New Modalities in TB Diagnosis

New Modalities in TB Diagnosis

The diagnosis in endemic countries depends more on the use of labour intensive, easy to use methodology with minimum infrastructure or equipment. The need is to find a viable alternative for smear microscopy. This method has to have these desirable features : Results within 2 hours. Simple training. Easy interpretation. Should function well in HIV +ve patients. Should allow start of treatment as early as possible.

Methods

Direct Methods Direct Microscopy (ZN, Kinyoun, Flurochrome). Culture (Traditional, Rapid methods). Detection of DNA or RNA of mycobacterial origin( PCR, LAMP, TAA / NAA, LCR, Fast Plaque).

Direct Microscopic Examination Hallmark of staining is Ziehl-Neelsen stained slides. Easiest & quickest diagnostic test. Limited sensitivity (46-78%) but specificity is virtually 100%. it lacks sensitivity, requiring 10,000 bacilli/ml of sputum to become positive.

Ziehl-Neelsen staining TB bacilli appear as straight/curved rods (1-4μ x 0.2-0.8μ) singly, in pairs or in clumps. The yield of microscopic examination correlates well with the extent of disease, the presence of cavitation, and the quality of specimen. It is a good marker for infectiousness & the response to the treatment.

flurochrome staining Centrifugation & flurochrome staining (auramine O) with UV microscopy markedly increase the sensitivity & a large number can be examined in a much shorter time. In general, the fluorescent method should be used by laboratories with large specimen numbers. It is more expensive than the conventional ZN staining requiring a fluorescent microscope

Current Guidelines: WHO + International Union Against Tuberculosis and Lung Disease Essential step investigation pts suspected pulmonary TB: microscopic exam at least 3 serial specimens for acid fast bacilli 1st + 3rd sputum specimens collected in the health facility (spot specimens) and 2 nd usually collected at home (early morning)

Traditional Culture: More sensitive & can be positive even when bacterial load is low (10-100 bacilli/ml). Sensitivity 80-85%; Specificity 98%.. 3 Types of media are used: Egg based: LJ, Petragnani and ATS. Agar based: Middlebrook 7H10 or 7H11. Liquid based: Kirschner s, Middlebrook 7H9. Growth is slow and takes 6-8 weeks. There after the same length of time is required for complete identification & sensitivity testing.

Liquid media compared to solid media Advantages compared to solid media: more rapid high quality of media fully automated system testing of 1 st, 2 nd, and new drugs safety: plastic tubes Disadvantages: expensive higher contamination rate dependency on a company no DST for Cycloserine

Broth Based Rapid Culture Methods: Radiometric (BACTEC). Septicheck AFB. Mycobacterial growth indicator tubes (MGIT). Substantial improvement in time to detection & total number of positive cultures can be realized from using broth based systems.

BACTEC Growth is ascertained by liberation of 14CO2 as metabolized by mycobacteria & detected by BACTEC 460 instrument & reported in terms of growth index (GI) value.

BACTEC Average time to recovery of M.tb from smear positive specimens is 8 days. When smear negative, culture positive samples are examined, mean time for detection is 14 days. More sensitive than traditional method. Can also be used for drug susceptibility testing.

Mycobacterial Growth Indicator Tube (MGIT) Rapid Method. Consists of round bottom tubes containing 4 ml of modified Middlebrook 7H9 broth which has an oxygen sensitive fluroscent sensor at the bottom.* When mycobacteria grow, they deplete the dissolved oxygen in the broth & allow the indicator to fluoresce.

Mycobacterial Growth Indicator Tube (MGIT) Positive signals are obtained in 10-12 days. MGIT can also be used as a rapid method for the detection of drug resistant strains of Mtb directly from acid-fast smear positive samples as well as from indirect drug susceptibility studies. Advantages over BACTEC Cheaper. No problem of radioactive waste disposal.

Rapid culture systems: TK Medium Colorimetric system Metabolic activity of growing mycobacteria changes the color, before bacterial colonies appear(red to yellow). The test can distinguish between mycobacteria and contamination (red to green).

Rapid culture systems: TK Medium TK Medium also enables susceptibility testing for drug resistance, and differentiation between M. tuberculosis and NTM TK Medium to be a practical, low-cost, simple test but published evidence on this test is limited. The test is not currently FDA approved. TK Medium s sensitivity was comparable to that (LJ) A multicenter study in Turkey: Time to detection with TK Medium was 2 weeks, as compared with 4 weeks LJ medium

Detection and identification of mycobacteria directly from clinical samples

Detection and identification of mycobacteria directly from clinical samples Genotypic Methods : PCR LAMP TMA / NAA Ligase chain reaction Phenotypic Methods : FAST Plaque TB

PCR IS6110 : It is a transposon which are self replicating stretches of DNA. Function not known. This sequence has been found in the M.tb complex organisms ( M.tb, M.africanum, M.microti, M.bovis). IS6110 sequence generally occurs only once in M.bovis but is found as often as 20 times in certain strains of M.tb, thus offering multiple targets for amplification.

PCR These tests can be used directly on clinical specimens (e.g., sputum) Detects nearly all smear + ve and culture +ve cases. PCR have high specificity for both pulmonary and extrapulmonary TB (but data on extrapulmonary TB are relatively limited) A positive PCR in a patient with a high pretest probability is fairly confirmatory of TB, particularly in those with positive smears. In contrast to specificity, sensitivity of PCR is lower and highly variable across studies.

