NEWER METHODS IN DIAGNOSIS OF TUBERCULOSIS

Views:
 
     
 

Presentation Description

NEWER METHODS IN DIAGNOSIS OF TUBERCULOSIS

Comments

Presentation Transcript

NEW TECHNOLOGIES IN DIAGNOSIS OF TUBERCULOSIS :

NEW TECHNOLOGIES IN DIAGNOSIS OF TUBERCULOSIS Dr.T.V.Rao MD 05-03-2016 Dr.T.V.Rao MD 1

A Tribute to Robert Koch The Mystery of Tuberculosis Continues? :

A Tribute to Robert Koch The Mystery of Tuberculosis Continues? 05-03-2016 Dr.T.V.Rao MD 2

Samples required for microbiological diagnosis of TB:

Samples required for microbiological diagnosis of TB 05-03-2016 Dr.T.V.Rao MD 3

Microscopy:

Microscopy Microscopy has been a diagnostic tool for TB for over a century, and still currently the most rapid diagnostic method. Standard light microscopy (LM) and fluorescent microscopy ( FM)are common methods 05-03-2016 Dr.T.V.Rao MD 4

Advancing Microscopy with Light Emitting Diodes LED :

Advancing Microscopy with Light Emitting Diodes LED The recent development of light emitting diodes (LED), with the appropriate fluorescent light output for FM and low power consumption, has led to the development of simple, robust LED FM microscopes, requiring minimal mains or battery power and no dark room requirement. 05-03-2016 Dr.T.V.Rao MD 5

Microscopy and Culturing Still feasible Alternatives :

Microscopy and Culturing Still feasible Alternatives Microscopic examination is suitable for laboratories at peripheral and higher levels and it can be done safely under minimal biosafety conditions. Mycobacterial culture is taken as the reference standard test for the diagnosis of TB, but this process is cumbersome and time-consuming. 05-03-2016 Dr.T.V.Rao MD 6

Antibiotic sensitivity testing by Conventional methods getting out dated :

Antibiotic sensitivity testing by Conventional methods getting out dated 05-03-2016 Dr.T.V.Rao MD 7

WHO Recommending the :

WHO Recommending the The WHO has recommended rolling it out as an alternative to LMs in resource-limited settings, based on studies that have shown comparable performance of LM and standard FM systems 05-03-2016 Dr.T.V.Rao MD 8

SCIENCE IS MOVING FROM BIOLOGY TO MICROBIOLOGY :

SCIENCE IS MOVING FROM BIOLOGY TO MICROBIOLOGY 05-03-2016 Dr.T.V.Rao MD 9

Molecular Biology advances with Nucleic acid based tests :

Molecular Biology advances with Nucleic acid based tests NAATs are molecular systems that can detect small quantities of genetic material (DNA or RNA) from microorganisms such as Mycobacterium tuberculosis. PCR is the most common among the variety of NAATs. NAATs are available as commercial kits and in-house tests and are used routinely in high-income countries for TB detection 05-03-2016 Dr.T.V.Rao MD 10

Molecular Biology advances with Nucleic acid based tests :

Molecular Biology advances with Nucleic acid based tests In-house PCR tests are widely used in the developing countries because these tests are less expensive than commercial kits. The line probe assays (LPA) are suitable only for national or regional level laboratories because of its complexity and bio safety requirements. 05-03-2016 Dr.T.V.Rao MD 11

What are Molecular tests:

What are Molecular tests In microbiology, the term molecular test is conventionally used to describe tests that detect pathogen nucleic acid (DNA or RNA). All molecular diagnostic tests have three stages: extraction, amplification and detection . 05-03-2016 Dr.T.V.Rao MD 12

Genotypic methods:

Genotypic methods Molecular techniques are aimed at the nucleic acid of the mycobacterium as the analyte. RibosomalrRNA is useful genetic target for the identification of organisms, since it often contains spesific sequences and is present in the cells and media in high quantity due to the growth of the mycobacteria. There are various applications of molecular techniques for the detection and identification of MTB. 05-03-2016 Dr.T.V.Rao MD 13

Extraction of Nucleic acid:

Extraction of Nucleic acid The extraction process should be carried out in a containment level 3 laboratories and will provide inactivation of organisms since Hazard Group 3 species may be present 05-03-2016 Dr.T.V.Rao MD 14

Amplification:

Amplification The nucleic acid in the sample or culture is amplified so that it can be detected. Polymerase chain reaction (PCR) is an example of a method for amplification, but a variety of other methods are available 05-03-2016 Dr.T.V.Rao MD 15

Polymerase Chain Reaction :

Polymerase Chain Reaction PCR is the common format of nucleic acid amplification tests (NAAT); othemethodologiesinclude ligase chain reaction, strain displacement amplification, loop-mediated isothermal amplifi cation (LAMP) and transcription mediated amplification. More recently, real-time 05-03-2016 Dr.T.V.Rao MD 16

RT PCR:

RT PCR RT PCR technologies based on fluorescent- probe detection or melting-curve analysis have been developed 05-03-2016 Dr.T.V.Rao MD 17

Detection:

Detection Methods of detection of the amplified nucleic acid also vary and include the use of electrophoresis, line probe assays or real time detection. Depending on the format, some tests may take two days for results to be available whereas others may produce results in a few hours 05-03-2016 Dr.T.V.Rao MD 18

Different types of molecular testing technology are available for TB:

Different types of molecular testing technology are available for TB 05-03-2016 Dr.T.V.Rao MD 19

Rules of the Lab Know before believe everything:

Rules of the Lab Know before believe everything No lab test is perfect Do not order a lab test if you are not ready to dealwith the result Treat the patient, not the lab testq For TB—There is a lot we still need to learn about DST and molecular detection of drug resistance 05-03-2016 Dr.T.V.Rao MD 20

NAAT for TB Diagnosis :

NAAT for TB Diagnosis Becoming standard of practice CANNOT replace clinical judgment or be relied on as the ONLY guide for therapy or isolation practices (less thanperfect sensitivity and specificity) Sensitivity>95 % for AFB smear-positive TB patients55–75 % of AFB smear-negative, culture-positive TB Patients Performance improves with increased clinical suspicion ofTB Potential benefits – impact on therapy, public healthmeasures , respiratory isolation, invasive procedures MMWR , 2009, 58:7-10 05-03-2016 Dr.T.V.Rao MD 21

Molecular Methods helps Detection Drug Resistance too :

Molecular Methods helps Detection Drug Resistance too These molecular techniques also aimed detecting resistance genes. Example includes; DNA probe and DNA sequencing of MTB gene such as catalase ( katG ) or RNA polymerase( rpoB ). Mutations in these genes have been associated with resistance to isoniazid and rifampicin respectively . 05-03-2016 Dr.T.V.Rao MD 22

Molecular Tests are Rapid, but expensive too:

Molecular Tests are Rapid, but expensive too The using of molecular primers in real- time PCR reaction can differentiate between the presence of the wild- type sequence and mutated sequence associated with drug resistance. Molecular tests are rapid (within few hours), highly sensitive and specific, but expensive, requires expertise and may not differentiate active infection as DNA from a dead organism during antibiotic treatment can be detected and amplified by PCR 05-03-2016 Dr.T.V.Rao MD 23

Line-probe assays :

Line-probe assays Line-probe assays and XPERT MTB/RIF: Line probe assays (LPAs) are actual molecular tests. Three main LPAs for the rapid diagnosis of TB and/or rapid detection of RMP resistance and MDR-/XDR-TB are currently available on the market: INNO- LiPA Rif. TB 05-03-2016 Dr.T.V.Rao MD 24

Line probe assays (LPAs) for tuberculosis were endorsed by the WHO in 2008 :

Line probe assays (LPAs) for tuberculosis were endorsed by the WHO in 2008 Line probe assays (LPAs) for tuberculosis were endorsed by the WHO in 2008 for molecular detection of drug resistance from smear-positive patients at risk of Multi Drug Resistant-Tuberculosis (MDRTB) Two commercial LPAs are currently available: 05-03-2016 Dr.T.V.Rao MD 25

Line Probe Assays for tuberculosis :

Line Probe Assays for tuberculosis Line Probe Assays for tuberculosis uses PCR/hybridization technique to identify members of the Mycobacterium Tuberculosis while simultaneously identifying drug-resistant strains by detecting the most common single nucleotide polymorphorisms (SNPs) associated with resistance. Meta-analyses have shown that LPAs are highly accurate for the detection of first-line drug resistance, especially in smear-positive sputum specimens. 05-03-2016 Dr.T.V.Rao MD 26

World Health Organization (WHO) recommends the use of Xpert MTB/RIF :

