Upload
unittbjknphg
View
487
Download
3
Tags:
Embed Size (px)
Citation preview
MYCOBACTERIUM : DIAGNOSIS AND LABORATORY TEST
9/4/2013
1
Dr. Azura HussinPathology Department
HRPZ II
Contents
9/4/2013
2
Introduction General characteristics of tubercle bacilli Lab diagnosis of tuberculosis - current technologies - newer technologies - problems in lab diagnosis - quality control Summary
Introduction
9/4/2013
3
Tuberculosis appears to be a disease as old as human history
Tuberculosis is a social disease with medical implications and it is a major public health problem
It remains the leading cause of death by infectious disease.
Tubercle bacillus discovered - more than a hundred years ago
First discovered in 1882 by Robert Koch - "Koch's bacillus
Introduction
12/09/14
4
According to the WHO, there are approximately 20 million active cases in the world today, and they infect 50-100 million people largely children annually.
The mortality is approximately 3 million annually (at least 80% from developing countries)
Malaysia, the number of cases detected per year has not declined substantially either.
Since 1989, 11,500 to 12,000 cases per year were detected
In 1995, 11,778 cases detected of which 6,500 cases are sputum positive and therefore are infectious.
9/4/2013
5
Why so high? HIV/AIDS Emergence of drug resistance TB Migration ? Early diagnosis of tuberculosis initiating optimal treatment - enable a cure curb the transmission of infection and
disease to others in the community.
9/4/2013
6
9/4/2013
7
DIAGNOSIS
Clinical :
- Symptoms (prolonged cough, fever, night sweats, LOA / W) and
- Signs (can be subtle as in minimal cases or obvious such as
consolidation, fibrosis or stony dullness due to pleural effusion)
Radiological and/ or
Bacteriological evidence
The tuberculin or Mantoux test
12/09/14
8
9/4/2013
9
TB is diagnosed by finding Myco ba c te rium tube rc ulo s is bacteria in a clinical specimen taken from the patient.
Other investigations may strongly suggest tuberculosis as the diagnosis, they cannot confirm it.
A definitive diagnosis of tuberculosis can only be made by culturing Myco ba c te rium tube rculo s is organisms from a specimen taken from the patient (most often sputum, but may also include pus, CSF, biopsied tissue, etc.).
A diagnosis made other than by culture may only be classified as "probable" or "presumed".
Transmission
9/4/201310
Kingdom BacteriaPhylum ActinobacteriaClass ActinobacteriaSubclass ActinobacteridaeOrder ActinomycetalesSuborder CorynebacterineaeFamily MycobacteriaceaeGenus Mycobacterium
unique genus
Species M. tuberculosisM. bovisM. africanumM. microti"M. canettii”M. capraeM. pinnipedii
Lineage of the agents of Mycobacterium
9/4/2013
11
Intro duc tio n - Members of Genus Myco ba c te rium
9/4/2013
12
Mycobacterium
9/4/2013
13
Mycobacterium Tuberculosis Complex (MTC)
species are closely related genetically differ in host, geographic range, certain
phenotypes and pathogenicity.
Among MTC – Mycobacterium Tuberculosis (MTB) is the most significant pathogen for human.
Caused by a bacteria - Mycobacterium tuberculosis
Rod shaped – slender or slightly curve
Size : 2-4 um x 0.2-0.5 um Non-motile Non-spore forming Obligate aerobe - lung Intracellular parasite
(monocytes/macrophages) Weak Gram positive
9/4/2013
14
Grows slowly (15-20 hrs)Optimum T : 37 C
Colonies take 4-6 weeks to become visible on LJ media.
can withstand weak disinfectants and can survive in a dry state for weeks
9/4/2013
15
Stains poorly by Gram Stain-”Ghosts”
Use heated Ziehl-Neelsen Carbol-fuchsin
Resists decolorizing by Acid-Alcohol → Acid-Fast
Colony Morphology Growth on LJ medium, colonies are rough, flat
with a raised centre, wrinkled
buff coloured, younger colonies are paler
Organisms tends to grow in parallel groups producing the colonial characteristics of serpentine coding
9/4/2013
16
# Colonies of Mycobacterium tuberculosis on Lowenstein Jensen medium. Colonies are small and buff coloured.
# In vitro-grown colonies often form distinctive serpentine cords. This observation was first made by Robert Koch who associated cord factor with virulent strains of the bacterium.
