Role of Laboratory Servicesin TB Control Dr Mohammad Rahbar

Professor of MicrobiologyDepartment Microbiology

Reference health Laboratory ,Tehran iran

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National Reference Laboratory

Province Laboratories

District Laboratories

Health Centers

Ministry of Health

National Laboratory Infrastructure

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Desirable Features of Desirable Features of Diagnosis Methods in TBDiagnosis Methods in TB

Sensitivity Specificity Predictive value Speed Reliability Reproducibility Cost Safety Easy to use Robustness Widest usage

What is new in the diagnosis of TB TRC/ICMR 3

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AFB smear

AFB (shown in red) are tubercle bacilli

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Aims of sputum microscopyAims of sputum microscopy

Diagnosis of patients with infectious tuberculosis Monitoring progress of patients on treatment

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Advantages of sputum Advantages of sputum microscopymicroscopy

More reliable than x-ray for the diagnosis of infectious TB Simple to perform Easy to read Minimal infrastructure required Inexpensive Quick Only tool to monitor and declare patients as “cured’’

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Diagnosis of pulmonary Diagnosis of pulmonary tuberculosistuberculosis

Three specimens optimalThree specimens optimal

Spot specimen on first visit;sputum

container given to patient

Early morning collection next day

Spot specimen during second visit

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Three sputum smears are optimalThree sputum smears are optimal

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93 100

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50

100

First Second Third

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lati

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osi

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A good smearA good smear Made from mucopurulent sputum

Spread evenly

3 cm x 2 cm in size

Not too thick

Thin enough to read newsprint through

Air dried before fixing

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False negative resultsFalse negative results Inadequate sputum collection

avoid – saliva, nasal discharge

collect – bronchial sputum from depth of chest Inadequate storage of sputum / stained smears

exposure to direct sunlight

radiation ( UV light )

excessive heat / humidity Not taking mucopurulent portion of sputum Inadequate smear preparation Inadequate smear examination Administrative & recording errors

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Consequences of false Consequences of false negative smear resultsnegative smear results

Patients with TB will not be treated, resulting in

suffering, spread of TB and death .

Intensive phase of treatment will not be

extended for the required duration, resulting in

inadequate treatment

Patient may lose confidence in the

programme

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False positive resultsFalse positive results

Food particles

Precipitated stains

Saprophytic AFB

Spores of B.subtlis

Fibres and pollen

Scratches on slide

Contamination through carry over of

AFB from one smear to another

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Consequences of false positive Consequences of false positive resultsresults

Patients are started on treatment unnecessarily

Treatment is continued longer than necessary,

in

follow-up examinations

Medications will be wasted

Patients lose confidence in the programme

QUALITY ASSURANCE PROGRAM (QAP )IN SPUTUM SMEAR

MICROSCOPY

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Quality Assessment

• Also called External Quality Assessment (EQA)

• A process that allows participant laboratories to assess their capabilities by comparing their results with those in other laboratories in the network .

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EQA

• Activities– On-site evaluation of the laboratory, – Panel testing and – Blinded cross checking or Random Blinded

Cross Checking (RBCC)

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Feedback

• A process of communicating results of EQA to the original laboratory, including suggestions for possible causes of errors and remedies.

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High False Results

• High False Negative (HFN).– A 1+ to 3+ positive smear that is misread as

negative. This is a major error.

• High False Positive (HFP).– A negative smear that is misread as 1+ to 3+

positive. This is a major error.

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Low False Results

This type of minor error occurs occasionally even in laboratories that are performing well.

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Low False Results

• Low False Negative (LFN).– A scanty (1- 9 AFB / 100 fields) positive smear

that is misread as negative.

• Low False Positive (LFP).– A negative smear that is misread as a scanty (1-

9 AFB / 100 fields) positive.

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Quantification Error (QE)

• Errors seen in grading of positive slides.

• Difference of more than one grade in reading a positive slide between examinee and controller.

• This is considered as a minor error that generally has no impact on case management.

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EQA

Three methods to evaluate laboratory performance:• On-site evaluation• Panel testing• Blinded rechecking

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EQA

Three methods to evaluate laboratory performance:• On-site evaluation• Panel testing• Blinded rechecking

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• Contents of a On-site evaluation checklist.

• General Information.

• Action required to be taken in the previous visit.

• Current visit particulars.

• Panel testing of slides, if required.

• Assessment of EQA responsibilities of lower level supervisor.

On-site Evaluation

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On-site Evaluation..

• Availability of patient slides.

• Proper storage for EQA.

• Adequate training of staff with refresher

courses.

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Panel TestingFor assessment of proficiency in staining

and or reading.

Least expensive and resource intensive of

the 3 methods of EQA.

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Panel Testing

• Preparation from known patients’ specimen.

• Manufacture of slides with required grade of smears.

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Manufacture of Panel Testing Slides

• To be done at National ReF Lab.

• Collection of negative and high grade positive patients specimen.

• Addition of Formalin to stabilize the pus cells and to render the specimen safe for handling.

• Liquefaction of specimen using NALC.

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Manufacture..

• Mixing of positive and negative specimens to obtain the a known number of AFBs and pus cells in the specimen.

• Preparation of slides.

• Validation of a sample of specimen for consistency of the results.– Results to match to an extent of at least 95%.

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Panel Testing..Limitation:

Checks only technicians ability to stain and/

or read smears not a useful means to assess

routine laboratory performance

Not required as a routine if Blinded

Rechecking is done.

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Panel Testing..• Panel testing is useful to:

– Supplement rechecking program.

– Provide some preliminary data on peripheral

laboratory capabilities prior to implementing a

rechecking program.

– Asses current status of performance or to quickly detect

problems associated with very poor performance.

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Panel Testing..

– Evaluate proficiency of laboratory

technicians following training.

– Monitor performance of individuals when

adequate resources are not available to

implement a rechecking program.

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Random Blinded Rechecking

• The most important method in EQA.

• Controller and reading from high level lab for

peripheral microscopy centers (MCs).

• A country wide program for blinded rechecking of

slides at regular intervals.

• A long term goal for optimal EQA.

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Lot Quality Assurance Sampling

• A blinded rechecking sampling method.

• Sample size depends on;– Total number of negatives slides processed each

year.– Slide positivity rate.– Expected performance (sensitivity and specificity)

compared to the controllers.– Maximum number of errors allowed before action

is taken.

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Summary

Establishment of a Laboratory network is an important function of a National level Program Manager

Internal Quality Control and External Quality Assurance are essential for ensuring proper diagnosis by sputum smear microscopy