This document discusses molecular assays for tuberculosis diagnosis and drug resistance testing. It begins with an introduction to the global burden of TB and importance of accurate diagnosis. It then describes various microbiological diagnostic methods including smear microscopy, culture techniques, nucleic acid amplification tests (NAATs), and molecular methods like whole genome sequencing (WGS). Specific NAATs discussed in detail are Xpert MTB/RIF and targeted sequencing approaches. Centralized high-throughput diagnostic tests are also mentioned. Overall the document provides an overview of established and emerging molecular methods that can improve TB diagnosis and detection of drug resistance.
This presentation is about lab diagnosis of tuberculosis. It highlights use of currently available diagnostic methods in identifying pulmonary and extrapulmonary tuberculosis.
This presentation is about lab diagnosis of tuberculosis. It highlights use of currently available diagnostic methods in identifying pulmonary and extrapulmonary tuberculosis.
Newer diagnostic methods in tuberculosis detectionApollo Hospitals
One-third of the world's population has been infected with Mycobacterium tuberculosis, with new infections occurring in about 1% of the population each year. However 90–95% of infections remain asymptomatic. Thus early diagnosis of tuberculosis and drug resistance improves survival and helps to promote contact tracing, implementation of institutional cross-infection procedures, and other public-health actions. There have been many advances and modifications to the methodology for tuberculosis diagnosis some of which are very promising. But these advances have not kept pace with the explosion of tuberculosis or the outbreak of drug resistant tuberculosis. This review describes some of the newer advances in tuberculosis diagnostics and the challenges they face.
Tuberculosis is a raging problem round the globe. Eradicating TB is a herculean task but is possible is efforts from all corners from the world. The diagnostics have taken a big leap and with effective medications, our dream of TB free world may come true. But unlimited efforts are need to reach our goal.
what is new in prevention, diagnosis and treatment of tuberculosis tb short.pptxPathKind Labs
Many changes have been made recently in Tuberculosis. The first important change is that instead of control now the focus is on eradication. for that to happen we need to change the way we detect, diagnose and treat tuberculosis.
RECENT ADVANCES IN DIAGNOSIS OF TUBERCULOSISANGAN KARMAKAR
TRADITIONAL TESTS AND RECENT DIAGNOSTIC MODALITIES FOR TUBERCULOSIS WITH EMPHASIS TO MOLECULAR DETECTION TECHNIQUES, DRUG SENSITIVITY ASSESMENT IN INDIAN PERSPECTIVE
High Resolution Outbreak Tracing and Resistance Detection using Whole Genome ...QIAGEN
In March 2014, a molecular cluster of five multidrug-resistant Mycobacterium tuberculosis was detected by the Austrian National Reference Laboratory. An investigation was initiated to determine if transmission had occurred within Austria. Epidemiological links to Germany and Romania prompted a multi-national joint investigation, tracing the outbreak. The results were published by Fiebig and coworkers in 2017.
Whole genome SNP analysis allowed for high resolution clustering of isolates. Whole genome sequencing further permitted simultaneous detection of resistance-causing variants. Using an improved variant detection pipeline, we identified novel variants undetected in the original study. We used functional analysis to explore if novel variants could be associated to antimicrobial resistance.
Using the data published by Fiebig at al. in 2017, we demonstrate the use of CLC Microbial Genomics Module for tracing pathogen transmission during outbreaks and for the detection and functional analysis of resistance-causing variants. The applied user-friendly tools and preconfigured workflows ensure ease of use and reproducibility.
Catridge based nucleic acid amplification test(CBNAAT) / RIF assay gene xpert POWER PONT. other normal tests versus CBNAAT. issues for cbnaat by WHO & CONCLUSION.
Newer diagnostic methods in tuberculosis detectionApollo Hospitals
One-third of the world's population has been infected with Mycobacterium tuberculosis, with new infections occurring in about 1% of the population each year. However 90–95% of infections remain asymptomatic. Thus early diagnosis of tuberculosis and drug resistance improves survival and helps to promote contact tracing, implementation of institutional cross-infection procedures, and other public-health actions. There have been many advances and modifications to the methodology for tuberculosis diagnosis some of which are very promising. But these advances have not kept pace with the explosion of tuberculosis or the outbreak of drug resistant tuberculosis. This review describes some of the newer advances in tuberculosis diagnostics and the challenges they face.
