significant bacteriuria

Significant Bacteriuria

Presented by

Dr. DK Kalra

Definition

Significant Bacteriuria term was intended by Kass in 1957 to provide

a means of differentiating between contamination in the freshly voided

specimen of urine and true urinary infection.

The numbers of bacteria (≥105 CFU/ml) greater than those likely to

result from contamination from the urethral meatus and its environs.

For urine collected via bladder catheterisation, the threshold is 100

CFU/ml.

The threshold is also 100 CFU/ml for women displaying UTI

symptoms.

Definition

Asymptomatic Bacteriuria:

If bacteria were isolated in quantitative counts of

≥105 CFU/mL in a voided urine specimen from

asymptomatic patients.

Common in pregnancy, patient with indwelling

catheter, and Diabetes Mellitus.

Urinary tract infection (UTI)

Definition:

urinary tract infections are characterized by the presence

of ≥105 CFUs/ml of a single bacterial species or multiple

organisms in two consecutive urine specimens, properly

collected from a person with symptoms or signs of a UTI.

Examination• Macroscopic - Appearance, Proteinuria• Microscopic – WBC, RBC, Bacteria• Chemical Tests – Nitrate Reduction, Leucocyte

Estrase, Glucose Oxidation tests• Culture Method – CLED & MacConkey Media• Quantitative Tests – Miles and Misra Method,

Pour Plate Method• Semi Quantitative Tests – Standard Loop, Filter

Paper, and Dip-Slide Tests.

Urine microscopy

Normal- <0-2 pus cells/hpf in males, <0-5/hpf in females.

UTI- Pus cells >10/μl.

Pyelonephritis- White blood cells + white cell casts.

Transitional epithelial cells- after catheterisation &

transitional cell carcinoma.

Presence of large number of squamous cells in urine-

contamination with vaginal fluid.

Cultural methods

• Loop: Double loop used Loop size of 4mm which carries 0.01 ml of urine Loopful of urine is spread over the surface of the agar

plate by making primary well (semiquantitative

purpose) & secondary well (separate colonies)

• Media used:

CLED and MacConkey

Chemical Tests for Significant Bacteriuria

Griess Nitrite Test:

Nitrites are not present in normal urine

Ingested nitrites nitrates and excreted in urine

If gram negative bacteria are present in urine with

enzyme nitrate reductase converts nitrates to nitrites

A typical commercial Griess reagent contains 0.2%

naphthylethylenediamine dihydrochloride, and

2% sulphanilamide in 5% phosphoric acid

Griess Nitrite Test…

• If Nitrites are present in urine they will give pink colour.

• Para-arsanilic acid or sulphanilamide + NO2 →Diazonium salt

In an acid medium• Diazonium salt + tetrahydrobenzoquinoline → Pink

azo dye

Griess Nitrite Test…

Some organisms like Staphylococcus, Enterococci do not reduce

nitrate to nitrite.

Also urine must be retained in the bladder for minimum 4hrs for

conversion of nitrates to nitrites so early morning sample is preferred.

The nitrite test has a 92% to 100% sensitivity for UTI but only a 35%

to 85% specificity.

Nitrite test is especially useful in patients with indwelling urinary

catheters to determine whether they are infected or not.

Leukocyte Esterase Test

Leukocyte esterase test: (Combur-Test UX strips - Roche)

It detects esterase enzyme released in urine from granules of

leukocytes, Positive in pyuria.

If this test is positive urine culture should be done.

75% to 96% sensitivity and a 94% to 98% specificity for detecting

pyuria. False-positive tests are usually caused by contamination,

often by vaginal secretions. False-negative test can be caused by

hypertonic urine.

Chemical Tests…

Reaction catalysed by leukocyte esterase Indolecarboxylic acid ester → Indoxyl + Acid In acid medium Indoxyl + Diazonium salt → Violet azole dye

Glucose Test Paper: enzymes used are glucose oxidase and peroxidase.

Normal value: 160-180 mg/dl. Catalysed by glucose oxidase Glucose + O2 → D-glucono-δ-lactone + H2O2

Catalysed by peroxidase H2O2 + Chromogen → Oxidised chromogen (coloured) + H2O

Quantitative methods: Miles & Misra Method

Miles & Misra method: (1938)It is used to determine the CFU in bacterial suspension or in urine.

Materials required: A calibrated dropping pipette delivering drops of 20μl. Petri dishes containing nutrient agar or other appropriate

medium. Phosphate Buffered Saline (PBS) or other appropriate diluent. Bacterial suspension or homogenate.

Quantitative methods

• CFU per ml = Average number of colonies for a dilution

X 50 X dilution factor.

• Advantages:

• Faster than other methods.

• Less bacterial contamination.

