This document summarizes updates in the diagnosis of tuberculosis (TB). Direct diagnostic methods include microscopic examination of samples by Ziehl-Neelsen staining or fluorescent dyes to identify Mycobacterium tuberculosis, as well as culture-based techniques like traditional culture, radiometric culture (BACTEC), and broth-based methods like mycobacterial growth indicator tubes (MGIT) that provide faster results. Newer molecular techniques like polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) can also directly detect M. tuberculosis DNA or RNA. Indirect methods involve detecting antibodies by tests like ELISA or antigens by methods like TB STAT-PAK.
Hello Guys,
This presentation consists of the updated guidelines under National tuberculosis elimination programme of India (MOHFW). The presentation includes case definitions and diagnostic algorithms for Pulmonary, Extrapulmonary and Drug resistant TB(MDR/ XDR TB) and the tratment protocols in pediatric cases.
Hope you find it useful.
Hello Guys,
This presentation consists of the updated guidelines under National tuberculosis elimination programme of India (MOHFW). The presentation includes case definitions and diagnostic algorithms for Pulmonary, Extrapulmonary and Drug resistant TB(MDR/ XDR TB) and the tratment protocols in pediatric cases.
Hope you find it useful.
what is community acquired pneumonia(CAP),what is the prevalence of (CAP) ,what are the risk factors and what are the causative agents ,what are the clinical presentations ,how to diagnose it,what are the needed investigations ,what is the management ,what are the procedures to decrease the incidence,
Catridge based nucleic acid amplification test(CBNAAT) / RIF assay gene xpert POWER PONT. other normal tests versus CBNAAT. issues for cbnaat by WHO & CONCLUSION.
what is community acquired pneumonia(CAP),what is the prevalence of (CAP) ,what are the risk factors and what are the causative agents ,what are the clinical presentations ,how to diagnose it,what are the needed investigations ,what is the management ,what are the procedures to decrease the incidence,
Catridge based nucleic acid amplification test(CBNAAT) / RIF assay gene xpert POWER PONT. other normal tests versus CBNAAT. issues for cbnaat by WHO & CONCLUSION.
This is a presentation giving an overview of the GeneXpert DX system for detection of MTB. The assay described in this presentation is the MTB/RIF test.
My Powerpoint on Tuberculosis, includes:
What is the incidence and prevalence?
What are the symptoms?
How is it diagnosed?
How is it treated?
What are the treatment guidelines?
The Evaluation of the Speed-Oligo® Mycobacteria Assay for Identification of M...CrimsonpublishersCJMI
The Evaluation of the Speed-Oligo® Mycobacteria Assay for Identification of Mycobacterium spp. from Smear Positive and Negative Sputum Samples by Gülnur Tarhan in ohesive Journal of Microbiology & Infectious Disease
Abdominal Tuberculosis – How Far are Our Diagnostics Illuminating?Apollo Hospitals
Tuberculosis can involve any part of the gastrointestinal tract from mouth to anus, the peritroneum, pancreas and the hepatobiliary system. Gastrointestinal tuberculosis mimics many clinical conditions and only a high degree of suspicion can help in the diagnosis otherwise there are chances of missing it leading to high morbidity and mortality. Various methods of diagnosis are available but which one is the right test for a particular patient needs to be ascertained. Culture remains the gold standard method of diagnosis. Fast track cultures like MGIT/M Bact Alert 3 D can give faster results with in few days to few weeks. Molecular tests are fastest and can be used as a supplementary test. Nested PCR can give results with in few hours.
Genotyping of 27 Human Papillomavirus Types by Using L1 Consensus PCR Product...Alberto Cuadrado
Amplification of human papillomavirus (HPV) DNA by L1 consensus primer systems (e.g., MY09/11 or
GP51/61) can detect as few as 10 to 100 molecules of HPV targets from a genital sample. However, genotype
determination by dot blot hybridization is laborious and requires at least 27 separate hybridizations for
substantive HPV-type discrimination. A reverse blot method was developed which employs a biotin-labeled
PCR product hybridized to an array of immobilized oligonucleotide probes. By the reverse blot strip analysis,
genotype discrimination of multiple HPV types can be accomplished in a single hybridization and wash cycle.
