This document discusses various laboratory methods for diagnosing tuberculosis (TB), including:
- Sputum smear microscopy to detect acid-fast bacilli, the most common initial diagnostic method.
- Nucleic acid amplification tests like PCR and GeneXpert that can rapidly detect TB in sputum through DNA amplification.
- Culture-based methods grown on solid or liquid media to isolate Mycobacterium tuberculosis from clinical samples, which is then tested for drug susceptibility.
- Immunological tests like interferon-gamma release assays that detect TB infection by measuring T-cell responses to TB antigens.
It provides details on the principles, advantages, and limitations of different microbiological, molecular,
The PPT is mainly all about Mycobacterium Tuberculosis. Agents causing the disease Tuberculosis, pathogenesis, laboratory diagnosis, treatment and prophylaxis. It was made for both BSc and MSc students.
The PPT is mainly all about Mycobacterium Tuberculosis. Agents causing the disease Tuberculosis, pathogenesis, laboratory diagnosis, treatment and prophylaxis. It was made for both BSc and MSc students.
Cryptococcosis also called as Torulosis is a subacute or chronic fungal infection caused by Cryptococcus neoformans. It leads to compications such as fatal meningoencephalitis. It is an opportunistic infection in HIV-infected patients. The PPT discuss on the morphology of the fungus, pathogenesis, laboratory diagnosis and treatment.
Catridge based nucleic acid amplification test(CBNAAT) / RIF assay gene xpert POWER PONT. other normal tests versus CBNAAT. issues for cbnaat by WHO & CONCLUSION.
Childhood TB was written to enable healthcare workers to learn about the primary care of children with tuberculosis. It covers: introduction to TB infection, the clinical presentation, diagnosis, management and prevention of tuberculosis in children.
Cryptococcosis also called as Torulosis is a subacute or chronic fungal infection caused by Cryptococcus neoformans. It leads to compications such as fatal meningoencephalitis. It is an opportunistic infection in HIV-infected patients. The PPT discuss on the morphology of the fungus, pathogenesis, laboratory diagnosis and treatment.
Catridge based nucleic acid amplification test(CBNAAT) / RIF assay gene xpert POWER PONT. other normal tests versus CBNAAT. issues for cbnaat by WHO & CONCLUSION.
Childhood TB was written to enable healthcare workers to learn about the primary care of children with tuberculosis. It covers: introduction to TB infection, the clinical presentation, diagnosis, management and prevention of tuberculosis in children.
Childhood TB was written to enable healthcare workers to learn about the primary care of children with tuberculosis. It covers: introduction to TB infection, the clinical presentation, diagnosis, management and prevention of tuberculosis in children
Undergraduate level Presentation on Childhood Tuberculosis based on WHO guidelines, local Myanmar guidelines, Nelson Textbook of Paediatrics and WHO training modules.It would be mostly appropriate for countries with high Tuberculosis burden.
Sources specified. The original sources of some photos could not be mentioned due to space limitations. I deeply apologize for that.
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
India has the largest burden of tuberculosis. The disease is gradually extending its storm into the paediatric age group, the manifest in which is severe and tortous. So a preventive approach is always better than a curative approach
Information about diagnostic methods and techniques for tuberculosis including microscopy, fluorescence microscopy, mycobacterial culture, molecular techniques (line probe assay, Xpert MTB/RIF), interferon gamma release assay (IGRA) and tuberculin skin test (TST)
Similar to Laboratory diagnosis of Tuberculosis gs (20)
CHAPTER 1 SEMESTER V - ROLE OF PEADIATRIC NURSE.pdfSachin Sharma
Pediatric nurses play a vital role in the health and well-being of children. Their responsibilities are wide-ranging, and their objectives can be categorized into several key areas:
1. Direct Patient Care:
Objective: Provide comprehensive and compassionate care to infants, children, and adolescents in various healthcare settings (hospitals, clinics, etc.).
This includes tasks like:
Monitoring vital signs and physical condition.
Administering medications and treatments.
Performing procedures as directed by doctors.
Assisting with daily living activities (bathing, feeding).
Providing emotional support and pain management.
2. Health Promotion and Education:
Objective: Promote healthy behaviors and educate children, families, and communities about preventive healthcare.
This includes tasks like:
Administering vaccinations.
Providing education on nutrition, hygiene, and development.
Offering breastfeeding and childbirth support.
Counseling families on safety and injury prevention.
3. Collaboration and Advocacy:
Objective: Collaborate effectively with doctors, social workers, therapists, and other healthcare professionals to ensure coordinated care for children.
