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TimoKouri

Urinalysis and Bacterial Culture

Investigations

  • A routine urinalysis consists of chemical screening with multiple test strips, also called dipsticks, and particle counting.
    • In chemical screening, there are several reagent pads on the strip, for detecting leucocytes (esterase activity), erythrocytes (haemoglobin peroxidase activity), bacteria (nitrite test to detect nitrate reducing pathogens) and protein (albumin) in urine. The relative density of urine is important for assessing the concentration of the sample. In addition, testing for glucose, ketones and pH may sometimes be useful.
      • If a urinary tract infection (UTI) is suspected, the sensitivity of the leucocyte and nitrite tests combined is only 40-50% compared with 103 colony-forming units (CFU)/ml in bacterial culture but approximately 80-90% compared with 105 CFU/ml. The sensitivity of a strip test in detecting pyuria (leucocyturia) in an infant with systemic signs or symptoms (bacteraemia) is 95%, if 5 x 104 CFU/ml is considered a significant result in urine culture, as it is in the USA.
      • The analytical sensitivity of the leucocyte test is about 20 x 106 cells/l (corresponding to 2-3 cells/high-power field, HPF, in traditional sediment microscopy).
      • The analytical sensitivity of an erythrocyte test done with an automatic counter is about 10 x 106 cells/l (1-2 cells/HPF; in traditional microscopy, some erythrocytes disappear during centrifugation of the sediment).
      • The sensitivity of the protein test is about 200 mg/l or 100 mg/l of albumin. A urine albumin-to-creatinine ratio (ACR) sensitive for at least 10 mg/l albumin should be used for screening for incipient albuminuria.
      • The relative density of urine is determined chemically on a dipstick. However, the dipstick reaction is insensitive for dilute samples (relative density of urine 1.000-1.005). Automated strip reading devices used in laboratories therefore measure the relative density by refractometry. The relative densities of isotonic and concentrated urine are 1.010-1.015 and > 1.015, respectively.
  • The basic particle count includes urine leucocytes, erythrocytes, various epithelial cells, casts and bacteria. The test is used for investigating UTI, haematuria and renal diseases. Both flow cytometric and automatic imaging devices provide more accurate results than dipstick tests. In smaller laboratories, urine particle counting is done visually by microscopy. A flow cytometer measures the conductivity of the sample, providing its approximate osmolality.
    • Reference limits: urinary leucocyte count below 10 × 106 /l (diagnostic grey zone 10-30 × 106 /l), urinary erythrocyte count below 10-20 × 106 /l (diagnostic grey zone 20-50 × 106 /l), bacteria in urine negative (the technical limits for a positive result vary by device). Less than 5 × 106 /l epithelial cells or casts in urine (specific limits vary by device).
    • In Finnish regional patient materials, the sensitivity of particle counting as compared to significant bacterial culture may be as high as 95%.
    • The concentration of urine, i.e. relative density or osmolality based on conductivity, is considered when interpreting the results. The osmolality of concentrated urine is > 400 mOsm/kgH2O.
  • The cause of the UTI and its antimicrobial sensitivity are confirmed by bacterial culture of urine. The culture should be requested from the first specimen obtained before prescribing antimicrobials. Random cystitis in otherwise healthy adult female patients need not be confirmed by urine culture; clinical confirmation by a questionnaire is sufficient (use locally available version of the Acute Cystitis Symptom Score http://www.acss.world). After receiving a positive culture result, a microbiological laboratory will proceed to identify significant bacterial species and to define antimicrobial sensitivity by further techniques.
    • Results of bacterial culture of urine have been received more rapidly over recent years for several reasons.
      • Based on urine particle counting (leucocytes and bacteria), laboratories may release probably negative results in bacterial culture without actual culture.
      • The primary culture plate for uropathogens used today is a chromogenic plate, where E. coli, for example, can be detected directly in the screening culture based on the colour of the colonies.
      • In large laboratories, most uropathogens can be detected by mass spectrometry for bacteria, i.e. MALDI-TOF MS (matrix-assisted laser desorption ionisation time-of-flight mass spectrometry).
  • A urine sample for cytological studies should be ordered when a malignant neoplasm is suspected; see Renal cancer Kidney Cancer, Bladder cancer Bladder Cancer. The sample should be taken from fresh urine (second sample in the morning, i.e. urine that has been retained in the bladder for 2-3 hours).