Twenty-seven HPV probe mixes, two control probe concentrations, and a single reference line were immobilized
to 75- by 6-mm nylon strips. Each individual probe line contained a mixture of two bovine serum albuminconjugated
oligonucleotide probes specific to a unique HPV genotype. The genotype spectrum discriminated on
this strip includes the high-risk, or cancer-associated, HPV genotypes 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 55,
56, 58, 59, 68 (ME180), MM4 (W13B), MM7 (P291), and MM9 (P238A) and the low-risk, or non-cancerassociated,
genotypes 6, 11, 40, 42, 53, 54, 57, 66, and MM8 (P155). In addition, two concentrations of b-globin
probes allowed for assessment of individual specimen adequacy following amplification. We have evaluated the
performance of the strip method relative to that of a previously reported dot blot format (H. M. Bauer et al.,
p. 132–152, in C. S. Herrington and J. O. D. McGee (ed.), Diagnostic Molecular Pathology: a Practical Approach,
(1992), by testing 328 cervical swab samples collected in Digene specimen transport medium (Digene Diagnostics,
Silver Spring, Md.). We show excellent agreement between the two detection formats, with 92%
concordance for HPV positivity (kappa 5 0.78, P < 0.001). Nearly all of the discrepant HPV-positive samples
resulted from weak signals and can be attributed to sampling error from specimens with low concentrations
(<1 copy/ml) of HPV DNA. The primary advantage of the strip-based detection system is the ability to rapidly
genotype HPVs present in genital samples with high sensitivity and specificity, minimizing the likelihood of
misclassification.
This is a research article presentation. I have prepared an original article for power point presentation, it will be helpful for you all to know how to present an original article on journal club.
Before doing research in any field it is very important to know the way of writing a research article . We should know which different points to remember at the time of research paper presentation .I have included all the headings which fulfill all the demands of a better way to present a research article on journal club.
Oxygen Therapy is not Beneficial in COPD Patients with Moderate HypoxaemiaGamal Agmy
A Randomized Trial of Long-Term Oxygen for COPD with Moderate Desaturation
The Long-Term Oxygen Treatment Trial Research Group*
N Engl J Med. 2016 October 27; 375(17): 1617–1627
Best Ayurvedic medicine for Gas and IndigestionSwastikAyurveda
Here is the updated list of Top Best Ayurvedic medicine for Gas and Indigestion and those are Gas-O-Go Syp for Dyspepsia | Lavizyme Syrup for Acidity | Yumzyme Hepatoprotective Capsules etc
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Muktapishti is a traditional Ayurvedic preparation made from Shoditha Mukta (Purified Pearl), is believed to help regulate thyroid function and reduce symptoms of hyperthyroidism due to its cooling and balancing properties. Clinical evidence on its efficacy remains limited, necessitating further research to validate its therapeutic benefits.
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
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
Basavarajeeyam is a Sreshta Sangraha grantha (Compiled book ), written by Neelkanta kotturu Basavaraja Virachita. It contains 25 Prakaranas, First 24 Chapters related to Rogas& 25th to Rasadravyas.
- Video recording of this lecture in English language: https://youtu.be/kqbnxVAZs-0
- Video recording of this lecture in Arabic language: https://youtu.be/SINlygW1Mpc
- 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
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
Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
Local Advanced Lung Cancer: Artificial Intelligence, Synergetics, Complex Sys...Oleg Kshivets
Overall life span (LS) was 1671.7±1721.6 days and cumulative 5YS reached 62.4%, 10 years – 50.4%, 20 years – 44.6%. 94 LCP lived more than 5 years without cancer (LS=2958.6±1723.6 days), 22 – more than 10 years (LS=5571±1841.8 days). 67 LCP died because of LC (LS=471.9±344 days). AT significantly improved 5YS (68% vs. 53.7%) (P=0.028 by log-rank test). Cox modeling displayed that 5YS of LCP significantly depended on: N0-N12, T3-4, blood cell circuit, cell ratio factors (ratio between cancer cells-CC and blood cells subpopulations), LC cell dynamics, recalcification time, heparin tolerance, prothrombin index, protein, AT, procedure type (P=0.000-0.031). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and N0-12 (rank=1), thrombocytes/CC (rank=2), segmented neutrophils/CC (3), eosinophils/CC (4), erythrocytes/CC (5), healthy cells/CC (6), lymphocytes/CC (7), stick neutrophils/CC (8), leucocytes/CC (9), monocytes/CC (10). Correct prediction of 5YS was 100% by neural networks computing (error=0.000; area under ROC curve=1.0).
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
2. Updates in Diagnosis of TB
By
Gamal Rabie Agmy , MD , FCCP
Professor of Chest Diseases ,Assiut University
ERS National Delegate of Egypt
3.