Objective: Advocate for the rights and best interests of their patients, especially when children cannot speak for themselves.
This includes tasks like:
Communicating effectively with healthcare teams.
Identifying and addressing potential risks to child welfare.
Educating families about their child's condition and treatment options.
4. Professional Development and Research:
Objective: Stay up-to-date on the latest advancements in pediatric healthcare through continuing education and research.
Objective: Contribute to improving the quality of care for children by participating in research initiatives.
This includes tasks like:
Attending workshops and conferences on pediatric nursing.
Participating in clinical trials related to child health.
Implementing evidence-based practices into their daily routines.
By fulfilling these objectives, pediatric nurses play a crucial role in ensuring the optimal health and well-being of children throughout all stages of their development.
The dimensions of healthcare quality refer to various attributes or aspects that define the standard of healthcare services. These dimensions are used to evaluate, measure, and improve the quality of care provided to patients. A comprehensive understanding of these dimensions ensures that healthcare systems can address various aspects of patient care effectively and holistically. Dimensions of Healthcare Quality and Performance of care include the following; Appropriateness, Availability, Competence, Continuity, Effectiveness, Efficiency, Efficacy, Prevention, Respect and Care, Safety as well as Timeliness.
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Global launch of the Healthy Ageing and Prevention Index 2nd wave – alongside...ILC- UK
The Healthy Ageing and Prevention Index is an online tool created by ILC that ranks countries on six metrics including, life span, health span, work span, income, environmental performance, and happiness. The Index helps us understand how well countries have adapted to longevity and inform decision makers on what must be done to maximise the economic benefits that comes with living well for longer.
Alongside the 77th World Health Assembly in Geneva on 28 May 2024, we launched the second version of our Index, allowing us to track progress and give new insights into what needs to be done to keep populations healthier for longer.
The speakers included:
Professor Orazio Schillaci, Minister of Health, Italy
Dr Hans Groth, Chairman of the Board, World Demographic & Ageing Forum
Professor Ilona Kickbusch, Founder and Chair, Global Health Centre, Geneva Graduate Institute and co-chair, World Health Summit Council
Dr Natasha Azzopardi Muscat, Director, Country Health Policies and Systems Division, World Health Organisation EURO
Dr Marta Lomazzi, Executive Manager, World Federation of Public Health Associations
Dr Shyam Bishen, Head, Centre for Health and Healthcare and Member of the Executive Committee, World Economic Forum
Dr Karin Tegmark Wisell, Director General, Public Health Agency of Sweden
Defecation
Normal defecation begins with movement in the left colon, moving stool toward the anus. When stool reaches the rectum, the distention causes relaxation of the internal sphincter and an awareness of the need to defecate. At the time of defecation, the external sphincter relaxes, and abdominal muscles contract, increasing intrarectal pressure and forcing the stool out
The Valsalva maneuver exerts pressure to expel faeces through a voluntary contraction of the abdominal muscles while maintaining forced expiration against a closed airway. Patients with cardiovascular disease, glaucoma, increased intracranial pressure, or a new surgical wound are at greater risk for cardiac dysrhythmias and elevated blood pressure with the Valsalva maneuver and need to avoid straining to pass the stool.
Normal defecation is painless, resulting in passage of soft, formed stool
CONSTIPATION
Constipation is a symptom, not a disease. Improper diet, reduced fluid intake, lack of exercise, and certain medications can cause constipation. For example, patients receiving opiates for pain after surgery often require a stool softener or laxative to prevent constipation. The signs of constipation include infrequent bowel movements (less than every 3 days), difficulty passing stools, excessive straining, inability to defecate at will, and hard feaces
IMPACTION
Fecal impaction results from unrelieved constipation. It is a collection of hardened feces wedged in the rectum that a person cannot expel. In cases of severe impaction the mass extends up into the sigmoid colon.
DIARRHEA
Diarrhea is an increase in the number of stools and the passage of liquid, unformed feces. It is associated with disorders affecting digestion, absorption, and secretion in the GI tract. Intestinal contents pass through the small and large intestine too quickly to allow for the usual absorption of fluid and nutrients. Irritation within the colon results in increased mucus secretion. As a result, feces become watery, and the patient is unable to control the urge to defecate. Normally an anal bag is safe and effective in long-term treatment of patients with fecal incontinence at home, in hospice, or in the hospital. Fecal incontinence is expensive and a potentially dangerous condition in terms of contamination and risk of skin ulceration
HEMORRHOIDS
Hemorrhoids are dilated, engorged veins in the lining of the rectum. They are either external or internal.
