Marie Pezzlo, M.A., MT(ASCP) F(AAM) Microbiology Consultant

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1 Marie Pezzlo, M.A., MT(ASCP) F(AAM) Microbiology Consultant 1

2 The Association of Public Health Laboratories adheres to established standards regarding industry support of continuing education for healthcare professionals. The following disclosures of personal financial relationships with commercial interests within the last 12 months as relative to this presentation have been made by the speaker: Marie Pezzlo, M.A., MT(ASCP) F(AAM) Consultant for POCARED Diagnostics 2

3 Describe the impact of UTI on the health care system and the laboratory List the frequency at which common and uncommon microorganisms cause UTIs Discuss current recommendations for interpretation of urine cultures Describe the utility of urinalysis and other rapid urine tests to assist with the diagnosis of UTIs 3

4 ~8 million office visits/yr in US costing $1 billion 1.5 million hospitalizations UTI represent 40% nosocomial infections (Selected by Centers for Medicare and Medicaid Services (CMS) for which hospitals no longer receive additional payment.) 1 Increased infections among immunocompromised patients 1 Saint, S et al Catheter-associated urinary tract infection and the Medicare rule changes. Ann Intern Med. 150:

5 Acute symptoms of infection Septic workup Monitoring catheters Prostatitis Symptoms of dysuria and frequency Pregnancy Follow-up to treatment Determine cause of protein, WBCs and blood in urine 5

6 Infection Type Clinical Manifestation Significant Colony Count (CFU/ml) Upper urinary tract infection: Pyelonephritis Lower urinary tract infection: Cystitis Acute: fever, chills, flank pain, nausea, vomiting, WBC casts Chronic: asymptomatic, mild or acute, WBC casts, lesions Symptomatic: dysuria, frequency Asymptomatic Prostatitis Urethritis Acute: fever, chills back pain Chronic: asymptomatic or acute Dysuria, frequency

7 CFU/ml WBC Clinical Condition Acute cystitis Acute pyelonephritis Asymptomatic bacteriuria /- +/- Symptomatic male Catheter-associated Acute dysuria in women Based on clinical studies 7

8 8

9 E.coli Enterococcus spp. Klebsiella pneumoniae Proteus mirabilis Enterobacter spp. Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus saprophyticus Streptococcus agalactiae Candida spp. 9

10 Aerococcus urinae Corynebacterium urealyticum Enterobacteriaceae other than common pathogens Acinetobacter spp. and other nonfermenters Aerococcus spp. other than A. urinae Mycobacterium spp. Mycoplasma hominis Ureaplasma urealyticum Chlamydia trachomatis Neisseria gonorrhoeae Trichomonas vaginalis 10

11 Type of Infection Uncomplicated UTI: occurs without underlying abnormality or impairment of urine flow, e.g., cystitis Complicated UTI: when there are underlying factors that predispose to ascending bacterial infection. Predisposing factors include urinary instrumentation (eg, catheterization, cystoscopy), anatomic abnormalities, and obstruction of urine flow Type of Collection Noninvasive: voided, indwelling catheter, pediatric bag Invasive: straight catheter, suprapubic bladder aspirate, cystoscopy, nephrostomy 11

12 Pathogen Gram-negative Escherichia coli Klebsiella pneumoniae Proteus mirabilis Enterobacter spp. Pseudomonas aeruginosa Uncomplicated Cystitis 70%-90% 2%-6% 2%-4% 0%-1% 0%-1% Complicated UTIs 40%-50% 10%-17% 5%-10% 5%-10% 2%-10% Gram-positive Staphylococcus saprophyticus Enterococcus spp. Streptococcus agalactiae (GBS) Staphylococcus aureus Candida spp. 5%-20% 1% -2% 0%-1% 0%-1% 0%-1% 0%-4% 1%-20 1%-4% 1%-2% 2%-5% Gaspari R, Bosker G. Urinary tract infection: risk stratification, clinical evaluation, and evidence-based antibiotic therapy-year 2003 update. Atlanta, GA: American Health Consultants. 2003;1-22. Available from: Last accessed April 29,

13 Represent a majority of specimens received for culture 60 70% are negative Of positives, ~50% are contaminated Therefore technical time is spent on 80-85% of cultures that are not clinically significant Time spent on a urine culture varies from 10 to 30 min Using 15 min and 50 urines/day = 12.5 hours or 1.5 FTE/day 13

14 14

15 Voided midstream urine Cleansing No cleansing Catheter urine, indwelling & nonindwelling Suprapubic bladder aspirate Foley Catheter Tip - NOT ACCEPTABLE 15

