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TimoKouri

Urinalysis and Bacterial Culture

Investigations

  • A routine urinalysis consists of chemical analysis (reagent strip or dipstick test) and particle counting (basic count or advanced differential count) performed either by microscopy (direct chamber counting or sediment) or using automated analysers.
    • The basic particle count includes urine leucocytes, erythrocytes, epithelial cells, cylinders and bacteria. Details in the examination sequences vary between laboratories.
    • The sensitivity of a multiple reagent strip test is approximately 20 × 106 cells/l compared with cell count (i.e. 2-3 cells per field of view) for leucocytes and for erythrocytes 10 × 106 cells/l (1-2 cells per field of view; part of erythrocytes disappears during the centrifugation of the sediment).
      • National or local adjustments may apply.
  • A bacterial culture is requested when a urinary tract infection is suspected. In particular cases, special cultures may be agreed on with the local microbiological laboratory in selected patients especially if particle counting is used to screen negative bacterial cultures. In addition, tuberculosis bacilli, chlamydiae and gonococci, among others, do not grow on an ordinary bacterial culture plate (these have their specific test procedures).
  • Urine sample for cytological studies is ordered when a malignant neoplasm is suspected (examined in a pathological laboratory).
    • Urine sample that is as fresh as possible (second sample in the morning, i.e. urine that has been retained in the bladder for 2-3 hours)
    • If the sample cannot be delivered to the laboratory within two hours, it should be centrifuged and the sediment fixed in 50% alcohol.
  • Investigations are chosen according to the suspected illness. Arrangements should be agreed upon locally.

Selection of investigations according to the suspected illness

Suspected urinary tract infection (UTI), bacteriuria and pyuria

  • The diagnosis of recurrent acute cystitis in an otherwise healthy adult female patient should be based on recognised symptoms. Laboratory investigations are not necessary (see picture 1). In other cases, a urine bacterial culture is indicated; see Urinary Tract Infections. In elderly persons, attention should be paid on the symptom picture in order to avoid unnecessary examinations and treatments.
  • A rapid test is used to establish the presence of pyuria and/or bacteriuria either by chemical screening (demonstration of leucocytes and nitrites) utilizing leukocyte esterase and bacterial nitrate reductase, or by standard particle counting through direct counting of leukocytes and bacteria.
    • It is important to remember the inadequate sensitivity of chemical screening in determining bacteriuria in a dysuric patient: only about 50-60% compared with the sensitivity of bacterial culture, if one considers even a small growth of uropathogens (from 103 colony-forming units [CFU]/ml) as significant. A 100-fold number of colony-forming units > 105 /ml can be detected with 85-95% sensitivity, depending on the type of bacteria, bladder time or patient group.
    • Staphylococcus saprophyticus and certain types of enterococci do not have nitrate reductase. The type of nutrition consumed by the patient (food contains no nitrates derived from vegetable origin or added nitrates) or the irritation of the bladder (short bladder time of the urine) do not always favour the production of nitrites in infections caused by other bacteria either.
    • A high vitamin C intake will give false negative readings for leucocytes in reagent strip testing.
    • The sensitivity of particle counting as compared to culture may be 95-100% as compared to CFU of 104 /ml in bacterial culture, and even up to the level of 103 CFU/ml in regional laboratory services. In addition, the result is more precise in automated counting, which is why it is recommended for hospital laboratories that provide emergency services. Leukocyte count > 50 × 106 /l and amount of bacteria classified as positive suggest urinary tract infection. The laboratory may also provide the clinical unit with information on the probability of a UTI calculated using age- and sex-specific reference values.
    • Delays in sample transportation, as well as centrifugation in microscopy (differential count), will lead to leucocyte and erythrocyte destruction, resulting in falsely low particle counts.
  • Diagnosing UTI in small, otherwise healthy children Urinary Tract Infection in a Child involves verifying the presence of pyuria followed by a bacterial culture of the urine, which is obtained, when aiming at a contamination-free result, via a bladder puncture Interpretation of Urine Test Results in Children. In detection of pyuria, the sensitivity of chemical analysis may be about 90% if only otherwise healthy infants with acute general symptoms are considered.
  • The limits of clinically significant counts of uropathogens and yeasts in urine depend on the patient's clinical picture, patient preparation as well as factors associated with the specimen; see table T1. The evaluation of the culture results calls for the consideration of the bladder time (whether the urine had been less than or over 4 hours in the bladder), the method of and success in collecting the sample together with the concentration (relative density, earlier known as specific gravity) of the urine.
  • Even substantial bacterial growth may be due to contamination. A positive result in the bacterial suggests contamination if
    • the patient is asymptomatic and particle counting is negative
    • the sample contains three or more species of bacteria (improperly collected sample, mixed microbial flora), or
    • the sample contains a large number of squamous epithelium cells.
    • The results of particle counting and bacterial culture may differ from each other also because of logistical reasons or a mix-up in the identification tags of the samples.
  • The aetiology of pyuria may also be other than an infection by a microbe that grows on a culture dish, such as another type of microbial infection or some other inflammation, for example glomerulonephritis Glomerulonephrites or interstitial cystitis Bladder Pain Syndrome (Interstitial Cystitis).

