MYCOBACTERIUM : DIAGNOSIS AND LABORATORY TEST

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Dr. Azura HussinPathology Department

HRPZ II

Contents

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Introduction General characteristics of tubercle bacilli Lab diagnosis of tuberculosis - current technologies - newer technologies - problems in lab diagnosis - quality control Summary

Introduction

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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

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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.

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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.

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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

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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

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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

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Intro duc tio n - Members of Genus Myco ba c te rium

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Mycobacterium

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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

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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

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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

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# 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

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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

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Mycobacterium Cell Wall

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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

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Network Of TB Laboratory (Organization of TB Laboratory Services)

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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

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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.

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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

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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

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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

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Tests offered - KKM

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Smears microscopic – ZN / IF Culture, sensitivity and identification PCR Line Probe Assay

Quality sample = Quality smearQuality smear = Quality lab result

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Specimen Quality

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Saliva/ Induced Sputum Blood Stained

Specimen Quality

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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

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SMEAR EXAMINATION

Z-N Stain

Fluorescent Stain

Light MicroscopeFluorescent Microscope

Specimen

Systematic Examination of Smears

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3 cm

2 cm

Uniform Smear Size

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Why We Do Sputum Smear Examination?

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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

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3 cm = 150 visual field

1

2

Microscopy

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Microscopy

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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

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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

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AFB in Single ArrangementAFB in Single Arrangement

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AFB - in Various Arrangements

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AFB in Clumps AFB in Clumps

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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

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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

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Culture

Advantages For susceptibility

testing Able to identify the

species of mycobacterium

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Disadvantages

Slow grower : delay in the lab diagnosis / treatment LaboriousExperience staffSpecial equipment Expensive

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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

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BACTEC MGIT 960 systemBacT/Alert 3DVersaTREK

Automated Mycobacteria Culture System

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1. Mycobacteria Growth detection.

2. Antimicrobial Susceptibility Testing (AST)

Myco ba c te rium tube rc ulo s is colonies on Middle Brook Agar.

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BIOCHEMICAL IDENTIFICATION FOR MYCO BACTERIUM TUBERCULO SIS

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Biochemical Test Result

1 Niacin Test Positive

2 Catalase 68º C/pH 6.8 Negative

3 Nitrate Reduction Test Positive

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Nitrate Reduction Test

Catalase Test 68ºC/pH 7

New technologies - Molecular techniques

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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

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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,

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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

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Serological diagnosis of Tuberculosis

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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

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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

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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

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TB Spot-TB

Skin Test

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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

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Identification

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Only done in MKAK Sg. BulohConventionalNAAT ( Nucleic acid amplification

test)Hybridisation gene probe

QC

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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

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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

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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

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Tuberculosis Reference Laboratory

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1. National Public Health Laboratory, Ministry of Health Malaysia, Sungai Buloh, Selangor.

Role of TB Reference Laboratory

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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) ?

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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

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Reporting

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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 +

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* Oil immersion fields

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KELEMAHAN PEWARNAAN

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Penyelengaraan Mikroskop yang baik

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Slide artefact - Excessive Heat Slide artefact - Excessive Heat Fixing of SmearFixing of Smear

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Slide artefact - Glass Slide Slide artefact - Glass Slide Scratch and AFBScratch and AFB

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Soot Deposit on Underside of Soot Deposit on Underside of SmearSmear

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What Are the Causes of Pale Staining AFB?

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Pale Staining AFBPale Staining AFB

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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

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Time Effect of Carbol Fuchsin on Intensity of AFB

2 min

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3 min

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5 min

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Effect of Not Heating the Carbol Effect of Not Heating the Carbol Fuchsin to SteamingFuchsin to Steaming

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What Are the Causes of Excessive Counterstaining ?

Leaving counterstain on slide for longer than one minute

Methylene blue concentration > 0.3%

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Good Quality CounterstainGood Quality Counterstain

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Excessive CounterstainExcessive Counterstain

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Effect of Excessive Counterstain-1Effect of Excessive Counterstain-1

c

Nuclei too dark

Background too strong

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Effect of Excessive Counterstain-2Effect of Excessive Counterstain-2

Background too strong

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Combination of Excessive Combination of Excessive Counterstain & Poorly Stained AFBCounterstain & Poorly Stained AFB

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What Are the Causes of Insufficient Decolorisation?

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Assessment of Smear QualityAssessment of Smear Quality

Uneven smear preparation and insufficient decolorisation

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Insufficient Decolourisation Insufficient Decolourisation

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Importance of Correct Importance of Correct DecolourisationDecolourisation

Insufficient Correct

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Summary

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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.

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