Requesting emergency tests depending on the availability of laboratory services

  • Urinalysis and bacterial culture are requested both in and out of hours. The choice of tests depends on what is being sought and on the availability of laboratory services (Table T1). The table also includes some further investigations not done out of hours.

Rapid urinary diagnosis out of hours

Urinary finding sought based on the clinical pictureRecommended test if an emergency laboratory is available or if a delayed response is sufficientRapid diagnosis at the treating unit if the delay of laboratory response is too long for the emergency process
BacteriuriaParticle counting and bacterial culture of urine* (from the same sample before medication)Chemical screening and bacterial culture of urine* (from the same sample before medication)
PyuriaParticle counting and bacterial culture of urine* Chemical screening and bacterial culture of urine*
HaematuriaParticle counting of urineChemical screening of urine
ProteinuriaChemical screening of urine, in specific cases also ACR** (sensitive albuminuria) or urine protein** (immunoglobulin light chains)Chemical screening of urine
GlucosuriaChemical screening of urineChemical screening of urine
KetonuriaChemical screening of urineChemical screening of urine
Urine concentrationChemical screening of urine (relative density), included also in particle counting with flow cytometer, in specific cases osmolality of urine** Chemical screening of urine
* A negative response to a request for bacterial culture of urine may be given based on particle counting (rapid diagnosis). If so, the regional laboratory will have specified how the tests are technically combined, i.e. whether a separate request is needed for particle counting and whether the sample should be divided between two tubes.
** Usually not available out of hours.
Selection of investigations according to the suspected illness

Suspected urinary tract infection (UTI), bacteriuria and pyuria Urine Dipstick Test for the Diagnosis of Urinary Tract Infection

  • The diagnosis of random acute cystitis in an otherwise healthy adult female patient should be based on recognised symptoms (see the ACSS above). Laboratory investigations are not necessary. In other and unclear cases, a urine bacterial culture is indicated. See Urinary Tract Infections.
    • Elderly persons often have asymptomatic bacteriuria. Therefore, it should first be decided based on the clinical symptoms whether antimicrobial treatment will be initiated in case of a positive culture result before requesting urine bacterial culture, to avoid unnecessary treatments.
  • Rapid tests can be used to establish the presence of pyuria and/or bacteriuria in adults either by chemical screening or by particle counting.
  • The following limitations of diagnostic tests should be kept in mind when interpreting the results.
    • A negative result with a multiple strip test will not exclude UTI.
    • If the patient has symptoms consistent with UTI, a leucocyte count > 30 x 106 /l and a positive bacterial count in urine suggest UTI. Exact limits for sensitive and specific screening are given in laboratory manuals. In addition to appropriate laboratory findings, the patient should have symptoms consistent with UTI.
  • In young children, pyuria is needed to diagnose UTI Urinary Tract Infection in a Child. After detecting leucocyturia, urine culture should be performed from a suprapubic bladder aspiration (SPA) specimen or other specimen that is as contamination-free as possible.
  • The cause of pyuria may not be infection by a microbe that grows on a culture plate, but another type of microbial infection (chlamydia Chlamydial Urethritis and Cervicitis, gonorrhoea Gonorrhoea, tuberculosis Diagnosing Tuberculosis) or an immunologic nephritis, such as glomerulonephritis Glomerulonephrites or interstitial cystitis Bladder Pain Syndrome (Interstitial Cystitis).

Interpretation of urine bacterial culture

  • In patients with consistent symptoms, a UTI is considered to be caused by a uropathogen or yeast growing in bladder urine. Whether the number of colony-forming units (CFUs) in the urine specimen is significant depends on the clinical picture, bladder symptoms and details of specimen collection; see Table T2.
    • The following factors should be considered
      • the bladder time (less than 4 h suggesting irritated bladder, in which case colony counts below 105 CFU/ml may be significant)
      • way of collection (bag/pad specimen, midstream urine or sterile, invasive sampling)
      • documentation on correct midstream collection
      • patient's age and sex, and
      • concentration of urine.
  • A positive culture result does not necessarily indicate a UTI because even high counts of bacterial colonies growing from the urine specimen may be due to contamination (physiological microbes from the skin or external genitals) or colonization (of uropathogens in the urethra or the bladder). Colonization of uropathogens in the bladder may cause similar growth in bacterial culture in an asymptomatic person as a uropathogen causing symptoms of infection in a symptomatic patient.
  • The prevalence of asymptomatic bacteriuria (105 CFU/ml or more) is 1-5% in otherwise healthy adult female patients, 2-10% during pregnancy, 10-20% in the elderly (male or female) and as high as 50% in care home residents. Bacterial growth at 103 -104 CFU/ml is more frequent than this in midstream urine specimens. Contamination or colonization is suggested by:
    • an asymptomatic patient with urine leucocyte concentration within healthy range in particle counting
    • a specimen containing polymicrobial growth in culture (3 or more species of bacteria), suggesting improperly collected midstream specimen or colonization of the bladder with several types of microbes
    • a specimen containing squamous epithelial cells (neither sensitive nor specific for contamination).
  • The results of particle counting and bacterial culture may also differ from each other for logistic reasons (disintegrated leucocytes or excessive bacterial growth during transportation) or sometimes due to mix-up of samples.