FLATULENCE
As gas accumulates in the lumen of the intestines, the bowel wall stretches and distends (flatulence). It is a common cause of abdominal fullness, pain, and cramping. Normally intestinal gas escapes through the mouth (belching) or the anus (passing of flatus)
FECAL INCONTINENCE
Fecal incontinence is the inability to control passage of feces and gas from the anus. Incontinence harms a patient’s body image
PREPARATION AND GIVING OF LAXATIVESACCORDING TO POTTER AND PERRY,
An enema is the instillation of a solution into the rectum and sig
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CRISPR-Cas9, a revolutionary gene-editing tool, holds immense potential to reshape medicine, agriculture, and our understanding of life. But like any powerful tool, it comes with ethical considerations.
Unveiling CRISPR: This naturally occurring bacterial defense system (crRNA & Cas9 protein) fights viruses. Scientists repurposed it for precise gene editing (correction, deletion, insertion) by targeting specific DNA sequences.
The Promise: CRISPR offers exciting possibilities:
Gene Therapy: Correcting genetic diseases like cystic fibrosis.
Agriculture: Engineering crops resistant to pests and harsh environments.
Research: Studying gene function to unlock new knowledge.
The Peril: Ethical concerns demand attention:
Off-target Effects: Unintended DNA edits can have unforeseen consequences.
Eugenics: Misusing CRISPR for designer babies raises social and ethical questions.
Equity: High costs could limit access to this potentially life-saving technology.
The Path Forward: Responsible development is crucial:
International Collaboration: Clear guidelines are needed for research and human trials.
Public Education: Open discussions ensure informed decisions about CRISPR.
Prioritize Safety and Ethics: Safety and ethical principles must be paramount.
CRISPR offers a powerful tool for a better future, but responsible development and addressing ethical concerns are essential. By prioritizing safety, fostering open dialogue, and ensuring equitable access, we can harness CRISPR's power for the benefit of all. (2998 characters)
3. Mycobacteria Tuberculosis
Non sporing, Non-capsulated. Weakly Gram Positive.
Strongly Acid Fast (due to mycolic acid in cell wall).
Lipid Rich Cell Wall (confers resistance to disinfectants,
detergents, common antibiotics and traditional stains).
Long Generation time.
Capable to grow intracellularly in unactivated alveolar
macrophages.
Primarily disease caused from host response to infection.
Humans are the only natural reservoir.
Person to person transmission, by infectious aerosols/droplets.
4. Lin P et al. J Immunol 2010
Clearance
Pulmonary TB
Low grade TB
“percolating
“Dormant infection
Septic TB
Miliary TB
Extrapulmonary TB
LTBI
active
TB
Spectrum of M. tuberculosis infection
5. Magnitude of the Problem
Source: WHO Geneva; WHO Report 2008: Global Tuberculosis Control; Surveillance, Planning and Financing
Global annual incidence = 9.1 million
India annual incidence = 1.9 million
India is 17th
among 22
High Burden
Countries (in terms of
TB incidence rate)
6. Evolution of TB Control in India
1950s-60s Important TB research at TRC and NTI
1962 National TB Control Program (NTCP)
1992 Program Review
only 30% of patients diagnosed;
of these, only 30% treated successfully
1993 RNTCP pilot began
1998 RNTCP scale-up
2001 450 million population covered
2004 >80% of country covered
2006 Entire country covered by RNTCP
16. Haematology
Complete Blood Count is usually Normal.
Usually moderate Normochromic or slightly
hypochromic anemia.
Anemia may be there due to the chronic
debiliting natureof the disease.
Megaloblastic Anemia (Macrocytes in blood) in
cases of abdominal TB with malabsorption.
Thrombocytosis may be seen.
ESR & CRP are Raised.
17. Tubercular Lymphadenitis
Needle aspiration(FNAC) is a good test for TB
lymphadenitis in HIV-infected persons
Can be done the same day in the health facility.
Has a low rate of adverse effects.
Has a high yield for diagnosing TB.
19. TB Meningitis
Subacute or chronic
Headache, fever, neck stiffness, decreasing mental
status
Lumbar puncture essential for diagnosis
CSF –
o clear or slightly turbid, forms fibrin coagulum on
standing.
o Raised white cell count (100-1000 lymp/ul)
o Elevated protein (>45mg/dl),
o Reduced glucose ( <50mg/dl)
20. Traditional Methods for
Diagnosis of Tuberculosis
Presumptive Definitive
1. Clinical
2. Radiological Isolation & Identification
3. AFB Microscopy of M. Tuberculosis
4. Tuberculin Test
5. Pathological
21. Mantoux Tuberculin Skin Test (TST)
0.1 ml of tuberculin purified protein derivative (-
PPD) into inner surface of forearm intradermally.