16 Urinary Tract Infection Contamination Improper Collection Techniques Improper Storage Delayed Processing Extended Incubation Time 16

17 Study of 242 women randomized in 3 groups 77- no cleansing or midstream 84 - cleansing + midstream collection 81 cleansing + vaginal tampon + midstream collection Results Contamination rates 29%, 32% and 31%, respectively Conclusion In young, outpatient women with symptoms suggestive of a urinary tract infection, mid-stream clean-catch urine does not decrease contamination rates Lifshitz, E. and L.Kramer Arch Intern Med Sep 11:160(16):

18 BD vacuatainer tube with Boric acid and sodium formate 18

19 Process within 2 h if not refrigerated or in a preservative Process within 24 h (preferably 8 h) when refrigerated Request repeat specimen in the absence of collection time Use preservative when refrigeration is not possible Specimens that cannot be transported immediately to the laboratory, or unable to be refrigerated if immediate transport is not possible, or do not have a bacteriostatic preservative, may undergo bacterial overgrowth leading to falsely elevated colony counts. 1 1 CLSI. Urinalysis; Approved Guideline Third Edition. CLSI Document GP16- A3. Wayne, PA: Clinical and Laboratory Standards Institute;

20 5% Sheep blood + CHROMagar - E.coli CNA+ MacConkey biplate 5% Sheep blood agar 5%Sheep blood + CHROMagar E. coli + Enterococcus 20

21 Typical Appearance of microorganisms E.coli dark pink to reddish Enterococcus turquoise blue Klebsiella, Enterobacter, Citrobacter metallic blue Proteus brown halo Pseudomonas cream, translucent S. aureus golden, opaque, small S. saprophyticus pink, opaque, small Advantages of CHROMagar Can easily presumptively identify the common uropathogens Easy to distinguish a mixed culture Can perform identification and AST from isolates, if appropriate 21

22 Significant finding: a single organism/oil immersion field from an uncentrifuged urine = 10 5 CFU/ml 22

23 23

24 Colony count determined by Clinical manifestation Method of collection Voided or 10 5 Catheterized or 10 4 Suprapubic or 10 3 Based on clinical guidelines and/or laboratory guidelines 24

25 American College of Obstetricians & Gynecology (2008) Scottish Intercollegiate Guidelines Network (2006) University of Michigan Health System (2005) Infectious Disease Society of America (IDSA) (2005, 2010, 2011) Consensus 1 Culture is not needed in most cases of initial uncomplicated cystitis Empirical treatment can be based on symptoms. Need to culture all upper and complicated UTI 1 Burd, EM and KS Kehl A critical appraisal of the role of the clinical microbiology laboratory in the diagnosis of urinary tract infection. JCM 49:9; S34-S38 25

26 Infectious Diseases Society of America UTIs IDSA Guidelines 26

27 McCarter, YS, EM Burd, GS Hall and M Zervos Cumitech 2C, Laboratory diagnosis of urinary tract infections. Coordinating ed., SE Sharp. ASM Press, Washington, DC 27

28 No of Isolates Colony Count (CFU/ml) Performing Definitive ID* (Yes/No) 1 10,000 < 10,000 2 Both 10,000 Both < 10,000 1 isolate 10,000 and 1 isolate < 10, isolate 100,000 Any other combination Yes, if appropriate No Yes, if appropriate No Yes only for the isolate 10,000, if appropriate Yes only for the isolate 100,000, if appropriate No McCarter, YS et al Cumitech 2C, Laboratory diagnosis of urinary tract infections. Coordinating ed., SE Sharp. ASM Press, Washington, DC 28

29 No of Isolates Colony Count (CFU/ml) Performing Definitive ID* (Yes/No) 1 1,000 < 1,000 Yes, if appropriate No 2 Both 1,000 Both < 1,000 1 isolate 1,000 and 1 isolate < 1, isolate 10,000 Any other combination Yes, if appropriate No Yes only for the isolate 1,000, if appropriate Yes only for the isolate 10,000, if appropriate No McCarter, YS et al Cumitech 2C, Laboratory diagnosis of urinary tract infections. Coordinating ed., SE Sharp. ASM Press, Washington, DC 29

30 Culture with no growth and.001ml was cultured: Report as sterile or <1,000 CFU/ml or no growth of 1,000 CFU/ml Culture containing organisms deemed acceptable for identification (ID): Report organism ID, colony count and AST (if appropriate) for 1 or 2 probable pathogens Culture with mixed species at various quantities Report the presence of mixed flora, quantities and request a repeat specimen 1 McCarter, YS et al Cumitech 2C, Laboratory diagnosis of urinary tract infections. Coordinating ed., SE Sharp. ASM Press, Washington, DC 30