4. Diagnosis of TB
Direct Methods
1-Direct Microscopy (ZN, Kinyoun, Flurochrome).
2-Culture (Traditional, Rapid methods).
3- Detection of DNA or RNA of mycobacterial
origin ( PCR, LAMP, TAA / NAA, LCR, Fast Plaque).
7. Direct Microscopic
Examination
*Hallmark of staining is Ziehl-Neelsen stained slides.
Easiest & quickest diagnostic test.
* Limited sensitivity (46-78%) but specificity is virtually
100%.
* Centrifugation & flurochrome staining (auramine O) UV
microscopy markedly increase the sensitivity & a large
number can be examined in a much shorter time.*
*Chest 1969;95:1193
8. Direct Microscopic Examination
ZN staining requires = 105 bacilli/ml.
TB bacilli appear as straight/curved rods (1-
4μ x 0.2-0.8μ) singly, in pairs or in clumps.
The yield of microscopic examination
correlates well with the extent of disease, the
presence of cavitation, and the quality of
specimen.
It is a good marker for infectiousness & the
response to the treatment.
9. Zeil-Neelson Staining
Wire 0.01 ml of specimen 200mm2 slide
Oil immersion field 0.02mm
Slide=10000 field=0.01ml specimen
10,000 organism/slide=1 AFB/field=1000,000 organism/ml
1000 organism/slide=1 AFB/10 field=100,000 organism/ml
100 organism/slide=1 AFB/100field=10,000 organism/ml
10.
11. QUANTITATION SCALE FOR ACID-FAST BACILLUS
SMEARS ACCORDING TO STAIN USED
Carbolfuchsin (× 1,000)
Fluorochrome
(× 250)
Quantity Reported
No AFB/300 fields No AFB/30 fields No AFB seen
1-2 AFB/300 fields 1-2 AFB/30 fields Doubtful, repeat
test
1-9 AFB/100 fields 1-9 AFB/10 fields Rare (1+)
1-9 AFB/10 fields 1-9 AFB/field Few (2+)
1-9 AFB/field 10-90 AFB/field Moderate (3+)
> 9 AFB/field > 90 AFB/field Numerous (4+)
12. Several approaches are being made to
enhance the
sensitivity of the smear microscopy :
* Concentration of sputum sample by centrifugation
enhances sensitivity to almost 100%.*
* Treatment of sputum samples with Zwitterionic
detergent, also known as C18 carboxyprophylbetaine(
CB18) interferes with the innate buoyancy of the
bacilli and enhances the result of sputum
microscopy.**
*J Clin Microbiol1999;31:2371
**J Clin Microbiol1998;36:1965
13. Traditional Culture
More sensitive & can be positive even when
bacterial load is low (10-100 bacilli/ml).
Sensitivity 80-85%; Specificity 98%.
Required for precise identification of causative
organisms.
3 Types of media are used:
* Egg based: LJ, Petragnani and ATS.
*Agar based: Middlebrook 7H10 or 7H11.
*Liquid based: Kirschner’s, Middlebrook 7H9.
Growth is slow and takes 6-8 weeks . There after
the same length of time is required for complete
identification & sensitivity testing.
14.
15. Broth Based Rapid Culture Methods
Micro colony detection on solid media.
Radiometric (BACTEC).
Septicheck AFB.
Mycobacterial growth indicator tubes (MGIT).
Substantial improvement in time to detection &
total number of positive cultures can be realized
from using broth based systems.
16. Micro colony Detection on Solid Media
Plates poured with thin layer of Middlebrook 7H11 agar
medium are incubated and examined microscopically on
alternate days for the first 2 days and less frequently
thereafter.
In less than 7 days micro-colonies of slow growing
mycobacteria such as M.tb can be detected.
17. BACTEC
Growth is ascertained by liberation
of 14CO2 as metabolized by
mycobacteria & detected by BACTEC
460 instrument & reported in terms of
growth index (GI) value
18. BACTEC
Average time to recovery of M.tb from smear positive
specimens is 8 days.
When smear negative, culture positive samples are
examined, mean time for detection is 14 days.
More sensitive than traditional method.*
Can also be used for drug susceptibility testing.
*J Clin Microbiol 1994;32: 918-925
19. BACTEC
A special procedure unique to BACTEC system
for identification of M.tb complex is based on
observation that p-nitro-α-acetylamino-β-
hydroxypropiophenone (NAP) will inhibit organisms
belonging to M.tb complex while having little or no
effects on other mycobacteria.