Tuberculosis is a raging problem round the globe. Eradicating TB is a herculean task but is possible is efforts from all corners from the world. The diagnostics have taken a big leap and with effective medications, our dream of TB free world may come true. But unlimited efforts are need to reach our goal.
what is new in prevention, diagnosis and treatment of tuberculosis tb short.pptxPathKind Labs
Many changes have been made recently in Tuberculosis. The first important change is that instead of control now the focus is on eradication. for that to happen we need to change the way we detect, diagnose and treat tuberculosis.
RECENT ADVANCES IN DIAGNOSIS OF TUBERCULOSISANGAN KARMAKAR
TRADITIONAL TESTS AND RECENT DIAGNOSTIC MODALITIES FOR TUBERCULOSIS WITH EMPHASIS TO MOLECULAR DETECTION TECHNIQUES, DRUG SENSITIVITY ASSESMENT IN INDIAN PERSPECTIVE
High Resolution Outbreak Tracing and Resistance Detection using Whole Genome ...QIAGEN
In March 2014, a molecular cluster of five multidrug-resistant Mycobacterium tuberculosis was detected by the Austrian National Reference Laboratory. An investigation was initiated to determine if transmission had occurred within Austria. Epidemiological links to Germany and Romania prompted a multi-national joint investigation, tracing the outbreak. The results were published by Fiebig and coworkers in 2017.
Whole genome SNP analysis allowed for high resolution clustering of isolates. Whole genome sequencing further permitted simultaneous detection of resistance-causing variants. Using an improved variant detection pipeline, we identified novel variants undetected in the original study. We used functional analysis to explore if novel variants could be associated to antimicrobial resistance.
Using the data published by Fiebig at al. in 2017, we demonstrate the use of CLC Microbial Genomics Module for tracing pathogen transmission during outbreaks and for the detection and functional analysis of resistance-causing variants. The applied user-friendly tools and preconfigured workflows ensure ease of use and reproducibility.
Catridge based nucleic acid amplification test(CBNAAT) / RIF assay gene xpert POWER PONT. other normal tests versus CBNAAT. issues for cbnaat by WHO & CONCLUSION.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
2. INTRODUCTION
• Tuberculosis is the leading infectious disease killer globally
• India accounts for 34% of estimated global 1.5 millon TB death in 2020
• According to WHO 5,04,000 deaths recorded in India in year 2020.
• Despite the severity of the epidemic, approximately 3 million people with TB were
deemed “missing” due to underdiagnosis as well as underreporting to national TB
programs.
• The World Health Organization (WHO) End TB Strategy calls for finding these
missing millions in order to meet the sustainable development goal of ending TB
by 2030
3. • The TB Elimination target seeks to reduce Indias tuberculosis death from 32
per lac population people in 2015 to below 3 per lac by 2025.
• Advancements in molecular methods for MTB detection has shortened the
time to diagnosis to a few days.
• To reduce the global TB burden - accurate and efficient diagnosis.
• With a disease like TB time is crucial.
• whereas diagnosis by conventional culture systems needs several weeks.
• Diagnostic delays and health sysytem failures often result in missed or late
diagnosis ,with serious consequences for TB patients.
4. MICROBIOLOGICAL DIAGNOSIS
SMEAR
MICROSCOPY
Z N Stain
Fluorescenc
e Stain
CULTURE
TECHNIQUES
SOLID
LIQUID
Mycobacterium
Growth
Indicator Tube
(MGIT)
MOLECULAR METHODS
PCR
Real Time PCR
CB NAAT/
GeneXpert
Line Probe Assay (LPA)
Whole genome Sequencing
TESTS FOR
LTBI
TST/Mantou
x test
Interferon
Gamma
Release
Assay
(IGRA)
OTHER
Lipoarabin
omannan
assay
(LAM)
Adenosine
Deaminas
e
(ADA)
7. Uses
• Used as an initial diagnostic test for the detection of AFB in PTB
• Monitors response to therapy
Benefits
• Can be done safely with low risk level and minimal biosafety precautions
• Same day results
• Inexpensive and widely available
Limitations
• Low sensitivity (50%), which is further reduced in HIV-positive individuals and
children
• Limited specificity; can detect non-tuberculous mycobacteria(NTMs) and does not
detect
drug resistance.
