Culturing of urine, Skills in microbiology

Culturing of urine skill based learning :

Dr.T.V.Rao.MD Culturing of urine skill based learning Dr.T.V.Rao MD 1

Over view of Urinary Tract Infections:

Over view of Urinary Tract Infections Most common infectious disease Most Numerous specimens are received in the Laboratory 30 to 40 % of specimens received in Microbiology laboratories are Urine specimens, to Identify the Infection. Diagnostic information is important for the clinician. Appropriate clinical information gives many clues for better diagnostic evaluations . Specimen collection is the primary objective in getting an ideal sample. Dr.T.V.Rao MD 2

What is urinary tract infection:

an infection of one or more structures in the urinary system. Most UTIs are caused by gram-negative bacteria, most commonly Escherichia coli or species of Klebsiella, Proteus, Pseudomonas, or Enterobacter, although other strains, such as Staphlyococcus and Serratia, are emerging What is urinary tract infection Dr.T.V.Rao MD 3

Urinary tract infections are more common in women:

The condition is more common in women than in men. UTI may be asymptomatic but is usually characterized by urinary frequency, burning pain with voiding, and, if the infection is severe, visible blood and pus in the urine. Fever and back pain often accompany kidney infections Urinary tract infections are more common in women Dr.T.V.Rao MD 4

Common Uropathogens:

Common Uropathogens Escherichia coli Other Enterobacteriaceae ( Klebsiella , Enterobacter , Proteus , Citrobacter ) Pseudomonas aeruginosa Enterococcus Staphylococcus saprophyticus Staphylococcus aureus Streptococcus agalactiae (group B) Candida Associated with staphylococcemia Denotes vaginal colonization in pregnant women Dr.T.V.Rao MD 5

Uncommon Uropathogens M.tuberculosis produces chronic infection:

Corynebacterium urealyticum 1 Haemophilus influenza and H. parainfluenzae 2 Blastomyces dermatitidis 3 Neisseria gonorrhaeae 4 Mycobacterium tuberculosis 5 Uncommon Uropathogens M.tuberculosis produces chronic infection Dr.T.V.Rao MD 6

Pathophysiology of urinary tract infection:

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 Dr.T.V.Rao MD 7

Collecting the urine:

There are several different methods for collection of a urine sample. The most common is the midstream clean-catch technique . Hands should be washed before beginning. For females, the external genitalia (sex organs) are washed two or three times with a cleansing agent and rinsed with water. In males, the external head of the penis is similarly cleansed and rinsed. The patient is then instructed to begin to urinate, and the urine is collected midstream into a sterile container. Collecting the urine Dr.T.V.Rao MD 8

Specimen Collection:

The urine collected in a wide mouthed container from patients A mid stream specimen is the most ideal for processing Female patients passes urine with a labia separated and mid stream sample is collected Specimen Collection Dr.T.V.Rao MD 9

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10 Female patients Urine collected with Instructions Spread labia, using plain soap or antiseptic wipe front to back, dry with tissues Retract prepuce, using plain soap or antiseptic clean glans. Dry with tissues. Male patients Begin passing urine Stop flow in midstream Pass several ml into pen container without touching rim Stop flow before it ends Recap container Pass remaining urine into lavatory Send specimen to laboratory immediately (refrigerate if prolonged transport time) Dr.T.V.Rao MD

Collection and preservation 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) Collection and preservation of Urine Specimens Dr.T.V.Rao MD 11

Collecting urine in infants and young children differs from adults:

In infants, a urinary collection bag (plastic bag with an adhesive seal on one end) is attached over the labia in girls or a boy's penis to collect the specimen. Collecting urine in infants and young children differs from adults Dr.T.V.Rao MD 12

Specimen collection in infants and young Children:

Non invasive methods are safe and ideal Follow the Broomhall et al method By tapping just above the pubis with two fingers place on suprapubic region after 1 hour of feed, tapping on at the rate of 1 tap/second for a period of 1 minute, if not successful tapping is repeated once aging. The child spontaneously pass the Urine and to be collected in a sterile container Specimen collection in infants and young Children Dr.T.V.Rao MD 13

catheterized urine:

Another method is the catheterized urine specimen in which a lubricated catheter (thin rubber tube) is inserted through the urethra (tube-like structure in which urine is expelled from the bladder) into the bladder. This avoids contamination from the urethra or external genitalia. catheterized urine Dr.T.V.Rao MD 14

Specimen can be collected after clamping the catheter :

