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Culturing of urine, Skill based learning


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Culturing of urine, Skill based learning

Culturing of urine, Skill based learning

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  • 2. OVER VIEW OF URINARY TRACT INFECTIONSMost common infectious diseaseMost Numerous specimens are received in the Laboratory30 to 40 % of specimens received in Microbiologylaboratories are Urine specimens, to Identify the Infection.Diagnostic information is important for the clinician.Appropriate clinical infor mation gives manyclues for better diagnostic evaluations.Specimen collection is the primary objective in getting anideal sample. Dr.T.V.Rao MD 2
  • 3. WHAT IS URINARY TRACT INFECTIONan infection of one ormore structures in theurinary system. Most UTIsare caused by gram-negative bacteria, mostcommonly Escherichiacoli or species ofKlebsiella, Proteus,Pseudomonas, orEnterobacter, althoughother strains, such asStaphlyococcus andSerratia, are emerging Dr.T.V.Rao MD 3
  • 4. 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 Dr.T.V.Rao MD 4
  • 5. COMMON UROPATHOGENS Escherichia coli Klebsiella, Enterobacter, Other Enterobacteriaceae ( Klebsiella , Enterobacter , Proteus, Citrobacter) Proteus , Citrobacter ) Pseudomonas aeruginosa Enterococcus Staphylococcus saprophyticus Staphylococcus aureus Streptococcus agalactiae (group B) CandidaAssociated with staphylococcemiaDenotes vaginal colonization in pregnant women Dr.T.V.Rao MD 5
  • 6. UNCOMMON UROPATHOGENS M.TUBERCULOSIS PRODUCES CHRONIC INFECTIONCorynebacteriumurealyticum 1Haemophilusinfluenza and H.parainfluenzae 2Blastomycesdermatitidis 3Neisseriagonorrhaeae 4Mycobacteriumtuberculosis 5 Dr.T.V.Rao MD 6
  • 7. PATHOPHYSIOLOGY OF URINARY TRACT INFECTIONAscending route of infection most commonColonization of urethra and periurethral tissue byuropathogens the initial event in urinary tract infectionUrinary tract infection more common in women than men dueto short female urethra with distention and turbulent flow thatwashes urethral organisms into the bladder during micturitionand in close proximity to perianal areasHospital infection associated with lower urinary tractinstrumentation (catheterization, cystoscopy)Once in the bladder uropathogens multiply, then pass up theureters (especially if vesicoureteral reflux present) to therenal pelvis and parenchymaSource of uropathogens: enteric bacteria Dr.T.V.Rao MD 7
  • 8. COLLECTING THE URINEThere are several different methods for collectionof a urine sample. The most common is themidstream clean - catch technique Hands should clean- washed before beginning. For females, theexternal genitalia (sex organs) are washed two orthree times with a cleansing agent and rinsed withwater. In males, the external head of the penis issimilarly cleansed and rinsed. The patient is theninstructed to begin to urinate, and the urine iscollected midstream into a sterile container. Dr.T.V.Rao MD 8
  • 9. SPECIMEN COLLECTIONThe urine collected in awide mouthed containerfrom patientsA mid stream specimen isthe most ideal forprocessingFemale patients passesurine with a labiaseparated and midstream sample iscollected Dr.T.V.Rao MD 9
  • 10. Urine collected with InstructionsFemale patients Male patients Begin passing urineSpread Stop flow in midstream Retractlabia, prepuce, Pass several ml into pen container without touching rim using plainusing plain soap orsoap or Stop flow before it ends antisepticantiseptic Recap container clean glans.wipe front to Pass remaining urine into lavatory Dry withback, tissues. Send specimen to laboratorydry with immediatelytissues (refrigerate if prolonged transport time) Dr.T.V.Rao MD 10
  • 11. COLLECTION AND PRESERVATION OF URINE SPECIMENSUrine collected in sterilespecimen container mustbe processed within 2hours, or refrigerated andprocessed within 24 hoursUrine collected in sterilespecimen container withborate preservativeshould be processedwithin 24 hours (norefrigeration required) Dr.T.V.Rao MD 11
  • 12. COLLECTING URINE IN INFANTS AND YOUNG CHILDREN DIFFERS FROM ADULTSIn infants, a urinarycollection bag (plasticbag with an adhesiveseal on one end) isattached over thelabia in girls or a boyspenis to collect thespecimen. Dr.T.V.Rao MD 12