Limits for significant uropathogen or yeast concentrations for identification of species. The clinical significance depends on clinical picture, way of specimen collection and the quantity of bacterial growth.

Clinical presentation or way of urine collectionLimits for significant growth (CFU/ml)
Asymptomatic bacteriuria
Catheter sample when changing indwelling catheter
Suspected UTI, midstream specimen (no knowledge of the quality of urine collection)		105
Suspected UTI, midstream specimen (good quality)* 103
Catheter specimen from female/male patient (in-and-out catheterization)104 /103
Bladder puncture specimen, quantitative culture* 102
Bladder puncture sample, enrichment cultureNo limit
* Growth at 103 -104 CFU/ml with 1-2 different bacterial species is significant if the patient has bladder symptoms and the specimen was collected properly (primary pathogens E. coli and Staphylococcus saprophyticus), or if an in-and-out catheter sample was cultured. An inoculum using a 10-µl loop is needed to detect 103 CFU/ml reproducibly, and a 100-µl loop to detect 102 CFU/ml (both equivalent to a minimum of 10 colonies on a plate).
Suspected haematuria
  • The clinical workup for haematuria is described elsewhere Haematuria. Haematuria may be caused by
    • a systemic illness (for example bleeding diathesis)
    • an illness of the kidneys or lower urinary tract (such as urolithiasis, a tumour or inflammation)
    • an artefact (menstruation, excessive cleansing [scrubbing] before taking the specimen)
    • physical strain (e.g. endurance sports).
  • Chemical testing with a reagent strip is sufficient for excluding haematuria.
    • The heme from myoglobin is also detected by the strip test (rhabdomyolysis); bacterial peroxidases likewise cause positive results.
    • Red urine is not always associated with blood. It may be due to factors such as eating beetroot, medication or urate deposit.
  • Identified haematuria should be confirmed by particle counting. The upper reference limit for the urinary erythrocyte count in healthy individuals depends on the method of analysis; it is approximately 10-20 × 106 /l. An erythrocyte count of > 50 × 106 /l is more specific for haematuria because of variability of urine concentration.
  • Even though cancer in the urinary tract is among the most severe causes of haematuria, haematuria does not always occur in cancer.

Testing for kidney-derived urinary particles

  • Urine particle counting is still valuable to diagnose or to confirm kidney diseases. Compared to specific urinary protein assays, urine particle counting is more specific for kidney diseases but less sensitive. Kidney diseases are suggested by the presence of erythrocytes, casts and renal tubular epithelial cells in urine.
  • In particle counting of urine, casts and non-squamous, or "small" epithelial cells are usually not differentiated due to the associated limitations of automatic counters.
    • A usual particle counting report includes the total number of casts (hyaline and pathological casts) and the number of "small epithelial cells" (renal tubular epithelial cells and the transitional epithelial cells of the lower urinary tract).
    • Healthy people have no casts or transitional or tubular epithelial cells in their urine (there may be some hyaline casts), i.e. the upper reference limit is below 3-5 particles × 106 /l.
  • More detailed classification of urine casts and epithelial cells is done by microscopy as clinically necessary, or to confirm the results of an automatic device. There are laboratory-specific differences in relevant request and report practices.
  • The morphology of erythrocytes can be investigated separately with a phase contrast microscopy if the patient has isolated haematuria in the absence of proteinuria. Erythrocytes originating from the kidneys (glomeruli) are dysmorphic. Dysmorphic erythrocytes amounting to > 80% of all erythrocytes suggest probable haemorrhage of renal origin, a fraction of 30-70% suggests possible haematuria of renal origin.

