Abdominal Tuberculosis – How Far are Our Diagnostics

Illuminating?

Review Article

INTRODUCTION

Abdominal tuberculosis can involve the luminal,gastrointestinal tract, liver, spleen, lymph node, peritoneumand the female genital tract; the most common site beingthe ileocaecal region [1]. The disease may developsecondary to primary focus elsewhere in the body usuallythe lungs or it may originate within intestinal tract fromswallowed sputum or rarely ingestion of cow’s milk [2].The management is largely dependent upon the correctdiagnosis and drug sensitivity testing for Mycobactertia.

CLINICAL SIGNS & SYMPTOMS

Symptoms

Abdominal pain, fever, weight loss, anorexia, diarrhea,constipation, nausea or vomiting, malena, haemetemesis [1].

Signs

Abdominal tenderness, abdominal distension, ascites,hepatomegaly, splenomegaly, abdominal mass, cervicallymhadenopathy, supraclavicular lymhadenopathy, pulsatilemass in aorto duodenal fistula , dysphagia and odynophagiain esophageal tuberculosis and anemia [2-4].

MICROBIOLOGICAL DIAGNOSIS

Samples

The following patient samples can be collected

ABDOMINAL TUBERCULOSIS - HOW FAR ARE OUR DIAGNOSTICS ILLUMINATING?

Shamma Arora* and Raman Sardana***Junior Consultant (Microbiology), ** Senior Consultant (Microbiology) & Additional Director Medical Services, Indraprastha

Apollo Hospitals, Sarita Vihar, New Delhi 110 076, India.

Correspondence to:Dr. Shamma Arora,Junior Consultant (Microbiology),Indraprastha Apollo Hospitals, Sarita Vihar,New Delhi 110 076, India.

e-mail: shamataluja@yahoo.com

Tuberculosis can involve any part of the gastrointestinal tract from mouth to anus, the peritroneum,pancreas and the hepatobiliary system. Gastrointestinal tuberculosis mimics many clinical conditions andonly a high degree of suspicion can help in the diagnosis otherwise there are chances of missing it leadingto high morbidity and mortality. Various methods of diagnosis are available but which one is the right test for aparticular patient needs to be ascertained. Culture remains the gold standard method of diagnosis. Fasttrack cultures like MGIT/ M Bact Alert 3 D can give faster results with in few days to few weeks. Molecular testsare fastest and can be used as a supplementary test. Nested PCR can give results with in few hours.

Key words: Abdominal tuberculosis, Automated methods for culture, Nucleic acid sequence basedamplification, ELISA for tuberculosis.

depending upon the clinical condition of the patient i.e.sputum, gastric aspirate, pus, ascitic fluid, lymphnode,gastric biopsy, omental culture and mesenteric lymphnode. The above mentioned samples can be subjected tostaining as well as culture.

Acid Fast Bacilli (AFB) smear

AFB staining provided preliminary confirmation ofdiagnosis although it can not differentiate Mycobacteriumtuberculosis from other acid fast bacilli. For a reliableresults smear requires approximately 10,000 organisms/mL. Therefore results may be negative in early stages ofdisease or when the organism load is less [5]. Staining foracid fast bacilli is positive in less than 3% of ascitic fluidsin tubercular peritonitis [6]. Two types of acid fast stainsare commonly used – Carbolfuchsin stains (ZN stain &Kinyon stain) and Fluorochrome stains (auromine O withor without rhodamine). Fluorochrome attains are moresensitive but dead mycobacteria will also give brighyyellow colour fluorescence with this stain. This feature hasto be remembered when using acid fast smears to assesstreatment efficacy and in that case if bright yellowcoloured bacilli are seen in fluorochrome stained smears;carbolfuchsin stain should be performed for confirmationof acid fast bacilli [5]. Besides many laboratories wouldnot have fluorescent microscope.

Culture

Various culture methods range from conventional

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culture system i.e. LJ media to automated systems ofBactec 9000 MB, MGIT 960, ESP Culture system, MB/BacT Alert 3D system. Besides this some semi automatedculture systems like i.e. MGIT, Septichek AFB, MB Redoxare also available [7]. It is known that M.tuberculosis canoccasionally be isolated in stool of persons with healthyconditions. Therefore special decontamination techniquesand BacTec technology must be used for culture [8].

In conventional culture system a positive culture isobtained in less than 20% of cases, and it takes 6-8 wk forthe mycobacterium colonies to appear. Singh, et aladvocated that processing of one liter of ascitic fluid mayyield up to 80% positive results [6].