Read between 48-72 hrs.
A positive tuberculin skin test result is supportive
evidence in the diagnosis of TB in areas of low
prevalence (or no vaccination); however, a negative
tuberculin skin test result may occur in
approximately one third of patients.
24. A Negative Test Result could result from the following:
(1) Anergy secondary to immunosuppression or malnourishment;
(2) Recent infection;
(3) Circulating mononuclear cells suppressing the specifically
sensitized circulating T-lymphocytes in the peripheral blood; or
(4) Sequestration of purified protein derivative specific reactive T-
lymphocyte.
However, results of a tuberculin skin test is repeated 6 to 8 weeks
later would usually be positive.
False positive in Atypical mycobacterial infection and previous
BCG vaccination.
25. Role of Radiography
Chest X-Ray (CXR) can support a diagnosis of PTB
Not used routinely for follow-up
PTB can exist with normal CXR
Must be interpreted with other information
History and exam
Sputum smear results
Also useful in diagnosing other types of TB,
especially in bones, joints, and spine
26. Chest X-Ray of Patient with Active
Pulmonary Tuberculosis
27.
28. Sputum Examination
CASE FINDING TOOLS
Sputum examination -:- sputum smear examination by
direct microscopy is the method of choice.
Collection of sputum –
Day 1 Sample 1 - Patient provide an “on the spot” sample
Day 2 Sample 2 - Patient bring early morning sample.
29.
30. Slide reporting
The number of bacilli
seen in a smear reflects
disease severity &
patient infectivity.
The table shows the
standard method of
reporting using 100X
magnification (WHO).
Number of bacilliNumber of bacilli ResultResult
reportedreported
NO AFB per 100 oilNO AFB per 100 oil
immersion fieldimmersion field
00
1-9 AFB per 100 oil1-9 AFB per 100 oil
immersion fieldimmersion field
ScantyScanty
10-99 AFB per 100 oil10-99 AFB per 100 oil
immersion fieldimmersion field
+ (1+)+ (1+)
1-10 AFB per oil1-10 AFB per oil
immersion fieldimmersion field
+ + (2+)+ + (2+)
>10 AFB per oil>10 AFB per oil
immersion fieldimmersion field
+ + + (3+)+ + + (3+)
31. Smear and Culture
Direct examination by Zeihl-Neelsen
staining requires bacillary densities of
5000-10,000/mL
Culture requires a minimum of 10 to 100
viable bacilli.
36. Mycobacterial Culture
Reasons to request mycobacterial culture:
• Patient previously on anti-TB treatment (Relapse,
Defaulter)
• Still smear-positive after intensive phase of treatment
or after finishing treatment
• Symptomatic and at high-risk of MDR-TB
• To test fluids potentially infected with M.
tuberculosis
• Investigation of patients who develop active PTB
during or after IPT.
• TB in health workers
37. Culture Based Methods
1. Liquid Culture (e.g., automated mycobacteria
growth indicator tube) – Faster and more sensitive
than solid media)
2. Microscopic Observation Drug Susceptibility
Testing (DST) – Yields fasterb culture and DST
results than do liquid or solid media and is
inexpensive(Requires Skilled Technician to interpret)
3. Thin Layer Agar Methodology (same as above)
4. Calorimetric DST methods using redox tetrazolium
slats, or a nitrate reductase assay – Lower cost, less
time (Potential Biohazard)
38.
39. Eight Week Growth of Mycobacterium
tuberculosis on Lowenstein-Jensen Agar
40.
41. DST performed on all cultures
Tests for isoniazid, rifampicin, ethambutol, and
streptomycin
If found to be multi-drug resistant, then send for
additional testing for susceptibility to second-line
medicines
TB Drug Susceptibility Testing
(DST)
42. BACTEC 460 TB System
(radiometric)
Developed in 1969 by Deland and Wagner.
Principle –
BACTEC 12B vial, utilize 14C labeled substrate (Palmitic acid).
On inoculation, mycobacteria, grow and release 14CO2.
The BACTEC instrument measures quantitatively the
radioactivity on a scale ranging from 0-999, as GI (Growth
Indicator).
The daiy increase in GI is proportional to growth in the medium.
DST (Drug Susceptibility Test) – When ATT is introduced in
the medium, reduced production of 14CO2 & decrease in GI.
43.
