Uti and lab diagnosis

DR.GARGI TIGNATH SHUKLA,
SAIIMS,INDORE,M.P

1. LABORATORY DIAGNOSIS OF URINARY TRACT INFECTIONS PRESENTED BY DR.GARGI TIGNATH GUIDED BY DR.PURTI AGRAWAL

2. Introduction: The urinary tract ,from the calyces of kidneys to the Urethra,is lined with a sheet of epithelium that is Continuous with that of skin. Protective factors: F low of Urine sloughing of these epithelial cells serve to protect the urinary tract from infection. M icro- organisms,particularly bacteria,may enter the urinary tract through the potential pathways of the epithelial surface to cause infections.

3. 3 Only lower part of urethra has a resident bacterial flora Rest of the urinary tract is normally sterile Flushing effect of urine flow Local phagocytic activity Mucosal IgA and secretions from prostatic and urethral glands Urinary Tract

4. Types of UTI: Upper UTI -Involves kidney or ureter Acute pyelitis : Infection of the pelvis of the kidney Acute pyelonephritis – infection of the kidney parenchyma. Lower UTI - Infection from the urinary bladder downwards Cystitis-infection of the urinary bladder. Prostatitis -infection of prostate. Urethritis -infection of urethra.

5. Uncomplicated UTI: Complicated UTI: Infection involving structurally and functionally abnormal urinary tract Infection involving structurally and functionally normal urinary tract (simple UTI)

6. Epidemiology and Risk Factors 50–80% of women in the general population- acquire at least one UTI during their lifetime—uncomplicated cystitis in most cases. Recent use of a diaphragm with spermicide , frequent sexual intercourse, and a history of UTI are independent risk factors for acute cystitis. Cystitis is temporally related to recent sexual intercourse, with a sixtyfold increase in the relative odds of acute cystitis in the 48 h after intercourse.

7. In healthy postmenopausal women, sexual activity, diabetes mellitus, and incontinence are risk factors for UTI. Many factors predisposing women to cystitis also increase the risk of pyelonephritis . Factors independently associated with pyelonephritis in young healthy women include frequent sexual intercourse, a new sexual partner, a UTI in the previous 12 months, a maternal history of UTI, diabetes, and incontinence. The common risk factors for cystitis and pyelonephritis : pyelonephritis typically arises through the ascent of bacteria from the bladder to the upper urinary tract. However, pyelonephritis can occur without clear antecedent cystitis.

8. predisposing factors : Age -Incidence increases with age. Except among infants and the elderly, UTI occurs far more commonly in females than in males. During the neonatal period, the incidence of UTI is slightly higher among males than females because male infants more commonly have congenital urinary tract anomalies. After 50 years of age, obstruction from prostatic hypertrophy becomes common in men, and the incidence of UTI is almost as high among men as among women. Between 1 year and ~50 years of age, UTI and recurrent UTI are predominantly diseases of females. The prevalence of ABU is ~5% among women between ages 20 and 40 and may be as high as 40–50% among elderly women and men.

9. Sex -sexually active women are prone to UTI due to -short urethra -proximity to the anus -urethral trauma during intercourse Pregnancy - - Dialation of ureters and renal pelvis -Stasis -Incompetence of vesicourethral valves -Hormonal changes .

10. Structural and functional abnormality of the urinary tract - -Obstruction due to urethral stricture,calculas,prostatic hypertrophy and tumour - Neurogenic bladder - Vesico -urethral reflux -Genital prolapse Metabolic- Diabetes mellitus Intervention - Instrumentation including catheterization and any surgical procedure Bacterial virulence - pilli and adherence to uroepithelium

11. Etiological Agents Of Urinary Tract Infections: Bacteria- Gram-negative bacilli- E-coli Proteus species Klebsiella Enterobacter Pseudomonas Gram-positive cocci - Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Enterococcus species

12. Gram negative cocci - Neisseria gonnorhoeae Others - Mycobacterium tuberculosis Salmonella species Gardnella Vaginallis VIRUSES - Adenovirus FUNGI - Candida albicans PARASITES - Tricomonas vaginalis Schistosomia hematobium Enterobius vermicularis

13. 14 Ability to adhere urinary epithelial cells Some strains of E coli ( uropathogens ) possess pili interacting with galactose containing receptor sites on epithelial cell surfaces Proteus sp . possess urease , which raises the pH and cause precipitation of phosphate crystals leading to stone formations Pathogenicity Entry of microorganisms By ascending route following colonization or periurethral area by enteric organisms Rarely hematogenous

14. clinical presentation Asymtomatic bacteriuria - About 5-7% of pregnant women have been reported to have urinary tract infections without symptoms. Undetected and untreated bacteriuria -symptomatic infection later in pregnancy- pyelonephritis and hypertension-prematurity- perinatal death.

