DR.T.V.RAO MD 1

Dr.T.V.Rao MD

MOLECULAR METHODS IN DIAGNOSIS OF

TUBERCULOSIS EMERGING TRENDS

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• 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

• 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

• 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

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• Re-emerging problem in industrialized countries

• • Infections in immuno-compromised patients Multi-drug resistant strains (MDR-TB)

CHALLENGES WITH TUBERCULOSIS INFECTION

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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 weight 1

•Responsible for many of this bacterium’s characteristic properties

•Acid fast—retains acidic stains

•Confers resistance to detergents, antibacterial

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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 RNA3

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 testingRifampin resistance(PCR oligohybridization sequencing)

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SYMPTOMSWhat 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 blood4.

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

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• 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

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• 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

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MOLECULAR METHODS IN DIAGNOSIS OF

TUBERCULOSIS

Several methods are available, mainly used as

Research tools

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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 DefinitionClinical Diagnosis

Molecular Disease DefinitionMolecular Diagnosis

in-vitro Diagnostics

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• 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?

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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

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• 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

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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

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• 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

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MYCOBACTERIUM TUBERCULOSISGENOME

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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

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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

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NAAS- VARIOUS TYPES

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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.

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• 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

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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

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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).

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REAL TIME PCR REPLACING OLDER METHODS

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• 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

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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

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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

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.2203Raviglione 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).

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• Reverse hybridization

• – Line probe assays

• • RNase Cleavage

• • Diagnostic Sequencing (Genotyping)

MOLECULAR METHODS DRUG RESISTANCE

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UNDERREPORTED THREAT OF MULTIDRUG-RESISTANT TUBERCULOSIS IN AFRICA

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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%

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Xpert MTB/RIF

EMERGING MOLECULAR METHODS MADE MORE AFFORDABLE

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• 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

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• 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

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• 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

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• 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

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WITHOUT INFORMATION, THE DOCTOR CANNOT ACT.

With information,

he cannot but act.Time to act to Prevent Spread of MDR Tuberculosis

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HL MENCKEN’S LAWEvery complex problem

has a simple solution.

And it is always wrong in Tuberculosis

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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