44. New Approach in Diagnosis of TB
Replication of M. Tuberculosis
1. Antigen Detection Tests –
LAM ELISA Urinary Antigen Test(ELISA BASED TEST, detect
LAM, Antigen 85 – LipoArabinoMannon) still developing
Sputum Antigen Test
2. Microscopic Visualization of bacteria –
LED Microscopy
Bleach Microscopy
3. Culture based Detection Tests –
Microscopic observation drug susceptibility assays (MODS)
Thin Layer Agar
Phage based tests
Calorimetric media
45. Replication of M. Tuberculosis
4. Nucleic Acid Amplification Tests (NAATs)-
LAMP
GeneXpert MTB
Transrenal DNA detection
Genotype MTBDRPlus
(High Specificity and Positive Predictive Value)
5. Volatile Organic Compounds (VOC) detection-
E-nose
Biosensors
(Emitted from the infected cells & released in exhaled breath through nanomaterial
biosensors or Gas Chromatography)
46. Immune Response to M. Tuberculosis
I. Cellular Immune Response -
INF-Y release assays(IGRA)
Quanti-FELON TB gold
T-SPOT TB
Rd ESAT-6 skin test
II. Humoral Immnune Response –
Antibody Detection Tests
Serological tests
New Approach in Diagnosis of TB
47. Polymerase Chain Reaction
Polymerase chain reaction (PCR) is based on
amplification of mycobacterial DNA fragments.
It can detect as few as 10 mycobacteria.
Advantages of PCR include rapid diagnosis,
improved specificity and sensitivity, and no
requirement of intact immunity.
48. Molecular Beacon Assay(at MDL)
Target: DNA
Realtime PCR
PCR to amplify target sequences
At the same time, Molecular beacon probes are used to
detect INH and RIF resistance mutations.
2 MBs for INH (targeting katG & inhA)
3 MBs for RIF (targeting core of rpoB)
49. Real-Time PCR
2 components
PCR to amplify target sequences.
A system to monitor PCR product.
Fluorophore-labeled probes
An optical device to detect
fluorescence
Software to record data
No post-PCR manipulations
Fast
when PCR is done, results are ready
for interpretation.
No amplicon contaminations
iCycler
IQ5
50. What is a Molecular Beacon?
←Loop (15-30 nt)
←Stem (5-7 nt)
Fluorophore→ ←Quencher
Hair-pin structure
51. Molecular
Beacon (off)
Hybrid (Molecular Beacon - On)
Detection of Mutations with a Molecular Beacon
(Loop portion containing wildtype SQ)
Mutant Sequence
Wildtype Sequence
Amplicon
Heat Light
+
Courtesy of Dr. Probert
Loop
QuencherFluorophore
Fluorophore
52. An Example of a Good MB
No mutations,
Susceptible
Mutant,
Resistant
R
F
U
Threshold
53. Causes of false-positive results include DNA
contamination or presence of nonviable
organisms
The disadvantages of PCR include high cost,
risk of contamination, and the technology
involved in the procedure does not permit routine
diagnostic use at present.
54. 55Limitations
Limited genes & sites are targeted.
• Some mutations are not detected.
Emerging resistance in mixed populations may not be
detected.
Some mutations do not confer resistance.
• Rare occurring, but lead to wrong interpretation.
• Silent mutation in rpoB: codon 514.
• Not a silent mutation but only cause little change in MIC.
Available for INH and RIF only.
New MBs for other drugs not developed yet.
Phenotypic drug susceptibility testing is still needed.
58. Line Probe Assays
Target: DNA
Traditional PCR (not realtime)
Amplify target sequences.
Reverse hybridization
Amplicons hybridize to probes immobilized on membrane
(strip).
Colorimetric detection of captured amplicons on strip.
Observation of bands. One probe for one band.
60. 61
Conjugate ctrl
rpoB wild-type,
5 segments
4 rpoB mutations
rpoB universal ctrl
MTBDR by HAIN Lifescience LiPA RIF.TB by
INNOGENETICS
katG universal ctrl
katG wild-type
2 katG mutations
Universal ctrl
MTBC
516
526
531
315
marker line
MTBC
More probes are added in MTBDRsl to detected 2nd
-line drug R-mutations.
61.
62. 63
Line Probes Features
Many controls; more objective
MTBDRsl (HAIN) added embB, gyrA, rrs (screen for
XDR).
Exact mutations are available for most prevalent
mutations only.
Some mutations are detected by lacking bands in wild-
type sequences.
Emerging resistance in mixed populations may not be
detected.
Phenotypic drug susceptibility testing is still needed.