Suspected proteinuria

  • Kidney disease is usually associated with proteinuria Proteinuria.
  • Screening using a conventional multiple test strip detects albumin from the concentration of about 100 mg/l (total protein about 200 mg/l), upwards.
  • More sensitive methods are required for the detection of moderate albuminuria associated with diabetes or hypertension (at least 10 mg/l of albumin should be detected): albumin is usually measured from a single-voided morning specimen and expressed as albumin-to-creatinine ratio (ACR) in urine. Albuminuria can be examined more accurately in timed collection of night urine.
  • The excretion of immunoglobulin light chains in myeloma cannot be detected with a multiple strip test. Myeloma is diagnosed by fractioning of serum and urine proteins (electrophoresis) and by quantitative assay of serum free light chains.

Other basic chemical investigations

  • Detecting an increased concentration of glucose in the urine with multiple test strips is particularly useful when out-of-hours diagnosis is needed in ill children or elderly patients. Blood glucose should always be checked if the patient's urine tests positive for glucosuria.
    • SGLT2 inhibitors increase glucose excretion in urine.
  • Ketone bodies can be detected with the inaccurate Legal's test on the strip.
    • It will turn positive already after 12 hours of fasting.
    • When monitoring diabetic ketosis at a hospital it is recommended to measure hydroxybutyrate from plasma (available also as a point-of-care test), because the strip only reacts to acetoacetic acid and acetone.
  • The pH of the urine is physiologically between 5 and 9.
    • Adjustment of pH may be attempted in some patients with kidney stones or when accelerated elimination of certain drugs is required.
    • Bacterial urease breaks urea down to ammonia, whereby the pH of the urine becomes more alkaline. The effectiveness of nitrofurantoin may decrease in an alkaline pH environment in some patients.
  • Osmolality of the urine provides the best estimate of the kidney concentrating capacity. The osmolality of plasma and of isotonic urine is approximately 300 mOsm/kg H2O. Urine osmolality may be approximated from urine conductivity or creatinine concentration.

Patient guidance

Preparing for giving a urine specimen

  • Patient instructions for providing midstream urine specimens are available in laboratory handbooks or multilingual versions on laboratory websites. See also local guidelines for instructing patients on how to collect laboratory specimens.
  • When preparing patients for urine collection, the factors listed below should be discussed in addition to the collection technique.
    • Urine concentration
      • Refraining overnight from excessive drinking will ensure sufficient concentration of urine to detect bacterial growth and protein. The patient may drink, at the most, a glass of water in the morning.
      • As untimed specimens will be examined out of hours, urine concentration must be considered; at night, 20-50 ml of water is excreted per hour but in daytime, water excretion may be as high as 200-500 ml/h (the usual variation in daily diuresis being 500-3 000 ml).
    • Physical exercise
      • If needed, the ACR from another specimen collected during the day (record exact time) can be compared with that from the first morning specimen to examine orthostatic proteinuria or exercise-induced proteinuria.
    • Bladder time
      • To ensure bacterial growth, the urine should be retained in the bladder for more than 4 hours before specimen collection (for E. coli, the duration of the cell cycle is approximately 2 hours). The limit of significant growth in a concentrated morning urine specimen is 105 CFU/ml, whereas in patients with dysuria as little as 103 CFU/ml may be significant if the midstream specimen was collected successfully (Table T1).
    • Medication
      • Any current antimicrobial medication should be recorded in (electronic) referrals for urine bacterial culture, to help planning of antimicrobial sensitivity testing of grown uropathogens.

Urine samples: methods of collecting single-voided specimens

  • A midstream urine specimen (known earlier as clean catch urine) will decrease bacterial contamination from the outer genitalia and skin. Cleansing before collecting a midstream urine specimen is recommended, despite opposite opinions in published literature on small patient groups.
    • A sample for urethritis investigations, such as chlamydia test, should be collected from a ”first-catch” urine specimen (collected no sooner than two hours after last voiding, see Chlamydial Urethritis and Cervicitis).
  • In preschool-aged children, a collection cup should be attached with blue tack in the front of the potty if they can already control urination (potty sample).
  • The most recommended way of collecting a specimen from an infant is a free clean-catch; the chances of success can be improved by stimulating the pubic area. Alternatives include specimens taken with pads or bags which are reliable only in ruling out an infection.
  • An infant's UTI should be confirmed with a suprapubic aspiration (SPA) of bladder urine (Video Urine Aspiration Sampling in a Child) or by in-and-out catheterization.
  • The best way to collect specimens from patients with indwelling catheters is by inserting a new catheter and letting the sample flow through it into the collection container. The specimen must not be collected from the old indwelling catheter, since a positive result often suggests merely colonisation of the catheter.
    • In the case of a suprapubic percutaneous catheter (such as Cystofix® ), the specimen should preferably be taken by puncturing the catheter at the point especially provided for this purpose, if available.
  • In-and-out catheterization is recommended in bladder function disorders and in incontinent elderly patients instead of microbiologically poor specimens taken from the bedpan.
  • In special cases, specimens collected during cystoscopy or urostomy or other invasive procedures can be examined. The procedures of culturing and interpretation of these results should be agreed with the local microbiological laboratory.