  • 13. 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 Dr.T.V.Rao MD 13
  • 14. 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. Dr.T.V.Rao MD 14
  • 15. SPECIMEN CAN BE COLLECTED AFTER CLAMPING THE CATHETERIf the patient already has aurinary catheter in place, aurine specimen may becollected by clamping thetubing below the collectionport and using a sterileneedle and syringe toobtain the urine sample;urine cannot be taken fromthe drainage bag, as it isnot fresh and has had anopportunity to grow bacteriaat room temperatur Dr.T.V.Rao MD 15
  • 16. SUPRAPUBIC PUNCTURE ATECHNICALLY 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 Dr.T.V.Rao MD 16
  • 17. SENDING THE SPECIMEN TO LABORATORYIf delivery of the urinespecimen to thelaboratory within onehour of collection is notpossible, it should berefrigerated. The healthcare provider should beinformed of anyantibiotics currently orrecently taken. Dr.T.V.Rao MD 17
  • 18. DIAGNOSIS OF URINARY TRACT INFECTIONStep 1Microscopy of Urine fordetection of Pyuria,Leucocytes should befound in numbers of atleast as great as 10 4 /ml before the pyuria isestablished Dr.T.V.Rao MD 18
  • 19. WET FILM EXAMINATION OF URINEAll wet films to be examinedwith high power ( x 40 )objective.Prepare the drop of urine aftermixing the urine withoutcentrifugationTransfer 0.05 ml on the middleof the microscope slide andcover slip is applied.The prepared specimen show asmall excess of fluid along theedges of the cover slip.A approximate finding of 1leukocyte / 7 high power fieldscorresponds to presence ofpyuria. Dr.T.V.Rao MD 19
  • 20. INOCULATING THE CULTURE PLATE WITH URINEPlate: provide largesurface for isolation andobservation of coloniesUsing a sterile loop or asterile swab streak yoursample on the petri plateImportant let yoursterilized loop cool beforeyou pick up your sample
  • 22. 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
  • 23. CULTURING OF URINE FOR ISOLATION OF BACTERIAL PATHOGENSSemiquantitativecultureSelect the MediaFor common isolates Mac Conkey’s agarhelps in differentiationof Lactose fermentingorganisms from nonlactose fermentingpathogens Dr.T.V.Rao MD 23
  • 24. CHOOSING MEDIA TO SUIT MICROORGANISMS IS IMPORTANTMacConkey agarutilized as selectivedifferential agar forgram-negativebacteria,Colistin nalidixicacid agar asselective agar forgram-positivebacteria, andchocolate agar forfastidious gram-negative bacteria(Haemophilus) Dr.T.V.Rao MD 24
  • 25. INOCULATION OF URINEInoculation of urine forquantitative culture(colony formingunits→cfu’s) performedwith a calibrated 0.001mL and 0.01 mL plasticor wire loopSheep blood agar (SBA)utilized for quantitativeurine culture Dr.T.V.Rao MD 25
  • 26. 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
  • 27. ENRICHED CULTURE MEDIA FOR ISOLATIONBlood agar helps in isolation offastidious, extractingstrains May extendedincubation for isolationof pathogens for morethan 48 hours withadded atmosphere of 5– 10 % co 2 Dr.T.V.Rao MD 27
  • 28. 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 fecalisOn few occasions even counts 10 3 are proved significant Dr.T.V.Rao MD 28
  • 29. IDENTIFICATION OF GRAM + ORGANISMS All colonies identified morphologically as Staphylococcus to be characterized as Staphylococcus aureus Staphylococcus saprophyticus Staphylococcus epidermidisEnterococci - fecal group of organisms Dr.T.V.Rao MD 29
  • 30. 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 Dr.T.V.Rao MD 30
  • 31. INTERPRETATION OF ENTEROBACTERIACEAEA single species ofEnterobacteriaceaerecovered at >10 5 cfu’s/mLurine: with patientssymptomatic for urinary tractinfection, 95% probability oftrue bacteriuriaA single species ofEnterobacteriaceaerecovered at 10 4 -10 5 cfu’s/mLurine: with patientssymptomatic for urinary tractinfection, 33% probability oftrue bacteriuria Dr.T.V.Rao MD 31
  • 32. 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 Dr.T.V.Rao MD 32
  • 33. WHAT IS SIGNIFICANT BACTERIURIA Significant bacteriuria in anasymptomatic patient is100,000 or more colonies permilliliter of urine from amidstream, clean-catchspecimen; yet, a colony countof 200 Escherichia coli per mlmay be significant in amidstream male void orcatheterized female. About95% of all positive UTIcultures will produceessentially pure cultures ifurine is collected carefully andthe media inoculatedpromptly. Dr.T.V.Rao MD 33
