diagnosis of genitourinary tuberculosis dr. jayesh dhabalia dr. ulhas sathe consultant urologist...

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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

Bibliography Das P, Ahuja A, Gupta SD. Incidence, etiopathogenesis and pathological aspects

of genitourinary tuberculosis in India: A journey revisited. Indian J Urol 2008;24:356-61

Debra L. Piddington et al; Infection and Immunity, August 2000, p. 4518-4522, Vol. 68, No. 8

10th Edn, Topley and Wilson. Bacteriology; Vol. 2, pg 1190 Negi SS, Khan SF, Gupta S, Pasha ST, Khare S, Lal S. Comparison of the

conventional diagnostic modalities, bactec culture and polymerase chain reaction test for diagnosis of tuberculosis. Indian J Med Microbiol 2005;23:29-33

Nguyen Van Hung et al; Fluorescence microscopy for tuberculosis diagnosis ; The Lancet Infectious Diseases - Volume 7, Issue 4 (April 2007)

A. K. Hemal, N. P. Gupta et al ; Polymerase Chain Reaction In Clinicall Suspected Genitourinary Tuberculosis: Comparison With Intravenous Urography, Bladder Biopsy, And Urine Acid Fast Bacilli Culture ; UROLOGY 56: 570–574, 2000

Venkataswamy MM, Rafi W, Nagarathna S, Ravi V, Chandramuki A. Comparative evaluation of bactec 460tb system and lowenstein-jensen medium for the isolation of M. tuberculosis from cerebrospinal fluid samples of tuberculous meningitis patients. Indian J Med Microbiol 2007;25:236-40

P Anargyros, D S Astill and I S Lim; Comparison of improved BACTEC and Lowenstein-Jensen media for culture of mycobacteria from clinical specimens ; J Clin Microbiol. 1990 June; 28(6): 1288-1291

9. H.P. Chien; M.C. Yu; M.H. Wu; T.P. Lin; K.T. Luh; Comparison of the BACTEC MGIT 960 with Löwenstein-Jensen medium for recovery of mycobacteria from clinical specimens ; The International Journal of Tuberculosis and Lung Disease, Volume 4, Number 9, September 2000 , pp. 866-870(5)

10. L. Heifets, T. Linder, T. Sanchez, D. Spencer, and J. Brennan; Two Liquid Medium Systems, Mycobacteria Growth Indicator Tube and MB Redox Tube, for Mycobacterium tuberculosis Isolation from Sputum; Journal of Clinical Microbiology, March 2000, p. 1227-1230, Vol. 38, No. 3 Specimens

11. W. K. Chew1, R. M. Lasaitis, F. A. Schio and G. L. Gilbert; Clinical evaluation of the Mycobacteria Growth Indicator Tube (MGIT) compared with radiometric (Bactec) and solid media for isolation of Mycobacterium species; J Med Microbiol 47 (1998), 821-827

12. Wong SH, Lau WY, Poon GP, et al: The treatment of urinary tuberculosis. J Urol 131: 297–301, 198

13. Gow JG: Genito urinary tuberculosis, in Walsh PC, Retik AB, Stamey TA, et al (Eds): Campbell’s Urology, 6th ed. Philadelphia, WB Saunders, 1992, vol 1, pp 951–981

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