• Can not detect DRTB in HIV+ individuals.
8. Uses
• Used as an initial diagnostic for TB as well as to isolate cultures for DST
• Monitors MDR-TB treatment
Benefits
• High sensitivity and specificity test for the detection of MTBC
• Provides an isolate for DST (phenotypic testing)
• Can assess treatment progress
Limitations
• Requires a high level of biosafety precautions
• Requires trained staff
• Automated liquid culture is more expensive than solid culture
• Solid culture is slow; takes 4–8 weeks to detect
MTBC
CULTURE TECHNIQUES
9. SOLID (CONVENTIONAL)
• Egg Based -Lowenstein -Jensen Media
• Agar Based - Middlebrook 7H10 OR 7H11 media
LIQUID (AUTOMATED)
• Mycobacterium Growth Indicator Tube
(MGIT)
• BacT/Alert
10. Uses
• Detection of resistance to anti-TB drugs
Benefits
• Culture-based, phenotypic DST remains essential for drugs for which there
are not
yet reliable molecular tests
• Phenotypic DST for second-line agents is required to confirm or exclude
XDR-TB
Limitations
• Requires a high level of biosafety precautions, highly skilled staff and strict
quality
control
• Culture-based phenotypic DST can take weeks to months to generate
results
Phenotypic (culture-based) DST
11. DST remains essential for
drugs
for which there are not yet
reliable molecular tests.
12. WHO-endorsed and emerging molecular tests
for TB and drug resistance.
Outlined in blue are WHO-endorsed NAATs,
• LPAs
• Xpert Ultra
• LAMP
• Truelab
Tests that are not yet WHO endorsed but are
under development or evaluation are outlined in
orange.
• DST, drug sensitivity testing;
• GX, GeneXpert;
• POC,NAATs point of care; NAAT
13. we will discuss the role of the
• Nucleic acid amplification test (NAAT).
• Whole-genome sequencing (WGS) in the diagnosis
of TB.
• Mycobacterial load detection assays.
• Serological diagnosis of active TB.
• Molecular diagnostic of multi-drug resistant TB (MDR-
TB).
14. MOLECULAR METHODS
NAAT - NUCLEIC ACID AMPLIFICATION TEST
TYPES
• REAL TIME PCR
• LOOP MEDIATED AMPLIFICATION [LAMP]
• LINE PROBE ASSAY [LPA]
• WHOLE GENOMIC SEQUENCING
15. NUCLEIC ACID AMPLIFICATION TEST (NAAT)
• Because the conventional bacteriological diagnosis of TB has several
limitations, the NAAT has emerged as a potential alternative.
• The NAAT systems, with rapid turn-around times, facilitate testing and
treatment initiation in the same visit and, therefore, loss to follow-up
cases can be reduced.
• Most NAAT assays detect the mycobacterial insertion element IS6110 for
the identification of the MTB complex organisms.
• NAAT detects MTB ribosomal RNA or DNA directly from sputum
specimens, both the acid-fast bacilli (AFB) smear-positive and AFB
smear-negative.
• The NAAT showed very high sensitivity in sputum smear-positive patients
and around 61 to 76% sensitivity in patients with smear-negative sputum.
16. Currently, the NAAT that is endorsed by the WHO is the
• Xpert/RIF MTB assay.
The other two NAATs, approved by the Food and Drug Administration (FDA) for testing respiratory
AFB smear-positive specimens are;
• Amplified Mycobacterium Tuberculosis Direct (MTD) Test
(Gen-Probe, Inc),
• Amplicor Mycobacterium tuberculosis Test (Roche Molecular
Systems, Inc),
Other commercial NAATs are also available, including the
• Loop-mediated isothermal amplification-based MTB detection
system,
• Cross-priming amplification-based TB diagnostic system,
• GenedriveⓇ Mycobacterium tuberculosis iDⓇ.
17. • In multi-bacillary diseases with a high mycobacterial load, a positive AFB smear with a
positive NAAT would indicate active tuberculosis
• whereas a positive AFB smear with a negative NAAT in the absence of inhibitors would
indicate nontuberculous mycobacterial (NTM) disease.
