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Molecular methods in diagnosis of Tuberculosis tuberculosis

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Diagnosis of Tuberculosis Molecular Based Methods

Diagnosis of Tuberculosis Molecular Based Methods

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  • 1. Dr.T.V.Rao MD
    Molecular methods in Diagnosis of Tuberculosisemerging trends
    Dr.T.V.Rao MD
    1
  • 2. Historical Background
    Neolithic Time
    2400 BC - Egyptian mummies spinal columns
    460 BC
    Hippocrates, Greece
    First clinical description: Phthisis / Consumption
    (I am wasting away)
    500-1500 AD
    Roman occupation of Europe it spread to Britain
    1650-1900 AD
    White plague of Europe, causing one in five deaths
  • 3. Historical Background
    1800-1900
    Industrial revolution (Europe)
    50 mil. Infected & 7 mil. Dying annually
    1844
    Half of England’s population infected with TB
    1900’s
    Approximately all of Europe’s adult population infected with TB
    1850-1952
    Sanatorium Movement ( Brehmer and Trudeau)
    Emphasis on rest, good nutrition, and fresh mountainous air
    Isolation led to decrease in transmission
  • 4. 24th March 1882 (Robert Koch)TB Day
    Discovery of staining technique that identified Tuberculosis bacillus
    Definite diagnosis made possible and thus treatment could begin
    1890 (Robert Koch)
    Tuberculin discovered
    Diagnostic use when injected into skin
    1895 (Roentgen)
    Discovery of X-rays
    Early diagnosis of pulmonary disease
    Diagnostic discoveries
  • 5. Nine million people suffer from tuberculosis
    Two million people die each year.
    Tuberculosis accounts for one-third of Aids deaths world wide every year.
    Globally, there have been just 347 identified cases of XDR-TB, mainly in the former USSR and in Asia
    Global Status
  • 6. Re-emerging problem in industrialized countries
    • Infections in immuno-compromised patients Multi-drug resistant strains (MDR-TB)
    Challenges with tuberculosis Infection
    Dr.T.V.Rao MD
    6
  • 7. Mycobacterium tuberculosis-Characteristics
    Gram positive
    Obligate aerobe
    Non-spore-forming
    Non-motile rod
    Mesophile
    0.2 to 0.6 x 2-4um1
    Slow generation time: 15-20 hours
    • May contribute to virulence1
    Lipid rich cell wall contains mycolic acid—50% of cell wall dry weight1
    • Responsible for many of this bacterium’s characteristic properties
    • 8. Acid fast—retains acidic stains
    • 9. Confers resistance to detergents, antibacterial
    Dr.T.V.Rao MD
    7
  • 10. Diagnostics of Mycobacterium
    Initial screening:
    • TB skin test (Purified Protein Derivative).
    Drawbacks: BCG injected subjects are positive, 3 days delay for result
    • QFT-G test (measures INF- response to TB specific antigen)
    TB tests Active, depending on the suspected location of bacterium:
    • 3-5 samples of sputum
    • 11. multiple gastric aspirate
    • 12. urine (UTI)
    • 13. CSF (meningeal)2
    Cultures
    Samples are processed for fast acid stain (FAS smear positive indicates Mycobacterium) and cultured after alkali decontamination (30s in 1-2% NaOH)
    Molecular methods use species-specific genes, including light and heave ribosomal RNA3
    Clinical specimen/
    decontamination
    culture
    Direct detection:
    Species identification:
    • 16S rRNA hybridization (MTB and MAC)
    • 16. 16S rRNA gene PCR sequencing (NTM)
    • 17. restriction fragment length polymorphism
    Susceptibility testing
    Rifampin resistance
    (PCR oligohybridization sequencing)
    Dr.T.V.Rao MD
    8
  • 18. Symptoms
    What are the symptoms of TB?
    Symptoms of pulmonary TB include:
    • Coughing
    • 23. Pleurisy (pain when taking deep breaths)
    • 24. Coughing up blood4.
    Dr.T.V.Rao MD
    9
  • 25. Effective laboratory Diagnosis
    Sputum smear examinations – rapid classification of species (atypical mycobacteria common in AIDS)
    Culture examinations
    Rapid drug sensitivity testing
    Emerging Molecular Methods are trend setters in rapid Diagnosis of TB
  • 26. Tuberculosis continues to be, as it has been for centuries, one of the most prevalent infectious diseases of humans and is the leading cause of mortality from a single infectious disease worldwide . Laboratory methods play a crucial role in establishing the diagnosis, monitoring therapy, and preventing transmission of tuberculosis.
