1. Diagnosis of Genitourinary Tuberculosis
Dr. Jayesh Dhabalia Dr. Ulhas Sathe
Consultant Urologist Consultant Urologist
Professor & Head Sahyog Speciality Hospital
Department of Urology, & Lithotripsy Centre
LTMG Hospital , Mumbai Jhamnagar

2. INTRODUCTION
General Incidence – India1
One fifth of world TB population
1.8 million new cases / year
8 lac infectious cases
One death every 90 sec , 45 / hour , 1080 / day , 3.88 lack /year

3. INTRODUCTION
General Incidence – India – GUTB1
Most common extra pulmonary TB
30 % of all extrapulmonary TB
18% of infertile women
11% of hematospermia
5-25 year after primary pulmonary TB
Primary GUTB - anecdotal cases in females

4. MICROBIOLOGICAL BACKGROUND
Classification
M tuberculosis complex
M tuberculosis – GUTB
M bovis – Very rare
M microtii- X
M africanum - X
DIAGNOSTIC PROBLEM
Atypical / NTM / MOTT
NO GUTB
FALSE POSITVE
Mycobacteria
leprae

5. MICROBIOLOGICAL BACKGROUND
Bacterial characteristics – diagnostic difficulties
Slow growing - divides every 15–20 hours
- Delayed growth on culture
Can survive in a dry state for weeks
- Infectivity & contamination
Obligate aerobic
Acid-fast -waxy coating on the cell surface makes the cells impervious to Gram staining

6. GUTB - DIAGNOSTIC DIFFICULTIES
Characteristics of M. tuberculosis bacteria
Difficult and delayed lab diagnosis
Index of suspicion not high enough

7. GUTB - DELAYED DIAGNOSIS
After significant irreversible anatomical changes -
major surgical procedure
( thimble bladder , multiple ureteric strictures, multiple infundibular stenosis)
Irreversible loss of kidney function
Infertility in both gender
Early diagnosis in India – high index of suspicion

8. INVASIVE – Endoscopy / Biopsy / Dye study
GUTB – DIAGNOSTIC MODALITIES
CLINICAL
RADIOLOGICAL
LABORATORY
Reliability increases
with
Progression of
disease
Multisystem
involvement
Early changes
Needs confirmation
Advanced disease
Almost diagnostic
Valuable for
Early disease
diagnosis
Rapid diagnosis
Drugs sensitivity
INVASIVE – Endoscopy / Biopsy / Dye study

9. GUTB - CLINICAL EVALUATION
SPECIFIC - Genitourinary tract
Lower urinary tract – 50 to 80 %
Burning , frequency , urgency , urge incontinence
Dysuria , hematuria
Suprapubic pain / perineal discomfort
Decreased stream , straining, ineffective voiding
Slough in urine

10. GUTB - CLINICAL EVALUATION
Upper urinary tract symptoms
Pain - kidney and ureter region
Gross hematuria- 10 %
Genital – Male
Hematospermia - 10 %
Azoospermia
S/S of chronic
epididymorchitis
Genital – Female
Menstrual irregularities
Pelvic pain syndrome
Infertility – 18 %

11. GUTB - CLINICAL EVALUATION
Other systems
Respiratory % patients
Gastrointestinal %
Lymphoreticular
Constitutional to 15 % 1
Evening rise of temperature
Weight loss
Anorexia

12. GUTB - LABORATORY DIAGNOSIS
Supportive
Urine – R&M
Tuberculin test
Serodiagnosis
Potentially diagnostic
ZN staining
Culture
PCR – urine, tissue
Histological diagnosis

13. GUTB - PROBLEMS IN LAB DIAGNOSIS
Paucibacillary
Intermittent bacterial shedding
Fastidious / slow growth – difficult to culture
Diagnostic difficulty due to atypical mycobacteria / MOTT / NTM
Sensitivity of all tests extra pulmonary TB

14. GUTB - LAB DIAGNOSIS SUPPORTIVE TESTS
Urine – routine and microscopy
Acidic urine , sterile pyuria , microscopic hematuria
Guide for further investigation, especially in pauci-symptomatic patients
Montaux Test (Robert Koch in 1890)
If Positive – supports the diagnosis
If Negative – can not exclude extrapulmonary TB
Response – HIV, Immunocompromised , Post-transplant pts
Problems in India
Invariably positive - Exposure in childhood , BCG vaccination

