Diagnosis of Genitourinary Tuberculosis

Dr. Jayesh Dhabalia Dr. Ulhas SatheConsultant Urologist Consultant UrologistProfessor & Head Sahyog Speciality Hospital Department of Urology, & Lithotripsy CentreLTMG 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

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

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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

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 %

Other systems • Respiratory - 12 % patients• Gastrointestinal - 10 %• Lymphoreticular

Constitutional - 10 to 15 % 1

– Evening rise of temperature – Weight loss– Anorexia

GUTB - CLINICAL EVALUATION

GUTB - LABORATORY 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

1. 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

2. T cell–based cellular immune response – Different antigens detects different types of TB• Test kit not available

II. Direct detection of bacterial antigens & metabolites • Test kit not available

GUTB – SERODIAGNOSISPROBLEMS

• 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 DIAGNOSTICLAB TESTS

COLLECTION OF SAMPLESUrine • 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

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 overZN – 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%

Diagnostic ProblemsFew Indian strains lack copy of IS 6110IS 6110- present in M.tb complex bacteria

( tuberculous / africanum / microti / bovis)False positive – old cases of TB, contaminationFalse negative – extremely paucibacillary cases

( detection limit up to 10 copies / ml )

Cannot differentiate dead from live bacteria25

MOLECULAR TECHNOLOGY POLYMERASE CHAIN REACTION (PCR)

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

MOLECULAR TECHNOLOGY - PCR

• Availability - Limited• Sensitivity - 75% - 95%6

• Specificity - 95 - 97 %6

• Processing time - One day• Cost - Rs 1600 • Antibiotic sensitivity - NA

TEST UTILITY EVALUATION

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 CULTUREMEDIA

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

Bactec 460 TB system

MGIT

MB Redox

M.TUBERCULOSIS CULTURESOLID MEDIA

• 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

LOWENSTEIN-JENSEN MEDIUM

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

• More sensitive and specific • Shorter processing time• Variable availability• Simultaneous differentiation of atypical v/s

typical

LIQUID CULTURE MEDIA

M.TUBERCULOSIS CULTURE RADIOMETRIC

BACTEC 460 TB systemPrinciple – 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

M.TUBERCULOSIS CULTURELIQUID MEDIA

(1) Mycobacteria Growth Indicator Tube (MGIT)

• Sensor Contains 4 ml of modified Middlebrook 7H9 broth with an oxygen quenching- based fluorescent sensor.

Nonradiometric Liquid Culture Media

M.TUBERCULOSIS CULTURELIQUID 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

Nonradiometric Liquid Culture Media

M.TUBERCULOSIS CULTURE SOLID V/S LIQUID MEDIA

Solid media Liquid media

Availability Good Variable

Sensitivity 40-70 % 80-95 %

Specificity 95 - 97% 95 - 97%

Processing time 4-8 weeks 12-15 days

Cost 1500 1500

Antibiotic sensitivity + +

Hence liquid media is preferred

TEST UTILITY EVALUATION RADIOMETRIC V/S NONRADIOMETRIC

Radiometric Non radiometric

Bactec MGIT MB redox

Availability Variable Most common Uncommon

Sensitivity 90-95 % 70 - 80% 80%

Specificity 95-97% 95-97% 95-97%

Processing time 12-14 days 17 days 16 days

Cost 1600 1600 -

Antibiotic sensitivity

+ + +

Technical difficulty

Labor intensiveRadiation hazards

GUTB DIAGNOSISENDOSCOPY & 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

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 infection47

GUTB DIAGNOSIS ENDOSCOPY & BIOPSY

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

• Bivalved resected specimen shows two foci of caseous necrosis in the upper pole (arrows).

GUTB DIAGNOSISENDOSCOPY & BIOPSY

DIAGNOSTIC LAB TESTSCOMPARISON

AFB Stain PCR LJ Culture Bactec MGIT MB redox

Availability Universal Limited Common Limited Limited Uncommon

Sensitivity 40 – 60 % 95% 40-70 % 80-95 % 70 - 80% 80%

Specificity 95 - 97 % 95 - 97 % 95 - 97 % 95 - 97 % 95 - 97 % 95 - 97 %

Processing time

45 mins one day 4-8 weeks 12-14 days 17 days 16 days

Cost Rs 500 Rs 1600 Rs 1600 Rs 1500 Rs 1500 -

Antibiotic sensitivity

NA 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 testsRecommendation – PCR with Bactac

Radiometric Culture51

• 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

Specific

NonSpecific

IVP

Urine-PCR/AFB stain & Culture

Positive

AKT

Negative

Endoscopy

MorphologySpecific /Non-Specific

Biopsy –HPE Tissue PCR

Positive

NegativeClinical Decision

AKT

?

Normal

No Biopsy

DIAGNOSTIC ALGORITHM

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