Molecular methods in Diagnosis TuberculosisT

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Molecular methods in Diagnosis of Tuberculosis emerging trends :

Dr.T.V.Rao MD Molecular methods in Diagnosis of Tuberculosis emerging trends Dr.T.V.Rao MD 1

Historical Background:

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

Historical Background:

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

Diagnostic discoveries:

24 th 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

Global Status:

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

Challenges with tuberculosis Infection:

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

Mycobacterium tuberculosis-Characteristics:

Mycobacterium tuberculosis - Characteristics Gram positive Obligate aerobe Non-spore-forming Non-motile rod Mesophile 0.2 to 0.6 x 2-4um 1 Slow generation time: 15-20 hours May contribute to virulence 1 Lipid rich cell wall contains mycolic acid—50% of cell wall dry weight 1 Responsible for many of this bacterium’s characteristic properties Acid fast—retains acidic stains Confers resistance to detergents, antibacterial Dr.T.V.Rao MD 7

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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 multiple gastric aspirate urine (UTI) 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 RNA 3 Clinical specimen/ decontamination culture Direct detection: Microscopy PCR MTB rifampin resistance Species identification: 16S rRNA hybridization (MTB and MAC) 16S rRNA gene PCR sequencing (NTM) restriction fragment length polymorphism Susceptibility testing Rifampin resistance (PCR oligohybridization sequencing) Dr.T.V.Rao MD 8

Symptoms:

Symptoms What are the symptoms of TB? Fever Fatigue Weakness Weight loss Night sweats Symptoms of pulmonary TB include: Coughing Pleurisy (pain when taking deep breaths) Coughing up blood 4 . Dr.T.V.Rao MD 9

Effective laboratory Diagnosis:

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

Tuberculosis continues to be global health problem:

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

Tuberculosis A disease of growing Importance:

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

Molecular Methods in Diagnosis of Tuberculosis:

Molecular Methods in Diagnosis of Tuberculosis Several methods are available, mainly used as Research tools Dr.T.V.Rao MD 13

Medical Progress: Evolution or Revolution?:

14 Medical Progress: Evolution or Revolution ? … Genetics Clinical expertise Classical epidemiology Differential diagnosis Risk assessment - prevention Historic Drivers of Medical Progress More differentiated, molecular understanding of pathology and drug action Clinical Disease Definition Clinical Diagnosis Molecular Disease Definition Molecular Diagnosis in-vitro Diagnostics

Molecular Diagnostics Why?:

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 Diagnostics Why? Dr.T.V.Rao MD 15

molecular tests for detection of nucleic acids:

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

Beginning of Gene amplification methods:

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

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Dr.T.V.Rao MD 18

Molecular diagnosis of tuberculosis. :

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

Breakthrough with molecular technologies :

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

Mycobacterium tuberculosis genome :

Mycobacterium tuberculosis genome Dr.T.V.Rao MD 21

Nucleic acid amplification assays :

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

NAAs- various types:

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

NAAs- various types:

NAAs- various types Dr.T.V.Rao MD 24

AMPLICOR M. TUBERCULOSIS assay:

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

DNA Amplification Assay:

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

Direct Detection of M. tuberculosis in sputum by DNA amplification:

Direct Detection of M. tuberculosis in sputum by 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

Target amplification :

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

Real Time PCR replacing older Methods:

Real Time PCR replacing older Methods Dr.T.V.Rao MD 29

DNA fingerprinting of M. tuberculosis:

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

NAA- summary:

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

NAA- Limitations:

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

Alarming Rise of Resistant Tuberculosis:

Alarming Rise of Resistant Tuberculosis WHO Report on Anti-TB Drug Resistance 490,000 new cases of MDR-TB each year, with >110,000 deaths 1 Accounts for 5% of 9 million new cases of TB 2 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 XDR 4 Around 40,000 XDR-TB cases emerge every year 1 1 Tuberculosis: MDR-TB & XDR-TB—The 2008 Report. The Stop TB Department, WHO. 2 Hargreaves S. http://infection.thelancet.com , Vol 8, April 2008, p.220 3 Raviglione MC. NEJM 2008;359:636-8. 4 Anti-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).

Molecular Methods Drug Resistance:

Reverse hybridization – Line probe assays • RNase Cleavage • Diagnostic Sequencing (Genotyping) Molecular Methods Drug Resistance Dr.T.V.Rao MD 34

Underreported Threat of Multidrug-Resistant Tuberculosis in Africa :

Underreported Threat of Multidrug-Resistant Tuberculosis in Africa Dr.T.V.Rao MD 35

Molecular methods for drug resistance:

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

Emerging molecular methods made more affordable:

Dr.T.V.Rao MD 37 Xpert MTB/RIF Emerging molecular methods made more affordable

Affordable PCR TB diagnostic tool developed :

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

Xpert MTB/RIF:

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

Xpert MTB/RIF:

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 tes t. Xpert MTB/RIF Dr.T.V.Rao MD 40

Rapid TB test gets nod of approval from WHO :

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

Without information, the doctor cannot act. :

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

HL Mencken’s Law:

43 HL Mencken’s Law Every complex problem has a simple solution. And it is always wrong in Tuberculosis Dr.T.V.Rao MD

Follow me for Articles of Interest on Microbiology ..:

44 Follow me for Articles of Interest on Microbiology ..

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Created by Dr.T.V.Rao MD for ‘ e ‘ learning resources for Medical Microbiologists in the Developing World Email doctortvrao@gmail.com 45