Laboratory Diagnosis of Urinary Tract Infections

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

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. UTI

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. UTI

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. UTI

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. UTI

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

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 UTI

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 , prostaticsecretion ,urethral material ) 4-Timingand number of specimens 5-Specimen transport 6-Specimen examination : a) Screening procedures b) Urine culture c) Antimicrobial susceptibility teting UTI

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

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. UTI

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. UTI

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. UTI

13. Etiologic Agents UTI

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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. UTI

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

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 ) UTI

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 105 /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). UTI

20. Gram-stain Presence of at least one organism per oil immersion field( examining 20 fields) correlates with significant bacteriuria ( >105CFU/ml The gram stain should not be relied on for detecting polymorphonuclear leukocyte in urine. UTI

21. Nitrate test A positive test indicatesthat 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 < 6, urobilinogen ,non fresh specimen , collected urine during the day or by catheters. UTI

22. 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 > 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. UTI

23. Screening methods Presence of more than 8PMN,s/mm3 correlateswell 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. UTI

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 > 20 / hpf or leukocyte casts , clumps in urine sediment is abnormal. In women , the acute urethral syndrome or dysuria – pyuria syndrome is associated with > 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. UTI

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26. 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 UTI

27. Culturemedia 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 UTI

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

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. UTI

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. UTI

31. for inserting calibrated loop into the urine. UTI

32. Method for streaking UTI

33. Incubation • Once plated ,urine cultures are incubated overnight at 35°C .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. UTI

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 UTI

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. UTI

36. Interpretation. UTI

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

41. 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. 6In positive urine cultures: we should request another culture after 48 -72 h. we should request another culture after therapy. ( Test of cure specimen ) - UTI

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44. Genital Specimens UTI

45. 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 UTI

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 UTI

47. 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 UTI

48. Urethritis 1- GU : sympthomatic or nonsympthomatic 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 ,withoutgram- 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. UTI

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50. 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,.. ) UTI