Title: Laboratory Diagnosis of Urinary Tract Infections
1Laboratory 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.
3Infections 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.
6predisposing factor for UTI
- Age , sex
- Pregnancy
- Diabetes
- Renal disease
- Kidney stones
- Renal transplantation
- urinary catheters
- Immune defficency
- Structural and neurological abnormalities
7Clinical presentation of UTI
- The clinical presentation of UTIs may vary
ranging from asymptomatic infection to
pyelonephritis. - Urethritis
- Cystitis
- Acute uretheritis syndrome
- Prostatis
- Pyelonephritis
8SPECIMEN
- 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.
12Resident 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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16 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.
17SCREENING PROCEDURES
- Gram Stain
- Pyuria
- Nitrate Reductase test
- Leukocyte Esterase test
- Catalase
18- 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 ) -
19 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.
21Nitrate 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. -
22Leukocyte 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. -
23 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.
24PYURIA
- 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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26URINE 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
27Culture 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
28Culture Media
- CHOC TSB , THIO Use for surgically collected
kidney urine or specimens collected by cystoscopy
or after prostatic massage.
29 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.
30 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. -
31 for inserting calibrated loop into the
urine.
32 Method for streaking
33Incubation
- 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.
34Interpretation 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
- 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.
36 Interpretation.
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40Significant low colony counts
- New bornes , children
- Antibiotic therapy
- Excess use of water , dilution of urine
- Random urine samples
- Obstructive uropathy ( tumor , stones,.. )
41WHO 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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43 44 Genital Specimens
45GENITAL 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
46GENITAL 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
47GENITAL 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
48Urethritis
- 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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50Cervicitis
- 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,.. )
51Chlamydia 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.
52Continue..
- 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
53Trichomonas 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.
54Bacterial 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 .
55Who 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
56BV 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
57Interpretation 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
58Clue Cell of BV
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