• If the culture was positive in the above case, the physician could consider the patient as a
bacteriologically confirmed case of TB.
• A NAAT could determine whether AFB smear positive patients had TB or not.
• Moreover, if the NAAT result is positive but the AFB smear result is negative, the
decision to begin anti-TB treatment would rely on the clinical judgment while awaiting
culture
results.
• According to Centers for Disease Control (CDC), if the sputum is smear-negative and the
NAAT also negative, an additional specimen should be tested with NAAT.
• However, if the culture results detected MTB bacteria growth, then the patient could also be
classified as bacteriologically confirmed for having pulmonary TB
20. Xpert MTB/RIF
Endorsed by WHO in 2010
Based on NAAT
Updated recommendations in 2013 for diagnosis of
• Pulmonary TB
• Paediatric TB
• Extrapulmonary TB
• Rifampicin resistance ( MDR TB)
21. • HAVE SENSITIVITY OF 99% IN SPUTUM POSITIVE
• AND MORE THAN 80% IN SPUTUM SMEAR NEGATIVE CASES.
• According to the WHO in 2013, a Xpert MTB/RIF assay could be used for: an add-on
test following microscopic TB examination;
• A replacement examination for AFB smear microscopy; detection of MTB in both AFB
smear-positive and smear-negative culture-positive cases.
• The Xpert MTB/RIF assay detects rifampicin resistance by PCR amplification of the
81-bp fragment of the MTB rpoB gene and subsequent probing of this region for
rifampicin resistant-associated mutations and the results can be obtained within 2
hours.
• The WHO recommends subjects who are at high risk of MDR-TB should always have
their sputum checked using the Xpert MTB/RIF test.
22. ADVANTAGES
1. Better sensitivity &
specificity
2. Rapid result (2hrs)
3. No tech expertise
required
LIMITATIONS
1. Blood ,stool ,and urine are not
recommended
2. Low sensitivity in pericardial
,ascitic,synovial fluid and pleural fluid
3. Expensive
4. Susceptibility test for other drugs cannot
be performed
5. Not advised for follow up cases
6. Infrastructure is required.
26. • Whole-genome sequencing (WGS) is becoming an affordable and accessible method that can
identify microevolution within MTB lineages as they are transmitted between hosts.
• The WGS can detect various types of mutations better than the Xpert MTB assay.
• There are two classes of sequencers that exist: the first generation sequencer and the second
generation
• (widely known as the next-generation sequencer [NGS]).
• The first generation sequencer is relatively slow, but has a high throughput and low cost
(approximately $65 per bacterial genome).
• The second generation has a lower throughput, higher cost (approximately $150 per genome in the
case of the IlluminaⓇ MiSeq) and is able to sequence multiple genomes in less than a day.
27. First generation
sequencer
Next-generation
sequencer
Principles
and features
This first generation of DNA
sequencers are essentially automated
electrophoresis systems that detect
the migration of labelled DNA
fragments.
Rapid generation of data by sequencing
massive amounts of DNA in parallel using
diverse methodologies.
Capable of genotyping of genetic
markers where only the length of a
DNA
fragment(s) needs to be
determined.
Constitute various strategies that rely on a
combination of template preparation,
sequencing and imaging, and genome
alignment and assembly
methods.
28. • ADVANTAGE ;WGS could avoid false positives when a polymorphism in the
rifampicin-resistance determining region (RRDR) of rpoB is detected.
• LIM ;WGS has not been used as a routine diagnostic tool for TB, partly because of
the need to culture MTB for several weeks until an adequate amount of DNA can
be extracted.(LIM)
• Culturing slow-growing MTB before DNA extraction is a major time-consuming
step in WGS , therefore, workflow to extract DNA from frozen isolates without re-
culturing is important.
• There is a reliable and low-cost DNA extraction method from 1 mL of early
positive mycobacterial growth indicator tube (MGIT) cultures that is enough for the
WGS to identify mycobacterial species and predict antibiotic resistance in clinical
samples.
29. • Recently, an important method that .allows WGS without prior
specimen culture has been discovered.
• The method utilizes MTB DNA-specific biotinylated RNA baits to
capture full MTB genomes directly from non-cultured sputum
samples.
