Laboratory Diagnosis of TB

• Presenter:WATI VUKIALAU-VULAVA

LT/NTP - NORTH

Objectives

1. History of TB

2. elements of DOTS

3. What the bacteria looks like

4. Lab component of diagnosing TB

5. Sputum collection procedures

6. What good and poor sputum quality is

7. Available Lab tests in Fiji to detect TB

• TB has affected humans for millennia

• Historically known by a variety of names, including:– Consumption– Wasting disease– White plague

• TB was a death sentence for many

History of TB

• Until mid-1800s, many believed TB was hereditary

• 1865 Jean Antoine-Villemin proved TB was contagious

• 1882 Robert Koch discovered M. tuberculosis, the bacterium that causes TB

Mycobacterium tuberculosisImage credit: Janice Haney Carr

Cont…History of TB Scientific Discoveries in 1800s

• Political commitment to TB control

• Case detection by sputum smear microscopy• Regular, uninterrupted supply of high quality

anti-TB drugs

• Directly Observed Treatment (DOT)

• Standardised recording and reporting

Elements of DOTS Strategy

* long, slender, straight or curved, acid fast bacilli

Mycobacterium tuberculosis

* slow growers, obligate aerobes, intracellular

bacterium* structure composed of high

molecular weight acidic waxes, mycolic acid, cord

factor

WHY DO WE COLLECT SPUTUM?

1. To detect & diagnose TB (mycobacterium TB)

•Smear-positive patients are 4-20 times more infectious

•Untreated, a smear-positive patient may infect 10-15 persons/year

•Smear-positive patients are much more likely to die if untreated

2. To monitor effectiveness of treatment

When is the best time??

• The best time, but not the only time to collect a sputum specimen is upon awakening in the morning

• When more than 1 specimen is required they will be collected on different days

• Any specimens collected in one day will count as the first (1) specimen

Sputum Collection

• 3 specimens considered

3 sputum samples are optimal

81%

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These data have helped form the basis of the current three-specimen requirement for ruling out pulmonary TB. The probable rationale behind this

recommendation is that some patients shed mycobacteria irregularly and in small numbers and,

thus, increasing the number of specimens would increase the yield. With regard to the

recommendation for early morning specimens, the assumption is that M. tuberculosis would be present in maximum concentration in sputum after pooling

overnight in the respiratory tract.

Specimen collection timing

• Spot-Morning-Spot WHO/IUATLD recommendation

• Spot - 1st visit to the clinic• Early morning- first sputum in the morning• Spot - 2nd visit to the clinic

Instructions to Patients

Importance of sputum examination for TB

diagnosis

Need for real sputum, not saliva

How to produce good sputum

How to open and close the container

How to avoid

contamination of

outside of the

container

Give clear instructions to patients on how to collect the sputum:

Patient education:the best specimen comes from the lungs

• Sputum should be collected under direct observation, at least for the first time. This is to insure that the patient is being properly coached and is giving a good coughing effort, as well as insuring that uncooperative patients are producing their own sputum for examination.

INSTRUCTION FOR SPUTUM COLLECTION

1. Specimen collection should be done without the client rinsing the mouth or brushing teeth

2. If possible, go outside or open a window before collecting the sputum sample. This helps protect other people from TB germs when they cough.

3. The cup is very clean. Don’t open it until you are ready to use it.

4. Inhale 2-3 times, breathe out hard each time

5. Cough deeply from the chest

6. Place the container close to the mouth to collect the specimen

CON’T7. Spit the sputum into the plastic cup. Avoid contaminating

the inside of the container and lid

8. Keep doing this until the sputum reaches the 5 ml line (or more) on the plastic cup. This is about 1 teaspoon of sputum.

9. Screw the cap on tightly so it doesn’t leak.

10. Write on the specimen bottle the patient’s name, hosp number, the number of sputum collected, the date of collection

11. Put the cup into the specimen bag and into a specimen box

12. Submit this to the laboratory ASAP

• If the patient cannot cough up sputum, advice him/her to try breathing steam from a hot shower or a pan of boiling water.