Drawbacks :
Cost.
Problem of disposal of radioactive waste.
20.
21. Septicheck AFB
Combines broth & solid media into a single device
(biphasic culture approach).
Contains 30ml of modified Middlebrook 7H9 broth
in CO2 enriched culture bottle & a peddle with agar
media- one side of peddle covered with Middlebrook
7H11; other side contains Middle brook 7H11 with NAP.
Requires 3 weeks of incubation
Advantage: Simultaneous detection of Mtb. NTM, other
respiratory pathogen & even contaminant.
22. Mycobacterial Growth Indicator Tube (MGIT)
Rapid Method.
Consists of round bottom tubes containing 4 ml of
modified Middlebrook 7H9 broth which has an oxygen
sensitive fluroscent sensor at the bottom.*
When mycobacteria grow, they deplete the dissolved
oxygen in the broth & allow the indicator to fluoresce
brightly in a 365nm UV light.
* J Clin Microbiol 1999;37: 748-752
23.
24. Mycobacterial Growth Indicator Tube (MGIT)
Positive signals are obtained in 10-12 days.
MGIT can also be used as a rapid method for the
detection of drug resistant strains of Mtb directly from acid-fast
smear positive samples as well as from indirect drug
susceptibility studies.
Advantages over BACTEC
Cheaper.
No problem of radioactive waste disposal.
J Clin Microbiol 1999;37: 45-48
25. Detection and identification of
mycobacteria directly
from clinical samples
Genotypic Methods :
PCR
LAMP
TMA / NAA
Ligase chain reaction
Phenotypic Methods :
FAST Plaque TB
26. Polymerase Chain Reaction (PCR)
Essentially PCR is a way to make millions of identical
copies of a specific DNA sequence , which may be a
gene, or a part of a gene, or simply a stretch of
nucleotides with a known DNA sequence, the function of
which may be unknown.
A specimen that may contain the DNA sequence of
interest is heated to denature double stranded DNA.
27. Polymerase Chain Reaction (PCR)
Specific synthetic oligonucleotide primers bind to the
unique DNA sequences of interest and a heat stable
DNA polymerase (Thermus aquaticus) extends the
primer to create a complete & complimentary strand of
DNA.
This process is repeated sequentially 25-40 times,
thereby creating millions of copies of target
sequence.
28. Polymerase Chain Reaction (PCR)
Kd antigen 65(HSPs):
Used earlier
Heat shock protein believed to be distinct from other
bacterial HSPs.
This gene is identical in all species of mycobacteria.
Therefore unsuitable for detecting M.tb, particularly in
areas where species like M.avium or M.kansasii are
prevalent.
29. IS6110 :
It is a transposon which are
self replicating stretches of DNA.
Function not known.
This sequence has been found
in the M.tb complex organisms
(M.tb, M.africanum, M.microti,
M.bovis).
IS6110 sequence generally
occurs only once in M.bovis but is
found as often as 20 times in
certain strains of M.tb, thus offering
multiple targets for amplification.
30. Polymerase Chain Reaction (PCR)
With recent modification PCR can detect even a fraction
of a bacilli.
Role in pulmonary TB :
Detects nearly all smear +ve and culture +ve cases.
Useful technology for rapid diagnosis of smear –ve cases.
Able to identify 50-60% of smear -ve cases; this would
reduce the need for more invasive approaches to smear - ve
TB.
31. Distinguish M.tb from NTM in smear +ve cases as
IS6110 sequence is not found in NTM.*
Should not be used to replace sputum microscopy.
Sensitivity, specificity, & PPV for PCR is 83.5%,
99% & 94.2% respectively.**
*Am Rev Respir Dis 1991; 144:1160
** J Clin Microbiol 1999;31: 2049-2055
32. Polymerase Chain Reaction (PCR)
Role in Extrapulmonary TB
Limited Role
No comprehensive large series comparing the yield of
PCR with other available approaches has been published.
But at present, it is valuable adjunct in the diagnosis
of TB pleurisy, pericardial TB & other condition in which
yield of other tests are low.
33. Polymerase Chain Reaction (PCR)
Disadvantages :
Very high degree of quality control required.
Variation from lab to lab remain significant.
In pts. on ATT, PCR should not be used as an
indicator of infectivity as this assay remains +ve
for a greater time than do cultures.*
High false +ve results in patients previously treated with
ATT in contacts of sputum +ve active cases.
High Cost.