# It takes 4-6 weeks to get visual colonies 9/4/2013
17
MYCOLATE
Mycobacteria Gram-negative organisms
arabinogalactan
lipids lipid bilayer peptidoglycan
acyl lipid + LPS
lipid bilayer
Mycobacteria produce a thick mycolate-rich outer covering which functions as an exceptionally efficient barrier.
Cell wall
Unique cell wallMore than 60% lipid
Mycolic acids are hydrophobicCord Factor is toxic to mammalian cells
inhibits migration of Polymorphic Neutrophils9/4/2013
18
Mycobacterium Cell Wall
9/4/2013
19
High concentration of lipids cause:Impermeability to stains/dyesResistance to many antibioticsResistance to kill by acidic and
alkaline environments, even intracellularly
Resistance to osmotic lysis by Complement Fixation
Resistance to lethal oxidants
Makmal adalah komponen Makmal adalah komponen utama dalam -TB Controlutama dalam -TB Control
TIADA MAKMAL
TIADA DIAGNOSIS
TIADA PENGUBATAN
TIADA DOTS
TIADA TB CONTROL12/09/1420
Peranan MakmalPeranan Makmal
• Mengesan penyakit berjangkit
• Memantau kesan perawatan
• Memastikan kesembuhan pesakit
12/09/1421
Network Of TB Laboratory (Organization of TB Laboratory Services)
9/4/2013
22
District Hospitals
General Hospitals (State)
Institute of Respiratory Medicine
(MKAK Sungai Buloh)
Specimen collection centers/ some are examination centers
Examination centers (D/S)
Examination centers (D/S)
AFB Culture centers
TB Reference Laboratory
D/S, C/S, ID, BCG Viability Tests
Data compilation
Training / Set procedures
Health Center
Laboratory diagnosis
9/4/2013
23 There are numbers of diagnostic tests : simple AFB microscopy to complex molecular
techniques; The diagnostic modalities desirable features: sensitivity, specificity, reproducibility, cost effectiveness, safety, simplicity and easy application for wider use Quantitative - so that the infectiveness of the
individual cases can be measured
Specimen Collection - TB
Best specimen comes from the lung.
Saliva or nasal secretions are unsatisfactory.
Collect in open space- not toilets / dark unventilated rooms
Remove dentures and rinse mouth with water.
Inhale deeply 2–3 times, breathe out hard each time.
Cough deeply from the chest.
Place the open container close to the mouth to collect the specimen.
9/4/2013
24
What is a good sample and how to obtain it?
Samples for TB smear and culture Types of samples Sputum* CSF Gastric washing* Lymph nodes Tissues Stool urine
9/4/2013
25
Number of samplesAt least 2 or 3 fresh purulent samples from lower respiratory tract collected as spot – morning - spot Transportation
Ideally within one working day
* Induction sputum : smear negative / unable to produced sputum* Gastric lavage/Bronchoscopy : may considered in unsuitable sputum induction
Three samples required (Different day): Clinic patient:
Spot– morning – spot
Hospitalized patient: 3 morning specimens (better)
Yield increases rapidly after three specimens
9/4/2013
26Examples of containers
Reduction of number of smears for the diagnosis of pulmonary TB, 2007
Can be reduced from three to two, in places where a well-functioning external quality
assurance (EQA) system exists, where the workload is very high and human resources are limited.
WHO recommends the number of specimens to be examined for screening of TB cases
12/09/14
27
Tests offered - KKM
9/4/2013
28
Smears microscopic – ZN / IF Culture, sensitivity and identification PCR Line Probe Assay
Quality sample = Quality smearQuality smear = Quality lab result
9/4/2013
29
Specimen Quality
9/4/2013
30
Saliva/ Induced Sputum Blood Stained
Specimen Quality
9/4/2013
31
PurulentMucoid
Contents of a Sputum Specimen
Saliva (soluble in saline)
Mucous (from trachea & bronchus)
Sputum cup
Matters from pulmonary lesion(++++ bacil l i)Cheesy -yellowishComposition of a good
Sputum Specimen9/4/201332
Quality of Sputum % AFBSALIVA 4.9SALIVA + MUCUS 7.7MUCOPURULENT 19.2PURULENT 39.1
Distribution of AFB in the sputum specimen
9/4/2013
33
9/4/2013
34
SMEAR EXAMINATION
Z-N Stain
Fluorescent Stain
Light MicroscopeFluorescent Microscope
Specimen
Systematic Examination of Smears
9/4/2013
35
3 cm
2 cm
Uniform Smear Size
9/4/2013
36
Why We Do Sputum Smear Examination?
