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Laboratory Diagnosis of Urinary Tract Infections

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SCREENING PROCEDURES Gram Stain Pyuria Nitrate Reductase test Leukocyte ... children Antibiotic ... antimicrobial sensitivity testing. Infections of Urinary ... – PowerPoint PPT presentation

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Title: Laboratory Diagnosis of Urinary Tract Infections


1
Laboratory Diagnosis of Urinary Tract Infections
  • Dr S. Hekmat MD.CAP
  • Reference Laboratories of Iran
  • Microbiology Department

2
Introduction
  • The aim of microbiology laboratory in the
    management of urinary tract infection (UTI) is to
    reduce morbidity and mortality through accurate
    and timely diagnosis with appropriate
    antimicrobial sensitivity testing.

3
Infections of Urinary Tract
  • Epidemiology
  • UTIs are among the most common bacterial
    infections that lead patients to seek medical
    care. Approximately 10 of humans will have a UTI
    at some time during their lives.

4
  • Urine sample make up a large proportion of
    samples submitted to the routine diagnostic
    laboratory. A large laboratory may examine
    200-300 urine sample each day .This heavy
    workload reflects the frequency of UTI both in
    general practice and hospital settings.

5
  • Although optimal specimen collection ,processing
    ,and interpretation should provide the clinician
    with a precise answer ,no single evaluation
    method is fool proof and applicable to all
    patients group.

6
predisposing factor for UTI
  • Age , sex
  • Pregnancy
  • Diabetes
  • Renal disease
  • Kidney stones
  • Renal transplantation
  • urinary catheters
  • Immune defficency
  • Structural and neurological abnormalities

7
Clinical presentation of UTI
  • The clinical presentation of UTIs may vary
    ranging from asymptomatic infection to
    pyelonephritis.
  • Urethritis
  • Cystitis
  • Acute uretheritis syndrome
  • Prostatis
  • Pyelonephritis

8
SPECIMEN
  • 1- Specimen collection
  • 2- Foley catheter tips should not be submitted or
    accepted for culture since they are always
    contaminated with members of urethral flora.
  • 3-Types of specimen ( urine , prostatic secretion
    , urethral material )
  • 4-Timing and number of specimens
  • 5-Specimen transport
  • 6-Specimen examination
  • a) Screening procedures
  • b) Urine culture
  • c) Antimicrobial susceptibility teting

9
Rejection criteria
  • 1-Request a repeat urine specimen when there is
    no evidence of refrigeration and the specimengt2h
    old
  • 2- Request a repeat specimen when the collection
    time and method of collection have not been
    provided
  • 3-Reject 24-h urine collection

10
Rejection criteria
  • 4-Reject Foley catheter tips as unacceptable for
    culture, they are unsuitable for the diagnosis of
    urinary tract infections.
  • 5-Reject urine from the bag of the catheterized
    patients
  • 6- Reject specimens that arrive in leaky
    container.

11
Rejection criteria
  • 7-Exept for suprapubic bladder aspirates, reject
    request for anaerobic culture.
  • 8-If an improperly collected transported, or
    handled specimen can not be replaced ,document in
    the final report that specimen quality may have
    been compromised and who was notified. generally
    urine from inpatients is easily recollected.

12
Resident Microflora of the Urethra
  • Coagulase negative staphylococci( excluding s.
    saprophyticus )
  • Viridans and nonhemolytic streptococci
  • Lactobacilli
  • Diphtheroids
  • Nonpathogenic Neisseria spp.
  • Anaerobic cocci
  • Anaerobic gram- negative bacili
  • Commensal Mycobacterium spp.
  • Commensal Mycoplasma spp.

13
Etiologic Agents
Organism Community Hospital
E. Coli 58 50.8 P. mirabilis 4.3 5.1 Klebsiella /Enetrobacter 4.7 5.1 Enterococcus sp 5.5 11.9 Staphylococcus sp 4.0 8.4 P. aeruginosa 0.5 11.1 others 12.1 5.4 (beta-hemolytic strep ,chlamydia, u. urealyticum, acinetobacter,.. )
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Screening Procedures
  • AS many as 60 to 80 of all urine specimens
    received for culture by the acute care medical
    center laboratory may contain no etiologic agents
    of infection or contain only contamination.
  • Procedures developed to identify quickly those
    urine specimens that will be negative on culture
    and circumvent excessive use of media,
    technologist time and overnight incubation.