15. Symptomatic UTI: Urgency Frequency of micturation Associated with pain and dyscomfort . Pyelonephritis (upper UTI)- -loin pain,tenderness,high grade fever and rigour . Cystitis/ lower UTI- - Dysuria,fever with chills and increased frequency.

16. Collection and transport of specimen SUPRAPUBIC ASPIRATE Children,infants,older women EMU TB of urinary tract Initial flow Urethritis,prostatitis

17. Collection: Mid Stream Urine(MSU)- Appropriate for bacterial culture.

18. 19 Sterile specimen container Female patients Instruction for collection of mid stream urine 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)

19. Catheter sample urine( CSU): If the patient has been catheterized,the sample is collected as follows: Area over the catheter is first cleaned with alcohol,after donning clean gloves. With the help of a sterile syringe and needle,the urine sample is drawn and put into the universal container. Urine should be never collected from the urobag or by opening the draining tube.

20. Suprapubic Aspirate: Sample for culture. suprapubic aspirate is the gold standard for obtaining urine specimens for culture. Any growth of pathogenic bacteria in an SPA specimen is felt to be significant. It is a simple, safe, rapid and effective technique in infants/pediatric age group.

21. Indications: Young unwell infants for whom there is a need to obtain specimens as part of a septic work-up where  antibiotic administration should not be delayed while awaiting a clean-catch urine. For a child (< 2 years) when it is deemed important to confirm a UTI. Eg in a child with recurrent urinary tract infections with positive cultures but minimal cellular response. Children with previous UTIs with unusual or resistant organisms Children on prophylactic antibiotics

22. Contraindications: Bleeding diathesis Abdominal distension Complications: Microscopic haematuria is common. rare complications include: macroscopic haematuria , bladder haematoma , bladder haemorrhage , intestinal perforation.  

23. Equipment  One assistant to hold the infant (not parent) Specimen jar for urine 23G needle (25G for premature infants) 3ml or 5 ml syringe Ultrasound/Bladder scanner and gel  

24. SPA Method Assistant to hold infant supine with legs extended and together. To prevent voiding in boys, the shaft of the penis should be squeezed to occlude the urethra. Identify insertion point   –  midline, lower abdominal crease   .Wipe the overlying skin with an alcohol swab. Insert needle perpendicular to the skin in all directions. Do not aim the tip of the needle down into the pelvic region. (The bladder in a baby is predominantly an abdominal rather than pelvic organ). The skin should be punctured quickly as if popping a balloon with a needle. Insert needle to the hub and aspirate. If urine is not immediately aspirated, continue aspirating as the needle is withdrawn.

25. If unsuccessful, withdraw the needle to just under the skin, and advance at an angle with the needle aimed further away from the pelvis. Do not repeat this procedure more than once. If urine is obtained, remove needle and place urine into sterile urine jar.

26. Factors increasing the likelihood of a successful SPA: History of no voiding in the past 30 minutes, and the presence of a dry nappy Prehydration Bladder dull to percussion

27. Early Morning Sample (EMU) Indicated if renal tuberculosis is suspected. Three urine samples are collected on consecutive days. Entire morning sample is collected

28. Transport: Sample must be transported at room temperatute with in half an hour. OR Refrigerated at 4 degree Celcius upto 4 hrs Beyond 4hrs urine should not be processed for bacterial culture. If the sample is from a patient who has no immediate healthcare facility-a special container with 1.8% boric acid is provided,and urine can be kept for upto 24 hrs

29. Approach to diagnosis of UTI SEMI-QUANTITAVE METHOD ANTIBIOTIC SENSITIVITY

30. Laboratory Examination Of Urine DAY-1 1.Describe the appearance of urine colour of specimen- whether it is Cloudy or clear

31. APPEARENCE POSSIBLE CAUSE CLOUDY-(Possibly urine hasan unpleasent smell and containsWBCS ) Bacterial urinary infection. RED AND CLOUDY(Due to red cells) Urinary schistosomiasis Bacterial infection BROWN AND CLOUDY(due to haemoglobin ) Black water fever Other conditions-causing intra vascular haemolysis YELLOW BROWN/GREEN BROWN(Due to bilirubin ) Acute viral hepatitis Obstructive jaundice YELLOW ORANGE( due to urobilin -oxidized urobilinogen ) Hemolysis Hepatocellular jaundice MILKY WHITE(Due to chyle ) B ancroftian filariasis

32. Points to remember: 1.Colour of urine can be caused by the ingestion of certain foods,herbs ,drugs especially vitamins. Freshly passed urine is clear and pale yellow to yellow depending on concentration. When urine left to stand-cloudiness may develop- 1.Due to precipitation of urates in acidic urine(pink orange colour ). 2.Phosphates and carbonates in an alkaline urine.