31 Definition: Contamination may be defined as 3 organisms at 10 4 CFU/mL (noninvasive collection) or 10 3 CFU/mL (invasive collection) present in equal quantities; Exception: If 3 organisms are present, and one is predominant ( 2 log difference), the predominant organism should be identified if it is a probable pathogen. 1 Q-Probes study of 127 laboratories contamination rate 2 14,739 urines studied; rates varied from 0.8% to 41.7% with a median rate of 15% Overall lower contamination rate is associated with Specimen refrigeration Providing patient collection instruction 1 McCarter, YS et al Cumitech 2C, Laboratory diagnosis of urinary tract infections. Coordinating ed., SE Sharp. ASM Press, Washington, DC 2 Bekeris, LG et al Urine culture contamination: a College of American Pathologists Q- Probes study of 127 laboratories. Arch Path Lab Med. 132:

32 Routine screening for asymptomatic bacteriuria is recommended Screen for GBS in concentrations of 10 4 CFU/ml Identify GBS when present at 10 4 CFU/ml in pure culture or mixed with a second organism Revised Guidelines from CDC, MMWR 59(RR10);1-32, Nov. 19, 2010 Note: 1996 Guidelines did not specify a colony count threshold for defining GBS bacteriuria; 2002 Guidelines recommended reporting GBS in any concentration 32

33 Aerococcus urinae Risk factors old age; urologic conditions, e.g. prostatic hyperplasia Can result in endocarditis % isolation compared to other uropathogens 0.3 to 0.8 Gram stain: gram-positive cocci in tetrads and clusters Catalase- negative; PYR 2 - negative: LAP 3 positive No CLSI (or other) standardized method for AST Corynebacterium urealyticum Alkaline urine ph, struvite crystals, leukocytes and erythrocytes Important in renal transplant patients with post-transplant UTI Gram stain: pleomorphic, diphtheroid-like, small gram-positive rod Strict aerobe requiring incubation beyond 24 hours, preferable 48-72H Multi-drug resistant 1 Versalovic, J., K. C. Carroll, G. Funke, J. H. Jorgensen, M. L. Landry, and D. W. Warnock Manual of Clinical Microbiology, 10th ed. ASM Press, Washington, DC. 2 pyrrolidonyl aminopeptidase (pyrrolidonyl arylamidase); 3 leucine aminopeptidase 33

34 What to Do and Not Do 34

35 Drugs Commonly Prescribed for UTIs Pyelonephritis uuti Acute Severe (hospitalized) Nitrofurantoin TMP-SMX Fosfomycin Fluoroquinolone β-lactam (oral) Fluoroquinolone TMP-SMX Ceftriaxone Aminoglycoside Fluoroquinolone (IV) The following +/- Aminoglycoside Ampicillin Ext-spectrum cephalosporin Ext-spectrum penicillin Carbapenem uuti = uncomplicated urinary tract infection Gupta et al CID. 52:e103 (IDSA Guideline) Sanford Guide Slide Courtesy of Janet Hindler 35

36 Urine Isolates Subjected to AST UCLA Health System 2012 (N = 11,203) Enterococcus Other GN Citrobacter E. cloacae P. aeruginosa P. mirabilis K. pneumoniae Staphylococci 8,681 from OP or ER Urine ASTs represent 52% of all ASTs E. coli Other GP and Yeast <2% Slide courtesy of Janet Hindler 36

37 Direct AST Routine use not recommended 1 Do not report 2 Erythromycin Clindamycin Chloramphenicol Tigecycline 3 1 Smyth, M., J.E. Moore and C.E. Goldsmith Urol Nurs. 26(3) ; Urinary tract infections: role of the clinical microbiology laboratory. 2 CLSI M100-S23 Table 1A 3 Nix et al J Antimicrob Chemother. 65:

38 Essential that appropriate coding be used in the laboratory; important to apply all codes that reflect the work performed. CPT codes for bacterial urine cultures routine culture and colony count; only code to be used if there is no growth for each isolate that is presumptively identified for each isolate definitely identified and/or for AST CMS coding Medicare national coverage determinations (NCD) coding policy manual and change report, revision 1. nual pdf 2009 update Screening for asymptomatic bacteriuria not indicated refer to ctrs.htm 38

39 39

40 The Answer Will it impact patient care Will results be available within 2 h of collection Can it detect 10 4 CFU/ml Can it detect >90% of common urinary pathogens 40