9/4/2013
37
Diagnosis -sign and symptoms - Monitoring
Positive sputum –smear = Infectious case
This in not a sensitive technique- but rapid and specific
OBSERVATION OF STAINED SMEAR-International Standard-
Examine the smear under x100 objective with the 10 eye piece lens
Read at least 300 visual fields to give a report a negative
9/4/2013
38
3 cm = 150 visual field
1
2
Microscopy
9/4/2013
39 the simplest and most rapid procedure currently available to detect AFB in clinical specimens by ZN staining method.
Microscopy
9/4/2013
40
Albert e t a l., 2002. Sensitivity: 61.3-63.4%
Highest – patients with cavitary disease Lowest – patients with weak cough / less
advanced disease Specificity: 97.3-97.4% AFB smear microscopy in symptomatic
patients has high specificity (98%) Concentration of sputum sample by
cytocentrifugation has been found to enhance the sensitivity to almost 100%.
Microscopy - limitation
9/4/2013
41
impossible to distinguish different mycobacterial species.
Sputum : 5 000 – 10,000 tubercle bacilli/ml – smear positive.
Smear negative on expectorated sputum→ Cannot rule-out TB (ie extra pulmonary TB)
Smear negative/ unable to produce sputum: Sputum induction Fiberoptic bronchoscopy Gastric washing
Good Quality Staining of AFB
9/4/2013
42
AFB in Single ArrangementAFB in Single Arrangement
9/4/201343
AFB - in Various Arrangements
9/4/2013
44
AFB in Clumps AFB in Clumps
9/4/201345
12/09/1446
AFB Stain-LED Fluorescence Microscope
LED Fluorescent microscope Advantages: Covers 15 times as many fields
compared to ZN Same area can be covered at
a shorter time Examination of slides for 1.41
min by FM equivalent to 2.48 min by ZN
Heating not required More sensitive - low bacillary
content Increase sensitivity of
conventional light microscope of about 10%
Less expensive ( maintenance, dark room)
Disadvantages:
Handling & maintenance of optical equipment require advanced skill Periodic replacement of
bulbs Continuous supply of power High cost of microscope and
maintenance Doubtful smears to be re
examined by ZN
12/09/14
47
METHOD ADVANTAGES DISADVANTAGESTechniques;Ziehl-NeelsenKinyounFlourochrome
Rapid Simple/Easy to read Specificity >98%. Minimal infrastructure
required to set up Indicator of infectious
case Treatment monitoring Retrospective checking – cultures growing are AFB
Tedious Less sensit ive (45-60%) (>100,000 bacil l i /ml) Trained &
experience microscopist
Presumptive diagnosis of mycobacterial dz Can’t
differentiate between species Can’t be use for
DST
Microscopy
9/4/201348
Culture
Advantages For susceptibility
testing Able to identify the
species of mycobacterium
9/4/2013
49
Disadvantages
Slow grower : delay in the lab diagnosis / treatment LaboriousExperience staffSpecial equipment Expensive
9/4/2013
50
Culture should be attempted1. For Surveillance of drug resistance as an
integral part of evaluation of control program2. Smear negative pulmonary & extra
pulmonary3. Childhood tuberculosis4. Follow up of Tuberculosis cases who fail to
respond to standard treatment5. Investigations of high risk individuals who
are symptomatic– HIV +ve cases– History of exposure to MDR cases– Laboratory workers
CULTUREMycobacterial culture can be
performed on:
Conventional : egg based solid medium Lowenstein-Jensen
medium Ogawa medium
Automated : Liquid based medium Kirschner™s or
Middlebrook 7H9 broth. Middle brook 7H12
9/4/2013
51
BACTEC MGIT 960 systemBacT/Alert 3DVersaTREK
Automated Mycobacteria Culture System
9/4/2013
52
1. Mycobacteria Growth detection.
2. Antimicrobial Susceptibility Testing (AST)
Myco ba c te rium tube rc ulo s is colonies on Middle Brook Agar.