15. GUTB – SERODIAGNOSIS
Basis – by detecting
I. Specific immunological host response
Humoral (serological) antibody immune response to M. tb 
Ig G – High levels in active TB
Ig M – Immediate appearance, disappears later on
Commercial ELISA test kit available
T cell–based cellular immune response –
Different antigens detects different types of TB
Test kit not available
II. Direct detection of bacterial antigens & metabolites

16. GUTB – SERODIAGNOSIS PROBLEMS
Low sensitivity and specificity
At present supportive at best
Potential role in future
Early diagnosis
Response measurement to treatment
Early detection of relapse
LITTLE OR NO ROLE IN DIAGNOSIS

17. GUTB - POTENTIALLY DIAGNOSTIC LAB TESTS
COLLECTION OF SAMPLES
Urine
Physiologically pooled ( overnight) early morning 1stsample
Three consecutive days , ? 5 days
Volume – 10 ml for AFB, culture , PCR
Immediate processing – if not feasible – refrigerate at 4-8o C
Alkalinisation of urine - ? Increased yield2
Tissue
In neutral transport media – avoids dessication
Swab specimen – not suitable

18. GUTB - POTENTIALLY DIAGNOSTIC LAB TESTS
TEST UTILITY EVALUATION CRITERIAS
1. Availability
2. Sensitivity
3. Specificity
4. Cost
5. Time of processing
6. Antimicrobial sensitivity

19. GUTB - DIAGNOSTIC LAB TESTS ZN / AFB SMEAR EXAMINATION
First described by Franz Ziehl (1859 to 1926), a bacteriologist and Friedrich Neelsen (1854 to 1894)
Requiring ≥104 bacilli ml−1 of sample to achieve a positive result3
Can not differentiate between live versus dead bacteria

20. GUTB - DIAGNOSTIC LAB TESTS ZN / AFB SMEAR EXAMINATION
1. Take sediment and make a smear
2. Carbol-fuchsin solution - allow slides to stand in hot solution for 5 minutes.
3. Wash in running tap water.
4. 1% Acid alcohol until light pink and color stops running.
5. Wash in running tap water for 5 minutes...
7. Working methylene blue for 30 seconds.
8. Rinse in water.
9. Dehydrate, clear, and cover slip.
RESULTS:
Acid-fast bacilli bright red
Background blue

21. ZN - AFB STAIN TEST UTILITY EVALUATION
Availability - Universal
Sensitivity – 40 %4
Specificity % 4
Processing time mins
Cost Cheap
Antibiotic sensitivity - NA

22. GUTB - DIAGNOSTIC LAB TESTS FLUORESCENCE MICROSCOPY
Microscopy with fluorochrome dyes such as auramine O or auramine-rhodamine
To increase sensitivity over
ZN – AFB staining

23. TEST UTILITY EVALUATION
Availability Poor
Sensitivity % > ZN staining5
Specificity Similar
Processing time Less than ZN staining
Cost Significantly > ZN staining
Antibiotic sensitivity - NA

24. MOLECULAR TECHNOLOGY POLYMERASE CHAIN REACTION (PCR)
Principle by detection of species specific DNA
Technique – to amplify a single or few copies of a piece of DNA to generate millions of copies of a particular DNA sequence
Targeting different gene sequences of M. tuberculosis , showed different positivity
IS % - Most commonly targeted6
65kDa - 75%, 38 kDa - 72% ,85B protein - 73%

25. MOLECULAR TECHNOLOGY POLYMERASE CHAIN REACTION (PCR)
Diagnostic Problems
Few Indian strains lack copy of IS 6110
IS present in M.tb complex bacteria
( tuberculous / africanum / microti / bovis)
False positive – old cases of TB, contamination
False negative – extremely paucibacillary cases
( detection limit up to 10 copies / ml )
Cannot differentiate dead from live bacteria

26. MOLECULAR TECHNOLOGY - PCR
Universal Sample Processing (USP) Technology
AIM - To false negative rate by removing inhibitors of DNA amplification
Potent inhibitor-heparin, hemoglobin, phenol & sodium dodecyl sulfate4
Detected in 0 to 20% of the clinical specimen4

