Laboratory Evaluation of Urinary Tract Infection

1
Laboratory Evaluation of Urinary Tract Infection

• Dr. John R. Warren
• Department of Pathology
• Northwestern University
• Feinberg School of Medicine
• June 2007

2
Essential Elements of Urine Cultures

• Pathophysiology of urinary tract infection
• Microbiology of urinary tract infection
• Clinical signs and symptoms of urinary tract
  infection

3
Essential Elements of Urine Cultures

• Technical variables in specimen collection and
  transport
• Interpretation of urine cultures
• Quality management

4
Pathophysiology of urinary tract infection

• Ascending route of infection most common
• Colonization of urethra and periurethral tissue
  by uropathogens the initial event in urinary
  tract infection
• Urinary tract infection more common in women than
  men due to short female urethra with distention
  and turbulent flow that washes urethral organisms
  into the bladder during micturition and in close
  proximity to perianal areas
• Hospital infection associated with lower urinary
  tract instrumentation (catheterization,
  cystoscopy)
• Once in the bladder uropathogens multiply, then
  pass up the ureters (especially if vesicoureteral
  reflux present) to the renal pelvis and
  parenchyma
• Source of uropathogens enteric bacteria

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Pathophysiology of urinary tract infection

• Cystitis localized infection of the bladder with
  superficial neutrophilic inflammation of the
  mucosa (lower urinary tract infection)
• Pyelonephritis infection of the kidney with
  acute suppurative inflammation of the pelvis,
  medullary and cortical tubules, and
  corticomedullary intersititum (upper urinary
  tract infection)
• Urosepsis bacteremia due to pyelonephritis
• Papillary necrosis complication of
  pyelonephritis in diabetes and urinary tract
  obstruction with coagulative necrosis of renal
  pyramids and an intense inflammatory response
  between preserved and necrotic tissue
• Sloughing of necrotic pyramids complication of
  papillary necrosis that can cause urinary tract
  obstruction (in some instances sloughed portions
  voided and recovered in urine)
• Perinephric abscess associated with obstruction
  of an infected kidney with abscess formation in
  the pernephric space due to extension of
  bacterial infection across the renal capsule

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Pathophysiology of urinary tract infection

• Uncomplicated urinary tract infection Bacterial
  or yeast infection in a structurally and
  neurologically normal urinary tract
• Complicated urinary tract infection Bacterial or
  yeast infection in a urinary tract with
  functional or structural abnormalities

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Risk factors in complicated urinary tract
infection

• Indwelling catheters
• Urinary calculi
• Neurogenic bladder
• Prostatic enlargement
• Uterine prolapse
• Urologic instrumentation or surgery
• Renal transplantation
• Diabetes mellitus

8
Bacterial virulence factors in urinary tract
infection

• Escherichia coli strains expressing O-antigens
  O1, O2, O4, O6, O7, O8, O75, O150, and O18ab
  cause high proportion of infections
• Capsular K1, K5, and K12 antigens of E. coli
  associated with clinical severity
  (antiphagocytic)
• P-fimbriae enhance mannose-resistant attachment
  of E. coli to globoseries glycosphingolipid
  receptors (gal-gal) of uroepithelial cells
  (P-fimbriated E. coli dominant as cause of
  pyelonephritis and urosepsis)
• Type 1 fimbriae enhance mannose-susceptible
  adherence of E. coli to uroepithelial cells
  (virtually all cystitis-producing E. coli strains
  express type 1 fimbriae)
• Motile bacteria ascend the ureter against urine
  flow

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Bacterial virulence factors in urinary tract
infection

• Bacterial urease (Proteus, Corynebacterium
  urealyticum) splits urinary urea with generation
  of ammonium ion that alkalinizes urine with loss
  of acid pH as natural defense barrier against
  infection, stone formation with ureteral
  obstruction and survivial of bacteria deep within
  stones resisting eradication by antibiotic, and
  alkaline-encrusted cystitis
• Gram-negative endotoxin decreases ureteral
  peristalsis
• Hemolysin produced by many uropathogens damages
  renal tubular epithelium and promotes invasive
  infection
• Aerobactin (a siderophore) present at increased
  frequency in uropathogenic strains of E. coli
  promoting intracellular iron accumulation for
  bacterial replication

10
Host protective factors in urinary tract infection

• Flushing mechanism of micturition a major
  protective factor
• Low vaginal pH (3.5-4.5) (due to lactic acid
  produced by action of Lactobacilli on glycogen of
  sloughed vaginal epithelial cells) suppresses
  colonization by uropathogens
• Normal acid pH of urine (4.6-6) anti-bacterial
• Urinary Tamm-Horsefall protein (secreted by
  ascending loop of Henle) binds to
  mannose-sensitive fimbriae and blocks E. coli
  attachment to uroepithelial cells
• Chemotactic interleukin-8 released upon bacterial
  attachment to uroepithelial cells with
  recruitment of phagocytic neutrophils and
  eradication of bacteriuria

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Immune responses in urinary tract infection