World Health Organization (WHO) recommends the use of Xpert MTB/RIF World Health Organization (WHO) recommends the use of Xpert MTB/RIF assay for rapid diagnosis of tuberculosis (TB) and detection of rifampicin resistance. This systematic review was done to know about the diagnostic accuracy and cost-effectiveness of the Xpert MTB/RIF 05-03-2016 Dr.T.V.Rao MD 27

WHO Endorses New Testing Method in 2011:

WHO Endorses New Testing Method in 2011 In early 2011, WHO endorsed a novel, rapid, automated, molecular diagnostic test, the Xpert MTB/RIF assay that can simultaneously detect TB and rifampicin resistance. Xpert MTB/RIF provides result within 2 h, thus the patients can be started with proper treatment even on the same day. 05-03-2016 Dr.T.V.Rao MD 28

GenoType®MTBDR/MTBDRplus and Geno-Type:

GenoType®MTBDR/ MTBDRplus and Geno-Type GenoType®MTBDR/ MTBDRplus and Geno-Type® MTBDRsl (both HainLifescience , Germany). These assays are based on the targeted amplification (PCR) of specific fragments of the MTB genome, followed by hybridisation of PCR products to oligonucleotide probes immobilised on membranes 05-03-2016 Dr.T.V.Rao MD 29

Newer Innovative Methods in Diagnosis of Tuberculosis :

Newer Innovative Methods in Diagnosis of Tuberculosis INNO- LiPA Rif TB detects only RMP resistance, GenoType MTBDR/MTBDR plus detects both RMP and INH resistance, and GenoType MTBDRsldetects resistance to fluoroquinolones, injectable second-line drugs and ethambutol. These tests are designed for detection the MTB isolates in respiratory specimens. Xpert® MTB/RIF (Cepheid Inc, USA) is a fully automated RT-PCR based assay for the detection of TB bacteria and resistance to RMP in direct clinical specimens 05-03-2016 Dr.T.V.Rao MD 30

Molecular Methods are not routinely Used:

Molecular Methods are not routinely Used Genotypic methods are not routinely used in the mycobacterium laboratory; they are essentially for research purposes 05-03-2016 Dr.T.V.Rao MD 31

Many Molecular Methods in offing :

Many Molecular Methods in offing Multiple commercial tests are available and new tests are also being developed. Some laboratories have developed local “in-house” tests. The choice of test depends on clinical need and local laboratory factors such as numbers of specimens received by the laboratory, access to molecular platforms and expertise. 05-03-2016 Dr.T.V.Rao MD 32

Line probe assays :

Line probe assays Line probe assays and the Cepheid MTB/RIF test which is run on the GeneXpert platform are commercial systems that are recommended by the WHO, for resource poor countries – where financial support is available for the cost of tests. In developed countries this format of test is convenient but no more accurate or reliable than other test systems and may be more expensive. 05-03-2016 Dr.T.V.Rao MD 33

Limitations and Considerations on Molecular Methods :

Limitations and Considerations on Molecular Methods Sensitivity § Reduced for smear negative specimens and some specimen types? § Do you want to “rule in” or “rule out”? § Platform dependent q Specificity § Platform dependent q Does not replace need for culture § Culture still needed for conventional DST, genotyping q Amplicon cross contamination in open systems q Cost and sustainability § Expense can limit utilization 05-03-2016 Dr.T.V.Rao MD 34

Attention of Viewers :

Attention of Viewers I am thankful to many in the world who made me to achieve my desired goals faster than I thought, having > 3-5 million health professionals share and utilize my knowledge for the benefit of mankind, Today I wish to be freelancer to the world to create interest in Medical, Clinical and Diagnostic Microbiology with more emphasis on Infectious diseases and Hospital associated Infection wish to be your partner in educating many millions who know well the importance of Infectious diseases You can visit many web sites of mine www.medmicrobes.com www.slidehsare.com www.authourstream.com www,scribd.com Be a friend on Facebook with tummalapalli venkateswararao access Rao’s Microbiology Rao’s Infection care Microbiology connected Travancore Medical College For any assistance on INFECTION REALTED ISSUES CONTACT ME AT doctortvrao@gmail.com Mob +91 7204113154 05-03-2016 Dr.T.V.Rao MD 35

Slide36:

Program Created by Dr.T.V.Rao MD for Benefit of Medical and Paramedical Professionals in the Developing World Created from World Wide R esources Email doctortvrao@gmail.com 05-03-2016 Dr.T.V.Rao MD 36

authorStream Live Help