Pour Plate Method• In pour plate method bacterial counts are performed

using 8-10 tubes and dilutions up to 108 or 1010 are made.

• One millilitre of the diluted urine was mixed with nutrient agar 10-15 ml melted and cooled to 50 centigrade, incubated at 37°C. and examined after 24, 48, and 72 hours.

• Colony count is multiplied by dilution factor and will

give viable count/ml of bacteria.

Other Methods• Roll Tube Method – 2ml nutrient agar melted,

cooled to 50 centigrade, with 0.02 ml pipette one drop is added to two tubes.

Tubes rolled in horizontal position under cold water tap to make uniform agar layer, incubated and colonies counted.

• Surface Method - Various dilutions of specimen, drop with 0.02 ml pipette on surface from height less than ½” and incubate for 24 hrs and count colonies.

Semi quantitative methods

• Standard loop method:

0.001 ml loopful of urine yields 100 colonies.

The count/ml will be 105 (significant bacteriuria).

• Filter paper method: (Leigh & Williams 1964).

Count of 25 colonies of bacilli or 30 colonies of cocci

approximately corresponds to 105 bacteria/ml.

• Dip-slide method: With a slide coated with CLED or

MacConkey medium, good for peripheral hospitals.

Standard Loop Method

• A 4mm loop would pick up .01 ml of a liquid. 0.001 ml of urine if yields 100 colonies, then the count per ml will be 105 (significant).

• Can be tested and standardized by diluting an overnight culture of E.coli (109 org/ml) to 105 org/ml, 104 org/ml and 103 org/ml and culturing on CLED agar.

• Colonies are counted for each dilution and multiply by dilution factor (50).

Filter Paper Method

• Leigh & Williams (1964)

• 6mm wide strip of fluffless blotting or filter paper is bent into L shape with 12 mm long foot (area 12 x 6 mm) & sterlilized.

• Dip end & foot into urine, press on to the surface of agar medium.

• Incubate and afterwards count the colonies, 105 bacteria/ml corressponds to 25 bacilli or 30 cocci colonies

Dip-Slide Method

• Dip-slide is a small plastic tray with agar medium, opposite sides may carry different mediums.

• Mid stream urine is collected and dip-slide is immersed in urine.

• It is sent to lab and incubated and viable counts are estimated by representative charts.

• Convenient for peripheral labs.

• Does not provide cellular contents

Interpretation of urine culture

Urine specimen &

patientCount LE

Not likely to be significant

Additional data suggesting that

isolate is significant

Midstream,female with cystitis

>102 CFU +Potential pathogen

< contaminant-

Midstream, female with pyelonephritis

>105 CFU +Potential pathogen

< contaminant

Gram stain demonstrates potential pathogen in neutrophils and/or casts

Midstream, asymptomatic bacteriuria

>105 CFU -<105 CFU of

potential pathogen;

Confirm by repeating urine culture when clinically indicated

Interpretation of urine culture

Urine specimen &

patientCount LE

Not likely to be significant

Additional data suggesting that isolate is

significant

Midstream, male with UTI

>103CFU +<103CFU of

potential pathogen;


Straight catheter, all patients

>102CFU for symptomatic

patients+

<102CFU potential

pathogen/ml, urine LE is negative


Indwelling catheter, all patients

>103CFU +/-

Bacteriuria detected in

asymptomatic patients

No reason to culture unless patient is symptomatic

Significant Bacteriuria importance

• Significant bacteriuria is applicable for only

Enterobacteriaceae group.

• Multiple (three or more) species of gram negative

bacteria- contamination in such case significant

bacteriuria is not considered.

Asymptomatic Bacteriuria importance

• The Infectious Diseases Society of America (IDSA) established

guidelines for the screening and treatment of asymptomatic bacteriuria

(Nicolle et. al., 2005).

• The optimal management depends significantly on specific patient

characteristics, co-morbidities, and risk factors.

• Asymptomatic bacteriuria was consistently harmful in all populations

and necessitated antibiotic treatment.

• Antibiotic treatment of asymptomatic bacteriuria in pregnant women

resulted in a significantly reduced incidence of pyelonephritis.

Conclusion

• Culture done- Nitrite test or LE test positive (Reflexive

urine test).

• The absence of bacteria on Gram's stain of spun urine

sediment has a high negative predictive value for

significant bacteriuria.

• Urine cultures add enormous specificity to the

diagnosis of UTI and continue to be the gold standard for

diagnosis.

Conclusion

• A positive microscopic examination- 2 microorganisms

uniformly distributed per oil immersion field, after

observation of at least 20 fields, according to the criteria

of Washington et. al.

• Multiple (three or more) species of gram negative

bacteria- contaminated.

THANK YOU