63. Loop Mediated Isothermal
Amplification(LAMP)of DNA
Small Heating Device
Runs at High Temperature(avoids nonspecific amplifications)
Multiple primers sets (increased specificity and speed)
Direct from Sputum.
Closed System (No risk of contamination)
Minimal instrumentation
Fast (Less than 2hrs total)
Visual Detection(no instrumentation- Mg2P2O7 ppt - white)
Test Under Development still…….
64.
65. Cytokine Assays
T- cell Interferon-Gamma Release Assay (IGRA)
INF- y produced by T-lymphocytes, is capable of
activating macrophages, increasing their bactericidal
capacity against M tuberculosis and is involved in
granuloma formation.
Elevated concentrations of INF-y in TB is related to
increased production at the disease site by effectors T
cells.
The sensitivity of an elevated level varies from 78 to
100% and specificity from 95 to 100%
66. IGRA is useful in targeted strategy for latent TB
infection(LTBI) detection in low TB incidence settings
More specific than Tuberculin Skin Test
Can’t distinguish active from treated TB or LTBI.
False positive results in-
Hematologic malignancies
Empyema.
Note :- Immunosuppressed patients (HIV or after renal
transplant) had INF- Y levels similar to immunocompetent
individuals retaining its efficacy as a diagnostic test
67. Methods for detection of IFN-y
Two new blood tests
1. T-SPOT.TB [Oxford Immunec] – directly count the no of
IFN-y secreting T cells.
2. QuantiFERON-TB Gold [Cellestis Limited] –
measures the concentration of IFNy secretion.
Both tests based on detection of IFN-y in blood have been found to be more accurate than the
tuberculin skin test in the diagnosis of latent TB infection. Future research should focus on
the potential efficacy of quantification of specifically activated lymphocytes in body fluid
and blood using IFN-Y release assay in the diagnosis of TB.
68.
69. Immunodiagnosis
Serological tests are simple to perform & can be developed
into a rapid method for wide screening.
Immuno- chromatographic card test –
Sensitivity 80 % & specificity 88 %.
Principle - the test employs antihuman Ig-G, A, M antibody
rabbit dye conjugate highly purified antigen A60 from
Mycobacterium bovis stain BCG (cell culture), fixed in the
test line, & anti-rabbit antibodies in the control line. As the
sample flows through the absorbent pad, human
immunoglobulins are bound by the antihuman Ig- dye
conjugates to form an immunocomplex. This binds to the
A60 proteins in the test line & produce a red violet test
line, if the anti- Tb antibodies were present in the sample.
In control line excess conjugate react with the anti rabbit
antibodies forming second red violet line to demonstrate the
correct function of the reagent.
70. Cytokines:
Significantly higher levels of IL-6 have been
demonstrated in TB (A Potent
Biomarker/Biosignature).
Furthermore, the serum/pleural fluid IL-6 ratio was
significantly higher in TB.
IL-1b and Tumor necrosis factor(TNFa), produced
predominantly by mononuclear phagocytes, have been
shown to be present in TB.
Levels of soluble IL-2 receptors, IL-18,
immunosuppressive acidic protein, and IL-12p40 are all
significantly elevated in TB
71. Adenosine DeAminase (ADA) in
Pleural Fluid
ADA levels in Pleural fluid are measured by
colorimetric method.
Increased ADA levels (>36IU/L) observed in
Tubercular Pleural Effusion.
Quick test.
Adjunct test to help rule in or rule out tuberculosis in
pleural fluid.
72.
73. Lysozyme
Lysozyme, a bacteriolytic enzyme, have been
found to be higher in patients with TB.
However, a fluid to serum lysozyme ratio > 1.2 has
been found to be a better tool for the diagnosis of
TB.
74. Conclusion
Newer Technologies offer a significant time savings. However
these tests have limitations –
1. Costly.
2. Complex & Cumbersome.
3. Only smear positive (50% of culture positives)
4. Recommended only in special cases.
5. Add-on tests.
Culture is still the Gold Standard.
As recommended by CDC/WHO
* Whenever possible, use liquid culture & DST.
* Rapid testing and reporting essential for TB control.
75. Progress in TB Diagnosis
Past Present
Koch discovered tubercle bacillus 133
yrs back
No major discovery
Except TB Genome, IS6110, BACTEC
460 (liquid media)
TB diagnosed by symptoms - prehistoric Still the same practice in many High
Bruden Countries (HBCs)
Tuberculin test - > 100yrs Still Commonly used
Egg based media –almost 100yrs Still most commonly used
AFB Smear for diagnosis – 133 yrs back Still the major diagnostic tool in many
countries
Radiological Diagnosis Still Important (X-ray, CT Scan)
Earlier known as Consumption (severe wt loss)/ phthisis pulmonaris and the white plaque( extreme pallor in infected) .