Clinically significant limits for uropathogen and yeast concentrations. The clinical significance and the amount of bacterial growth may be greatly dependent on the sample collection method and the patient's condition, and hence the limits presented in the table below primarily provide guidance for the ordering of further bacterial cultures.

Clinical presentation or sample typeLimits of significant concentration in laboratories (colony forming units, CFU/ml)
Asymptomatic bacteriuria (if examination is warranted in the first place)
Suspected UTI (no knowledge of the quality of the midstream sample)
105
Catheter sample from a female patient104
Catheter sample from a male patient103
Symptomatic patient with suspected urinary tract infection and a good quality midstream sample*103
Bladder puncture sample, quantitative culture102
Bladder puncture sample, enrichment cultureNo limit
* 103 -104 CFU/ml with 1-2 different species may be a significant growth if the patient is dysuric and the sample collection succeeded properly (primary pathgens E. coli and Staphylococcus saprophyticus).

Suspected haematuria

  • Haematuria Haematuria may be caused by:
    • a systemic illness (for example bleeding diathesis)
    • an illness of the kidneys or lower urinary tract
    • an artefact (menstruation)
    • physical strain (e.g endurance sports).
  • Chemical testing with a reagent strip is sufficient for screening. The method is based on the pseudoperoxidase activity of haem and is clinically adequate for exclusion. The haem from myoglobin is also detected by the strip test (rhabdomyolysis); bacterial peroxidases likewise cause positive erythrocyte results.
  • Identified haematuria should be controlled by particle counting, due to issues in specificity.
  • The upper reference limit for urinary erythrocyte count in healthy individuals depends on diuresis and on the method of analysis; it is approximately 10-20 × 106 /l. Value > 50(-100) × 106 /l suggests haematuria. Detected haematuria requires diagnostic investigations and follow-up before it is regarded as benign. On the other hand, haematuria does not always occur in cancer of the urinary system.

Testing for urinary particles of renal origin

  • A basic urinary particle count will give only the total number of urinary particles of renal origin.
    • Particle count includes the total number of casts and the number of "small epithelial cells" (= non-squamous epithelial cells) that include the renal tubular epithelial cells and the transitional epithelial cells of the lower urinary tract.
    • In the automatic count, the total amount of epithelial cells includes the squamous epithelial, transitional epithelial and tubular epithelial cells.
  • A nephrological differential count on the urinary sediment will differentiate in more detail between tubular epithelial cells and those originating from the transitional epithelium and identify different cast types. In addition, leucocytes (both granulocytes and lymphocytes) and erythrocytes may be of renal origin. As an indication of an acute kidney injury (AKI), the sensitivity of urinary casts and tubular epithelial cells is 60-80% and their specificity 70-90%. In unselected population of an emergency department the sensitivity is only about 20% and consequently the examination is applicable in targeted investigation of kidney injury.
  • The morphology of erythrocytes can be investigated separately if the patient has isolated haematuria. Erythrocytes originating from the kidneys are dysmorphic. Availability of the investigation should be confirmed from the laboratory.
  • The upper reference limits for adult fasting early morning urine (sediment microscopy).
    • Women: 3-4 granulocytes, 1-2 epithelial cells, 1-2 erythrocytes per field of view
    • Men: 1-2 granulocytes, 1-2 erythrocytes per field of view
    • No casts or transitional or tubular epithelial cells should be present.

