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Significant bacteriuria

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D
Dinesh Kumar

This document discusses significant bacteriuria and how it is defined as ≥105 CFU/ml of bacteria in a urine specimen, or ≥100 CFU/ml for catheterized specimens. It outlines methods for urine examination including microscopy, culture, and chemical tests. Microscopy looks for white blood cells and casts. Culture methods include loop, pour plate, and dip-slide techniques to quantify bacteria. Chemical tests detect nitrites and leukocyte esterase. The interpretation of urine cultures depends on factors like specimen type and patient symptoms. Asymptomatic bacteriuria requires treatment in pregnant women to prevent pyelonephritis.

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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 & patient Count 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 & patient Count LE Not likely to be significant Additional data suggesting that isolate is significant Midstream, male with UTI >103CFU + <103CFU of potential pathogen; Gram stain demonstrates potential pathogen in neutrophils and/or casts Straight catheter, all patients >102CFU for symptomatic patients + <102CFU potential pathogen/ml, urine LE is negative Gram stain demonstrates potential pathogen in neutrophils and/or casts 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.
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Significant bacteriuria

  • 2. 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.
  • 3. 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.
  • 4. 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.
  • 5. 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.
  • 6. 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.
  • 7. 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
  • 8. 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
  • 9. 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
  • 10. 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.
  • 11. 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.
  • 12. 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
  • 13. 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.
  • 14. 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.
  • 15. 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.
  • 16. 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.
  • 17. 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.
  • 18. 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).
  • 19. 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
  • 20. 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
  • 21. Interpretation of urine culture Urine specimen & patient Count 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
  • 22. Interpretation of urine culture Urine specimen & patient Count LE Not likely to be significant Additional data suggesting that isolate is significant Midstream, male with UTI >103CFU + <103CFU of potential pathogen; Gram stain demonstrates potential pathogen in neutrophils and/or casts Straight catheter, all patients >102CFU for symptomatic patients + <102CFU potential pathogen/ml, urine LE is negative Gram stain demonstrates potential pathogen in neutrophils and/or casts Indwelling catheter, all patients >103CFU +/- Bacteriuria detected in asymptomatic patients No reason to culture unless patient is symptomatic
  • 23. 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.
  • 24. 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.
  • 25. 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.
  • 26. 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.