PCR sensitivity estimates have been lower in paucibacillary forms of TB (extrapulmonary TB and smear-negative pulmonary disease) A negative PCR in a patient with a high index of clinical suspicion should prompt continued workup. Useful technology for rapid diagnosis of smear ve cases of active TB. Able to identify 50-60% of smear -ve cases; this would reduce the need for more invasive approaches to smear - ve cases.

PCR Commercial kits include: M. tuberculosis Direct Test (MTD; Gen-Probe Inc., CA, USA), the Amplicor M.tuberculosis (MTB) tests (Roche Molecular Diagnostics, CA, USA), and the BD ProbeTec ET assay (BD Biosciences, MD, USA The Amplicor and MTD tests are currently US FDA approved In general, the accuracy of in-house PCR tests has been more heterogeneous than commercial kits

PCR In summary, available NATs have not realized their early promise. Therefore, efforts are underway to simplify testing protocols and increase their accuracy.

Loop-mediated isothermal amplification It is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. LAMP is used for detection of M.tb complex, M.avium, and M.intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT) or on a solid medium. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA.

Loop-mediated isothermal amplification The amplification efficiency is high, and DNA can be amplified 10 9 10 10 times in 15 60 min Currently, there is limited evidence on the accuracy of LAMP for the detection of TB

INNO-LiPA Rif TB Strip assay (line probe assay PCR-based Detect MtbC Detect RIF R (rpob gene) Not FDAapproved for US

Principle of the Line Probe Assays Chromogen (MBT/BCIP) Alkaline Phosphatase Colour reaction Streptavidin Biotin DNA-probe Biotin-labelled single stranded amplified target Nitrocellulose strip

GenoType MTBDR Hain Lifescience, Germany Strip techology (line probe assay) PCR based Detect MtbC Detect RIF R (rpob gene) Detect INH R (katg gene) Not FDA-approved for US

Commercial line probe assays for detection of M. tuberculosis complex & mutations associated with drug resistance Genotype MTBDR from Hain Lifescience Detects presence of TB complex and mutations associated with rifampin & INH resistance Application for U.S. FDA clearance in process INNO-LiPA Rif TB from Innogenetics Detects presence of TB complex & mutations associated with rifampin resistance (91% of rif-resistant isolates are also INH-resistant)

Review Article re Inno-LiPA Rif TB Morgan, Kanantri, Flores, and Pai. 2005.BMC Infectious Diseases 5:62-70 Reviewed 4 studies in which LiPA was applied directly to clinical specimens Sensitivities ranged from 80 to 100% 3/4 showed sensitivity >90% for Rif All 4 studies had specificities of 100%

Drug Susceptibility Testing Solid Media Löwenstein-Jensen (Middlebrook) 3-4 weeks Liquid Media BACTEC 460 TB MGIT 7-10 days Molecular based Methods InnoLipa GenoTypeMTBDR Xpert MTB home made - methods Hours 1day

MDR-TB diagnosis using conventional solid culture and DST Microscopy Solid culture 1st line DST 2nd line DST 24h 6-8w 3-4w 3-4w MDR-TB diagnosis after 9 to 12 weeks MDR-TB diagnosis using liquid culture and DST Microscopy Liquid culture 1st line DST 2nd line DST 24h 2-3w 1-3w 1-2w MDR-TB diagnosis after 3 to 5 weeks MDR-TB diagnosis using line probe assay, liquid culture and DST Microscopy 24h Line probe assay 24h Line probe assay 24h + MDR-TB 2nd line diagnosis DST after 1 to 2 days 1-2w 1st Liquid line culture DST 2nd 1st line DST - 1-2w 2-3w 1-2w 1-3w MDR-TB diagnosis after 3 to 5 weeks

Cepheid Xpert MTB/RIF Assay

Assay Procedure for the MTB/RIF Test

GeneXpert System The system requires the use of a single use disposable GeneXpert cartridge that hold the PCR reagents and host the PCR process. Because the cartridges are selfcontained, cross contamination between the samples in the analyzer are eliminated. The Xpert reagents include sample processing controls and probe check controls. There are other internal checks the system must pass for proper analysis. The final detection and interpretation of the results are measured by the fluorescent signals that are interpolated in the specific targeted nucleic acid sequence.

5 20 80 500-1000 Samples per shift

Principle of Molecular Beacons Probe target is amplified in a real time PCR Molecular Beacon Probe forms a hairpin No fluorescence because of close proximity of reporter dye to quencher dye Hybridization to target separates reporter dye from quencher resulting in fluorescence Xpert MTB/ RIF utilizes 5 probes for wildtype RRDR in rpob and 1 probe for amplification control B. globigii Detection of WT sequence, not a specific mutation

Xpert MTB/RIF Assay At least 2 of 5 rpob probes positive within 2 cycles and amplification control positive= M. tuberculosis complex Failure of proper hybridization of one or more rpob probes= rifampin resistance

MTB / Rif-resistance test Workflow sputum simple 1-step external sample prep. procedure time-to-result < 2 h throughput: >16 tests / day / module no need for biosafety cabinet integrated controls

Rifampin Susceptible Sample

Rifampin Resistant Sample

Bacteriophage-based assays

Bacteriophage-based assays Direct phage assay demonstrate promise, but require further validation and field testing in high-burden settings

Sensitivity and Specificity of the MTB/RIF Test for the Detection of Rifampin and Multidrug Resistance, as Compared with Phenotypic Drug-Susceptibility Testing Alone and in Combination with Sequencing of Discrepant Cases, According to Site