33. 2.Microscopic Examination: Wet preparation : To detect- Significant pyuria ,(WBCs in excess of 10 cells/µl of urine.counting -1WBCs per low power field-correspond to 3 cells per/ µl ). Red casts Yeast cells T.vaginalis motile trophozoites S.haematobium eggs Bacteria(provided urine is freshly collected).

34. Reporting Of Wet Preparation : 1. Bacteria -seen as rods,but sometimes cocci or streptococci -bacteria is usually accompanied by pyuria (pus cells in urine). 2. WBC -Round 10-12µm in diameter,cells that contain granules .(in UTI- found in clumps). -In urine sediments,WBCs /hpf40X)- Few upto -10WBCs/ hpf Moderate-11-40 WBCs/ hpf Many-40 WBCs/ hpf

35. Significant pyuria ->10 WBCs/µl Bacteriuria without pyuria - Diabetes Enteric fever Bacterial endocarditis Contaminated urine Pyuria with a sterile routine culture- Renal tuberculosis Gonnococcal urethritis C.Trachomatis Leptospirosis Patient of UTI treated with antimicrobials

36. Red cells: Smaller and more refractile than white cells,have definate outline and contain no granules. Isotonic urine ringed appearance. Hypertonic urine-small and crenated . Glomerulonephritis-dysmorphic (vary shapes and sizes). Causes of hematuria - acute glomerulonephritis Bacterial infections Urinary schistosomiasis . Red cells in urine of women –may be due to menstruation

37. Casts : Solidified protein and are cylindrical in shape.(formed in kidney). Hyaline casts - colourless and empty,-damage to the glomerular filter membrane Seen following strenous exercise or during fever. Waxy casts -hyaline casts remained in kidney for longer time,appear thick ,dense ,indented or twisted,may be yellow in colour . Cellular cast- WBCs-inflammation of kidney,pelvis or tubules Red cells casts-appear orange red –indicates hemmorhage into the renal tubules or glomerular bleeding.

38. Granular casts : Irregular sized granules originating from degenerate cells and protein. Seen in renal damage. Epithelial cells : Nucleated vary in size and shape. Reported as few,moderate and many. Normally few are seen in urine. Large no. indicates inflammation of urinary tract. Vaginal contamination of the specimen.

39. Yeast cells: oval in shape and some show single budding Can be differentiated with RBCs-run a drop of dil.acetic acid under cover slip-red cells will be haemolysed but not the yeast cells. Seen in women with vaginal candidiasis In diabetic and immunocompromised patient.

40. Trichomanas vaginalis : Littile larger than white cells. motile,move by flagella and an undulating membrane. seen in Acute vaginitis Eggs of s.haematobium : Large size(145x55µm) Spine at one end.

41. Eggs of schistosomia

42. CRYSTALS: Characteristic refractile appearance. Indicator of urinary tract calculi. Type of crystal Acidic urine Calcium oxalate crystals Colourless,small,octahedral or oval spheres or biconcave disc( dumbell shape).

43. examination of Gram stained smear: Prepare and examine a Gram stained smear of the urine when bacteria or WBCs seen in wet preparation. Transfer a drop of the urine sediment to a slide and spread it to make a thin smear, heat fix/methanol fix and stain it by Gram technique.

44. Look for bacterias associated with urinary infections –gram negative rods. Occasionally,Gram positive cocci and streptococci may be seen. Single type of organism-uncomplicated acute UTI. More than one type of organisms-chronic or reccurrent infections - In acute urethritis of male To make presumptive diagnosis of gonnorhea -gram negative intracellular dipplococci in pus cells

45. 3.BIOCHEMICAL TESTS Protein Nitrate Leucocyte esterase

46. Proteins : Sulphosalicylic acid reagent test Protein reagent strip test Significance of the test: Proteinuria is found in most bacterial urinary tract infections . Other causes include: glomerulonephritis,nephrotic syndrome,ecclampsia,urinary schistosomiasis,hypertension and severe febrile illness.

47. Griess test or nitrite reagent strip test Principle: urinary pathogens:E coli(commonest cause of UTI), proteus species and klebsiella species are able to reduce the nitrite ,normally present in urine to nitrite. Used to screen UTI in pregnancy in antenatal clinics. Test is false negative : when infection is caused by pathogens that do not reduce nitrite, e.g-enterococcusfaecalis , pseudomonas,staphylococcus,candida organisms. Occasionally person is on diet lacking vegetables. Bacterias are very few in urine.

