Laboratory Diagnosis of UTIPractical part

Dr. Shler Ghafour Raheem

BSc., MSc., PhD Medical Microbiology

shler.ghafour@tis.edu.iq

• Urinary tract infection (UTI) in the United States is the most common

bacterial infection, and urine cultures often make up the largest

portion of workload for a hospital-based microbiology laboratory.

• Urine culture contamination can be reduced with proper techniques for

urine collection, preservation, storage, and transport,

• Nearly half of all women will experience at least one UTI during their

lifetime

• The most common nosocomial infection is catheter-associated UTI.

Specimen Collection

• Preventing contamination bynormal vaginal, perianal, andinterior urethral flora is the mostimportant consideration whencollecting a clinically relevant urinespecimen. Voided Midstream SpecimenCollection : The urine is contaminated withbacteria from the urethra unless the first portion ofthe voided specimen is discarded.

• The urine collected in a wide mouthed container from patients

Transport of Urine for Culturing

• All collected specimens of urine tobe transported to laboratory without delay. Delay of 1 – 2 hour deter thequality of diagnostic evaluations.

• If the delay is anticipated thespecimens are at preserved at 40c

• In field conditions Boric acid can beadded at a concentration of 1.8 %

Chemical Tests for Significant Bacteriuria

1- Nitrite test:

• Nitrites are not present in normal urine.

• Ingested nitrites are converted to nitrate and excreted in urine.

• If gram-negative bacteria (e.g. E.coli,Salmonella, Proteus, Klebsiella, etc.) are present in

urine. Nitrites are then detected in urine by reagent strip tests.

2- Leucocyte esterase test:

• It detects esterase enzyme released in urine from granules of leucocytes.

• Thus the test is positive in pyuria. If this test is positive, urine culture should be done.

• The test is not sensitive to leucocytes < 5/HPF.

Specimen Inoculations

All cultures processed by

• Semiquantitative method a loop ofstandard dimension of approximatelyknown volume is inoculated into selectedculture plate

• Calibrated loops of 0.01 mL should beused, not 0.001-mL (1 μL) loops, becausequantitation is difficult to obtain with a lowinoculum.

Culturing Procedure

Culture for Causative Agents of Urinary Tract Infections

• Generally, routine urine cultureshould include plating onto oneselective medium (e.g., MacConkeyagar) and one nonselectivemedium Blood agar (BA).

• CLED agar can be used as a solemedium, reducing the cost withoutcompromising the quality.

CLED (cysteine-, lactose-, and electrolyte-deficient) agar

• is a differential culture medium for use in isolating and enumerating bacteria in urine from the suspected cases of Urinary Tract Infection.

• Supports the growth of all potential urinary pathogens

• CLED agar can be used as a sole medium, reducing the cost without compromising the quality.

• It’s economy and convenience (less time, easy morphological differentiation). .wp.com/microbeonline.com/wp-content/uploads/2015/03/Lactose_non_lactose_fermenters_on_CLED_agar.jpg

Benefits and Disadvantages of Using CLED Agar for Urine Culture

• Good discrimination of gram-negative bacteria on the basis of lactose

fermentation and colony appearance;

• Inhibits swarming of Proteus spp (Proteus mirabilis and Proteus

vulgaris are frequently involved in urinary tract infection );

• Relatively low cost (compared with combined use of Blood Agar and

MacConkey agar for urine culture).

• Note: MacConeky medium containing bile salts also prevent the swarming of Proteus spp.

Disadvantages: poor growth of some gram-positive bacteria.

• After inoculation the cultureplates are incubated at 370cextending to > 18 hours are read

• Uropathogenic Escherichia coli (UPEC)is the predominant etiological agent ofuncomplicated urinary tract infection,manifested by inflammation of the urinary bladder, inhumans and is a major global public health concern.

Significance of Colony Counts:

• In women, vaginal flora may contaminatethe urine; many of the organisms thatcolonize the vagina are also implicated inUTIs. Therefore, it could be difficult to knowwhether growth in urine cultures was indicative ofinfection or contamination.

• In general > 100,000 bacteria/ml = infection

•Single organism

Reading the culture plates

• A true infection in the absence of prior antibiotic therapy the numberof bacteria is likely to be at least 105 or more.

• Contaminated specimens present with colony counts <104, howevereven less than 103

• Several studies prove counts >104 to be considered as presence ofUrinary tract infection with the supporting clinical history

• On some occasions more than one pathogen is isolated but shouldbe processed for all practical purposes eg E.coli along withStreptococcus fecalis

• On few occasions even counts 103 are proved significant

Interpretation of culture Plates

• A true infection in the absence of prior antibiotic therapy the number of bacteria is likely to be at least 105 CFU or more with patients symptomatic for urinary tract infection, 95% probability of true bacteriuria

Interpretation of culture Plates

• Several studies prove counts >104

to be considered as presence ofUrinary tract infection with thesupporting clinical history

• Ignore mixed urethral flora at <104

Reporting of negative urine cultures

• Negative culture resultsshowing no bacterialgrowth are available after24 hours.

Questions?

•Mention The Five I's of Microbiology

Lab Diagnosis of Enterobacteriaceae

Microscopic appearance(gram stain): Gram-negative rods

Culture characteristic:

• Blood agar

• Eosin methylene blue or MacConkey agar (differentiate lactose fermentation)

- Lactose fermenters (colored colonies): Citrobacter, Enterobacter, Escherichia, and Klebsiella

- Non–lactose fermenters (colorless colonies)

Non−Lactose Fermenters

Nonmotile, non-H2S producers

• Shigella, Yersina

Motile, H2S producers

• Proteus, Salmonella

Identification of Isolates Gram +ve isolates

The minimal tests to differentiate Gram + cocci include

1. Catalase

2. Coagulase test

3. Bile esculin testing

4. Bacitracin in

Streptococcus isolates

Classification of Gram (+) bacteria

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Lab diagnosis of staphylococci

A- Microscopic appearance(gram stain):

• Gram positive cocci, Grape-like arrangement (clusters)

B- Culture characteristics

Staphylococcus aureus Growing on

Blood Agar

Staphylococcus saprophyticus Growing on

Blood Agar

• Mannitol salt agar

S.aurues : yellowish

discoloration of media,

other Staphylococci.no

discoloration (remains pink

media).

References

• Luis M. de la Maza, Marie T. Pezzlo, Cassiana E. Bittencourt, Ellena M. Peterson. 2020. Color Atlas of Medical Bacteriology. (2020, Wiley) -libgen.lc.

• Robert W. Bauman, Todd P. Primm. 2018. Microbiology with Diseases

by Body System. Fifth edition, Pearson.

• Warren E. Levinson, Peter Chin-Hong, Elizabeth Joyce, Jesse Nussbaum, Brian Schwart. 2018. Review of Medical Microbiology & Immunology, 15th edition. McGraw-Hill Education..

• Patrick R. Murray, Ken S. Rosenthal, Michael A. Pfaller. 2020. Medical Microbiology. Elsevier