• Large numbers of submucosal IgA-producing plasma
  cells in bacterial cystitis
• IgM and/or IgG antibodies produced against
  O-antigen, K antigen, type 1 and P fimbriae, and
  lipid A
• Protective role of antibodies unclear, may limit
  damage within the kidney and prevent persistent
  colonization and thus recurrence of infection

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Pathophysiology of urinary tract infection

• Hematogenous seeding of renal cortex less
  frequent than ascending infection
• Kidney a common site of abscess formation in
  Staphylococcus aureus bacteremia, less often in
  candidemia, rarely with gram-negative bacteremia
• Hematogenous seeding of kidney also occurs with
  Salmonella (typhoid) and Mycobacterium
  tuberculosis
• Evidence for a role of periureteral and renal
  lymphatics in urinary infection lacking

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Common Uropathogens

• Escherichia coli
• Other Enterobacteriaceae (Klebsiella,
  Enterobacter, Proteus, Citrobacter)
• Pseudomonas aeruginosa
• Enterococcus
• Staphylococcus saprophyticus
• Staphylococcus aureus1
• Streptococcus agalactiae (group B)2
• Candida
• 1Associated with staphylococcemia
• 2Denotes vaginal colonization in pregnant women

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Uncommon Uropathogens

• Corynebacterium urealyticum1
• Haemophilus influenzae and H. parainfluenzae2
• Blastomyces dermatitidis3
• Neisseria gonorrhaeae4
• Mycobacterium tuberculosis5
• 1Colistin nalidixic acid (CNA) agar
• 2Chocolate agar
• 3Brain heart infusion, inhibitory mold, or
  Sabourad dextrose agar
• 4Enhanced recovery with chocolate agar
• 5Lowenstein-Jensen medium, Middlebrook broth or
  agar

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Commensal Microflora of the Urethra

• Coagulase-negative staphylococci (except S.
  saprophyticus)
• Viridans and non-hemolytic streptococci
• Lactobacilli
• Diphtheroids (Corynebacterium except C.
  urealyticum)
• Saprophytic Neisseria
• Anaerobic bacteria

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Common Risk Factors for Urinary Tract Infection
Women

• Urinary tract obstruction (including calculi)
• Catheterization (straight, indwelling)
• Pregnancy
• Urologic instrumentation or surgery
• Neurogenic bladder
• Renal transplantation
• Sexual intercourse
• Estrogen deficiency (loss of vaginal
  lactobacilli)

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Common Risk Factors for Urinary Tract Infection
Men

• Urinary tract obstruction (including calculi)
• Catheterization (straight, indwelling)
• Prostatic enlargement
• Urologic instrumentation or surgery
• Neurogenic bladder
• Renal transplantation
• Insertive rectal intercourse
• Lack of circumcision (children and young adults)

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Signs and Symptoms of Lower Urinary Tract
Infection

• Inflammatory irritation of urethral and bladder
  mucosa
• Frequent and painful urination of small volumes
  of turbid urine
• Occasional suprapubic pain or sensation of
  heaviness
• Fever generally absent

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Signs and Symptoms of Upper Urinary Tract
Infection

• Fever and chills (systemic reaction)
• Flank pain
• Lower urinary tract signs and symptoms
  (frequency, urgency, and dysuria)

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Asymptomatic Bacteriuria

• Presence of uropathogens by culture without signs
  or symptoms of urinary tract infection
• Clinically significant (should be treated) with
  preschool children (? vesicoureteral reflux,
  congenital urinary tract anomaly), pregnant
  women, and adults with obstructive uropathy
• Without clinical significance (should not be
  treated) for adults in absence of urinary tract
  obstruction

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Urinary Tract Specimens

• First-voided morning urine optimal (generally
  bacteria have been proliferating in bladder urine
  for several hours)
• Midstream urine specimens (initially voided urine
  contains urethral commensals)
• Indwelling catheters (freshly placed, urine
  aspirated by needle inserted into catheter)
  (Foley catheter tips not acceptable)
• Straight catheter specimens
• Suprapubic aspirates (infants or children,
  recovery of anaerobes)1
• Cystoscopic collection of urine
• 1Contamination-free specimen

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Collection of Urine Specimens

• Urine collected in sterile specimen container
  must be processed within 2 hours, or refrigerated
  and processed within 24 hours
• Urine collected in sterile specimen container
  with borate preservative should be processed
  within 24 hours (no refrigeration required)

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Inoculation of Urine

• Inoculation of urine for quantitative culture
  (colony forming units?cfus) performed with a
  calibrated 0.001 mL and 0.01 mL plastic or wire
  loop
• Sheep blood agar (SBA) utilized for quantitative
  urine culture
• With 0.001 ml loop, 1 colony on SBA equivalent to
  1,000 cfus per mL of urine
• With 0.01 ml loop, 1 colony on SBA equivalent to
  100 cfus per mL of urine
• MacConkey agar utilized as selective differential
  agar for gram-negative bacteria, colistin
  nalidixic acid agar as selective agar for
  gram-positive bacteria, and chocolate agar for
  fastidious gram-negative bacteria (Haemophilus)

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Interpretation of Urine Cultures General
Guidelines