Tightly cap labeled sputum bottle Cushion a box or carton with

cellulose(optional) Pack 3 specimens (well labeled) of

same patients in 1 biohazard specimen bag

Place bag of specimen upright in the prepared box

Packing/Transportation Specimens

Keep upright

Absorbent paper

Packing specimensPut several layers of absorbent paper in the bottom of the shipper.

• Place the request form in the pocket of the plastic

• Prepare a checklist and place it in a separate bag

• Seal and label the box of specimen

• Send to nearest Health facility/hospital or laboratory.

Packing/Transportation Specimens

Sputum sampleWhat is a good sample?

• 3-5 ml• Usually thick and mucous, but may be fluid with

pieces of purulent material• Color varies from opaque white to green, reddish to

brown when blood is present• Clear saliva is not suitable; but examine saliva if a

better specimen can not be produced, especially for follow-up examinations

• A specimen mainly containing blood should not be examined; patient should see a doctor for immediate management

Best use of Collection Container

• A new container per patient

• To obtain a good quality sample, several attempts by patient may be necessary.

• Use a new container on each attempt

• Patient to open container only when about to collect specimen

• Container once opened should be considered used (dispose accordingly)

Best container??

• Clean• Plastic• Transparent• Wide mouth• Good seal• Easily labelled

Good quality sputum( mucoid)

Good quality sputum(purulent)

Blood stained

Good Quality Sputum

Poor sputum quality

saliva or nasal secretions are unsatisfactory. Saliva from mouth is water and thin.

Sputum from lungs is usually thick and sticky

 

Good sputum quality

Where can you store the sputum?

• You can store the cup in the refrigerator overnight if necessary. Do not put it in the freezer or leave it at room temperature.

Diagnosis

1) smear microscopy – to look for the bacteria (AFB) microscopically

2) Sputum culture - to grow the bacteria Mycobacterium tuberculosis

3) GeneXpert MTB/RIF - cartridge-based, automated that can identify Mycobacterium tuberculosis(MTB) and resistance to Rifampicin(RIF) within 2hr period

In Fiji, TB is diagnosed using the following Lab methods

1. Direct Microscopy

M. tuberculosis is a acid–fast bacterium, rod-shaped bacterium measuring 2-4 x 0.2-0.5 μm. They appear as bright red rods against a contrasting background.

The Ziehl-Neelsen stain is used to demonstrate the presence of the bacilli in a smear. The technique is simple, inexpensive and detects those cases of tuberculosis who are infectious.

M. tuberculosis appearing as bright red bacilli (rods) in a sputum smear stained with the Ziehl-Neelsen stain

AFB MICROSCOPY

Advantages -Rapid - High specificity (AFB in sputum = TB)

• All mycobacterium are acid fast, no exception ; • > 98% for AFB in high burden countries

- Accurate diagnoses- Using simple and available equipment

Disadvantage Low sensitivity; Reported sensitivity ranging 25 to 65% when compared to

culture

Species differentiation impossible. False positive; Saprophytic mycobacteria.

Reporting on AFB Microscopy

Number of bacilli seen Result reported

None per 100 oil immersion fields Negative

1-9 per 100 oil immersion fields Scanty, reportexact number

10-99 per 100 oil immersion fields 1+

1-10 per oil immersion field 2+

> 10 per oil immersion field 3+

3 smears = sensitivity of 1 culture

About 95% of infectious cases

Laboratory Diagnosis

1- Sputum smears stained by Z-N stain

Three morning successive mucopurulent sputum samples are needed to diagnoise pulmonary TB.

Advantage: - cheap – rapid - Easy to perform - High predictive value > 90% - Specificity of 98%Disadvantages: - sputum ( need to contain 5000-10000 AFB/ ml.) - Young children, elderly & HIV infected persons may

not produce cavities & sputum containing AFB.

Importance of AFB microscopy

• Sputum smear microscopy - the most efficient tools of case finding in a national tuberculosis control program because of its ability to identify and distinguish the cases with highest priority in tuberculosis control.

• It is dependable if sputum specimens are of GOOD QUALITY.

AFB smear-microscopy

Acid-fast bacilli (AFB) (shown in red) are tubercle bacilli

Acid fast smear showing TB bacilli

2. TB cultures

CultureCulture

Epidemiology 3

M. tuberculosis grows in Lowenstein Jensen medium or Ogawa medium, which contains inhibitors to keep contaminants from outgrowing the organism.