If the patient already has a urinary catheter in place, a urine specimen may be collected by clamping the tubing below the collection port and using a sterile needle and syringe to obtain the urine sample; urine cannot be taken from the drainage bag, as it is not fresh and has had an opportunity to grow bacteria at room temperatur Specimen can be collected after clamping the catheter Dr.T.V.Rao MD 15

Suprapubic puncture a technically demanding method:

On rare occasions, the health care provider may collect a urine sample by inserting a needle directly into the bladder (suprapubic tap) and draining the urine; this method is used only when a sample is needed quickly and technically competent staff are available Suprapubic puncture a technically demanding method Dr.T.V.Rao MD 16

Sending the specimen to Laboratory:

If delivery of the urine specimen to the laboratory within one hour of collection is not possible, it should be refrigerated. The health care provider should be informed of any antibiotics currently or recently taken. Sending the specimen to Laboratory Dr.T.V.Rao MD 17

Diagnosis of Urinary Tract Infection:

Diagnosis of Urinary Tract Infection Step 1 Microscopy of Urine for detection of Pyuria , Leucocytes should be found in numbers of at least as great as 10 4 / ml before the pyuria is established Dr.T.V.Rao MD 18

Wet Film examination of Urine:

Wet Film examination of Urine All wet films to be examined with high power ( x 40 ) objective. Prepare the drop of urine after mixing the urine without centrifugation Transfer 0.05 ml on the middle of the microscope slide and cover slip is applied. The prepared specimen show a small excess of fluid along the edges of the cover slip. A approximate finding of 1 leukocyte / 7 high power fields corresponds to presence of pyuria. Dr.T.V.Rao MD 19

Inoculating the Culture plate with urine:

Plate: provide large surface for isolation and observation of colonies Using a sterile loop or a sterile swab streak your sample on the petri plate Important let your sterilized loop cool before you pick up your sample Inoculating the Culture plate with urine

Selection of Loop for Semiquantitative Method:

Selection of Loop for Semiquantitative Method Dr.T.V.Rao MD 21

Specimen Inoculations:

Specimen Inoculations All cultures processed by Semiquantitative method a loop of standard dimension of approximately known volume is inoculated into selected culture plate In general a loop of SWG – 28 with a diameter of 3.26 mm internal diameter which can hold a drop of water or urine 0.004 ml. After inoculation the culture plates are incubated at 37 0 c extending to > 18 hours are read The colony counts are made, as each colony corropsdes to number of viable bacteria per ml of urine Dr.T.V.Rao MD 22

Culturing of urine for Isolation of Bacterial pathogens:

Culturing of urine for Isolation of Bacterial pathogens Semiquantitative culture Select the Media For common isolates Mac Conkey’s agar helps in differentiation of Lactose fermenting organisms from non lactose fermenting pathogens Dr.T.V.Rao MD 23

Choosing media to suit Microorganisms is important :

MacConkey agar utilized as selective differential agar for gram-negative bacteria, C olistin nalidixic acid agar as selective agar for gram-positive bacteria, and chocolate agar for fastidious gram-negative bacteria ( Haemophilus ) Choosing media to suit Microorganisms is important Dr.T.V.Rao MD 24

Inoculation of Urine:

Inoculation of urine for quantitative culture (colony forming units→cfu’s) performed with a calibrated 0.001 mL and 0.01 mL plastic or wire loop Sheep blood agar (SBA) utilized for quantitative urine culture Inoculation of Urine Dr.T.V.Rao MD 25

Culture Media for isolations CLED Medium:

Culture Media for isolations CLED Medium It is also an excellent universal culture medium owing to its wide spectrum of nutrients, lack of inhibitors and the fact that it allows a certain degree or differentiation between the colonies. It contains lactose as a reactive compound which, when degraded to acid, causes bromothymol blue to change its colour to yellow. Alkalinization produces a deep blue colouration. The lack of electrolytes suppresses the swarming of Proteus Laboratories which have difficulty in availability of blood agar, CLED medium is opted Dr.T.V.Rao MD 26

Enriched Culture Media for Isolation :

Enriched Culture Media for Isolation Blood agar helps in isolation of fastidious, extracting strains May extended incubation for isolation of pathogens for more than 48 hours with added atmosphere of 5 – 10 % co 2 Dr.T.V.Rao MD 27

Reading the Culture Plates:

Reading the Culture Plates A true infection in the absence of prior antibiotic therapy the number of bacteria is likely to be at least 10 5 or more. Contaminated specimens present with colony counts <10 4, however even less than 10 3 On several occasions the colonies are diverse species Several studies prove counts >10 4 to be considered as presence of Urinary tract infection with the supporting clinical history On some occasions more than one pathogen is isolated but should be processed for all practical purposes eg E.coli along with Streptococcus fecalis On few occasions even counts 10 3 are proved significant Dr.T.V.Rao MD 28