• A single species of Enterobacteriaceae recovered
  at gt105 cfus/mL urine with patients symptomatic
  for urinary tract infection, 95 probability of
  true bacteriuria
• A single species of Enterobacteriaceae recovered
  at 104-105 cfus/mL urine with patients
  symptomatic for urinary tract infection, 33
  probability of true bacteriuira
• Gram-positive, fungal, and fastidious
  uropathogens often present in lower numbers
  (104-105 cfus/mL urine)
• Urethral commensals recovered at lt104 cfus/mL
  urine

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Cumitech Guidelines for Inoculation of Urine
Cultures1

• Routine uncomplicated urinary tract infection in
  ambulatory outpatients (0.001 mL loop, SBA, MAC
  24 hr incubation)
• Surveillance neurogenic bladder, indwelling
  catheter, geriatric patents (0.001 mL loop, SBA,
  MAC, CNA 24 hr incubation)
• Special suprapubic aspirates or straight
  catheter specimens where previous cultures
  negative, unresponsive to therapy, or possibility
  of unusual urinary tract pathogen (0.001 and 0.01
  mL loop, BA, MAC, CHOC minimum 48 hr
  incubation)
• 1Clarridge, Johnson, Pezzlo, and Weissfeld, ASM
  Cumitech 2B, November 1998.

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Cumitech Guidelines for Interpretation of Routine
Urine Cultures1

• One isolate at gt104 Full ID and Susceptibility
• One or two gram-negative isolates at gt105 and
  other isolates at least 10X less Full ID and
  Susceptibility of gram-negative isolates
• Other patterns of isolates at gt104 Presumptive
  ID only
• Ignore mixed urethral flora at lt104
• 1Clarridge, Johnson, Pezzlo, and Weissfeld, ASM
  Cumitech 2B, November 1998.

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Cumitech Guidelines for Interpretation of
Surveillance Urine Cultures1

• One isolate at gt104 Full ID and Susceptibility
• One gram-negative isolate at gt105 with others at
  least 10X less Full ID and Susceptibility
• Other patterns of isolates at gt104 Presumptive
  ID only
• Ignore mixed urethral flora at lt104
• 1Clarridge, Johnson, Pezzlo, and Weissfeld,
  Cumitech 2B, November 1998

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Cumitech Guidelines for Interpretation of Special
Urine Cultures1

• One or two isolates at gt102 to 105 Full ID and
  Susceptibility
• 1Clarridge, Johnson, Pezzlo, and Weissfeld,
  Cumitech 2B, November 1998

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ASM Manual Guidelines for Urine Culture Results
Likely to Be Significant1

• Midstream, female with cystitis, gt102 with
  positive urine leukocyte esterase
• Midstream, female with pyelonephritis, gt105 with
  positive urine leukocyte esterase
• Midstream, asymptomatic, gt105 with negative urine
  leukocyte esterase (usually)
• Midstream, male with UTI gt103 with leukocyte
  with urine leukocyte esterase positive
• Straight catheter gt102 with urine leukocyte
  esterase positive
• Indwelling catheter gt103 with urine leukocyte
  esterase positive or negative
• 1Manual of Clinical Microbiology, 8th Edition,
  ASM, 2003

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NMH Guidelines for Interpretation of Urine
Cultures1

• Urine leukocyte esterase positive
• One or two organisms at gt103 Full ID and
  Susceptibility
• One organism at gt104 with others (2 or more)
  at least 10X less Full ID and susceptibility
  of predominant organism
• Report all group B ß-hemolytic streptococci
  for women lt 50 years
• 1Modified from ASM Cumitech, ASM Manual, and CDC
  MMWR 200251 (RR-11)1-22

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NMH Guidelines for Interpretation of Urine
Cultures1

• Urine leukocyte esterase negative
• One or two organisms at gt105 Full and
  Susceptibility
• One gram-negative organism (pure culture) at
  gt104 Full ID and Susceptibility
• Yeast in pure culture ID as Candida
• albicans or not C. albicans
• Report all group B ß-hemolytic streptococci for
  women lt50 years
• 1Modified from ASM Cumitech, ASM Manual, and CDC
  MMWR 200251 (RR-11)1-22

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Quality Monitor for Urine Cultures

• lt5 of urine specimens contaminated by multiple
  urethral commensals present at gt104/mL

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References

• Sobel and Kaye. Urinary Tract Infections. In
  Mandell, Douglas, and Bennetts Principles and
  Practice of Infectious Diseases, 6th edition,
  Elsevier, 2005, pp. 975-905.
• Clarride, Johnson, Pezzlo, and Weissfeld.
  Laboratory Diagnosis of Urinary Tract Infections.
  Cumitech 2B, ASM Press, 1998, pp. 2-19.
• Thomson, Jr. and Miller. Specimen Collection,
  Transport, and Processing Bacteriology. In
  Manual of Clinical Microbiology, 8th edition, ASM
  Press, 2003, pp. 286-330.
• Chapter 60. Infections of the Urinary Tract. In
  Bailey Scotts Diagnostic Microbiology, 11th
  edition, Mosby, pp. 927-938,