27. MOLECULAR TECHNOLOGY - PCR TECHNICAL PROCESSING
Thermal cycling
Consisting of cycles of repeated heating and cooling of DNA for melting
Enzymatic replication of the DNA.
Primers (short DNA fragments) containing sequences complementary to the target region
As PCR progresses, the DNA generated is itself used as a template for replication
DNA template is exponentially amplified

28. MOLECULAR TECHNOLOGY - PCR
TEST UTILITY EVALUATION
Availability - Limited
Sensitivity % - 95%6
Specificity %6
Processing time - One day
Cost Rs 1600
Antibiotic sensitivity NA

29. MOLECULAR TECHNOLOGY RNA PCR
DNA PCR cannot differentiates dead from live organisms
Principle – Reverse transcriptase copies the RNA target from DNA into a transcription complex, which is then amplified by RNA polymerase

30. MOLECULAR TECHNOLOGY TISSUE PCR
Sampling limitations
Availability - Limited
Sensitivity %
Specificity to 97 %
Processing time - One day
Cost Rs 1600
Antibiotic sensitivity - NA
TEST UTILITY CRITERIA

31. M.TUBERCULOSIS CULTURE
Gold standard ????
Highly specific and sensitive
Detects presence of live bacteria
Can differentiate between typical and atypical
Antimicrobial sensitivity
Biggest drawback – time required

32. M.TUBERCULOSIS CULTURE MEDIA
Solid
Egg-based - Petragnini medium and Dorset medium
Middlebrook 7H10 Agar
Middlebrook 7H11 Agar
Blood based -Tarshis medium
Serum based - Loeffler medium
Potato based - Pawlowsky medium
LJ media
Liquid
Dubos' medium
Middlebrook 7H9 Broth
Proskauer and Beck's medium
Sula's medium
Sauton's medium
Radiometric – Bactec
Nonradiomaetric – MGIT / MB Redox

33. M.TUBERCULOSIS CULTURE
Solid Media
LJ media
Liquid Media
Radiometric
Non Radiometric
Bactec 460 TB system
MGIT
MB Redox

34. M.TUBERCULOSIS CULTURE SOLID MEDIA
LOWENSTEIN-JENSEN MEDIUM
 Growth medium specially used for culture of Mycobacterium
Processing time – 2-8 weeks
Time increases in pauci bacillary specimen
Further 2-8 weeks in typical v/s atypical -? Simultaneous culture with inhibition of M.tb
Further 2-8 weeks for drug susceptibility

35. M.TUBERCULOSIS CULTURE LOWENSTEIN-JENSEN MEDIUM
Composition
Malachite green ,Glycerol, Asparagine , Potato starch, Coagulated eggs, Mineral salt solution (Potassium dihydrogen phosphate, Magnesium sulfate, Sodium citrate )
Penicillin and nalidixic acid -Inhibit growth of gram positive and gram negative bacteria, . Presence of malachite green in the medium inhibits most other bacteria.

36. M.TUBERCULOSIS CULTURE
LIQUID CULTURE MEDIA
More sensitive and specific
Shorter processing time
Variable availability
Simultaneous differentiation of atypical v/s typical

37. M.TUBERCULOSIS CULTURE RADIOMETRIC
BACTEC 460 TB system
Principle
Detection of the metabolism of the bacteria
Detects 14CO2 liberation during the decarboxylation of 14C labelled substrates palmitic acid
Differentiates typical and atypical mycobacteria 4
p-nitro benzoic acid (PNBA) test
The BACTEC NAP test

38. M.TUBERCULOSIS CULTURE NONRADIOMETRIC
Principle – detection of metabolism of bacteria
Types
Mycobacteria Growth Indicator Tube (MGIT)
MB Redox
If bacteria present
Oxygen consumed in metabolism
Fluorescence
Vial is positive

39. M.TUBERCULOSIS CULTURE LIQUID MEDIA
Nonradiometric Liquid Culture Media
(1) Mycobacteria Growth Indicator Tube (MGIT)
Sensor Contains 4 ml of modified Middlebrook 7H9 broth with an oxygen quenching- based fluorescent sensor.

