August 15, 2011 Innersense Voice 122 Laboratory Diagnosis of Genital Tuberculosis Despite decades of research and availability of a number of diagnostic tests and therapieutic regimens (TB) is an increasing public health concern worldwide. It is classified as pulmonary tuberculosis (PTB), tuberculosis of the lungs or extrapulmonary tuberculosis (EPTB), tuberculosis of organs, other than lungs. The EPTB sites most commonly involved are the lymph nodes, pleura, genitourinary tract, bones and joints, meninges, peritoneum, and pericardium. However, virtually all organ systems may be affected. As a result of hematogenous dissemination in HIV-infected individuals, EPTB is seen more commonly today than in past. The most common form of extrapulmonary TB is genitourinary disease, accounting for 27% (range, 14 to 41%) worldwide (9). The global prevalence of GTB is estimated to be 8-10 millions cases, with a rising incidence in the developing counties partly as a result of its association with HIV virus infection and emergence of multidrug resistance. However, the actual incidence may be under reported due to asymptomatic presentation of GUTB and paucity of investigations. In comparison to only pulmonary TB, which comprises around 68.4%, the incidence of combined pulmonary-extrapulmonary cases and extrapulmonary TB alone comprise 12% and 20-25% of the total disease burden respectively (8). The typical presentation of GUTB includes pelvic pain, menstrual irregularity, general malaise and infertility. Diagnosis of early TB is very difficult. Early diagnosis may be associated with a more favorable result before extensive genital damage occurs.[1] Female Genital Tract Tuberculosis (FGTB): It is estimated that 18% of infertile women, aged between 20-40 years in India suffer from GTB. The genital organs commonly affected are as follows: fallopian tube (95-100%), endometrium (50-60%), ovaries (20-30%), cervix (5-15%), myometrium (2.5%) and vulva/vagina (1%). [2] Male Genital Tuberculosis (MGTB): It is predominantly associated with tuberculosis of the kidney and prostate, seminal vesicle, epididymis, testes as well as scrotum may occasionally be affected. In men, the sites most commonly involved are epididymis, followed by the prostate. Testicular involvement is less common and usually is the result of direct extension from the epididymis. Tubercular prostatitis usually results from antegrade infection within the urinary tract. Tuberculous epididymo-orchitis has a considerable effect on fertility, the sperm count and motility may be reduced due to blockage of the vas and/or secondary atrophy. [2] Diagnosis of GTB The diagnostic dilemma arises because of varied clinical presentation of the disease confounded by diverse results on imaging, laparoscopy, histopathology, bacteriological and serological tests, each of which has its limitation in diagnostic sensitivity and specificity. Diagnosis of GTB has profound implications for asymptomatic women seeking fertility. Endometrial TB Diagnosis of GTB depends on following: The sensitivity of endometrial biopsy or curetting in the diagnosis of TB is low  Clinical Symptomatology (40%), as the granulomas are often focal and the functionalis layer is shed  CBC, ESR, RFT, CRP every four weeks (granulomas take two weeks to develop). So, if suspected, curettage/biopsy should be performed during the late secretory phase of the  Chest X-ray menstrual cycle. In a comparative study of sensitivity of detection of  Pelvic ultrasound / Hystero-salpingography genitourinary TB, smear microscopy, histopathological examination, (HSG). mycobacterial culture, nucleic acid amplification by PCR, or combination of  Laparoscopy culture and PCR were 87.5%, 82.3%, 91.6%, 96.4% and 100% respectively. While the specificity for the same were as follows, 86.36%,  Histopathology 84.6%, 88.88%, 100% and 100% respectively [2].  Microbiology o Mantoux test o QTG-T o AFB microscopy / culture  Endometrial aspiration / Endometrial biopsy/ Endometrial curettage/ Menstrual blood in females. August 15, 2011 o o Innersense Voice 122  Urine – 3 consecutive days Molecular tests HIV Photomicrograph of section of endocervix showing caseating granulomas and Langhans giant cells admist chronic inflammatory infiltrate. (H&E 400 X). Source: Journal of Clinical and Diagnostic Research. 