So, better understanding of how to use these tests in
conjunction with available clinical information is essential.*
Am J Respir Crit Care Med 1997;155: 1804-1854
34. LAMP*
Loop-mediated isothermal amplification.
It is a novel nucleic acid amplification method in which
reagents react under isothermal conditions with high
specificity, efficiency, and rapidity.
LAMP is used for detection of M.tb complex, M.avium,
and M.intracellulare directly from sputum specimens as well
as for detection of culture isolates grown in a liquid medium
(MGIT) or on a solid medium (Ogawa’s medium).
*Iwamoto T et al J Clin Microbiol 2003;41 :2616-2619
35. LAMP
This method employs a DNA polymerase and a set
of four specially designed primers that recognize a total
of six distinct sequences on the target DNA.
Species-specific primers were designed by
targeting the gyrB gene.
Simple procedure, starting with the mixing of all
reagents in a single tube, followed by an isothermal
reaction during which the reaction mixture is held at
63°C.
60- min incubation time.
36. LAMP
Due to its easy operation without
sophisticated equipment, it will be simple
enough to use in:
Small-scale hospitals,
Primary care facilities
Clinical laboratories in developing countries.
Difficulties :
Sample preparation
Nucleic acid extraction
Cross-contamination.
37. TMA / NAA
Transcription Mediated Amplification (TMA).
Nucleic Acid Amplification (NAA).
These techniques use chemical rather than
biological amplification to produce nucleic acid.
Test results within few hours.
Currently used only for respiratory specimens.
38. Ligase Chain Reaction
It is a variant of PCR, in which a pair of
oligonucleotides are made to bind to one of the DNA target
strands, so that they are adjacent to each other.
A second pair of oligonucleotides is designed to
hybridize to the same regions on the complementary DNA.
39. Ligase Chain Reaction
The action of DNA polymerase and ligase in the
presence of nucleotides results in the gap between
adjacent primers being filled with appropriate nucleotides
and ligation of primers.
It is mainly being used for respiratory samples, and has
a high overall specificity and sensitivity for smear +ve and –
ve specimens.
40. FAST Plaque TB
It is an original phage based test.
It uses the mycobacteriophage to detect the presence
of M.tb directly from sputum specimens.
It is a rapid, manual test, easy to perform and has a
higher sensitivity than microscopy, in newly diagnosed
smear +ve pts.*
*Int J Tuberc Lung Dis 1998;2: 160
45. TB STAT-PAK
Immuno-chromatographic test.
Has been evolved with a capability to differentiate
between active or dormant TB infection in whole blood,
plasma or serum.
Its value in disease endemic countries is yet to be
ascertained.*
*Eur Resp J 1995;8: 676
46. Antibody detection by ELISA
Several serodiagnostic tests, principally those using
ELISA methodology for measurement of IgG Ab are
available.
38-Kd Ag provides serodiagnostic test with most
favorable test characteristics described, but is limited by
the lack of purified Ag.
Serum IgG Ab are observed to rise during the first 3
months of therapy but fall after 12-16 months.
47. Antibody detection by
ELISA.
Other purified antigens to which
antibodies are detected
:
30 Kd protein antigen
16 Kd heat-shock antigen
Lipoarabinomannan(LAM) – LAM is a
complex glycolipid associated with cell
wall of mycobacteria & is produced in
substantial quantities by growing M.tb.
A60 antigen
ES31/41 antigen
48. Antibody detection by ELISA
.
IgM Ab levels have usually been found to be so low
that their reliable measurement has been difficult.
Serodiagnosis with crude Ag gives high false
positive results.
These tests lack specificity because polyclonal Ab
are used.
Use of monoclonal antibodies have increased their
specificity.
49. Antibody detection by ELISA
.
It takes several months after diagnosis for patients with
pulmonary TB to reach maximum antibody titers so that
serodiagnosis appears to be more useful in chronic
extrapulmonary disease (bone or joint) than in acute
forms (miliary, TBM).
Serodiagnosis also has limited utility in smear
negative patients with minimal PTB; In pediatric TB & in
disease endemic countries with high infection rates.
50. Insta test TB
It is a rapid in vitro assay for the detection of
antibody in active TB disease using whole blood or
serum.
The test employs an Ab binding protein
conjugated to a colloidal gold particle and a unique
combination of TB Ags immobilized on the
membrane.*
*Tuberc. Lung Dis 1998;2: 541
51. TB MPB 64 patch test
MPB 64 is a specific mycobacterial antigen for M.tb
complex.