17
SCREENING PROCEDURES
  • Gram Stain
  • Pyuria
  • Nitrate Reductase test
  • Leukocyte Esterase test
  • Catalase

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  • Factors considered for selecting urine screening
    tests
  • Accuracy , ease of performance , reproducibility
    , turn- around-time , .
  • Screening tests are insensitive at levels below
    105CFU/ml
  • They are not acceptable for urine specimens
    collected by suprapubic aspiration ,
    catheterization ,or cystoscopy.
  • They may fail to detect symptomatic patients with
    low colony counts ( Acute urethral syndrome )

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Gram Stain
  • The gram stain is the easiest ,least expensive,
    and probably ,the most sensitive and reliable
    screening method for identifying urine specimens
    that contain greater than 10 5 /mL . After a drop
    of well-mixed uncenterifuged urine is allowed to
    air dry, the smear is fixed, stained and examined
    under oil immersion (x100).

20
Gram-stain
  • Presence of at least one organism per oil
    immersion field( examining 20 fields) correlates
    with significant bacteriuria ( gt105CFU/ml The
    gram stain should not be relied on for detecting
    polymorphonuclear leukocyte in urine.

21
Nitrate test
  • A positive test indicates that bacteria reduce
    nitrate are present in significant numbers. if
    the test is positive , a culture should be
    considered , provided that specimen is collected
    and stored properly.
  • Methods for detection REAGENT STRIP
  • A fresh, first morning , clean midstream specimen
    is the best.
  • 70 overall positive results , when compared
    with cultures.
  • 93 for E.coli
  • Positive results most
    Enterobacteriaceaea
  • Negative results Enterococcus
  • False positive medication
  • False negative ascorbic acid
    , low pH lt 6, urobilinogen ,non fresh
    specimen , collected urine during the day or by
    catheters.

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Leukocyte Esterase Test
  • Evidence of a host response to infection is
    presence of PMNs in the urine.
  • Inflammatory cells produce Leukocyte Esterase .
  • An enzymatic, simple , inexpensive and rapid test
    is for measuring it., with reagent strip
    by using fresh clean catch or catheter urine
    specimens.
  • Positive results correlate with significant PMNs
    either intact or lysed., reliable for gt 10
    /microliter is used as an indication of pyuria.
  • Contamination with vaginal fluid may Positive
    results .
  • Interference Hematuria , bacteriuria affect the
    reaction.
  • Protein , ascorbic
    acid , formalin inhibits the test.
  • Oxidizing agents give
    false positive.
  • Trichomonas and
    eosinophils are sources of esterase
  • causing false
    positive.
  • It is not sensitive for pyuria in
    patients with acute urethral syndrome.
  • Confirmatory tests Microscopic urinalysis ,
    culture.

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Screening methods
  • Presence of more than 8PMN,s/mm3 correlates well
    with infection. This test can be performed using
    a hemocytometer ,but it is not easily
    incorporated into the work flow of most
    microbiologically laboratories .The standard
    urinanalysis includes an examination of
    centrifuged sediments of urine for enumeration of
    PMNs results of which do not correlate well with
    infections. Pyuria also can be associated with
    other clinical disease ,such as vaginitis ,and
    therefore not specific for UTIs.

24
PYURIA
  • Increased numbers of leukocytes especially PMNs
    in urine, during UTI , renal diseases ,or
    transiently during fevers , severe exercises.
  • The presence of many leukocytes gt 20 / hpf or
    leukocyte casts , clumps in urine sediment is
    abnormal.
  • In women , the acute urethral syndrome or dysuria
    pyuria syndrome is associated with gt 8 /
    microlitre PMNs. However bacterial colony counts
    are lower than expected.
  • Pyuria when in the wetmount of urine
    sedimentation there are
  • 5 10 leukocyte / hpf ( x 40 )., which
    indicates 50 -100 leukocyte / mm3.