Suspected proteinuria

  • Renal disease is usually associated with proteinuria Proteinuria.
  • Screening using a conventional multiple reagent strip detects albumin from the concentration of about 0.2 g/l, i.e. 200 mg/l, upwards.
  • More sensitive methods are required for the detection of microalbuminuria associated with diabetes or hypertension (at least 10 mg/l of albumin should be detected): measured from an overnight collection of urine or, more conveniently, from a single early morning sample (urine albumin/creatinine ratio). Sensitive strip tests are also available, if centralized regional quantitative analysis is not compatible with the care process.
  • The excretion of immunoglobulin light chains in myeloma cannot be detected with a reagent strip test. Myeloma is diagnosed by fractioning of the serum and urine proteins (electrophoresis).

Other basic chemical investigations

  • Detecting an increased concentration of glucose in the urine with reagent 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. The significance of assessing the amount of glucosuria has increased after the sodium-glucose co-transporter 2 inhibitors (SGLT2 inhibitors, gliflozins) were introduced in the treatment of diabetes.
  • Ketone bodies can be detected with the Legal's test of a reagent 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 on whole blood), because the Legal's test 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 renal stones or when the elimination of certain drugs requires acceleration. Bacterial urease breaks urea down to ammonia, whereby the pH of the urine changes to alkaline direction.
  • The measurement of relative density of the urine (earlier specific gravity) provides suggestive information on the concentrating ability of the kidneys. Due to the inaccuracy of the chemical reaction in the strip pad, large automated strip reading devices measure the relative density by refractometry.
    • Dilute urine: relative density 1.000-1.005
    • Isotonic urine: relative density 1.010-1.015
    • Concentrated urine: relative density 1.020 or more.
  • Urine bilirubin and urobilinogen are a standard part of the multi-reagent strips, which is why they are in international multicentre trials traditionally requested as "safety tests". Liver function can be investigated substantially better by blood tests.
  • Osmolality of the urine will provide the best estimate of the renal concentrating ability. Osmolality of plasma and of isotonic urine is approximately 300 mOsm/kg H2O. Urine osmolality may also be approximated on the basis of urine creatinine concentration.

Patient guidance

  • The main problems encountered with urine samples are associated with their collection, the filling and labelling of specimen tubes and the reliability of transport as well as relaying of sufficient patient history details in the electronic information systems on the success in taking the sample and on antimicrobial medications.
  • The patient must be given both oral and written instructions (multilingual as needed; local contact details and laboratory opening hours clearly indicated).
  • The division of duties and the procedures concerning patient guidance should be clearly agreed upon in the institutions providing health or other, e.g. social care.

Preparing for giving a urine sample

  • Interpretation of the urinalysis results depends on how the patient was prepared.
  • Concentration of the urine sample
    • Overnight fasting (if possible to arrange) will ensure sufficient concentration of urine. The patient may drink, at the most, a glass of water in the morning. Only patients who are clearly dehydrated may drink more. A dilute sample will yield false negative results.
    • To reveal even minor abnormalities in the urine, a concentrated morning sample should be obtained if the patient's acute symptoms do not prevent this. If the patient presents out-of-hours (bladder irritation, abdominal pain, acute haematuria, etc.), the sample is not timed and the impact of diuresis should be taken into account when interpreting the results (the water excretion of 20-50 ml/h during the night may increase to 200 ml/h or occasionally even to 500 ml/h during the day)
  • Physical exercise
    • Orthostatic proteinuria and exercise-induced proteinuria may be avoided by collecting a sample in the morning after the night's rest (albumin/creatinine ratio from the first morning urine). If needed, the albumin/creatinine ratio from another sample during the day (record exact time) can be compared with that of a sample obtained from the first morning urine.
  • Bladder time
    • To ensure bacterial growth, the urine should be retained in the bladder for 4-6 hours before sample collection (for E. coli, the duration of the cell cycle is approximately 2 hours).
    • In outpatient care, due to the available options for accessing the laboratory, it may be more practical to use the second concentrated morning sample (collected at or delivered to the laboratory) than a sample collected at home.
    • In patients with dysuria, samples that have been in the bladder for a shorter time than four hours are examined, which should be taken into account when interpreting the culture results: even a lesser bacterial growth may be significant due to the short bladder time, provided that the sample was given successfully.
  • Medication
    • The laboratory should be informed of any past or current antimicrobial medication to aid the sensitivity testing of any cultured uropathogens. Also in electronic referrals this information should be included.