• WGS data can be obtained several weeks before the drug
susceptibility test (DST) data is available. (ADV)
• DNA sequencing could also be used to confirm rifampin
resistance from Xpert MTB/RIF.(ADV)
• However, with the price in the hundreds of dollars for this
method, routine WGS for clinical specimens is limited to high-
income countries (LIM)
30. • WGS was performed using the Illumina platform.
• Drug resistance profiles and lineages were predicted in silico using the Total
Genotyping Solution for TB (TGS-TB).
• Using the phenotypic DST results as a reference, WGS-based prediction
demonstrated high concordance rates of
Isoniazid (95.0%),
Rifampicin (RIF) (98.0%),
Pyrazinamide (98.5%) and
Fluoroquinolones (FQs) (99.5%)
96.0% to 99.5% for second-line injectable drugs (SLIDs);
• whereas, lower concordance rates of ethambutol (87.5%), streptomycin (88.0%) and
ethionamide (84.0%).
31. TARGETED SEQUENCING
There are ongoing efforts by multiple stakeholders to validate
targeted sequencing as a complete end-to-end solution for DR-TB
detection,
From DNA extraction direct from respiratory samples (i.e., without
the need for first culturing and then isolating a specimen), targeted
library preparation and sequencing, to result reporting.
32. • Deeplex Myc-TB uses ultradeep sequencing of 24-plexamplicon mixes for
mycobacterial species identification, genotyping, and DST.
• Another newly developed targeted sequencing assay for DR-TB is DeepChek-
TB (Translational Genomics Research Institute, Flagstaff, USA), which has
recently been licensed by ABL (Luxembourg) Advanced Biological
Laboratories.
• Both tests are currently for research use only.
• Low TB Burden, including
United Kingdom (Public Health England),
Netherlands, and
New York state,
Have already transitioned from phenotypic culture to WGS for DST for first-line
drugs.
33. Sequencing is currently being successfully implemented for DR-TB
surveillance purposes in at least seven countries—
Azerbaijan,
Bangladesh,
Belarus,
Pakistan,
Philippines,
South Africa,
Ukraine
INDIA in year 2018, infrastructure and technical support for sequencing
were introduced at five national TB program laboratories.
It is hoped that this will be the beginning of the foundations of a clinical
diagnostic network in the future.
34. STRENGHTH
Whole-genome sequencing Targeted sequencing
Full genome sequenced Sequence directly from sample
No prespecified targets needed Large number of gene targets
Comprehensive solution Less expensive than WGS
Detect rare mutations and heteroresistance Simpler bioinformatics and storage
Detect rare mutations and heteroresistance
35. WEAKNESSES
Requires culture isolates Knowledge of targets required
Slower than targeted NGS Less information than WGS
Complicated bioinformatics
Expensive Expensive
36. High-throughput solutions:
Centralized diagnostic tests.
Recently, centralized, high-throughput NAATs for TB diagnosis and drug resistance detection have
been developed and are currently undergoing WHO evidence evaluation.
RealTime MTB (Abbott Molecular, Abbott Park, USA),
,
FluoroType MTB (Hain Lifescience, Nehren, Germany),
Cobas MTB (Roche, Rotkreuz, Switzerland),
Max MDR-TB (BD, Franklin Lakes, USA) assays
Run on established multidisease platforms that are already employed for such diseases
as human immunodeficiency virus (HIV), human papillomavirus, and hepatitis C virus.
37. ADVANTAGES
WHO technical expert group meeting reported that the centralized
assays’ performance for detecting resistance to INH and RIF was
similar to LPA
Centralized TB assays are promising due to their high diagnostic
accuracy and ability to run large numbers of samples
simultaneously,
Their automated nature reduces the hazard of contacting
infectious respiratory specimens for health care workers and
laboratory technicians.
39. Regarding sequencing for DST, centralized sequencing platforms have been
the
norm, but there is increasing interest in smaller and more portable
sequencing devices,such as
MinION (Oxford Nanopore, Oxford, UK)
iSeq from Illumina (San Diego,USA)
Validation for both is on-going.
40. MinION (Oxford Nanopore, Oxford, UK)
Its a nanopore sequencing panel for direct TB drug resistant prophylling
The panel targeted 10 resistance loci out of which 8 were succecfully amplified
from the majority for predicting first and second line drug resistance
Its a smaller , portable device .