  • 34. CONCEPT OF SIGNIFICANT BACTERIURIAUp to 10 4 /ml considered normali.e. Insignificant10 5 /ml and above considered tobe SignificantConcept valid only for voidedspecimen of urineExceptions - slow growingorganisms, patient on antibiotictherapy, diuretic therapy Dr.T.V.Rao MD 34
  • 35. 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 Dr.T.V.Rao MD 35
  • 36. GRAM POSITIVES AND FUNGI THE COUNTS MAY BE <10 5Gram-positive, fungal,and fastidiousuropathogens oftenpresent in lowernumbers (10 4 -10 5cfu’s/mL urine)Urethral commensalsrecovered at <10 4cfu’s/mL urine Dr.T.V.Rao MD 36
  • 37. IDENTIFICATION OF ISOLATES GRAM + ISOLATESThe minimal tests todifferentiate Gram +cocci include 1 Catalase 2 Coagulase test 3 Bile esculin testing 4 Bacitracin inStreptococcus isolates Dr.T.V.Rao MD 37
  • 38. 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
  • 40. CUMITECH GUIDELINES FOR INOCULATION OF URINE CULTURES 1 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. Dr.T.V.Rao MD 40
  • 41. CUMITECH GUIDELINES FOR INTERPRETATION OF ROUTINE URINE CULTURES 1One isolate at >10 4:Full ID andSusceptibilityOne or two gram-negative isolates at >10 5and other isolates atleast 10X less: Full IDand Susceptibility ofgram-negative isolates Dr.T.V.Rao MD 41
  • 42. CUMITECH GUIDELINES FOR INTERPRETATION OF ROUTINE URINE CULTURES 1 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. Dr.T.V.Rao MD 42
  • 43. CUMITECH GUIDELINES FOR INTERPRETATION OF SPECIAL OR UNCOMMON URINE CULTURES 1 One or two isolates at >10 2 to 10 5: Full ID and Susceptibility is essential before confirmed as uncommon isolates Dr.T.V.Rao MD 43
  • 44. ASM MANUAL GUIDELINES FOR URINE CULTURE RESULTS LIKELY TO BE SIGNIFICANT 1 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 negative1 Manual of Clinical Microbiology, 8 th Edition, ASM, 2003 Dr.T.V.Rao MD 44
  • 45. NMH GUIDELINES FOR INTERPRETATION OF URINE CULTURES 1 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) more ) at least 10X less: Full ID and susceptibility of predominant organism Report all group B β - hemolytic streptococci for women < 50 years1 Modified from ASM Cumitech, ASM Manual, and CDC MMWR 2002;51 (RR - 11):1- 22 (RR- 11):1 - Dr.T.V.Rao MD 45
  • 46. NMH GUIDELINES FOR INTERPRETATION OF URINE CULTURES 1Urine leukocyte esterase negativeOne or two organisms at >10 5: Full andSusceptibilityOne4 gram-negative organism (pure culture) at>10 : Full ID and SusceptibilityYeast in pure culture: ID as Candida albicans or notC. albicansReport all group B β-hemolytic streptococci for women <50 years1 M od ified from ASM Cumitech, ASM M anual, and CDC M M WR 2002;51 (RR - 11):1 -22 Dr.T.V.Rao MD 46
  • 47. ANTIBIOTIC SENSITIVIT Y TESTINGAll the isolatedbacteria identifiedas pathogenic to betested for AntibioticSensitivity/Resistancepattern by diskdiffusion methods Dr.T.V.Rao MD 47
  • 48. ANTIBIOTIC SENSITIVITYAlways use a puregrowth of the isolate.Perform testing forantibiotic sensitivitywith inoculum whichis comparable todefined McFarlandstandards Dr.T.V.Rao MD 48
  • 49. ANTIBIOTIC SENSITIVITY TESTINGHigh potency disksusedAntibiotics normallyactive in urineselectedAntibiotics found inhigher concentrationin urine preferredPrimary sensitivity testfrequently used Dr.T.V.Rao MD 49
  • 50. REPORTING OF CONTAMINATED SPECIMENSMicroscopy helps todetect pus cells andepithelial cells, aninsignificant growth withfew pus cells can ignoredas contaminantsIn females even thepresence of leucocyteswith Squamous epithelialcells without a definedsignificant growth shouldbe ignored. Dr.T.V.Rao MD 50
  • 51. REPORTING OF NEGATIVE URINE CULTURESNegative cultureresults showing nobacterial growth areavailable after 24hours. Positive resultsrequire 24-72 hours tocompleteidentification of thenumber and type ofbacteria found Dr.T.V.Rao MD 51
  • 52. CLINICAL PROBLEMS MANIFESTING AS URINARY TRACT INFECTIONSAn infection of theGenitourinary tractwhere the MicrobesDonot grow on selectedmedia as inMycobacteriumtuberculosis Gonococcus Nutritionally exacting or anaerobic bacteriashould be considered. Dr.T.V.Rao MD 52
  • 53. 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 Dr.T.V.Rao MD 53
  • 54. Created by Dr.T.V.Rao MD for ‘eLearning” resources forMicrobiologists in the Developing World Email Dr.T.V.Rao MD 54