Sample storage and transport

  • Multiple strip tests should be carried out at the emergency unit or in the laboratory only when the sample to be analysed has reached room temperature (+20°C), no earlier than 15-30 minutes from urine collection.
    • Urine at body temperature will give false positive results.
    • A specimen stored at room temperature can be tested within 8 hours.
  • Urine specimens should be collected and sent to the laboratory in specified containers (at room temperature), as agreed locally, or be stored at +4°C until analysed.
  • Samples divided for particle counting (basic count and erythrocyte morphology) should be sent to the laboratory in a test tube with preservative at room temperature, to be analysed within 24 hours from collection.
  • Samples divided for bacterial culture should also be sent in a tube with preservative at room temperature (at hospitals, they can be kept in the refrigerator without preservative for 24 h).
    • Urine samples should be inoculated on a culture plate as soon as possible because the time needed to preliminary culture results is 18-24 h (weak growth emerging only after 48 hours). Identification of the bacterial species and antimicrobial sensitivity testing may take an additional 24-72 hours.
    • A SPA (suprapubic aspiration) specimen is cultivated immediately either in a blood culture bottle (enrichment broth) or on an enriched agar plate as instructed by the local laboratory.

    References

    • Kouri TT, Hofmann W, Falbo R, et al. The EFLM European Urinalysis Guideline 2023. Clin Chem Lab Med 2024;(): [PubMed]
    • Tuokko S, Koskinen M-K, Kouri T, Saijonkari M, Sopenlehto K. [Succeed in laboratory tests - a recommendation on test selection, patient identification and guidance]. An NRF Guideline® . Helsinki: Nursing Research Foundation, 2021.Available in Finnish at http://hotus.fi/hoitosuositus/onnistu-laboratorionaytteissa-suositus-tutkimusten-valinnasta-potilaan-tunnistamisesta-ja-ohjaamisesta/.
    • Kouri T, Holma T, Kirjavainen V, et al. UriSed 3 PRO automated microscope in screening bacteriuria at region-wide laboratory organization. Clin Chim Acta 2021;516():149-156 [PubMed]
    • [Urinary tract infections.] Current Care Guideline. Working group appointed by the Finnish Medical Society Duodecim, the Finnish Society of Nephrology, the Clinical Microbiologists Society, the Infectious Diseases Society of Finland, the Finnish Medical Association of Clinical Chemistry, the Finnish Paediatric Society, the Finnish Urological Society, and the Finnish Association for General Practice. Helsinki: Finnish Medical Society Duodecim 2021 (accessed 16 June 2023). Available in Finnish at http://www.kaypahoito.fi/hoi10050.
    • Cavanaugh C, Perazella MA. Urine Sediment Examination in the Diagnosis and Management of Kidney Disease: Core Curriculum 2019. Am J Kidney Dis 2019;73(2):258-272 [PubMed]
    • LaRocco MT, Franek J, Leibach EK, et al. Effectiveness of Preanalytic Practices on Contamination and Diagnostic Accuracy of Urine Cultures: a Laboratory Medicine Best Practices Systematic Review and Meta-analysis. Clin Microbiol Rev 2016;29(1):105-47 [PubMed]
    • Schroeder AR, Chang PW, Shen MW, ym. Diagnostic accuracy of the urinalysis for urinary tract infection in infants < 2 months of age. Pediatrics 2015. DOI: 10.1542/peds.2015-0012
    • Devillé WL, Yzermans JC, van Duijn NP, et al. The urine dipstick test useful to rule out infections. A meta-analysis of the accuracy. BMC Urol 2004;4():4 [PubMed]