41.
42. INDIGINEOUS PRODUCT - True NAT (Molbio
Diagnostics)
• Point of Care
• Portable
• Rapid
• Automatic
• Battery Operated
43. It is Chip-based, micro real-time PCR-based
Assays for TB detection that produce results in 1 hour on the portable Truelab platform
(Molbio Diagnostics).
Already being rolled out in India,
Truenat is characterized as a more affordable alternative to Xpert that is made in India.
Truenat MTB,
Truenat MTB Plus,
Truenat MTB-Rif Dx (Molbio Diagnostics, Goa, India)
The govt of Andhra pradesh is first adopted of trueNAT under its RNTCP it improved TB case
notification rate by 30% in Andhra Pardesh
44. USES
• First WHO-recommended molecular test for TB and RIF resistance that can be used in peripheral
settings with limited infrastructure.
• Used as an initial diagnostic test for TB
Benefits
• Designed to be operated in peripheral laboratories with minimal infrastructure and minimally trained
Lab
technicians
• Battery-powered and uses room temperature stable reagents
• Can generate results for TB in one hour and for RIF resistance in one additional hour
Limitations
• Electricity still required for charging the batteries
• More manual steps than the Xpert MTB/RIF test
45. WHO RECOMMENDATION
In adults and children with signs and symptoms of pulmonary TB, the Truenat MTB or MTB Plus may
be used as an initial diagnostic test for TB rather than smear microscopy/culture.
In adults and children with signs and symptoms of pulmonary TB and a Truenat MTB or MTB Plus
positive result, Truenat MTB-RIF Dx may be used as an initial test for RIF resistance rather than
culture and phenotypic DST.
47. LIMITATIONS
• Requires at least 3 separate rooms to avoid
cross
contamination and moderate to high levels of
biosafety precautions( biosafety containment
level3)
• Cannot be used to monitor treatment
• Can not fully replace conventional culture
methods
• Expensive
• Requires well trained staff
Benefits
• Able to rapidly detect resistance to RIF, INH, FQs and
second –line injectables.
• Detects Mycobacterium Tuberculosis complex
(MTBC) and determines its drug sensitivity to RIF and
INH.
• Can perform multiple test at once
• Fast and accurate results under 48 hrs
• Able to provide guidance on treatment decision
Uses
• Detection of resistance to anti-TB drugs
LPA is recommended for use on smear-positive sputum
specimens and M. tuberculosis isolates as the initial test
instead
of Phenotypic culture-based DST to detect resistance to RIF
and INH.
WHO RECOMMENDATIONS
48. ADVANTAGES
1. QUICK RESULTS IN UNDER 48 Hrs
2. ACCURATE
3.WHO Recommended test for detection
of resistance against additional drugs.
4.Necessary for guiding treatment
decisions
LIMITATIONS
1. Not as fast as Xpert
2.Expensive
3.Requires well-trained staff in a
professional laboratory
4.Has high biosafety requirements ( can
only be used in certain labs)
49. • Endorsed by WHO in 2015
• Rapid test to detect mycobacterial
lipoarabinomannan(LAM) MTBC antigens in urine
• From urine of Active TB patients
• Used only in HIV+ Patients presenting with TB
symptoms and CD4 Count less than 100 per
microlitre.
50. BENEFITS
• Requires no equipment
• Point of care test
• Simple, easy and fast (25 mins)
• Easy to collect urine specimen
• Allows treatment to be started early
• Inexpensive
LIMITATIONS
• Does not provide any information on drug resistance
• Cannot be used to monitor treatment
• Low sensitivity
• Require follow up with other diagnostic test
• Used in limited patient population
• Can not distinguish MTB from other non mycobacteria
LAM
51. Uses
• Used as an initial diagnostic for TB
Benefits
• Requires minimal infrastructure and has biosafety requirements similar to smear microscopy
• Detection of amplified product is based on the turbidity visualized with the naked eye or under
ultraviolet light
• Requires less than one hour to perform
Limitations
• Most suitable for settings with low prevalence of HIV and MDR-TB
• Cannot be used to monitor treatment
• Does not detect RIF resistance
LAMP :LOOP MEDIATED ISOTHERMAL AMPLIFICATION
52.