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URINE CULTURE
  • Indications
  • UTI (symptomatic or non symptomatic patients )
  • Urinary tract obstruction
  • Bacteremia of unknown source
  • Follow up patients with indwelling catheter
  • Follow up patients after removal of indwelling
    catheter
  • Follow up of antibiotic therapy

27
Culture media
  • 1-Blood Agar Plate
  • 2-MacConkey agar
  • 3-Columbia colisitin nalidixic acid (CNA) or
    phenylethylalcohole agar (PEA) (optimal)
  • NOTE The advantages adding CNA agar is that
    it allows detection of gram-positive microbia
    when overgrown with gram-negative microbia.
  • 4-CLED

28
Culture Media
  • CHOC TSB , THIO Use for surgically collected
    kidney urine or specimens collected by cystoscopy
    or after prostatic massage.

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Culture
  • 1-Loop method
  • a-use either platinum or sterile plastic
    disposable loop.
  • B-Sizes
  • (1) 0.001-ml(1-µl) to detect colony count greater
    than 1,000CFU/ml
  • (2)0.01-ml(10-µl) loop to detect colony count
    between 100 and 1,000CFU/ml
  • (3)Dispsible loops are coded ,according delivery
    volume.

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Culture..
  • ( 2)Pipettor method
  • Sterile pipette tips and pipettor to deliver
    1- 10 µl urine
  • Before inoculation ,urine mixed thoroughly
    and the top container then removed .The
    calibrated loop is inserted vertically into the
    urine In a cup. Otherwise ,more than the desired
    volume of urine will be taken up, potentially
    affecting the quantitative. culture results.

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for inserting calibrated loop into the
urine.
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Method for streaking
33
Incubation
  • Once plated ,urine cultures are incubated
    overnight at 35C .For the most part ,incubation
    for a minimum of 24 hours is necessary to detect
    uropathogens. Thus some specimens inoculated
    later in day can not be read accurately the
    next morning.Thses cultures should either be
    reincubated until the next day or possible
    ,interpreted later in day. when 24 hours
    incubation has been completed.

34
Interpretation of Urine culture
  • As previously mentioned ,UTI may be completely
    asymptomatic ,produce mild symptoms, or cause
    life threatening infection. Of importance ,the
    criteria most useful for microbiologic assessment
    of urine specimens is depend not only on the type
    of urine submitted( e.g. voided ,..) but the
    clinical history of the patients or the
    patients(e.g sex, age, symptom, antibiotic
    therapy

35
Interpretation
  1. Ideally ,the clinician carrying for the patient
    should provide the laboratory with enough
    clinical information to allow specimen from
    different patients population to be identified.
    These specimen could be selectively processed
    using the guidelines.

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Interpretation.
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Significant low colony counts
  • New bornes , children
  • Antibiotic therapy
  • Excess use of water , dilution of urine
  • Random urine samples
  • Obstructive uropathy ( tumor , stones,.. )

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WHO procedures for urine specimens
  • 1- screening tests ,before urine culture
  • 2- urine culture for specimens with positive
    screening tests results, gram staining.
  • 3- If screening tests results are positive , but
    urine culture is negative , we should maintain
    specimen for 24 h later , ( after 48 h ) , then
    report.
  • 4- Performing AST for isolated uropathogenes.
  • 5- Monitoring , reexamination patients who had
    UTI before.
  • 6 In positive urine cultures
  • we should request another culture after 48
    -72 h.
  • we should request another culture after
    therapy.
  • ( Test of cure specimen )

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Genital Specimens

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GENITAL TRACT SPECIMENS
  • Patients in high risk situations
  • Patients known to have gonorrhea
  • Male patients with NGU, PGU, epididymitis, and
    prostatis
  • Females with mucopurulent cervicitis, urethral
    syndrome, endometriosis,
  • Neonates born to infected mothers
  • Infertility investigations

46
GENITAL TRACT SPECIMENS
  • For Females
  • Cervical specimens should be collected after
    removing excess mucous from the cervical and
    surrounding mucosa
  • Use a second swab to collect specimen by rotating
    the swab for 10 to 30 secs. in the endocervical
    canal
  • Collect vaginal specimens using a speculum
    without any lubricant

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GENITAL TRACT SPECIMENS
  • For males
  • Urethral specimens are collected by inserting a
    swab 2 to 4 cm. into the urethra and rotating the
    swab for 2 to 3 seconds

