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Molecular Diagnostics Why ?

MOLECULAR DIAGNOSIS OF TB AND IGRA PRESENTED BY : Dr. Kiran N. PG Student Chest & Tuberculosis Govt. Medical College, Patiala. Molecular Diagnostics Why ?. Detection and Diagnosis uncultivable or difficult to culture need for rapid diagnosis

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Molecular Diagnostics Why ?

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  1. MOLECULAR DIAGNOSIS OF TB AND IGRAPRESENTED BY :Dr. Kiran N. PG StudentChest & TuberculosisGovt. Medical College, Patiala

  2. Molecular Diagnostics Why? • Detection and Diagnosis • uncultivable or difficult to culture • need for rapid diagnosis • inadequacy of phenotypic methods (biochemical) • Prognosis and management • need for quantitative information (bacterial load) • susceptibility testing (drug resistance) without culture - Molecular resistance testing

  3. METHODS OF DIAGNOSIS OF PULMONARY TUBERCULOSIS 1)DIRECT METHODS: Detects mycobacteria and its products 2)INDIRECT METHODS : Antigen and Antibody Detection 3)RADIO-DIAGNOSIS :CXR,CT AND MRI DIRECT METHODS 1)Direct Microscopy -ZN stain, Kinyoun, Flurochrome. 2)Culture -Traditional, Rapid methods. 3) Detection of DNA or RNA of mycobacterial origin i,e (molecular methods) includes - PCR, LAMP, TAA / NAA, LCR, Fast Plaque.

  4. INDIRECT METHODS • 1)Antibody detection : • TB STAT-PAK • ELISA • Insta test TB • 2) Antigen detection : • TB MPB 64 patch test. • Quantiferon-GOLD test. • 3) Biochemical Assays : • ADA • Bromide Partition • Gas Chromatography

  5. MOLECULAR DIAGNOSIS OF TUBERCULOSIS • Rapid and sensitive tools for the diagnosis of tuberculosis are needed, due to the increased incidence of tuberculosis epidemics and the length of time required by classical diagnostic tests, especially among human immunodeficiency virus (HIV)-infected patients. In this context, the recent advances in cloning and characterization of M. tuberculosis genes has allowed the application of basic molecular biology techniques to the examination of clinical samples, such as sputum and bronchoalveolar lavage (BAL), for the molecular diagnosis of tuberculous infection. By using the polymerase chain reaction (PCR) for the amplification of mycobacterial nucleic acids and nonradiometric revelation techniques, the time required for the identification of mycobacteria has been considerably shortened (24-48 h), in comparison to the time required by microbiological tests

  6. Dr.T.V.Rao MD

  7. MOLECULAR TESTS FOR DETECTION OF NUCLEIC ACIDS • The majority of molecular tests have been focused on detection of nucleic acids, both DNA and RNA, that are specific to Mycobacterium tuberculosis, by amplification techniques such as polymerase chain reaction (PCR); and detection of mutations in the genes that are associated with resistance to anti tuberculosis drugs by sequencing or nucleic acid hybridization. Recent developments in direct and rapid detection of mycobacteria, with emphasis on M. tuberculosis species identification by 16S rRNA gene sequence analysis or oligohybridization and strain typing, as well as detection of drug susceptibility patterns, all contribute to these advance

  8. Polymerase Chain Reaction PCR was invented by Kary Mullis in 1983 (Nobel Prize for Chemistry in 1993) • PCR is a technique that takes a small amount of a specific DNA sequence and amplifies for further testing. • It`s like a “molecular photocopier”.

  9. Polymerase Chain Reaction (PCR) • Essentially PCR is a way to make millions of identical copies of a specific DNA sequence , which may be a gene, or a part of a gene, or simply a stretch of nucleotides with a known DNA sequence, the function of which may be unknown. • A specimen that may contain the DNA sequence of interest is heated to denature double stranded DNA. • Specific synthetic oligonucleotide primers bind to the unique DNA sequences of interest and a heat stable DNA polymerase (Thermusaquaticus) extends the primer to create a complete & complimentary strand of DNA.

  10. This process is repeated sequentially 25-40 times, thereby creating millions of copies of target sequence. The amplified sequence can then be detected by agarose gel electrophoresis.DNA sequence used include: 1) 65 Kd antigen (HSPs): • Used earlier • Heat shock protein believed to be distinct from other bacterial HSPs. • This gene is identical in all species of mycobacteria. • Therefore unsuitable for detecting M.tb, particularly in areas where species like M.avium or M.kansasii are prevalent.

  11. 2) 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 can detect even a fraction of a bacilli.

