitative method •But, is cumbersome for routine diagnostic work

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GRIESS NITRITE TEST

•It is based on nitrate reducing enzymes produced by bacteria present in urine.

•Gram -ve bacteria- NITRATES NITRITES

•The presence of nitrite detected by the test indicates UTI.

•Normal urine does not contain nitrite.

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TRIPHENYLTETRAZOLIUM CHLORIDE TEST (TTC TEST)

TTC is reduced by several frequently encountered urinary tract pathogens and produce a pink red precipitate.

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CATALASE TEST•Certain bacteria have an enzyme catalase which acts on hydrogen peroxide to release oxygen.

H2O2 H2O + O

[Nascent

oxygen]

•The presence of catalase is evident by formation of bubbles.•It indicates bacteriuria. (test present only in catalase +ve organisms.)

catalase

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GLUCOSE TEST PAPER

It is based on utilisation of the minute amount of glucose present in normal urine, by bacteria causing the infection.

Hence, it indicates bacteriuria.

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POLYMORPHONUCLEAR NEUTOPHILS(PMNs)

•PMNs are counted in uncentrifuged urine specimen with the help of haemocytometer.

8 PMN/mm3 is indicative of infection.

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LEUCOCYTE ESTERASEPresence of the enzyme in urine indicates infection.

DETECTION OF LIPOPOLYSACCHARIDE

This test helps in the detection of gram –ve bacteria in urine.

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ANTIBIOTIC SENSITIVITY TEST

•Esch. coli and other common urinary pathogens develop multiple drug resistance and of the transferable variety.

•Antibiotic sensitivity is necessary to administer proper antibiotic.

•Done by STOKES DISC DIFFUSION method.

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-Umang Juneja