    Tuberculosis continues to be global health problem
    Dr.T.V.Rao MD
    11
  • 27. In addition, the importance of the mycobacteriologist has grown, in view of a changing epidemiology (e.g. social factors, the acquired immune deficiency syndrome (AIDS pandemic) and an increasing resistance of M. tuberculosis to drugs
    Tuberculosis A disease of growing Importance
    Dr.T.V.Rao MD
    12
  • 28. Molecular Methods in Diagnosis of Tuberculosis
    Several methods are available, mainly used as
    Research tools
    Dr.T.V.Rao MD
    13
  • 29. 14
    Differential diagnosis
    Risk assessment - prevention
    Medical Progress: Evolution or Revolution?
    Historic Drivers of Medical Progress
    Clinical expertise
    …Genetics
    Classical epidemiology
    More differentiated, molecular understanding of pathology and drug action
    Molecular Disease Definition
    Molecular Diagnosis
    Clinical Disease Definition
    Clinical Diagnosis
    in-vitro Diagnostics
  • 30. Detection and Diagnosis
    – uncultivable or difficult to culture
    – need for rapid answer
    – inadequacy of phenotypic methods (biochemical)
    • Prognosis and management
    – need for quantitative information (viral load)
    – susceptibility testing (drug resistance) without culture
    • Molecular resistance testing
    Molecular DiagnosticsWhy?
    Dr.T.V.Rao MD
    15
  • 31. 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 ant 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
    Dr.T.V.Rao MD
    16
  • 32. Gene amplification can achieve the goal of reducing the generation time of microorganisms to minutes, and of replacing biological growth on artificial media by enzymatic reproduction of nucleic acids in vitro . The importance of nucleic acid amplification methods lies in their wide applicability in the life sciences, and their potential to revolutionize the practice of medicine. Examples are nucleic acid sequence analysis and genetic fingerprinting
    Beginning of Gene amplification methods
    Dr.T.V.Rao MD
    17
  • 33. Dr.T.V.Rao MD
    18
  • 34. 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
    Dr.T.V.Rao MD
    19
  • 35. The rapid development and availability of a variety of new molecular genetic technologies present the clinician with an array of options for the accurate diagnosis of infectious diseases. This is particularly the case for tuberculosis, since molecular methods have been rapidly introduced into all working areas of the mycobacteriology laboratory.
    Breakthrough with molecular technologies
    Dr.T.V.Rao MD
    20
  • 36. Mycobacterium tuberculosisgenome
    Dr.T.V.Rao MD
    21
  • 37. 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 IO bacilli from a given sample
    NAA assays are also quite specific for M. tuberculosis, with specificity in the range of 98% to 99%.
    Official statement of ATS and CDC, July 1999
    Dr.T.V.Rao MD
    22
  • 38. 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
    Dr.T.V.Rao MD
    23
  • 39. NAAs- various types
    Dr.T.V.Rao MD
    24
  • 40. AMPLICOR M. TUBERCULOSIS assay
    Cohen, R. A., 1998. Am. J. Respir. Crit. Care Med. 156:156–161.
    Bonington, A., 1998. J. Clin. Microbiol. 36:1251–1254.
    Al Zahrani, 2000. Am. J. Respir. Crit. Care Med. 162:1323–1329.
    Dr.T.V.Rao MD
    25
  • 41. Amplification of specific DNA sequences
    (eg. Polymerase Chain Reaction – PCR)
    • Provide rapid diagnosis
    • High sensitivity & specificity
    • Possible to use crude DNA e.g. boiling
    DNA Amplification Assay
    Dr.T.V.Rao MD
    26
  • 42. Direct Detection of M. tuberculosis in sputumby DNA amplification
    Automatic system
    • Roche (Cobas Amplicor) : PCR for 16S rRNA gene
    • Abbott (LCx) : PCR/LCR for PAB gene
    • Becton Dickenson (BD ProbeTec) : SDA
    • Gen-Probe : Transcription-Mediated Amplification
    (TMA) for rRNA
    • Manual method
    • QMH-single tube nested PCR for IS6110 gene
    Dr.T.V.Rao MD
    27
  • 43. Target amplification
    Target amplification
    PCR Thermal cyclic synthesis of dsDNA by hybridization of IS6110 Roche Molecular Systems
    specific oligonucleotides to ssDNA target, extension to 65 kDa protein gene (16S rRNA, Amplicor™
    dsDNA by a thermostable polymerase and denaturation 16S rDNA gene Testkit).
    of ssDNA, which serves as a new target for the next MPB64 gene Colorimetric, automated
    cycle [19–23]. 35 kDa protein gene sandwich-hybridization
    assay using horseradish
    peroxidase (Cobas Analyzer).
    Dr.T.V.Rao MD
    28
  • 44. Real Time PCR replacing older Methods
    Dr.T.V.Rao MD
    29
  • 45. Subtyping M. tuberculosis strains used to rely mainly on testing for one or several phenotypic markers, notably unusual drug susceptibility patterns, and on phage typing.
