Paramasivan, C N

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Indian Journal of TuberculosisThe Indian Journal of TuberculosisVol. 53 April,  2006        No. 2EditorialMOLECULAR METHODS IN THE DIAGNOSIS OF TUBERCULOSISMOLECULAR METHODS IN THE DIAGNOSIS OFTUBERCULOSIS[Indian J Tuberc 2006; 53:61-63]               Early diagnosis of tuberculosis (TB) and initiating optimal treatment would not only enablecure of an individual patient but will also curb the transmission of infection and disease to others in thecommunity. Of the several distinct components of TB control programme, case finding remains thecorner stone for effective control of the disease. Currently, sputum smear microscopy by Ziehl-Neelsen (ZN) staining method is the simplest and most rapid test available to detect acid fast bacilli(AFB) in clinical specimens. Although it is a robust technique and quite inexpensive with a specificityof almost 100%, it suffers from a low sensitivity ranging from 22 to 78% and requires at least 5x103bacilli per ml of sputum. In most of the disease endemic developing high burden countries (DEDC)such as India, smear microscopy at present is the only cost effective tool for diagnosing patients withinfectious pulmonary tuberculosis and to monitor the progress of treatment.                      The presence of active tuberculosis is also being diagnosed in DEDCs by other conventionallaboratory procedures including specimen digestion and decontamination, isolation by culture on solid,and/or liquid media and identification and drug susceptibility testing of recovered isolates by standardphenotypic methods. Because of the slow growth of mycobacteria, the above procedures may require aturn-around time of 8-12 weeks or even longer. Additional limitations in relation to culture is the verylimited availability of quality assured culture facilities in DEDC. Further more, the diagnosis of tuberculosisin children is especially difficult as they can not easily produce sputum and clinical features are non-specific and chest radiographs are often difficult to interpret. Though the demonstration of mycobacteriain various clinical specimens by culture remains as a gold standard, this is not always possible in childrendue to pauci bacillary nature of illness. The same is true in extra pulmonary manifestations and quite oftenalso in patients suffering from HIV and tuberculosis. To meet these special situations, new techniques arevery much needed and among them molecular amplification assays such as PCR have been shown to bepromising alternative even for developing countries1.                 Molecular amplification techniques are mainly indicated for detection of organisms that requireprolonged incubation and also that cannot be grown in vitro or the current culture techniques are tooinsensitive. Several molecular methods have been developed during the last decade for the direct detectionand identification of M. tuberculosis in clinical specimens. These methods are able to potentially reduce thediagnostic turn around time from weeks to days, which acquire a greater relevance under specialcircumstances. If PCR is used in the diagnostic process as well as to identify disease caused by Non-tuberculous Mycobacteria (NTM) and to directly detect drug resistance of M.tuberculosis complex inclinical specimens, it will become less cumbersome for the patient, and reduce the delay both in diagnosisand also in the start of treatment.              The basic principle of any molecular diagnostic test is the detection of specific nucleic acidIndian Journal of Tuberculosissequence by hybridization to a probe, a complementary sequence and followed by the detection of thehybrid. DNA and RNA fragments have been proposed for the amplification target in M.tuberculosis and themost frequently used targets are IS6110, a repetitive element, 16S ribosomal DNA (rDNA), and 16S rRNA.                Although a large number of studies have been published on the ability of nucleic acid amplificationassays (NAA) to detect directly M.tuberculosis from clinical specimens, it is nearly impossible to draw anydefinitive conclusions based on these studies due to their varied sensitivity, specificity and predictive values.Additional issues which include nucleic acid isolation procedures, gene targets for amplifications, samplesize, cycling parameters, the type of patient population and type of samples cannot be ignored. Consideringall the above mentioned issues, the US Food and Drug Administration (FDA) prescribed culture as amandatory requirement in conjunction with the performance of any amplification–based test.Many commercial direct amplification tests (CDATs) are available. These include: COBASAMPLICOR Mycobacterium system (Roche Molecular system, Branchburg, NJ) which uses the 16SrRNAgene; Amplified M.tuberculosis Direct (AMTD) assay (Gene Probe, Inc, San Diego,Calif) in which 16SrRNA is the target amplified and which is identified with an acridinium ester-labelled M,tb complex specificDNA probe; and LCx MTB assay (Abbott LCx probe system by Abbott Laboratories, Abbott Park, III)which uses a Ligase chain reaction