Bactec 460 TB

The detection time for M.tuberculosis averages 9-14days & it may be less than 7 days for some strains ofMycobacteria other than M.tuberculosis but disadvantagesof system include cost of instrumentation , the inability toobserve colony morphology and detect mixed cultures;over growth by contaminants ; need for disposal ofradioactive material and extensive use of needles [8].

Mycobacteria growth indicator tube (MGIT & MGIT960)

MGIT system consists of round bottom glass tubes anda fluorescent compound which is sensitive to dissolvedoxygen in the broth. The tubes are observed daily forfluorescence under wood lamp up to 6 weeks. MGIT 960 anon radiometric automated system that holds 960 plastictubes which are continuously monitored. Studies haveindicated that MGIT 960 had the shortest mean time todetection i.e. 13.3 compared to 14.8 days for BACTEC TB& 25.6 days for LJ media [8].

MB/ Bact Mycobacteria detection system

The system is based on continuous monitor of themicrobial generated CO2 . The mean time for detection is 16days. Again it is a non radiometric detection of mycobacteriaeliminating the need for handling and disposal ofradioisotopes [8].

ESP culture system II

The system is based on continuous monitor of the gaspressure due to metabolic activity of microorganisms inculture bottle. Studies have indicated that ESP detectspositive cultures three times more frequently than BACTEC460 system and again no need to handle radioactive waste[8].

Molecular techniques

PCR of the mucosal biopsy specimens diagnose TB in

45 to 64 % of cases [9,10]. Studies have indicated that thefaecal PCR based on IS6110 insertion element hassensitivity and specificity of 88% & 100% respectively. Thefaecal PCR was able to detect >10 copies of M.tuberculosis.Besides this unlike endoscopic biopsy faecal PCR is a noninvasive test [11].

Nested PCR

In this PCR there is double amplification of a fragmentof the insertion element IS6110 only present inM.tuberculosis genome[12]. The Xpert M.tuberculosisassay/Rifampicin assay uses three specific primers and fivemolecular probes to assure high degree of specificity. Fordetection of rifampicin resistance it targets the rpoB geneand the results are available with in two to three hours.

Nucleic acid sequence based amplification (NASBA)

Three steps are involved in the use of this assay:isolation of 23 S rRNA , amplification of RNA by theNASBA method, and the reverse hybridization of theamplified products on membrane strips using an automatedmethod. Genotype Mycobacteria Direct (GTDIR; Hain LifeScience, Nehren, Germany) is based on NASBA applied toDNA strip technology. The assay is used for the directdetection of M.tuberculosis and other Mycobacteria i.e.M.avium, M.intracellulare, M.kansasii and M.malmonsefrom the clinical samples [13].

Besides this there are certain assays like GenoTypeMTBDRplus(GTPLUS) that allows the direct detection ofM.tuberculosis in clinical samples as well as detection ofresistance to isoniazid (kat G and inh A) and rifampicin(rpoB) [13].

Both of GTDIR and GTPLUS showed highersensitivities. Besides this as both of them are RNA PCRthese can differentiate between live and killed bacilli whichDNA PCR can not. GTDIR should be used in areas withlow prevalence of tuberculosis and high incidence ofinfections by non tubercular Mycobacteria while GTPLUSshould be used in areas with higher incidence of tuberculosisso that simultaneously resistance to isoniazid & rifampicincan be detected. Testing for Ethambutol, fluoroquinolonesand aminoglycoside resistance can also be checked [13].

Antigen detection tests

ELISA based on antigens 85B and 85 C (Ag 85) complex,a 65–kDa protein that corresponded M. tuber-culosis heatshock protein 65 (65 kDa HSP ), 14 k Da heat shock proteinHSP and MTB heat shock protein 71 (71 kDa HSP) can beused a screening test [14]. Three types of antibody responseIg M, Ig G, Ig A are available. ELISA and Soluble antigenfluorescent antibody are not sensitive and non specific and

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can only suggest a probable diagnosis [15].

SUPPLEMENTAL TESTS

• γ – Interferon assay. The interferon – gamma assayhave been used for the diagnosis of latentTuberculosis and active tuberculosis. The γ -interferon assay is a cell mediated immunity basedresponse to peptide antigens that stimulatemycobacteria proteins –Early secreted antigenictarget; 6 kDa protein( ESAT- 6) & Culture filtrateprotein-10 (CFP10). The proteins are absent from allBCG strains and from most non tubercularmycobacteria with the exception of M.kansasii,M.szulgai & M.marinum. Individuals infected withM.tuberculosis complex usually have lymphocytesand recognize these and other mycobacterial antigens.The γ - interferon assay is more sensitive in caseswith active tuberculosis compared with tuberculinskin test and is not affected by BCG vaccination [16].A positive test would indicate the likelihood ofinfection but would not differentiate for diseasewhich is so common in endemic regions like India.