  12. Mycobacterium tuberculosisgenome Dr.T.V.Rao MD

  13. Ingredients of Polymerase Chain Reaction • Primers: µM 0.1-0.5 • Deoxy-nucleotides triphosphate (dNTPs): µM 200-250nucleotides • Co-factors: • Cations: MgCl2 mM 1.5-6 • Buffer pH 8.3-8.8 • DNA polymerase: 0.5-2.5 U • Target DNA:  1 µg

  14. Chromosome and Desoxyribo-Nucleic-Acid Chromosom chromosome Zellkern nucleus Zelle cell DNA DNA - double helix Doppelhelix Basenpaare base pairs

  15. Primers • Primers are target sequence specific oligonucleotides that serve as template for the DNA polymerase • Forward and reverse primers flanking the target sequence allow both DNA strands to be copied simultaneously in both directions.

  16. Three steps of PCR: Denaturation, annealing and extension

  17. Role of PCR in pulmonary TB : • Detects nearly all smear +ve and culture +ve cases. • 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 • Distinguish M.tb from NTM in smear +ve cases as IS6110 sequence is not found in NTM. • Should not be used to replace sputum microscopy. • Sensitivity, specificity, & PPV for PCR is 83.5%, 99% & 94.2% respectively

  18. Role in Extrapulmonary TB • Limited Role • No comprehensive large series comparing the yield of PCR with other available approaches has been published. But at present, it is valuable adjunct in the diagnosis of TBM, pleurisy, pericardial TB & other • condition in which yield of other tests are low

  19. Disadvantages • Very high degree of quality control required. • Variation from lab to lab remain significant. • In pts. on ATT, PCR should not be used as an indicator of infectivity as this assay remains +ve for a greater time than do cultures • High false +ve results in patients previously treated with ATT in contacts of sputum +ve active cases. • High Cost

  20. Real Time PCR replacing older Methods

  21. LAMP* • 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.tbcomplex, M.avium, and M.intracellularedirectly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT) or on a solid medium (Ogawa’s 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. • Species-specific primers were designed by targeting the gyrBgene. • Simple procedure, starting with the mixing of all reagents in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63°C. • 60-min incubation time.

  22. ADVANTAGES: Due to its easy operation without sophisticated equipment, it will be simple enough to use in: • Small-scale hospitals, • Primary care facilities • Clinical laboratorie in developing countries. Difficulties : • Sample preparation • Nucleic acid extraction • Cross-contamination

  23. TMA / NAA • Transcription Mediated Amplification (TMA) / Nucleic Acid Amplification (NAA). • These techniques use chemical rather than biological amplification to produce nucleic acid. • Test results within few hours. • Currently used only for respiratory specimens.

  24. Nucleic acid amplification assays • NAA assays amplify M. tuberculosis-specific nucleic acid sequences using a nucleic acid probe. • The sensitivity of the NAA assays currently in commercial use is at least 80% in most studies • Require as few as bacilli from a given sample • NAA assays are also quite specific for M. tuberculosis, with specificity in the range of 98% to 99%.

  25. NAAs- various types • AMPLICOR M. TUBERCULOSIS assay • Amplified M.tuberculosis Direct (AMTD2) assay • LCx MTB assay, ABBOTT LCx probe system • BD ProbeTec energy transfer (ET) system (DTB) • INNO-LiPA RIF.TB assay

  26. NAAs- various types Dr.T.V.Rao MD

  27. NAA- Limitations • They are able to detect nucleic acids from both living and dead organisms so in pts on ATT, PCR should not be used as an indicator of infectivity as this assay remains positive for a greater time, than do cultures • A major limitation of NAA tests is that they give no drug-susceptibility information. • NAA should always be performed in conjunction with microscopy and culture

  28. DISADVANTAGES : 1) Poor sensitivity in smear –ve samples 2) Labour intensive 3) Highly trained personnel required & dedicated labour space because it requires atleast 3 separate rooms to avoid cross contamination. Hence suitable only for IRL.

  29. Ligase Chain Reaction • It is a variant of PCR, in which a pair of oligonucleotidesare made to bind to one of the DNA target strands, so that they are adjacent to each other. • A second pair of oligonucleotides is designed to hybridize to the same regions on the complementary DNA. • The action of DNA polymerase and ligase in the presence of nucleotides results in the gap between adjacent primers being filled with appropriate nucleotides and ligation of primers. • It is mainly being used for respiratory samples, and has a high overall specificity and sensitivity for smear +veand –vespecimens.