53. MYCOBACTERIAL LOAD DETECTION ASSAY
Quantification of the bacillary load has an important prognostic role in TB patients.
There are several assays that can be used to quantify mycobacterial load, such as the
• BD ProbeTec system,
• AdvanSure TB/NTM real-time PCR kit.
Advan Sure TB/NTM real-time PCR could be used to detect Mycobacterium and distinguish whether it is MTB or
NTM because it used the IS6110 primer for the detection of MTB complex and rpoB gene specific primer and prob
for the detection of NTM.
However, this assay does not determine if there are viable Mycobacterium organisms that truly exist in the specim
54. MOLECULAR DIAGNOSTIC OF MULTIDRUG RESISTANCE TUBERCULOSIS
Knowledge of the full drug susceptibility profile would enable customized treatment to increase efficacy
and avoid exposure to ineffective toxic drugs.
The PSQ (PYROSEQUENCING)method consists of seven PSQ assays for the mutations detection of the genes
or promoter regions, which are commonly responsible for resistance to the first line and second line drugs.
In one study, the correlations between the PSQ results and the phenotypic DST results were
98.7% for rifampin,
94.3% for isoniazid,
99.2% for capreomycin,
99.2% for amikacin, 96.4% for kanamycin,
97.6% for quinolones (levofloxacin, ofloxacin, or moxifloxacin).
55. GenoType MTBDRsl (MTBDRsl) is the only molecular test that is commercially-available for
detecting resistance to ;
• The fluoroquinolones
• levofloxacin,
• ofloxacin,
• moxifloxacin.
• The second-line injectable drugs kanamycin, capreomycin, and amikacin).
56. MTBDRsl detects rrs and gyrA genes mutations that confers resistance to
injectable antibiotics and fluoroquinolones, and is also capable of detecting
embB mutations that confer resistance to ethambuthol.
The sensitivity and specificity of the MTBDRsl test were as follows:
87% and 96%, respectively, for fluoroquinolones;
77% and 100%, respectively, for kanamycin;
80% and 98%, respectively, for capreomycin;
100% and 100%, respectively, for amikacin;
57% and 92%, respectively, for ethambutol.
However, despite the advances in the resistance detection tools, key
challenges still exist including difficulties in documenting the impact on
programmatic performance, long-term financial support, and a limited
number of scientific publications.
57. EMERGING TECHNOLOGIES
Xpert XDR
Another PCR-based cartridge has been designed to run on the GeneXpert and Omni platforms
for the simultaneous detection of mutations associated with resistance to multiple first- and
second-line TB drugs or extensively drug-resistant TB (XDR-TB).
In July 2020, the Xpert MTB XDR-TBcartridge was launched, but further validation and WHO
review are pending
Future developments will need to focus on drugs that are now critical for MDR and XDR-TB
management, including bedaquiline, pretomanid, and linezolid, but developing highly accurate
molecular diagnostics to detect resistance to these drugs is currently impossible due to the lack
of knowledge on resistance mechanisms.
58. GeneXpert Omni and other point-of-care devices
The GeneXpert platform was originally designed for use at the district or subdistrict level.
POC GeneXpert Omni platform is a long-awaited development, as it will permit the use of Xpert MTB/RIF and
Ultra assays in decentralized locations (e.g., primary care centers).
Omni promises to be a real POC platform with a 2-day battery life and no tablet or computer requirement
Other such POC NAATs are also under development.
For example,
Q-POC from QuantuMDx (Newcastle-upon-Tyne, United Kingdom) is a POC battery-operated PCR
system that promises to deliver TB testing results in less than 30 min. It has been evaluated in combination
with oral swabs as a sample, where its sensitivity and specificity, in preliminary studies, were similar to that of Xpert
59. CONCLUSION
TB Screening and diagnosis requires a COMBINATION of different methods and techniques to accura
test people
There is no single TB test that can do it all.
CULTURE remains GOLD standard for the definative diagnosis of TB in smear negative Patients.
Culture techniques should be used for monitoring treatment response or follow up cases.
Molecular methods are promising with a very low turn around time.
Suspected MDR/XDR cases first Diagnostic choice should be CBNAAT OR LPA.