2010 Feb;(4):2083-2086. Interferon Gamma Release Assays (IGRAs) IGRA is a coupling of the discovery of antigens ESAT-6 and CFP-10, which are relatively specific to M. tuberculosis, and the development of simplified technologies of measuring interferon-gamma (IFN-g) [4]. IGRAs measure a person’s immune reactivity to M. tuberculosis. White blood cells from most persons that have been infected with M. tuberculosis will release IFN-g when mixed with antigens derived from M. tuberculosis. They do not help differentiate latent tuberculosis infection from tuberculosis disease [5]. Two IGRAs approved by the U.S. Food and Drug Administration (FDA) are:  QuantiFERON®-TB Gold In-Tube test (QFT-GIT) - measures interferon-g in IU/mL using an enzyme-linked immunosorbent assay (ELISA).  T-SPOT®.TB test (T-Spot) - Counts the cells releasing IFN-G visualized as spots with the enzyme-linkedimmunospot (ELISPOT) technique. Clinical Utility:  Can detect both latent and active pulmonary and extra-pulmonary cases.  Useful for screening person who has symptoms of TB.  Screening suspected Extra-Pulmonary tuberculosis eg: GTB Role of Molecular Methods in Diagnosis of GTB A definite diagnosis can be made by positive mycobacterial culture and by demonstrating specific histopathological lesion in the specimen of suspected case of GTB. However, these methods have low detection rates and limitations as GTB is paucibacillary, also substantial number of TB lesions of the genital tract are bacteriologically mute. The low rate of positivity in culture may also be due to the presence of a bacteriostatic substance which inhibits the growth of the bacilli [7]. Hence, no single test can stand alone as the diagnostic test for genital tuberculsis. In recent years, PCR technique has evolved as useful and rapid technique for the diagnosis of pulmonary and extrapulmonary tuberculosis. Any method that is used to diagnose GTB should be highly sensitive to diagnose the disease reliably in its early stage, so that treatment may improve the prospects of cure, and in females before the tubes are damaged beyond recovery. Fundamentally all the available molecular tests are based on the principle of PCR. Advantages:  High specificity and sensitivity, requires only < 10 bacteria/mL of specimen to achieve a positive report.  Rapid method, results are available within a day of the DNA being extracted from the specimen.  Can be applied to sterile fluids like peritoneal fluid where the culture is difficult due to a low bacterial load. Disadvantages:  False Negative - absence of even a single AFB in the sample collected, and high salt concentration of a specimen which interferes with the PCR results.  False Positive- PCR cannot distinguish between live and hiked bacilli and there is a risk of false positive results. Depending upon the test requests received at SRL, the following can be a suggested step wise guideline for TB diagnosis in genital tuberculosis Clinical suspicion of genital TBSite specific sample for TB-PCRif negative, laparoscopic removal of fluid/tissue for histopathology and or culture. August 15, 2011 Innersense Voice 122 Case Study - Tubercular Cervicitis Clinically Mimicking As Carcinoma Cervix A 38 year old female presented in the gynaecology OPD with the complaint of menometrorrhagia. Her pelvic examination revealed a friable papillary growth on the cervix. Pap smear revealed a mixed inflammatory infiltrate along with atypical cells of uncertain origin (ASCUS). A punch biopsy was taken for diagnostic confirmation. The clinical differentials included neoplastic and viral aetiologies. The histopathological examination revealed endocervicitis with well formed epithelioid cell granulomas along with Langhans giant cell formation and caseation necrosis. A provisional diagnosis of tuberculosis of the cervix was made. AFB stain did not reveal any bacilli. Further investigations were done to rule out any comorbid conditions. A re- biopsy of the growth was done and the tissue was sent for tubercular polymerase reaction (TB-PCR), which was positive for Mycobacterium tuberculosis. The patient was started on antitubercular therapy. The growth regressed after four months. A repeat biopsy revealed the absence of granulomatous inflammation. Till the last follow up, the patient was symptom free [6]. As seen by the above case, diagnosis of GTB is based on collective investigations. Clinical symptomatology, radiography, and laboratory investigations together aid a physician to come a definitive diagnosis. However, early diagnosis and early treatment is the key to prevent the aftermath. Frequently Asked Questions (Endometrial Tuberculosis) 1) What is the clinical utility of TB PCR in Gynecological TB? Ans. TB PCR aids in confirming the diagnosis of genital TB infection in infertile patients with high index of suspicion. 