  30. MDR TB • MDR-TB is defined as resistance to isoniazid and rifampicin, with or without resistance to other first line drugs (FLD). • In the Global TB Report 2011, WHO estimated that among the 1.5 million RNTCP-notified cases of pulmonary TB in India in 2010, approximately 64,000 cases of MDR TB could be diagnosed. • MDR TB is important because patients with this type of drug resistance respond extremely poorly to standard anti-TB treatment with first-line drugs. MDR TB requires relatively costly laboratory diagnosis and treatment for at least two-years with drugs that are expensive, toxic, and not particularly potent. • A case of MDR TB is about 20-40 times more expensive to manage than a case of drug-sensitive TB, and patient suffering is magnified.

  31. MDR Suspect Criteria Criteria A- • All features of new TB cases. • Smear +ve previously treated cases who remain smear +ve at 4th month onwards. • All pulmonary TB cases who are contacts of known MDR TB case. • Criteria B – in addition to Criteria A : • All smear +ve previously treated pulmonary TB cases at diagnosis. • Any smear +ve follow up result in new or previously treated cases. • Criteria C – in addition to Criteria B : • All smear –ve previously treated pulmonary TB cases at diagnosis. • HIV TB co-infected cases at diagnosis.

  32. Molecular methods for drug resistance • Rifampin (RIF) • – Binds to β subunit of RNA polymerase (rpoB) • – 96% of resistant Mtb isolates have mutations in 81-bp region. • – Four (4) mutations . 75% of resistant clinical isolates • • Isoniazid (INH) . two genes – katG and inhA . 75-85% • • Pyrazinamide . pncA . 70% • • Streptomycin . rpsL . 65-75% • • Ethambutol .embB . 70%

  33. Causes of Drug Resistance in TB • Lack of understanding of why long term therapy with multiple drugs is necessary • Non-adherence to therapy by patients • Incorrect drug prescribing by providers • Poor quality drugs • Erratic supply of drugs • Malabsorption of drugs primarily due to symptoms of HIV/AIDS

  34. Development of drug resistance in M. tuberculosis • The mycobacterial cell is surrounded by a specialized, highly hydrophobic cell wall that results in decreased permeability to many antimicrobial agents. • Resistance of M tuberculosis to antimycobacterial drugs is the consequence of naturally occuring, spontaneous mutations in genes that encode either the target of the drug, or enzymes that are involved in drug activation. • Resistance-associated mutations have been described for all first-line drugs (isoniazid, rifampin, pyrazinamide, ethambutol, and streptomycin).

  35. Development of multi-drug resistant tuberculosis • No single genetic alteration has yet been found that results in the MDR phenotype (defined as resistance at least to INH and RMP). • MDR develops by sequential acquisition and selection of mutations at different loci, usually because of inappropriate patient treatment. • Inappropriate treatment may lead to disease progression. • Disease progression will increase the bacterial load and the risk of naturally occurring mutations. • Because MDR strains are the result of cumulative mutations, growth of M tuberculosis can successfully be controlled in the host by concomitant treatment with more than one drug. • Thus, treatment regimens that consist of three to four drugs are used routinely to treat patients with tuberculosis.

  36. Isoniazid resistance and katG • INH is a pro-drug that requires activation in INH-susceptible mycobacterial species. • the activation of INH results in a number of highly reactive compound that are capable of damaging the mycobacterial cell wall. • INH-resistant clinical isolates frequently loose their catalase-peroxidase activity (Middlebrook et al., 1954) • Association of this enzyme with INH activation was proven when the mycobacterialcatalase-peroxidase gene (katG) was cloned and sequenced. (Zhang et al., 1992) • Mutations in this gene were found in 70-80% of high INH-resistant clinical isolates. • The most common mutation that was found was the Ser315Thr mutation.

  37. Isoniazid resistance and virulence • The Ser315Thr mutation results in an enzyme without the ability to activate INH, but retains approximately 50% of its catalase-peroxidase activity. • This altered catalase-peroxidase provides high-level resistance to INH, while retaining a level of oxidative protection against host antibacterial radicals. • Isolates that carry other, mutations in katG are exhibiting varying levels of INH-resistance and catalase-peroxidase activity. Lysosomes with free oxigene radicals Phago-lysosomes with inactivated MTB MTB Phagosome with viable MTB

  38. Isoniazid resistance and inhA • INH blocks the synthesis of cell-wall mycolic acids, the major components of the envelope of M tuberculosis. • One intracellular target of the drug is fatty-acid enoyl-acyl carrier protein reductase (InhA). (Basso et al., 1998) • This enzyme is involved in synthesis of mycolic acids. • Mutations in the promoter region of the gene (inhA) encoding this enzyme result in over-expression of the protein. • The over-expressed enzyme may counter-balance the effect of INH and will result in a low-level resistance to the drug.