Identification of Gram + organisms:

Identification of Gram + organisms All colonies identified morphologically as Staphylococcus to be characterized as Staphylococcus aureus Staphylococcus saprophyticus Staphylococcus epidermidis Enterococci - fecal group of organisms Dr.T.V.Rao MD 29

Counting the colonies:

With 0.001 ml loop, 1 colony on SBA equivalent to 1,000 cfu’s per mL of urine With 0.01 ml loop, 1 colony on SBA equivalent to 100 cfu’s per mL of urine Counting the colonies Dr.T.V.Rao MD 30

Interpretation of Enterobacteriaceae:

A single species of Enterobacteriaceae recovered at >10 5 cfu’s/mL urine: with patients symptomatic for urinary tract infection, 95% probability of true bacteriuria A single species of Enterobacteriaceae recovered at 10 4 -10 5 cfu’s/mL urine: with patients symptomatic for urinary tract infection, 33% probability of true bacteriuria Interpretation of Enterobacteriaceae Dr.T.V.Rao MD 31

Interpretation of Enterobacteriaceae:

A single species of Enterobacteriaceae recovered at >10 5 cfu’s/mL urine: with patients symptomatic for urinary tract infection, 95% probability of true bacteriuria A single species of Enterobacteriaceae recovered at 10 4 -10 5 cfu’s/mL urine: with patients symptomatic for urinary tract infection, 33% probability of true bacteriuria Interpretation of Enterobacteriaceae Dr.T.V.Rao MD 32

What is Significant bacteriuria :

Significant bacteriuria in an asymptomatic patient is 100,000 or more colonies per milliliter of urine from a midstream, clean-catch specimen; yet, a colony count of 200 Escherichia coli per ml may be significant in a midstream male void or catheterized female . About 95% of all positive UTI cultures will produce essentially pure cultures if urine is collected carefully and the media inoculated promptly . What is Significant bacteriuria Dr.T.V.Rao MD 33

Concept of Significant Bacteriuria:

34 Concept of Significant Bacteriuria Up to 10 4 /ml considered normal i.e. Insignificant 10 5 /ml and above considered to be Significant Concept valid only for voided specimen of urine Exceptions - slow growing organisms, patient on antibiotic therapy, diuretic therapy Dr.T.V.Rao MD

What can be a significant count:

A single species of Enterobacteriaceae recovered at 10 4 -10 5 cfu’s/mL urine: with patients symptomatic for urinary tract infection, 33% probability of true bacteriuria What can be a significant count Dr.T.V.Rao MD 35

Gram positives and fungi the counts may be <105:

Gram-positive, fungal, and fastidious uropathogens often present in lower numbers (10 4 -10 5 cfu’s/mL urine) Urethral commensals recovered at <10 4 cfu’s/mL urine Gram positives and fungi the counts may be <10 5 Dr.T.V.Rao MD 36

Identification of Isolates Gram + isolates:

Identification of Isolates Gram + isolates The minimal tests to differentiate Gram + cocci include 1 Catalase 2 Coagulase test 3 Bile esculin testing 4 Bacitracin in Streptococcus isolates Dr.T.V.Rao MD 37

Biochemical tests in Gram - ve bacilli:

Biochemical tests in Gram - ve bacilli Catalase test Oxidase test Nitrite reduction test Indole test Methyl red test V P test Citrate test Decarboxylation tests Lysine, ornithine, Arginine tests Dr.T.V.Rao MD 38

Peer reviewed urine culture interpretations:

39 Dr.T.V.Rao MD Peer reviewed urine culture interpretations

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 1 Clarridge, Johnson, Pezzlo, and Weissfeld, ASM Cumitech 2B, November 1998. Cumitech Guidelines for Inoculation of Urine Cultures 1 Dr.T.V.Rao MD 40

Cumitech Guidelines for Interpretation of Routine Urine Cultures1:

One isolate at > 10 4 : Full ID and Susceptibility One or two gram-negative isolates at > 10 5 and other isolates at least 10X less : Full ID and Susceptibility of gram-negative isolates Cumitech Guidelines for Interpretation of Routine Urine Cultures 1 Dr.T.V.Rao MD 41

Cumitech Guidelines for Interpretation of Routine Urine Cultures1:

Other patterns of isolates at > 10 4 : Presumptive ID only Ignore mixed urethral flora at < 10 4 1 Clarridge, Johnson, Pezzlo, and Weissfeld, ASM Cumitech 2B, November 1998. Cumitech Guidelines for Interpretation of Routine Urine Cultures 1 Dr.T.V.Rao MD 42

Cumitech Guidelines for Interpretation of Special or uncommon Urine Cultures1:

One or two isolates at > 10 2 to 10 5 : Full ID and Susceptibility is essential before confirmed as uncommon isolates Cumitech Guidelines for Interpretation of Special or uncommon Urine Cultures 1 Dr.T.V.Rao MD 43

ASM Manual Guidelines for Urine Culture Results Likely to Be Significant1:

Midstream, female with cystitis, >10 2 with positive urine leukocyte esterase Midstream, female with pyelonephritis, >10 5 with positive urine leukocyte esterase Midstream, asymptomatic, >10 5 with negative urine leukocyte esterase (usually) Midstream, male with UTI: >10 3 with leukocyte with urine leukocyte esterase positive Straight catheter: >10 2 with urine leukocyte esterase positive Indwelling catheter: >10 3 with urine leukocyte esterase positive or negative 1 Manual of Clinical Microbiology, 8 th Edition, ASM, 2003 ASM Manual Guidelines for Urine Culture Results Likely to Be Significant 1 Dr.T.V.Rao MD 44

NMH Guidelines for Interpretation of Urine Cultures1:

Urine leukocyte esterase positive One or two organisms at >10 3 : Full ID and Susceptibility One organism at >10 4 with others (2 or more ) at least 10X less: Full ID and susceptibility of predominant organism Report all group B β -hemolytic streptococci for women < 50 years 1 Modified from ASM Cumitech, ASM Manual, and CDC MMWR 2002;51 (RR-11):1-22 NMH Guidelines for Interpretation of Urine Cultures 1 Dr.T.V.Rao MD 45

NMH Guidelines for Interpretation of Urine Cultures1:

NMH Guidelines for Interpretation of Urine Cultures 1 Urine leukocyte esterase negative One or two organisms at > 10 5 : Full and Susceptibility One gram-negative organism (pure culture) at >10 4 : Full ID and Susceptibility Yeast in pure culture: ID as Candida albicans or not C. albicans Report all group B β -hemolytic streptococci for women <50 years 1 Modified from ASM Cumitech, ASM Manual, and CDC MMWR 2002;51 (RR-11):1-22 Dr.T.V.Rao MD 46

Antibiotic Sensitivity Testing:

Antibiotic Sensitivity Testing All the isolated bacteria identified as pathogenic to be tested for Antibiotic Sensitivity/Resistance pattern by disk diffusion methods Dr.T.V.Rao MD 47

Antibiotic Sensitivity:

Antibiotic Sensitivity Always use a pure growth of the isolate. Perform testing for antibiotic sensitivity with inoculum which is comparable to defined McFarland standards Dr.T.V.Rao MD 48

Antibiotic Sensitivity Testing:

High potency disks used Antibiotics normally active in urine selected Antibiotics found in higher concentration in urine preferred Primary sensitivity test frequently used 49 Antibiotic Sensitivity Testing Dr.T.V.Rao MD

Reporting of Contaminated specimens:

Microscopy helps to detect pus cells and epithelial cells, an insignificant growth with few pus cells can ignored as contaminants In females even the presence of leucocytes with Squamous epithelial cells without a defined significant growth should be ignored. Reporting of Contaminated specimens Dr.T.V.Rao MD 50

Reporting of negative urine cultures:

Negative culture results showing no bacterial growth are available after 24 hours. Positive results require 24-72 hours to complete identification of the number and type of bacteria found Reporting of negative urine cultures Dr.T.V.Rao MD 51

Clinical Problems manifesting as Urinary tract Infections:

Clinical Problems manifesting as Urinary tract Infections An infection of the Genitourinary tract where the Microbes Donot grow on selected media as in Mycobacterium tuberculosis Gonococcus Nutritionally exacting or anaerobic bacteria should be considered. Dr.T.V.Rao MD 52

Urinary tract infection are over diagnosed :

Most over diagnosed condition, both by the clinicians and microbiologists. Makes the physicians to miss the existing condition. Unnecessary antibiotic prescription lead to drug resistance, loss of resources Urinary tract infection are over diagnosed Dr.T.V.Rao MD 53

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Created by Dr.T.V.Rao MD for ‘eLearning” resources for Microbiologists in the Developing World Email doctortvrao@gmail.com Dr.T.V.Rao MD 54