2) How do TB PCR correlates with other routine diagnostic methods in Gynecological TB cases? Ans. Various studies have indicated strong association of endoscopic features of genital TB with positive PCR results in endometrial specimens. Culture methods have limited detection rate (usually < 10%), and therefore they rarely correlate with TB PCR. Histopathology have higher detection range than culture methods. However due to secondary nature of the genital tuberculosis, the infecting organisms are sparse in number or in some cases the sample site may not represent the infected area or sometimes the cellular changes suggestive of TB may be due to certain pyogenic bacteria. In such scenarios, correlation between histopathology and PCR is very rare. Recent studies have highlighted that positive TB PCR results should be given due importance particularly in clinically suspected cases. In presence of positive PCR results, patients with infertility should be considered as having Genital TB and should be treated. 3) What type of gynecological specimens can be processed for TB PCR, which specimen is preferred? Ans. Endometrial Biopsies, endometrial aspirate (EA), fluid from the pouch of Douglas (POD), and Menstrual Blood are the most common specimens. In Gynecological TB, fallopian tube is the initial site of involvement, affected in almost all cases, followed by endometrium in 50-90% of cases. In as many as 50% of cases infection may be limited to the fallopian tube. Moreover, due to the cyclical shedding of the endometrium, granulomas do not have enough time to form, so the endometrium may not show evidence of tuberculosis in all the cycles. However despite these facts, endometrial biopsy is the preferred specimen for Gyecological TB. Though menstrual blood specimen offers a non-invasive means for diagnosis of Gynecological TB, presently there is limited evidence on its reliability mainly considering the cyclic shedding of TB bacilli. Tests offered at SRL Test Test Code GAMMA INTERFERON (TBFERON) 2405 TB DETECTION by Microscopy 1122 TB SPECIATION by Microbiology tests 2419ID TB CULTURE POSITIVE REFLEX MDR BY PCR SEQUENCING 9215RFX Test Test Code MYCOBACTERIA SPECIATION by PCRsequencing AFB SUSCEPTIBILITY, BACTEC : 10 DRUG PANEL AFB SUSCEPTIBILITY, BACTEC : 5 DRUG PANEL 1464R2 AFB SUSCEPTIBILITY, BACTEC : SIRE PANEL 1490 2402 1464R1 August 15, 2011 References: 1. 2. 3. 4. 5. 6. 7. 8. 9. Innersense Voice 122 TB MONITORING PANEL (CBC,ESR,SGPT/SGOT/CRE ATININE,ANA,AFB SMEAR) 5010 AFB SUSCEPTIBILITY, BACTEC : SIREP PANEL 5649 TB PANEL (MYCO3 PCR, ADA, GAMMA INTERFERON) 2440 AFB SUSCEPTIBILITY; MIC TESTING FOR RAPIDLY GROWING MYCOBACTERIA 1464RGM TB PCR (MYCOREAL) 2434 TB-Spot RD1323 TB PCR (MYCOTECT) 2439 AFB Culture (MGIT 960) 1464EP MYCO3PLEX (AFB Fluorescent Stain, Culture, PCR) 2435 Puri S, Bansal B. Diagnostic Value of Polymerase Chain Reaction in Female Tuberculosis Leading to Infertility and Conception Rate After ATT. JK Science Journal of Medical Education and Research. Jan-Mar 2009. Vol. 11(1). Das P, Ahuja A, and Datta-Gupta S . Incidence, etiopathogenesis and pathological aspects of genitourinary tuberculosis in India: A journey revisited. Indian J Urol. 2008 Jul-Sep; 24(3): 356–361. doi: 10.4103/0970-1591.42618 Jassawalla MJ. Genital tuberculosis - A diagnostic dilemma. J Obstet Gynecol India Vol. 56, No. 3 : May/June 2006 Pg 203-204 Lange C and Mori T. Advances in the diagnosis of tuberculosis. Respirology (2010) 15, 220–240. doi: 10.1111/j.14401843.2009.01692.x http://www.cdc.gov/tb/publications/factsheets/testing/IGRA.htm. Updated: July 25, 2011 Bhalla A, Mannan R, Khanna M, Bhasin TS. Tubercular Cervicitis Clinically Mimicking As Carcinoma Cervix: Two Case Reports. Journal of Clinical and Diagnostic Research. 2010 Feb;(4):2083-2086. Thangappah R, Paramasivan CN, Narayanan S. Evaluating PCR, culture & histopathology in the diagnosis of female genital tuberculosis. Indian J Med Res [serial online] 2011 [cited 2011 Aug 11];134:40-6. Available from: http://www.ijmr.org.in/text.asp?2011/134/1/40/83325 Alan JW, Louis RK, Andrew CN, et al., editors. Campbell-Walsh Urology. 9th ed. New York: Saunders, Elsevier; 2006. Marjorie PG, Holenarasipur RV. Extrapulmonary tuberculosis: An overview. Am Fam Physician. 2005;72:1761–8