Because of its slow growth, it takes 6-8 weeks before small buff-coloured colonies are visible on the medium.

Typical small, buff coloured colonies of M. tuberculosis on Lowenstein Jensen medium

Identification from solid media

• Rate of growth: visible isolated colonies in 2–4 weeks.

• Colony morphology:– buff-coloured (never pigmented)

– rough– waxy

– appearance of bread crumbs or cauliflower.

From colonies , ZN staining should be

performed 46

M. tuberculosis colonies

47

3. GeneXpert MTB/RIF Test

TEST CRITERIAXpert MTB/RIF test should be done on;1.Symptomatic patients with AFB negative (negative on 3 specimens)2.Relapses suspect cases3.HIV patients4.Cases from high burden (MDR) countries

OTHER REQUIREMENTS•Request forms for TB testing should be used•Clinicians should complete request form with clinical diagnosis•Specimen collected should be of good quality•Lab Technicians should only do GeneXpert test if diagnosis falls on any of the criteria above.1.AFB positive not converting at 3 months of treatment

How could GeneXpert help?

• Low sputum smear positivity of PTB in HIV patient is common due to:– Poor immunity to localize the infection– High possibility for TB dissemination to other organs

GeneXpert can detect more TB among PLHIV

GeneXpert - Facts and Background

• Endorsed by WHO : 8 DEC 2010• 18 months of rigorous assessment

of its field effectiveness in:Early diagnosis of TB, as well as MDR-

TB

TB complicated by HIV infection, which are more difficult to diagnose

• The test could revolutionize TB care!

How does the test work?

• Detects DNA sequences specific for Mycobacterium Tuberculosis and Rifampicin resistance by PCR

• Based on Nucleic Acid Amplification Test (NAAT). The Xpert® MTB/RIF

– purifies

– concentrates

– amplifies (by real-time PCR) and

– identifies targeted nucleic acid sequences in the Mycobacterium tuberculosis genome,

TB point of care testing• Simple • Minimum 3 steps for sample

preparation

• Rapid• 2 hours result availability

• Accurate• Sensitivity : adult PTB

Smear+Culture+ = 95% • Smear- Culture+ = 80% • Specificity : adult PTB = 95%

GeneXpert test is available in Fiji

• 3 GeneXpert diagnosis sites in the microscopic centres of National TB Programm supported by WHO/ Global Fund Grant, Fiji: Labasa Hospital, Lautoka and PJ Twomey Hospital.

• Testing done for:

Sputum Smear suspects caseMDR suspect cases and PLHIV

Take-home messages

• Sputum smear microscopy - the most efficient tools for detecting TB with highest priority in TB control hence is dependable if sputum specimens are of GOOD QUALITY.

• Sputum collection procedures needs to be stregthened

THANK YOU

•Any burning questions????

SPUTUM SALIVARY DATA, 1st QtrHEALTH

FACILITYTOTAL SPECIMENS POOR SAMPLE QUALITY

% salivaryDIAGNOSTIC / FOLLOW UP

DIAGNOSTIC / FOLLOW UP

LABASA 155 58

SAVUSAVU 46 22 48

LEKUTU 2 0

WAINUNU 2 1

SEAQAQA 6 3

NAVAKAKA N/STATION

2 2

DREKETI 3 2

NABOUWALU 8 5

WAINIKORO 8 4

TAVEUNI 39 23

UNSPECIFIED 1 0

TOTAL 272 120

SPUTUM SALIVARY DATA, 2nd Qtr

HEALTH FACILITY

TOTAL SPECIMENS POOR SAMPLE QUALITY

% SalivaryDIAGNOSTIC

FOLLOW UP DIAGNOSTIC

FOLLOW UP

LABASA 113 21 38 8

SAVUSAVU 39 2 12 0 31LEKUTU 1 0 0 0

NADURI3 3 0 3 0

SEAQAQA 6 0 1 0

DREKETI 2 0 0 0

RABI 8 1 2 1

WAINIKORO 10 0 6 0

TAVEUNI 5 1 2 0

TOTAL 187 25 64 9