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15/03/2012

CDC TB workshop, East London

Basic microbiology of TB & “traditional” diagnosis of TB

Dr Natalie Bey lisNHLS My cobacterial Ref erral Laboratory Johannesburg011 489 9182 / 9262natalie.bey lis@nhls.ac.za

MYCOBACTERIA

Mycobacterium tuberculosiscomplex

Mycobacterium otherthan M.tuberculosis

complex: NTM / MOTT

Mycobacterium leprae

M.tuberculosis

M.bov is

M.bovis BCG

5 others

exposed not infected

infected

Immune response

Active diseaseContain disease

Latent infxn

10%90%

2-23% lifetime risk10% / y with HIV infection

Cell wall richin fatty acids (mycolic acids)

Contents of cell includinggenes (DNA)

• Slow growing bacteria• Special stains needed for visualization of bacteria in specimens• Require special conditions for optimal growth• Damaged by heat – refrigerate specimens if delays expected

WHO IS MYCOBACTERIUM TUBERCULOSIS ?

Latent tuberculosisActive tuberculosis

The TB diagnostic pipeline

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Primary stain binds to mycolic acids

virulence

factor

Prevents osmotic lys is

60% of cell wall

Virulence factor:

prevents attack in phagocytic vacuoles

1. Young HIV infected women has been losing weight, is lethargic and coughs

occasionally.What would you do?

a) This patient probably does not have TB

b) This patient may have TB. I will send another sputum sample for mycobacterial culture

c) This patient may have TB. I will send another sputum sample for microscopy for acid-fast bacilli (AFB)

Sputum microscopy

• Detects most infectious cases

• Used for monitoring

• Cheap, available

• LIMITATION– Low sensitivity : 25-65%

– Lower in HIV+, children, paucibacillary disease, extrapulmonary disease

• Auramine (fluorescent) 10% more sensitive than ZN

The delayed TB diagnosis

Sl ide courtesy of FIND: Foundation for Innovative New Diagnostics

Reminder: sensitivity & specificity

Sensitivity = true pos / (true pos + false neg)

Specificity = true neg / (true neg + false pos)

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MICROSCOPY CULTURESENSITIVITY 25-65%

(5000 – 10 000 bacilli / ml)

ie many f alse negativ es

80-85%(10 bacilli / ml)

ie f ew f alse negativ es

Diagnosis of Tuberculosis Factors that contribute to improved sensitivity of both microscopy and

culture?

• Adequate specimen collection

• No. of specimens

• Optimal specimen transport

• Optimal specimen processing

• Certain clinical scenarios are less likely to yield positive smear and culture results

SPECIMEN COLLECTION

• Clean, sterile containers • Request forms completed• Safe packaging materials to

minimize risk of leakage / contamination

• Transport rapidly to laboratory – Infrastructure of nationwide

transport networks• Refrigerate if cannot be

processed immediately – Prevents overgrowth by

other bacteria• Collect before chemotherapy

started

2. A young HIV infected women who has been losing weight, is lethargic and coughs

occasionally.

What would you do?

a) Treat the patient for tuberculosis

b) Send another sputum for microscopy for AFBs

c) Send another sputum for mycobacterial culture

“A case of pulmonary TB is considered to be smear-positive if one or more sputum smear specimens at the start of treatment are positive for AFB (provided that there is a functional EQA system with blind rechecking1).

The definition of a new sputum smear-positive pulmonary TB case is based on the presence of at least one acid fast bacillus (AFB+) in at least one sputum sample in countries with a well functioning EQA system. (See www.who.int/tb /dots/laboratory/policy/en/index1.html.)”

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3. What would you do?

a. Phone the laboratory in frustration

b. Remain calm knowing that a certain amount of contaminated cultures is expected from the laboratory

c. Phone the laboratory and ask them to identify the contaminant as this is likely the cause of the lower respiratory tract infection and will assist with treatment choice

Culture of M.tuberculosis Fluorescence

indicates utilization of O2 by bacilli

320 specimens per drawer

Culture of mycobacteria

• Advantages: – Much more sensitive than microscopy

• 80 - 85% • LOW limit of detection: able to detect as few as

10 bacteria /ml– Allows

• Precise species identification• Drug susceptibility testing (DST) • Genotyping - identify epidemiological links

between patients

• Only definitive diagnosis and considered the “Gold Standard” microbiological test for TB

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4. What do you do now?

a.Start TB treatment

b.Do not start TB treatment; wait for final identification

c.Assess clinically & Rx for TB if indicated

d. Withhold TB treatment pending identification of AFB even if patient is ill

e. Assess clinically; if patient is stable & asymptomatic withhold Rx until identification is available

5. If you chose option a for Q4, what do you do now?

a.Stop TB treatment as this patient does not have TB

b. Continue TB treatment & send another specimen

c. Stop TB Rx & send another specimen

d. Assess clinically, send another specimen & start empiric TB Rx if indicated

e. Request a GeneXpert on another specimen

6. What does this result mean?

a. Laboratory reporting error

b.The lab failed to detect M.tb

c.The AFBs in this culture are non tuberculous mycobacteria (NTM)

d.This represents contamination of the specimen

e.The patient does not have TB