41 Advantages Eliminate culture for negative specimens Microbiologist focuses on positive specimens Results in Labor/Cost Savings Disadvantages Low sensitivity at <10 5 CFU/ml Multi-step test procedures Incubation period disrupts workflow Increased false-positives Growth requirement Cost/test 41

42 Microscopic Enzyme dipstick/tube Filtration Bioluminescence Chemiluminescence Spectrophotometry Flow Cytometry Fluorometry Electrochemical Molecular assay 42

43 Sensitivity (%) of Rapid Screens* *Pezzlo, M.T Comparison of rapid urine screens. Clin. Micro. Rev. Method 10 5 CFU/ml 10 4 CFU/ml 10 3 CFU/ml Microscopic Enzymatic Filtration Bioluminescence Photometry

44 Patient has 1 urinary symptoms Dysuria, frequency & urgency, hematuria, flank pain Urinalysis results Leucocyte esterase positive 8-10 WBC/HPF Note: A standard urinalysis will detect pyuria, defined as 10 leukocytes per milliliter; however, pyuria alone is not a reliable predictor of infection. The presence of both pyuria and bacteriuria microscopically markedly increases the probability of UTI. 1 1 National Guideline Clearinghouse Guideline synthesis: diagnosis and management of uncomplicated urinary tract infection. 44

45 Presence of nitrates is highly specific for bacteriuria Sensitivity is low between 10 3 and 10 5 CFU/ml Staphylococcus, Enterococcus and Pseudomonas are nitrite negative 45

46 Presence of WBCs Nitrate reductase (gramnegatives) Sensitivity = 10 5 CFU/ml; 95% if WBCs were present and 10 3 CFU/ml Pezzlo et al JCM 30:

47 47

48 iq 200 UF 1000i Sysmex Analyzer 48

49 UF-1000i is an automated, urinary screening system which bases its objective analysis on both physical and chemical particle properties. It uses: UF 1000i Sysmex Analyzer Specific fluorescent dyes for bacteria and sediment Results within minutes down to bacteria ranges between 10 3 /ml to 10 4 /ml Measures: Size, structure and fluorescence Based on Laser Flow Cytometry for particle counts previously unachievable visually In a study of 1,577 urines, when a gold standard of no growth at <10 2 was applied the sensitivity was 95%, specificity of 43%. This resulted in 20% (315 urines) workload reduction although 14% (44) of these were false-negatives. If the negative gold standard was increased to <10 5 CFU/ml, the workload reduction increased to 52%. Authors conclusion: A negative gold standard of <10 2 did not warrant its use. Broeren, MAC et al Screening for urinary tract infection with the Sysmex UF-1000i urine flow cytometer. J Clin Microbiol. 49:

50 The Auto-Particle Recognition (APR ) software, a highly trained neural network, uses size, shape, contrast and texture features to classify each image into one of 12 categories: Flow Cell Digital Imaging Auto-Particle Recognition (APR ) RBCs, WBCs, WBC Clumps, Hyaline Casts, Unclassified Casts, Squamous Epithelial Cells, Non-squamous Epithelial Cells, Bacteria, Yeast, Crystals, Mucus and Sperm. A recent study of 1000 urine specimens compared results from the iq200 to culture at 10 4 CFU/ml. Sensitivity (Sens) and negative predictive value (NPV) were 89% and 94%, respectively. When compared to clinical diagnosis, both Sens and NPV increased to 99% for both. Conclusion: system is reliable as a urine screen for culture. Parta, M et al IRIS iq200 workstation as a screen for performing urine culture. Diagn Microbiol Infect Dis. 75:5-8 50

51 Study performed to determine the ability of MALDI-TOF MS to identify organisms directly from urine specimens 260 samples positive by UF-1000i were included Sample extraction was necessary for reliable results 235 were positive by culture 205 (87%) MALDI-TOF results correlated with culture 2 (1%) had a different ID than culture 28 (12%) had a negative or non-reliable ID compared to culture Overall correlation with culture at 10 5 CFU/ml: 92% Problem MALDI-TOF organism IDs: E. faecalis (67%), K. oxytoca (78%), E. cloacae (83%) Ferreira L, et al Direct identification of urinary pathogens from urine samples by matrix-assisted laser desorption ionization-time of flight mass spectroscopy. J Clin Microbiol 48:

52 Currently available in Canada and Europe; FDA approval anticipated in 3-6 months for availability in U.S. Boric acid and sodium formate 52

53 Microbial Detection, Enumeration/ Colony Count, Identification Optical analyzer Specimen processor Processing fluid & Waste container Carousel with cuvettes Each microorganism has unique intrinsic fluorescence 53

54 54

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