    These markers have been replaced by more powerful DNA-typing methods, since the discovery and characterization of repetitive DNA in M. tuberculosis, such as direct repeat (DR) sequences and insertion sequences (IS6110 and IS1081), in the early 1990s
    DNA fingerprinting of M. tuberculosis
    Dr.T.V.Rao MD
    30
  • 46. NAA- summary
    Useful technology for rapid diagnosis of smear negative cases of active TB
    Able to identify 50-60% of smear -ve culture +ve cases
    Distinguish M.tb from NTM in smear +ve cases
    Should not be used to replace sputum microscopy as an initial screen & culture remains the gold standard
    Very high degree of quality control required
    Dr.T.V.Rao MD
    31
  • 47. 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
    Dr.T.V.Rao MD
    32
  • 48. Alarming Rise of Resistant Tuberculosis
    WHO Report on Anti-TB Drug Resistance
    490,000 new cases of MDR-TB each year, with >110,000 deaths1
    Accounts for 5% of 9 million new cases of TB2
    MDR-TB rates higher than ever (up to 22.3%), particularly in former Soviet Union countries
    XDR-TB reported by as many as 49 countries (by June 2008)3
    Recent WHO/IUATLD Global Surveillance report indicated 7.5% (301/4012) of MDR TB to be XDR4
    Around 40,000 XDR-TB cases emerge every year1
    1Tuberculosis: MDR-TB & XDR-TB—The 2008 Report. The Stop TB Department, WHO.
    2Hargreaves S. http://infection.thelancet.com, Vol 8, April 2008, p.220
    3Raviglione MC. NEJM 2008;359:636-8.4Anti-TB Drug Resistance in the World: Report No. 4. The WHO/IUATLD Global Project on Anti-Tuberculosis Drug Resistance Surveillance 2002-2007. World Health Organization, 2008 (WHO/HTM/TB2008.394).
  • 49. Reverse hybridization
    – Line probe assays
    • RNase Cleavage
    • Diagnostic Sequencing (Genotyping)
    Molecular Methods Drug Resistance
    Dr.T.V.Rao MD
    34
  • 50. Underreported Threat of Multidrug-Resistant Tuberculosis in Africa
    Dr.T.V.Rao MD
    35
  • 51. 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 . well-studied
    – 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%
    Dr.T.V.Rao MD
    36
  • 52. Dr.T.V.Rao MD
    37
    Xpert MTB/RIF
    Emerging molecular methods made more affordable
  • 53. Researchers have developed an automated PCR diagnostic test that can detect the presence of Mycobacterium tuberculosis (MTB) and the resistance to rifampin (RIF), an antibiotic used to treat it. The test, which was described in paper published in the New England Journal of Medicine, promises to help the public health sectors of low-income countries, where the occurrence of multidrug-resistant pulmonary tuberculosis (TB) is high.
    Affordable PCR TB diagnostic tool developed
    Dr.T.V.Rao MD
    38
  • 54. The new PCR-based TB diagnostic test—called Xpert MTB/RIF—is fast, sensitive, and automated. An accurate diagnosis can be obtained in less than 2 hours by adding a reagent to a sputum sample and, 15 minutes later, pipetting it into a cartridge that is inserted into the diagnostic instrument for 1–2 minutes.
    Xpert MTB/RIF
    Dr.T.V.Rao MD
    39
  • 55. Dual PCR reactions— sample-processing PCR is followed by hemi-processing PCR —increase the test’s sensitivity and specificity: according to the results published in NEJM, the PCR test was 98.2% sensitive in patients with smear-positive, culture-positive TB. And, because it is automated, there is little technical training needed to administer the test.
    Xpert MTB/RIF
    Dr.T.V.Rao MD
    40
  • 56. The World Health Organization (WHO) announced Wednesday its support of the Xpert MTB/RIF for rapid diagnosis of tuberculosis, multidrug resistant TB (MDR-TB) and TB in HIV-infected individuals. The results of the test demonstration study were announced in September of this year in the New England Journal of Medicine, producing results for many patients in approximately 100 minutes.
    Rapid TB test gets nod of approval from WHO
    Dr.T.V.Rao MD
    41
  • 57. Without information,the doctor cannot act.
    With information, he cannot but act.
    Time to act to Prevent Spread of MDR Tuberculosis
    Dr.T.V.Rao MD
    42
  • 58. 43
    HL Mencken’s Law
    Every complex problem
    has a simple solution.
    And it is always wrong in Tuberculosis
    Dr.T.V.Rao MD
  • 59. 44
    Follow me for Articles of Interest on Microbiology ..
  • 60. Created by Dr.T.V.Rao MD for ‘ e ‘ learning resources for Medical Microbiologists in the Developing World
    Email
    doctortvrao@gmail.com
    45