• Mantoux test: The test is considered to be significantif induration is more than 20 mm done by using PPD 5TU. One of the major limitation is possibleoccurrence of false positive results in individualsvaccinated with BCG [16]. It is a non specific testand has a lower value than interferon- γ assay.However it is much cheaper than other test.

• Adenosine deaminases (ADA) levels: ADA; anenzyme is distributed in mammalian tissue particularlyin T- lymphocytes. ADA is increased in tuberculousascites due to stimulation of T- cells bymycobacterial antigens [4]. Increased levels arefound in tuberculosis. Therefore it serves asimportant marker of tuberculosis. It can be raised invarious infections like infectious mononucleosis,typhoid, Viral hepatitis, initial stages of HIV infectionand malignant tumor. In countries with high incidenceof Tuberculosis and in high risk patients measurementof ADA in ascitic fluid might be a useful screeningtest. However in population with low prevalence oftuberculosis and high incidence of cirrhosis asciticfluid ADA is poor in sensitivity as well as specificity[17-19]. Studies have indicated that in peritonealtuberculosis ADA estimation has a sensitivity of100%, specificity of 96%, positive predictive value of91.6 %, negative predictive value of 100% [20].

• ESR: The ESR varies from 0-15 mm in first hour inmales and 0-20 mm in first hour in females. The ESRis elevated in 90 % of cases [21].

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6. Singh MM, Bhargava AN, Jain KP. Tuberculousperitonitis. An evaluation of pathogenetic mechanisms,diagnostic procedures and therapeutic measures. NEngl J Med 1969; 281: 1091-1094 .

7. Peyfeer GE. Welscher HM, Kissling P, et al. Comparisonof Mycobacteria growth indicator tube with radiometricand Solid culture for recovery of Acid fast bacilli. J clinMicrbiol. 1997; 364-368.

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9. Gan HT, Chen YQ, Quang HB, Yang XY. Differentiationbetween intestinal tuberculosis and Crohn’s disease inendoscopic biopsy specimens by polymerase chainreaction. Am J Gastroenterol 2002; 97: 1446-1451.

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11. Balamurugan R, Venkataraman S, John KR, Rama-krishna BS. PCR Amplification of the IS6110 Insertionelement of Mycobacterium tuberculosis in fecal samplesfrom patients with intestinal tuberculosis. J ClinMicrobiol. 2006; 1884-1886.

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13. Neonakis LK, Gitti ZG, Baritaki S, Petinaki E, Baritaki M,Spandidos DA. Evaluation of GenoType Mycobacteriadirect assay in comparison with Gen- ProbeMycobacterium tuberculosis Amplified direct Test andGenoType MTBRPLUS for direct detection ofMycobacterium tuberculosis complex in clinicalsamples. J Clin Microbiol, 2009; 47(8):2601-2603.

14. Kashyap RS. Saha SM. Khushboo JN, et al. Diagnosticmarkers for Tuberculosis ascites: A preliminary study.Biomarkers insights 2010; 5: 87-94.

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of enzyme-linked immunosorbent assay using myco-bacterial saline-extracted antigen for the serodiagnosisof abdominal tuberculosis. Am J Gastroenterol. 1992;87:105-108.

16. Hotta K, Ogura T, Nishi K, et al. Whole blood Interferon –gamma assay for baseline tuberculosis screeningamong Japanese Healthcare Students. PloS ONE.2007; 8 [Serial No.1-8].

17. Bhargava DK, Nijhawan S, Gupta M. Adenosinedeaminase and Tuberculous peritonitis. Lancet. 1989;1: 1261.

18. Bhargava DK, Gupta M, Nijhawan S, Dasarathy S,Kushwaha AKS. Adenosine deaminase deaminase in

peritoneal tuberculosis: Diagnostic value in ascitic fluidand serum: Tubercle 1990; 71: 1121-1126.

19. Hillebrand DJ, Runyon BA, Yashmineh WG, Rynders GP.Ascitic fluid adenosine deaminase insensitivity indetecting tuberculous peritonitis in the United States.Hepatology.1996; 24(6):1408-1412.

20. Gupta BK, Bharat V, Bandyopadhyay D. Sensitivity,Specificity , Negative and Positive Predictive Values ofAdenosine deaminases in patients of Tubercular andnon- tubercular Serosal effusion in India. J Clin Med Res.2010; 2(3):121-126.

21. Bhargava DK. Abdominal Tuberculosis : Current Status.Apollo medicine. 2007; 4(4): 287-291.

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