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Urethritis
  • 1- GU sympthomatic or non sympthomatic males
  • 6-10 WBC /hpf ,intracellular gram- negative
    diplococci
  • Purulent discharge
  • 2- NGU chlamydia . T ( 30-50 NGU ), U.
    urealyticum , Trichomonas
    .V
  • More than 10 WBC /hpf ,without gram- negative
    diplococci
  • Gram staining has 98 sensivity ,
    specifity..
  • Specimen collection , culture of gonorrhea
  • a) Urethral sampling by sterile swab or plastic
    loop.
  • b) streak directly on culture media ( TM ,MTM
    ,NYC GC, Choc with isovitalix ) in 35 c , 10-15
    CO2 or transfer into transport media ( Ameis or
    Stwart ) 12h 25c or refrigerate.

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Cervicitis
  • 1- N. gonorrhoeae
  • Direct exam in men Culture in women (
    80-90 sen )
  • 2- Chlamydia .T
  • 3- cervicovaginal specimens should be cultured
    for bacterial spp.( staph .aureus, strep.
    Agalactiae, listeria , colestridium,.. )

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Chlamydia trachomatis
  • C. T is an obligatory intracellular bacteria ,
    needs cell cultures for growth.
  • Life cycle EB (pathogen ) , IB inclusion body
  • Men urethritis ,sympthomatic or asympthomatic
    like
  • GU..
  • Women usually asympthomatic ,have both
    urethritis ,endocervicitis,with mucopurulent
    cervical discharge , erythema , edema.
  • It causes sterile pyuria. ( culture negative )
  • Lab diagnosis Sampling , culture
  • 1- Sampling with swab in men ( urethra ) ,with
    swab( cervical ,urethral or cytology brushes )
    in women.

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Continue..
  • Transport media 2- sucrose phosphate ( 2-SP )
    .
  • They should be refrigerated if they can not be
    immediately
  • Long term preservation -70c freezer
  • Then it can be sonicated to realease EB.
  • Diagnosis
  • 1- Presumptive diagnosis based on clinical
    symptoms , EIA , DFA , PCR
  • 2- Definitive diagnosis full culture ( cell
    cultures like HeLa , McCoy ) and identification
    of inclusion bodies by cythologic smears

53
Trichomonas vaginalis
  • Common sexually transmitted disease
  • Disease associations and adverse outcomes
  • Vaginitis,itching with frothy yellow greenish
    discharge.
  • Urethritismen (asympthomatic with milky
    discharge )
  • Treatment of both partners is suggusted ,
    reinfection can occur.
  • Laboratory diagnosis
  • 1-Finding trophozoite in wetmount urine or
    vaginal or prostatic secretions., seeing jerky
    motility.( 50 sen )
  • 2- Stained smears ,seeing pear-shaped organisms.
  • 3- Culture is the most sensitive method of
    detection, hold 7-days.

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Bacterial Vaginosis
  • G. vaginalis occurs in almost 100 of women with
    BV.
  • Amore important bacteria in BV is Mobiluncus is
    gram negative anaerobic bacilli.
  • Watery noninflammatory exudate lacking PMN s
    vaginosis not vaginitis.
  • Vaginal fluid has increased pH gt 5 , ( because of
    decreasing lactobaclli
  • clue cells ( gt20 EPITHELIAL CELLS IN WETMOUNT
    )
  • Foul odor of exudate whiff test.
  • Diagnosed rapidly by clinician .

55
Who Should be Screened for BV?
  • Women with vaginal symptoms
  • especially. if failed therapy
  • Pregnant women at high risk of preterm birth
  • Pregnant women with genital symptoms
  • rule out trichomoniasis as well
  • Women with gynecologic surgery

56
BV Scored Gram Stain
TYPE Number seen/OIF Number seen/OIF Number seen/OIF Number seen/OIF Number seen/OIF
None lt1 1-5 6-30 gt30
Lacto 4 3 2 1 0
Gard/Bact 0 1 2 3 4
Curved GNR 0 1 2 3 4
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Interpretation of Scored Gram Stain
  • 4-6 Intermediate
  • may indicate trichomoniasis, GC or CT
  • abnormal gram stain, but not consistent with BV,
    repeat test later
  • 7-10 Consistent with Bacterial Vaginosis

0-3 Normal
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Clue Cell of BV
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