method wherein detection is performed by a micro particle enzymeimmune assay with the LCx fluorimetric analyzer. Another test is the BD Probe Tec energy transfer systemDTB (Becton Dickinson Biosciences, Microbiology Products, Sparks, Md.), in which the large sequencesof IS6110 and 16S rRNA gene are both co-amplified. Detection is subsequently made by the increase influorescence polarization. Some CDATs are also available for the detection of both M.tuberculosis and itsresistance to rifampicin, e.g. the INNO-LiPA Rif.TB assay.However it should be noted that many of these tests have not yet been approved by the appropriateregulatory agencies. In 2000, the Centers for Disease Control and Prevention updated its recommendationsfor the use of CDATs in the diagnosis of active TB. Accordingly, it was recommended that sputum specimensbe collected on three different days for AFB microscopy and culture.  If AFB smears are negative, CDATis performed on the first specimen collected, otherwise it is performed on smear positive specimens. Ifsmear and CDAT are positive, that confirms TB. If the smear is positive and CDAT is negative, a test forinhibitors needs to be performed. If inhibiting substances are not detected on at least two consecutivesmear-positive CADT negative samples, the patient is presumed to have NTM. If a specimen is smearnegative and CDAT positive, and the same result is obtained from an additional specimen, the patient can bepresumed to have TB. Lastly, if both the smear and CDAT are negative in two consecutive specimens, thepatient can be presumed to be free from active TB. It also recommends that the decision regarding therapydepends upon clinical status of the patients2,3.        In diagnostic Mycobacteriology, the introduction of PCR or CDAT was considered as an excitingnew milestone. The probabilities of confirming a diagnosis of tuberculosis in paucibacillary situations arehigher with PCR than with the routine diagnostic procedures such as sputum smear microscopy followedby Chest X-rays 4. The advantage of the test is that it can be performed from a single specimen and yieldsresults within a day. This makes the diagnostic process shorter and more patient-friendly, and in turn it mayreduce transmission of infection. However, the disadvantage of PCR is that it may also detect nonviabletubercle bacilli and PCR negative data must be considered more carefully because of false negatives due tothe presence of inhibitors of PCR in some samples. These techniques, however, should be performedalways in conjunction with microscopy and culture, and the results should be always interpreted with thepatients’ clinical data.EDITORIAL62Indian Journal of TuberculosisAlthough molecular testing methods have enhanced diagnostic potential in detecting M.tuberculosisfrom clinical specimens, to differentiate between mycobacterial species and also detect drug resistance,the application and cost effectiveness of such technically demanding testing methods in large DEDC suchas India with limited resources both in terms of man power with adequate technical expertise and laboratorieswith technical infrastructure, requires to be studied extremely carefully. However, in the future after theestablishment of quality ensured culture laboratories at the State level, the National Reference Laboratoriesin India, along with laboratories in the major referral teaching hospitals and laboratories in the corporatesector, may be able to take up molecular based methods in special situations as described earlier. Adequatequality assurance of each of these molecular tests would have to be ensured. Until then, molecular baseddiagnostic methods wherever it is practised at present in India should be used  only as an added adjunct inthe diagnosis of tuberculosis under special circumstances and always in conjuction with the existingestablished diagnostic techniques i.e. sputum microscopy and culture, and X-ray.C.N. ParamasivanREFERENCES1. Molecular diagnosis of Mycobacterial infections:  A.Forbes, Gaby Pfyffer and Kathleen D Eisenach, 2005;Chapter.6, p 95-98in the book “Tuberculosis and the Tubercle Bacillus” Edited by Stewart Cole et al, ASM Press, Washington DC.2. Claudio Piersimoni and Claudio Scarparo. Relevance of Commercial Amplification methods for Direct Detection of Mycobacteriumtuberculosis complex in clinical samples.  J  Clin Microbiol  2003; 41: 5355-53653. Centers for Disease Control and prevention.. Update: nucleic acid amplification tests for tuberculosis. Mor. Morta .Wkl Rep2000; 49: 950–952.4. Lydia Kivihya - Ndugga, Maarten van Cleeff, Ernest Juma, Joseph Kimwomi, Willie Githui, Linda Oskam, Anja Schuitema,Dick van Scoolingen, Lucy Nganga, Daniel Kibuga, Joseph Odhiambo, and Paul Klatser.  Comparison of PCR with the routineprocedure for diagnosis of tuberculosis in a population with high prevalence of tuberculosis and Human ImmunodeficiencyVirus. 2004. 42: 1012-1015EDITORIAL 63

Molecular methods in the diagnosis of tuberculosis

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Molecular methods in the diagnosis of tuberculosis

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