40. M.TUBERCULOSIS CULTURE LIQUID MEDIA
Nonradiometric Liquid Culture Media
(2) MB Redox Tube
Four ml of modified, serum-supplemented Kirchner medium with a colourless tetrazolium salt as a growth indicator.
During bacterial growth, the tetrazolium salt is reduced to a pink-, red-, or violet-colored formazan.
Contains a special vitamin complex which provides for a considerable acceleration of the growth of mycobacteria

41. M.TUBERCULOSIS CULTURE SOLID V/S LIQUID MEDIA
Solid media
Liquid media
Availability
Good
Variable
Sensitivity
40-70 %
80-95 %
Specificity %
Processing time
4-8 weeks
12-15 days
Cost
1500
Antibiotic sensitivity +
Hence liquid media is preferred

42. TEST UTILITY EVALUATION RADIOMETRIC V/S NONRADIOMETRIC
Bactec
MGIT
MB redox
Availability
Variable
Most common
Uncommon
Sensitivity
90-95 % %
80%
Specificity
95-97%
Processing time
12-14 days
17 days
16 days
Cost
1600 -
Antibiotic sensitivity +
Technical difficulty
Labor intensive
Radiation hazards

43. GUTB DIAGNOSIS ENDOSCOPY & BIOPSY
Indications
Suspected GU Koch's with equivocal radiology and lab test
Ureteric evaluation ( RGP) and stenting
Risks
Bladder perforation, septicemia
Problems
- Morphology - If normal no biopsy
- Changes - specific / nonspecific biopsy
? Need – Highly suggestive radiological findings

44. VIDEO

45. GUTB - ENDOSCOPY & BIOPSY
Classical Histological features
Granuloma formation
Caseous necrosis
Cavitation
Chronic inrterstital inflammation

46. HISTOPATHOLOGY

47. GUTB DIAGNOSIS ENDOSCOPY & BIOPSY
Differential diagnosis -
Fungal infection: histoplasmosis & cryptococcus
Infections:
Cat-scratch fever (caused by bartonella henselae)
Chronic pyelonephritis , sarcoidosis
Wegener's granulomatosis:
Drugs
Very rare compared to M.tb infection

48. GUTB DIAGNOSIS ENDOSCOPY & BIOPSY
Availability - Universal
Sensitivity to 56 % 6,12,13
Specificity to 97 %
Processing time - four days
Cost - Rs 500
Antibiotic sensitivity - NA

49. GUTB DIAGNOSIS
ENDOSCOPY & BIOPSY
Bivalved resected specimen shows two foci of caseous necrosis in the upper pole (arrows).

50. DIAGNOSTIC LAB TESTS COMPARISON
AFB Stain
PCR
LJ Culture
Bactec
MGIT
MB redox
Availability
Universal
Limited
Common
Uncommon
Sensitivity
40 – 60 %
95%
40-70 %
80-95 % %
80%
Specificity %
Processing time
45 mins
one day
4-8 weeks
12-14 days
17 days
16 days
Cost
Rs 500
Rs 1600
Rs 1500 -
Antibiotic sensitivity NA +

51. GUTB LAB DIAGNOSIS
PROBLEMS - No Gold Standards
Current Literature
- Not all test done in all patients
- Different samples from different systems
Sensitivity & Specificity
- Maximum with combination of tests
Recommendation – PCR with Bactac
Radiometric Culture

52. Urine-PCR/AFB stain & Culture
DIAGNOSTIC ALGORITHM
Persistent Irritative voiding &/OR genital symptoms
History of recurrent UTI, past H/O TB, Failed symptomatic t/t
Urine – sterile pyuria, acidic , microscopic hematuria
USG
NonSpecific
Urine-PCR/AFB stain & Culture ?
Specific
Positive
Negative
IVP
Endoscopy
AKT
Morphology
Specific /
Non-Specific
Biopsy –HPE Tissue PCR
Normal
Clinical Decision
Negative
No Biopsy
AKT
Positive

53. Bibliography
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10th Edn, Topley and Wilson.  Bacteriology; Vol. 2, pg 1190
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Nguyen Van Hung et al; Fluorescence microscopy for tuberculosis diagnosis ; The Lancet Infectious Diseases - Volume 7, Issue 4 (April 2007)
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55. THANK YOU