  39. Rifampin resistance • One of the main reasons for treatment failure and fatal clinical outcome in tuberculosis patients is resistance to RMP. • RMP exhibits a significant early bactericidal effect on metabolically active M tuberculosis, and excellent late sterilizing action on semidormant organisms undergoing short bursts of metabolic activity. • While monoresistance to INH is common, monoresistance to RMP is rare. • RMP resistance occurs most often in strains that are also resistant to INH, thus, RMP resistance can be used as a surrogate marker for MDR. • RMP inhibits mycobacterial transcription by targeting DNA-dependent RNA polymerase.

  40. Rifampin resistance and rpoB • Resistance to RMP is due to mutations in a well-defined, 81 base pair (27 codons) central region of the gene that encodes the β-subunit of RNA polymerase (rpoB). • More than 96% of the rifampin-resistant strains contain a mutation in this 81 bp region of rpoB. • The most common mutations (65–86%) alter either codon 526 or codon 531, and result in high-level resistance to RMP. • Alterations in other codons result in low-level resistance. • Rare mutations associated with rifampin resistance have also been found in the amino-terminal region of rpoB.

  41. MOLECULAR METHODS OF DIAGNOSING DRUG RESISTANT TB • Phenotypic methods: in liquid/solid media • Genotypic methods: Gene xpert/LPA • PHENOTYPIC METHODS 1)Commercial methods • Done on liquid or solid media • Can be direct/indirect type. • Indirect type has 3 variants: absolute concentration/resistant ratio/proportion method • Can be used for 1st line/2nd line DST

  42. 2)Non commercial methods • Less expensive than commercial systems but are prone to errors due to lack of standardization and local variations in methodology. These include a)MODS (microscopicobservation drug susceptibility)a microcolony method in liquid culture(middlebrook 7H9 broth) based on inoculation of specimens into drug-free and drug-containing media, followed by microscopic examination of early growth Recommended as direct or indirect tests for rapid screening of patients suspected of having MDR-TB.

  43. b)COLORIMETRIC METHODS Indirect testing methods based on the reduction of a coloured indicator added to liquid culture medium on a microtitre plate after exposure of M. Tuberculosis strains to anti-TB drugs in vitro. Recommended as indirect tests on M. tuberculosisisolates from patients suspected of having MDR-TB, although the time to detection of MDR is not faster (but less expensive) than conventional DST methods with commercial liquid culture or molecular LPA. The indicators that have been used to date include tetrazolium salts (XTT and MTT), Alamar blue and resazurin.

  44. A direct or indirect method on solid culture based on the ability of M.tuberculosisto reduce nitrate, which is detected by a colour reaction Recommended as direct or indirect tests for screening patients suspected of having MDRTB, although the time to detection of MDR in indirect application is not faster than conventional DST methods with liquid culture. c)NITRATE REDUCTION ASSAY (NRA)

  45. GENOTYPIC METHODS LPA Gene Xpert/RIF

  46. LPA(Line Probe Assay) It is a genotypic method use PCR and reverse hybridization with specific oligonucleotide probes fixed to nitrocellulose strips in parallel lines therefore often reffered to as “strip tests”. which detects resistance to Both H&R • Sensitivity for R is 97%, H is 90% • Specificity for R& H is 99% • Results within 48 hours

  47. Differences • HainGenotTypeMTBDRplus • It targets 23s rRNA gene space region. • It detects both R and H resistance. InnoLiPA Rif • It targets 16s - 23s rRNA gene space region. • It detects only R resistance.

  48. TB Molecular Identification AVAILABLE SYSTEMS (Commercial): • HainGenotTypeMTBDRplus • InnoLiPARif ADVANTAGES of molecular testing: • Rapid results • Specific information • Can be done in presence of contaminants • Less biohazard risk involved DISADVANTAGES of molecular testing: • Expensive • Dedicated equipment • Technical expertise required

  49. Rapid molecular detection of INH and RMP resistance: Line – Probe Assay Methodology • DNA isolated from processed original specimen or from cultured bacterial cells • Multiplexed amplification (PCR) of mycobacterial DNA using biotin labeled primers • Denaturation of the amplified DNA products into single-strands • Hybridization of the denatured DNA to probes on a membrane strip • Enzyme-mediated detection of bands where DNA products have bound to strip

  50. Polymerase Chain Reaction

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