This test becomes +ve in 3-4 days after patch
application and lasts for a week.
Specificity~100%, Sensitivity~98.1%.*
This promising test has been reported so far only in
one setting in Philippines and needs to be carried out in
other settings.
*Ind J Tuberc Lung Dis 1998;2: 541
52. Quantiferon-GOLD
Due to advances in
molecular biology and
genomics, an alternative has
emerged for the first time in the
form of a new class of in vitro
assays that measure interferon
(IFN-γ) released by sensitized T
cells after stimulation by
M. tuberculosis antigens.
Measures immune reactivity
to M.tb.
53. Quantiferon-GOLD
Interferon-γ assays measure cell-mediated
immunity by quantifying IFN-γ released from sensitized
T cells in whole blood/PBMCs incubated with TB
antigens.
54. Early assays employed PPD (same specificity problems
as the TST).
Newer assays (e.g., QFT-Gold) employ TB-specific
antigens: ESAT-6 and CFP-10.
Proteins encoded within the region of difference 1 of
M.tuberculosis.
Not shared with the BCG sub-strains and most NTM
(except: M. kansasii, M. szulgai, M. marinum and nonpathogenic
M.bovis).
Quantiferon-GOLD
55. Improved specificity: able to distinguish between TB and
NTM, BCG infection.
Studies in contacts, HIV infected and children underway.
Recommended for use in “ALL circumstances in which the
tuberculin skin test is currently used”.*
Includes contact investigations, immigrant evaluation,
surveillance (e.g. healthcare workers).
*Mazurek et al MMWR 2005;54:15
Quantiferon-GOLD
56. IGRAs Vs. TST
TST
In vivo
Single antigen
Boosting
2 patient visits
Inter-reader variability
Results in 2-3 days
Read in 48-72 hrs
IGRAs
In vitro
Multiple antigens
No boosting
1 patient visit
Minimal inter-reader variability
Results in 1 day
Stimulate w/i 12 hrs
57. IGRAs Vs. TST
QFT-g vs. TST Agreement = 83.6%*
Factors associated with discordance :
– Prior BCG
– Non-tuberculous mycobcateria immune reactivity
– Site bias in reading TST
– TB Treatment
*Mazurek et al JAMA 2001;286:1740
58. Biochemical markers of Diagnosis
Adenosine deaminase. (ADA)
Bromide partition test.
Gas chromatography of mycobacterial fatty
acids (Tuberculostearic acid).
59. Adenosine Deaminase (ADA)
It is an enzyme of purine metabolism. The level of this
enzyme is 10 times higher in lymphocytes (T cells >B
cells) than in RBC.
Whenever there is cell mediated immune response to an
antigenic stimuli, the ADA levels are the highest.
ADA is measured by the colorimetric method of Giusti.
60. enzymatic reaction is:
Adenosine + H2O + ADA = Inosine + NH3 +ADA
The amount of ammonia liberated is measured by
the colorimetric method.
Cut-off Sensitivity Specificity
Pleural Fluid 50 IU/ml 95% 100%
Ascitic Fliud 32.3 IU/ml 89% 98%
CSF 9 IU/ml 100% 100%
61. Bromide Partition Test
The partition of bromide ion between serum and CSF
after a loading dose reflects the integrity of the blood
brain barrier.
Either by direct chemical measurement or by using an
isotopic tracer, the ratio of bromide in serum to that in
CSF can be estimated.
Values <1.6 are characteristic of TBM.
62. In different studies the sensitivity and specificity of this
test has been found to be near 90%.*
It may be false +ve in herpes simplex, listeria, mumps,
measles, pyogenic meningitis and hypothyroidism.
With the availability of better tests, this test has been
given up.
*Taylor J et al. J Clin Microbiol 1999; 34: 56-59
63. Tuberculostearic Acid (TBSA)
TBSA is found in the cell wall of mycobacterium.
It is identified by gas chromatography or mass
spectrophotometry.
It is a costly investigation and requires complex
analytical equipment. (Seldom used)
Sensitivity >95%,Specificity>99%.*
*French M et al. J Clin Microbiol 1998; 54: 987-990
64.
65. CT Scan and MRI Scan in the
diagnosis of TB
The advent of CT and MRI imaging in the last
two decades has redefined the approach in analysis
of various diseases including TB.*
CT and MRI have shown several advantages
over conventional radiology in early diagnosis and
follow-up of TB in different parts of the body.
*Buxi TBS Indian J Pediatr 2002;69:965-972