9/4/2013
53
9/4/2013
54
BIOCHEMICAL IDENTIFICATION FOR MYCO BACTERIUM TUBERCULO SIS
9/4/2013
55
Biochemical Test Result
1 Niacin Test Positive
2 Catalase 68º C/pH 6.8 Negative
3 Nitrate Reduction Test Positive
9/4/2013
56
Nitrate Reduction Test
Catalase Test 68ºC/pH 7
New technologies - Molecular techniques
9/4/2013
57
Conventional PCR Real time PCR – direct detection from
samples # identification / sensitivity # detecting MDR / mutants gene associated with antibiotic resistant
Detection and identification of mycobacteria directly from clinical samples - an alternative for smear microscopy
9/4/2013
58
Line Probe Assay (LPA) - can be used to screen smear-positive sputum specimens for resistance to rifampicin and isoniazid in 1-2 days.
has the potential to substantially reduce the turnaround time of DST results.
training, supervision and adherence to stringent laboratory protocols to ensure high quality results during routine implementation
Tests take only hours to perform and may provide a rapid diagnosis, within 1-2 days of receipt of a sputum sample
Cannot replace culture and drug sensitivity tests Too expensive to be use routinely sensitivity : < 60% - > 95% <60% in smear negative (but positive for TB
culture) > 95% samples acid fast smear positive sputum
samples,
12/09/14
59
9/4/2013
60
9/4/2013
61
Lane 1: DNA Ladder MarkerLane 2: Sample 1Lane 3: Sample 2Lane 4: Sample 3Lane 5: Sample 4Lane 6 Sample 5Lane 7: PCR Pos ConLane 8: PCR Neg ConLane 9: Pos Ext ConLane 10: Neg Ext Con
9/4/2013
62
Serological diagnosis of Tuberculosis
9/4/2013
63
Should allow a rapid diagnosis, simple, cost effective
Most of the serological tests : low turn around time, high negative predictive value and are useful as
screening tests. Variable or low sensitivity : o 76% in smear positive cases, 59% in smear
negative cases, 64% in LN TB and 46% in pleural TB
expensive, require trained personnel and often have difficulty in distinguishing between M. tube rc ulo s is a nd NTM.
WHO (2011) : Patient safety- commercial serological assay should not be used to diagnose pulmonary or extra pulmonary TB
Some commercially available antibody tests for diagnosis of pulmonary TB
9/4/2013
64
Name of the assays Antigen used
MycoDot (Dot-blot) Lipo arabino mannan(LAM)
Detect-TB (ELISA) Recombinant protein PeptidePathozyme Myco(ELISA)
38 kDa (recombinant Ag) and LAM
Pathozyme TB(ELISA) 38 kDa (recombinant)Antigen A60 (ELISA) Antigen -60ICT diagnostics(membrane based)
38 kDa (recombinant)
Serological test - FOR LTBI
9/4/2013
65
Blood tests : T-SPOT.TB , QuantiFERON-TB Gold 393 consecutive with suspect TB had both tests –
blood and skin testPositive result: T-SPOT.TB of 38% vs QuantiFERON-TB Gold 26%
(p < 0.0001). Both blood tests showed similar overall agreement
with the skin test Blood tests were better than the skin test in
distinguishing BCG-vaccinated individuals from those with true positive results.
Indeterminate results 11% with QuantiFERON-TB Gold 3% with T-SPOT.TB (p < 0.0001).
Lancet 2006;367:1328-1334.
Detection of activated T-cells – producing gamma interferon
enable more people to be diagnosed and treated while their infection is still dormant / immune deficient
? thus a powerful new tool to help health authorities curb and control the TB epidemic
faster (results within 24 hours)
detecting antibodies induced by an infection
Painful, scarring False positive :- exposure / contact
with TB case- Post vaccination 3- 7 days to read the
induration 9/4/2013
66
TB Spot-TB
Skin Test
9/4/201367
High sensitivity (~90%) has been shown in culture confirmed TB patients
Specificity is also very high (>98%) even in BCG vaccinated individuals.
Additional procedure /diagnostic tests Extra pulmonary TB : histology or cytology TB lymphadenitis : FNA – cytology Pleural TB : thoracoscopy – histology/culture/
Adenosin Deaminase (ADA) TB meningitis : PCR / ADA
9/4/2013
68
Identification
9/4/2013
69
Only done in MKAK Sg. BulohConventionalNAAT ( Nucleic acid amplification
test)Hybridisation gene probe
QC
9/4/2013
70
Internal QC – staining materials
External QC Slides rechecking
Within same lab Within labs from same district / state
MKAK Sg. Buloh RCPA Australia
Problems in Lab. Dx
9/4/2013
71
The bacteria – slow growers Sample quality / criterion Wrong technique – STM not available Poor staining material Staff competency & attitude Equipment problem – microscope
Poor samples Improper
techniques Number of
organisms too small
Partially suppressed by antibiotics
Mycobacterial contamination of water or solutions
Transfer from +ve smears during staining or examination
Staining artefacts Insufficient
destaining of non AFB organisms
Other Acid fast organisms9/4/2013
72
False negative False positives
Delays of up to 2-3 weeks in starting anti-TB treatment is common
•Late intensive care admissions•In-hospital mortality•Nosocomial transmission•Inappropriate treatment
Impact of smear negative TB
9/4/2013
73
Tuberculosis Reference Laboratory
9/4/2013
74
1. National Public Health Laboratory, Ministry of Health Malaysia, Sungai Buloh, Selangor.
Role of TB Reference Laboratory
9/4/2013
75
To perform DST and Identification tests for AFB isolated from peripheral Laboratory
Standardization of Lab Techniques- To conduct training to technical staff (MLT) Compilation and monitoring data
Why Drug Susceptibility Tests (DST) ?