48. Leucocyte esterase test LEUCOCYTE ESTERASE TEST(LE) This is an enzyme specific for polymorphonuclear neutrophil (pus cells). Detects the enzyme from active and lyzed WBCs An alternative method of detecting pyuria when it is not possible to examine fresh urine microscopically for white cells. when the urine is not fresh and likely to contain mostly lyzed WBCs. LE-using a reagent strip test such as the BM-Test-LN( Boehringer strip) –detects both nitrite and leucocyte esterase.

49. False negative strip tests: Urine contains boric acid. Excessive amount of protein (>500mg/100ml). Excessive amount of glucose(>2g/100ml).

50. Urine culture : cultures are indicated in the following situations: – Complicated UTI including pyelonephritis – UTI in past 3 weeks indicating possible relapse or the presence of symptoms for > 7 days Recent hospitalization or catheterization indicating possible nosocomial infection – Transplant patients – MS patients – Prostatitis patients – Pregnancy – Diabetes Indications

51. Conventional: 5 % sheep BAP and MAC – CLED: Cysteine lactose electrolyte deficient medium: inhibit Proteus swarming and accommodate common pathogens • Paddles or “ Dip”type devices – SOLAR-CULT( Solar Biologicals , NY) – OnSite ™(Trek Diagnostics, Cleveland, OHIO) – DipStreak ( Novamed , Israel) – Other • Chromogenic media – BBL CHROMAgar – bioMerieuxCPS ID2 – RemelSpectra UTI • CCF: BAP and MAC is usual

52. Routine or non-invasive: Clean-Catch mid-void; indwelling catheter or pediatric “bag” – 0.001 ml calibrated loop onto BAP and MAC, Streak down center; spread out from there. – > 16 hr incubation, 35 °in an O2 incubator before reading plates initially; most laboratories discard as no growth at ~ 24 hrs.

53. Invasive collection methods: straight catheter, suprapubic aspirate, cystoscopy , nephrostomy ; “low colony count urine . – 0.01ml calibrated loop onto BAP and MAC – Streak down center and spread out from there – Consider > 24 -48 hr* incubation, 35 °C in a O2incubator – May want to include the 0.001 ml inoculums well for easier CFU determination We need to know what type of urine it is! *incubation time may vary if specific organisms.

54. Normal specimens may contain small no. organisms,,usually less than10,000 per /ml of urine. Urine from aperson with untreated acute urinary tract infection usualy contains 100,000 or more bacteria/ml. Approximate no. of bacteria per ml of urine,can be estimated by using acaliberated loop or a measured piece of filter paper Single colony represents-1 organisms. E.g if an innoculum of 1/500 ml produces 20 colonies, the number of organisms represented in 1/500 ml of urine is 20 or10,000 in 1 ml.

55. Cystein lactose electrolyte-deficient agar Mix the urine(freshly collected clean-catch specimen) by rotating the container. Using a sterile caliberated wire loop e.g one that holds 1/500 ml,inoculate a loopful of urine on a quarter plate of CLED agar,if microscopy shows many bacteria,use a half plate of medium. Incubate the plate aerobically at 35-37 degree overnight

56. Growth of both Gram positive and Gram negative pathogens. Indicator in CLED agar- bromothymol blue-therefore lactose fermenting colonies appear yellow. The medium is electrolyte deficient to prevent swarming of proteus species.

57. E. Coli colonies on mac conkey agar

58. DAY-2 EXAMINE AND REPORT THE CULTURE: Negative urines (no growth) – 0.01 ml inoculum • Sterile or < 100 CFU/ml OR • No growth of >100 CFU/ml – 0.001 ml inoculum • Sterile or < 1000 CFU/ml OR • No growth of >1000 CFU/ml Positive cultures: colony count reported along with ID (with or without AST) • Mixed cultures: reported as such with note about calling for consultation and/or further work-up • Unusual pathogens/isolates – Bring it to attention of supervisors – Call clinician or other health care provide

59. Reporting of urine culture Count approximate no. of colonies=Estimate the number of bacteria,that is colony-forming units per ml of urine. <10,000 organisms/ ml,not significant. 10,000-100,000,doubtful significance. >100,000/ ml,significant bacteuria .

60. Most commonly used Quantitative Criteria for UTI • Symptomatic women – >103 CFU bacteria/ml = most likely cause of the UTI• IDSA: 1000 CFU/ml: 80% sensitivity and 90% specificity– >10 2 CFU/ml= 95% sensitivity and 85% specificity for cystitis in women • Symptomatic men – >103CFU bacteria/ mL • Catheterized patients >103CFU bacteria/ mL • Asymptomatic individuals – >105CFU bacteria/ mL (IDSA recommends 2 urine samples) • Usually any growth of a pathogen in a suprapubic aspirate or intraoperatively obtained sample is considered significant

61. Antimicrobial sensitivity testing Indications- Urine with significant bacteriuria,particularly from patient with reccurent UTI. Complicated UTI.