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

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

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

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

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

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

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

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

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

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 %

GUTB - CLINICAL EVALUATION Other systems Respiratory - 12 % patients Gastrointestinal - 10 % Lymphoreticular Constitutional - 10 to 15 % 1 Evening rise of temperature Weight loss Anorexia

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

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

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

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

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

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

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

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

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

ZN - AFB STAIN TEST UTILITY EVALUATION Availability - Universal Sensitivity - 30 – 40 %4 Specificity - 95% 4 Processing time - 45 mins Cost - Cheap Antibiotic sensitivity - NA

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

TEST UTILITY EVALUATION Availability - Poor Sensitivity - 10 % > ZN staining5 Specificity - Similar Processing time - Less than ZN staining Cost - Significantly > ZN staining Antibiotic sensitivity - NA

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 IS6110 - 77% - Most commonly targeted6 65kDa - 75%, 38 kDa - 72% ,85B protein - 73%

MOLECULAR TECHNOLOGY POLYMERASE CHAIN REACTION (PCR) Diagnostic Problems Few Indian strains lack copy of IS 6110 IS 6110- 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

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

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

MOLECULAR TECHNOLOGY - PCR TEST UTILITY EVALUATION Availability - Limited Sensitivity - 75% - 95%6 Specificity - 95 - 97 %6 Processing time - One day Cost - Rs 1600 Antibiotic sensitivity - NA

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

MOLECULAR TECHNOLOGY TISSUE PCR Sampling limitations Availability - Limited Sensitivity - 95% Specificity - 95 to 97 % Processing time - One day Cost - Rs 1600 Antibiotic sensitivity - NA TEST UTILITY CRITERIA

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

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

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

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

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.

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

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

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

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.

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

M.TUBERCULOSIS CULTURE SOLID V/S LIQUID MEDIA Solid media Liquid media Availability Good Variable Sensitivity 40-70 % 80-95 % Specificity 95 - 97% Processing time 4-8 weeks 12-15 days Cost 1500 Antibiotic sensitivity + Hence liquid media is preferred

TEST UTILITY EVALUATION RADIOMETRIC V/S NONRADIOMETRIC Bactec MGIT MB redox Availability Variable Most common Uncommon Sensitivity 90-95 % 70 - 80% 80% Specificity 95-97% Processing time 12-14 days 17 days 16 days Cost 1600 - Antibiotic sensitivity + Technical difficulty Labor intensive Radiation hazards

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

VIDEO

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

HISTOPATHOLOGY

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

GUTB DIAGNOSIS ENDOSCOPY & BIOPSY Availability - Universal Sensitivity - 18 to 56 % 6,12,13 Specificity - 95 to 97 % Processing time - four days Cost - Rs 500 Antibiotic sensitivity - NA

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

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 % 70 - 80% 80% Specificity 95 - 97 % 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 +

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

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

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