Pott’s disease (Egyptian mummies spinal tb) by archaeologist.
Mycobacterium tubercule bacilli demonstrated by Robert Koch(coke) in 1882. (Koch’s bacilli)—Nobel prize 1905.
Every third person is infected.
Generation time is simply the time it takes for one cell to become two.
M. Tb is slow growing, a nonchromogen, doesn’t grow at 25c or in PNB medium.
Follows iceberg phemomenon.. As only tip of iceberg is visible.. Part below the iceberg indicates a lot of dormant infection in individuals, requiring urgent need of screening clinical findings and investigations for finding the dormant infections
In 2008 WHO report-9.4 million new cases equivalents to 139 cases per 100,000 population of TB globally. 1.98 million were estimated to have occurred in India (0.87 million---infectious cases, fifth of the global burden of TB. About 40% of Indian population is infected with TB bacillus.
TRC- Tuberculosis research Centre, Chennai
NTI – National Tuberculosis Institute, Bengalaru
Still a third of world’s population has been exposed so called THE CAPTAIN OF ALL MEN OF DEATH(19TH century)
Red Arrow – GW parenchymal focus under the pleura(lower part – upper lobe)
Blue Arrow – Hilar lymph nodes with caseation
Characteristic Tubercle (Central granular caseation surrounded by epitheloid and MN giant cells) at -
Lower Magnification
Higher Magnification
C. Tubercular Granuloma in Immunocompromised patient with no central caseation
D. Sheets of foamy macrophages packed with mycobacteria.
Upper parts of both lungs are riddled with GW areas of caseation and multiple areas of softening and cavitation.
Cut surface show numerous GW to GY tubercles.
Isoniazid may cause sideroblastic anemia showing hypochromic microcytes to normochromic macrocytes, often with few stippled red cells.
Thrombocytosis due to increase no of small megakaryocytes in the marrow.
ESR depends more on amount of fibrinogen than the amount of globulins present in the plasma.
TB is the most common cause of adenopathy among HIV-infected patients in sub-Saharan Africa. When biopsy is necessary, excisional biopsy is preferred because incisional biopsies in the setting of TB carry a high risk for poor wound healing and/or chronic fistula formation
Epithelioid histiocytes (Epithelioid cells) are activated macrophages resembling epithelial cells: elongated, with finely granular, pale eosinophilic (pink) cytoplasm and central, ovoid nucleus (oval or elongate), which is less dense than that of a lymphocyte. They have indistinct shape contour, often appear to merge into one another and can form aggregates known as giant cells.
The test for cryptococcal meningitis is Indian Ink stain
Pleural or pericardial fluids are not very sensitive samples for the detection of M. tuberculosis.
Tuberculosis bacilli is a great IMITATOR & may simulate many other diseases like sarcoidosis, pneumoconiosis, neoplasms, lung abscess and fungal infection.
Only the induration which is hard, dense raised formation is measured. Erythema area is not measured.
Disseminated TB have negative mantoux b/c of release of large amount of tuberculoproteins from endogenous lesions maskinh Hypersensitivity rxn.
Anergy d/t sarcoidosis, viral, hodgkins ds, fulminant tb
Radiography has more than 90% sensitivity but only 65-70% specificity for detecting PTB. A chest x-ray is an important tool in supporting the diagnosis of PTB in symptomatic individuals whose sputum smears are negative for AFB but it is not possible to diagnose PB using chest x-rays only.
Therefore, always request sputum smear examination for all TB suspects.
Roentgen discovered Xray in 1895 (Nobel prize in 1901)
Radiographic appearances suggestive of active TB:
Shadows in one or both upper zones. Cavities in one or both upper zones. Miliary pattern. Persistent shadows after pneumonia treatment. Pleural effusion. Intrathoracic adenopathy. Pericardial effusion
A combination of any of the above, especially in HIV-infected patients
Earlier by fluoroscopy without film. Later with Xray films(around 1935s)
CT by Godrey in 1972(Nobel prize in 1979)
Later HRCT developed
Because increment yields from sputum specimens are small, WHO recommends examining 2 smears(workload less, less time diagnosis, drop out less)
A Minimum 5.0 ml of Sputum Improves the Sensitivity of Acid-fast Smear for Mycobacterium tuberculosis
ZN Technique 5min heated stain till cooling. Wash with clean water.5min for acid alcohol..1-2min for methylene blue..