Sample collection

  • A midstream urine sample (instructed, known earlier as clean-voided urine) will decrease microbiological contamination from the outer genitalia. Providing patients with guidance may significantly reduce the share of contaminated samples (see above).
  • The most common method of collecting a sample from an infant is the clean-catch whereby a sterile specimen jar is held in the urine stream while the child urinates; alternatives include samples taken with pads or bags, which are, however, reliable only in ruling out an infection.
  • An infant's UTI should be confirmed with a bladder puncture sample (video Urine Aspiration Sampling in a Child). ”In-and-out” catheterization is used in some European countries.
  • The best way to collect samples from patients with permanent catheters is by inserting a new catheter and letting the sample flow through it into the tube. The sample should not be collected by letting urine drip from the tip of the old permanent catheter, since positive result may merely indicate colonisation of the catheter. From a suprapubic percutaneous catheter, the sample is preferably taken by puncturing the catheter through the point especially constructed for this purpose.
  • In-and-out catheterization is recommended in bladder function disorders and in incontinent elderly patients instead of microbiologically poor samples taken from the bedpan.
  • Special samples are also collected from a cystostomy or during cystoscopy.
  • The method, time and success of sample collection (especially concerning important midstream samples with high risk of contamination), bladder time (more or less than four hours) and possible antimicrobial medication should be indicated in the data system so that the person who interprets the results has the possibility to use the background information when evaluating the findings. It should be borne in mind that especially the culture result will be assessed in relation to the quality of obtaining the sample.

Sample storage and transport

  • A disposable specimen jar provided by the laboratory is used to collect the urine specimen which is then transferred to transport tubes supplied by own laboratory (patient's own jars or tubes should no longer be used).
    • When only a reagent strip test is performed at the care unit, the tubes are not required.
    • The specimen containers should be shaken before transferring the urine into the tubes and before testing.
    • The handling of bladder puncture specimens should follow local laboratory guidelines.
  • Chemical analysis (strip test) is carried out at the health care facility as a point-of-care test when the sample has reached room temperature (+20°C). This will take at least about 15-30 minutes from sample collection.
  • The sample should be sent to the laboratory using transport tubes with preservatives (allowing room temperature storage and transport), as agreed with the laboratory, or be stored at +4°C (refrigerator or a cool box) until testing, if a preservative is not used (intra-house transport).
  • Reagent strip testing can be performed on a sample without a preservative within 8 hours even if the sample was kept at room temperature, and within 72 hours if kept in a refrigerator.
  • A sample collected for particle counting (basic or differential count, erythrocyte morphology) or for specific microscopic examination should be stored with a preservative at room temperature due to possible delays in transport.
  • A urine sample should be cultured as soon as possible, because the time to preliminary culture results alone is 18-24 hours (rich bacterial growth becomes discernible in a shorter time, weak growth will emerge during 48 hours). Identification of the bacterial species and antimicrobial sensitivity testing will take an additional 24-72 hours.
    • The sample for bacterial culture will keep in a refrigerator for 24 hours; or 48 hours if kept in preservative containing tubes (for Pseudomonas aeruginosa only 24 hours).
    • A bladder puncture specimen should either be cultivated immediately in a blood culture bottle (enrichment broth) or by dropping a few drops on a chocolate agar plate. Check the instructions of your laboratory particularly regarding out-of-hours samples.

References

  • European Confederation of Laboratory Medicine. European urinalysis guidelines. Scand J Clin Lab Invest 2000; Suppl 231:1-96. [PubMed]
  • Perazella MA. The urine sediment as a biomarker of kidney disease. Am J Kidney Dis 2015;66(5):748-55. [PubMed]
  • Monsen T, Ryden P. A new concept and a comprehensive evaluation of SYSMEX UF-1000i flow cytometer to identify culture-negative urine specimens in patients with UTI. Eur J Clin Microbiol Infect Dis 2017;36(9):1691-1703. [PubMed]