9/4/2013
76
DST done for 3 main purpose;I. As guidance in the choice of the first
course of chemotherapy.
II. To confirm emergence of drug resistance in a patient who failed to show bacteriological response to treatment and may guide the choice of further course.
III. May be employed to estimate prevalence of ;
- Primary Drug Resistance &
- Acquired Drug Resistance in the community
9/4/201377
Reporting
9/4/2013
78
TAT for slide smear shall be 24 hours from the time of specimen receipt (to be reviewed later)
TAT for identification : 3 days after specimen receipt at MKAK Sg Buloh
TAT for sensitivity : preliminary – 17 days final – 31 days
WHO Quantification scale Ziehl Neelsen
Number of AFB Number of fields* examined
What to report
No AFB in 300 fields 300 fields
No Acid Fast Bacilli seen
1–9 AFB in 100 fields 100 fields
Record exact figure (1 to 9 AFB per 100 fields)
10– 99 AFB in 100 fields 100 fields
1 +
1– 10 AFB in each field 50 fields
2 +
More than 10 AFB in each field 20 fields
3 +
9/4/2013
79
* Oil immersion fields
9/4/201380
9/4/201381
9/4/201382
9/4/201383
KELEMAHAN PEWARNAAN
9/4/2013
84
Penyelengaraan Mikroskop yang baik
12/09/14
85
Slide artefact - Excessive Heat Slide artefact - Excessive Heat Fixing of SmearFixing of Smear
9/4/201386
Slide artefact - Glass Slide Slide artefact - Glass Slide Scratch and AFBScratch and AFB
9/4/201387
Soot Deposit on Underside of Soot Deposit on Underside of SmearSmear
12/09/1488
What Are the Causes of Pale Staining AFB?
12/09/14
89
Pale Staining AFBPale Staining AFB
12/09/1490
Causes of Pale Staining AFB Concentration of CF <0.3%:
Staining time less than 5 minutes
Inadequate heating of carbol fuchsin on the slide
Excessive exposure time with an acid alcohol decoloriser may remove the carbol fuchsin from the AFB
12/09/14
91
Time Effect of Carbol Fuchsin on Intensity of AFB
2 min
12/09/14
92
3 min
12/09/1493
5 min
12/09/1494
Effect of Not Heating the Carbol Effect of Not Heating the Carbol Fuchsin to SteamingFuchsin to Steaming
12/09/1495
What Are the Causes of Excessive Counterstaining ?
Leaving counterstain on slide for longer than one minute
Methylene blue concentration > 0.3%
12/09/14
96
Good Quality CounterstainGood Quality Counterstain
12/09/1497
Excessive CounterstainExcessive Counterstain
12/09/1498
Effect of Excessive Counterstain-1Effect of Excessive Counterstain-1
c
Nuclei too dark
Background too strong
12/09/1499
Effect of Excessive Counterstain-2Effect of Excessive Counterstain-2
Background too strong
12/09/14100
Combination of Excessive Combination of Excessive Counterstain & Poorly Stained AFBCounterstain & Poorly Stained AFB
12/09/14101
What Are the Causes of Insufficient Decolorisation?
12/09/14
102
Assessment of Smear QualityAssessment of Smear Quality
Uneven smear preparation and insufficient decolorisation
12/09/14103
Insufficient Decolourisation Insufficient Decolourisation
12/09/14104
Importance of Correct Importance of Correct DecolourisationDecolourisation
Insufficient Correct
12/09/14105
Summary
9/4/2013
106
Tuberculosis still stands as the most important infectious disease in humans despite of the advances in treatment.
Early and accurate detection of active cases remains an important objective for improved implementation of chemotherapy and for reduction in the spread of the disease.
The identification of the mycobacteria will depends on the quality of sample and the lab techniques
Several limitations to the traditional techniques used.
New techniques / diagnostic tests : to improved the diagnosis and shortened the testing time in the laboratory.
9/4/2013107