Processing of sputum – use of NaOH 40g/L then centrifugation(18-23% more sensitive).
Scanty may be due to contamination from tap water/deionized water.
Never write negative.. Report as NO AFB SEEN
Cant reliably distinguish MTB from NTM
Red,straight slightly curved rods occurring singly or in small groups.. may appear beaded..
Fluorescence microscopy is 10% more sensitive than conventional microscopy.
Used to determine viability of M.Tb (auramine O stained) in follow up sputum specimens to treatment failure.
LED(light-emitting diode) light source used. Cheaper, last longer.Attractive alternative to mercury vapour lamps. Hazards of dye toxicity.
LJ egg medium – protein enriched media with optimum temp of 35-37c.
LJ is an egg based media with addition of salts, 5% glyecerol and Malachite green.
With the Lowenstein-Jensen (L-J) method, a positive result (growth of mycobacteria) is usually apparent after three weeks. If there is no growth by 8 weeks, the result is negative. Approximately 4 weeks from receipt of specimen to culture results
An additional 3-4 weeks for susceptibility results
Therefore minimum of 7-8 weeks for DST results
(Botswana National Tuberculosis Programme Manual, page 33-34)
Raised, dry cream (aka buff,rough and tough) coloured colonies. Visible colonies are usually produced –weeks after incubation but culture should be incubated for upto 6weeks before being discarded.
Nitrate reduction and niacin production are definitive for M.tb
DST is a routine performed on all positive cultures
MDR = specimen shown to be resistant to isoniazid AND rifampicin +/- any other drugs
Specimens are cultured in Mycobacteria Growth Indicator tube (MGIT) with liquid media (Middle brook7H9 broth base) containing C14 labelled palmitic acid, OADC enrichment and PANTA antibiotic mixture.
Specially designed to accommodate Mycobacteria Growth Indicator Tube and incubate them at 37c.Scans tubes every 60min for increased fluorescence.
CONTINUOUS MONITORING BLOOD CULTURING INSTRUMENTS.
MGIT 960 is a nonradimeteric system.
Antigen detection tests are almost absolute now
CBNAATs used in MYH.
LAMP – Loop Mediated Isothermal Amplification of DNA
(Phenotypic)Fast Plaque TB principle – mycobacteriophage based assay. Detects only live bacteria.
Immunochromatographic tests (Serological test) are absolute now.
Molecular beacon test goes beyond what MTD & Amplicor can offer. It provides Identification of MTB and drug susceptibility of INH and RIF.
There is tremendous impact on TB patient management and control.
I will discuss this in detail on the 2nd part of my talk.
Bought a backup iCycler, newer version, named iQ5.
MB is an oligonucleotide probe with a hair-pin or loop-and-stem structure.
When the MB is at “rest”, the fluorophore is at the close proximity of the quencher, therefore, it is not emitting fluorescence.
When the MB is in “action”, that is, when the target is 100% complementary to MB loop SQ, the MB will undergo a conformational change, from hairpin to linear structure, and hybridizes to its target. The conformational change forces the fluorophore away from the quencher and therefore, fluorescence will be emitted.
This structure creates a competition for annealing between the arms and between the MB and the target SQ. The MB loses its competition when mismatches exist between its loop and the target. This unique property renders MB a great discriminary power to detect a single-nucleotide mismatch.
Identify the bacilli while simultaneously identify drug resistamt strains by detecting the most common Single Nucleotide Polymorphisms(SNPs) associated with resistance.
Marketed as TB Gold.. Low specificity of IGRA and consequent unnecessary t/t. WHO discouraged this test in developing countries.
IGRA discontinued after 2012 as it cant differentiate Active from latent tb.
Absolute now
LAM in cell wall of M.Tb induce IL-6.
IL-6 is the most potent stimulator for hepatic synthesis of CRP which also has thrombopoietic activity.
ADA is an enzyme involved in purine metabolism. Asso with greater lymphocyte proliferation.
&gt;100IU/L is exclusively seen in tubercular pleural effusion.
ADA needed for breakdown of adenosine from food and for the turnover of nucleic acids in tissues.
Tulip Diagnostic ADA kit in MYH
IHC with an antibody to MPT64, a secreted antigen specific to the M. tuberculosiscomplex, is a specific and sensitive technique for diagnosis of EPTB.
We are still in need of a costeffective, handy and accurate test for the diagnosis of latent tb cases.
GeneXpert gave a hope.. Awaiting for even better technologies..