TB Susceptibility

1
Internal Quality Control (QC) and External
Quality Assessment (EQA) for M. tuberculosis
Testing
John Ridderhof, DrPH
Division of Laboratory Systems/PHPPO Centers for
Disease Control and Prevention
2
(No Transcript)
3
TB Testing Services in LIFE Countries

• Direct AFB smear microscopy
• Case detection
• Monitoring treatment
• Culture and Drug susceptibility testing (DST)
• Surveillance
• Assess treatment failures
• Smear negative diagnosis?
• Other Nucleic acid amplification tests

4
(No Transcript)
5
(No Transcript)
6
The Keys to successful Quality Control are

• Adequately trained, interested, and committed
  staff
• Common sense use of practical procedures
• A willingness to admit and rectify mistakes
• Effective communication
• Ref Laboratory Services in Tuberculosis
  Control, WHO

7
(No Transcript)
8
(No Transcript)
9
Culture and DSTSpecimen Collection

• In most countries specimen collection is a
  laboratory responsibility

10
(No Transcript)
11
(No Transcript)
12
Culture and DST Specimen Viability

• Sputum should not be stored at room temperature
  for longer than 3 days for culture
• Can be stored up to 4 weeks without loss of
  smear positivitybut not recommended to to loss
  of ability to fix smear

Paramasivan CN et al, Tubercle 1983 64(2) 119-24
13
Variables Affecting the Quality of Culture

• Quality of specimen (sputum versus saliva)
• Transport time and conditions
• Digestion/decontamination of specimen
• Method Petroff, NALC/NaOH
• Centrifugation sufficient G-force
• Technique timing, use of vortex, decanting

14
QC of Culture Monitors

• Contamination rate (2 - 5)
• Correlation between smear and culture
• Percentage of culture isolates from smear
  positive specimens
• Isolation rate for saprophytic nontuberculous
  mycobacteria (NTM, e.g. M. gordonae)

15
Whats the problem?Correlation of Smear and
Culture
Culture
Smear
16
QC indicators for DST

• Weekly drug-susceptible control strain
• Drug resistant control strains?
• New culture media and drugs
• Internal comparison of methods if more than one
  method is available
• Exchange isolates with supranational laboratory

17
External Quality AssessmentCulture

• College of American Pathologists
• 10 samples/yr, 10 isolation and identification,
  2 DST, 2 AFB smears
• Other Proficiency testing programs
• NEQAS (UK)
• South Africa Medical Research Council

18
External Quality Assessment -- DSTWHO/IUATLD
Supranational Network

• Required as part of WHO surveillance for drug
  resistance
• 23 supranational laboratories
• Supranational laboratories receive 20 cultures/yr
  for assessing performance
• gt50 national or regional laboratories
• National laboratories share a sample of cultures
  with supranational laboratory to compare
  performance

19
External Quality AssessmentDrug Susceptibility
Testing-M.tb

• CDC Performance Evaluation Program
• M. tuberculosis and NTM drug susceptibility
• 150 Laboratories 17 International
• 10 cultures/yr 6 M.tb, 4 NTM

20
Direct AFB Microscopy

• Internal Quality Control
• External Quality Assessment (EQA)
• onsite evaluation
• rechecking
• proficiency testing

21
(No Transcript)
22
(No Transcript)
23
Internal Quality ControlFor AFB Microscopy

• Microscope
• preventative maintenance
• routine cleaning
• Stains
• expiration dates stains and chemicals
• Staining
• positive and negative controls

24
Proposed International GuidelinesEQA AFB
Microscopy

• Co-sponsored by WHO, IUATLD, KNCV, APHL, and CDC
• Consensus of 15 member workgroup including
  representatives from LIFE countries Uganda,
  Senegal, South Africa, and India

25
EQA -- AFB MicroscopyOn-site Evaluation --
Background

• Most countries lack the resources for annual
  visits of peripheral laboratory by central
  laboratory staff
• Laboratories in most countries are visited by a
  non-laboratory District supervisor
• Optimum evaluation is performed by trained
  laboratory staff in supervisory role

26
On-site Evaluation Results - Uganda
BEFORE
AFTER
Aziz, M. and G. Bretzel, Unpublished Data
27
EQA - AFB MicroscopyRechecking - Background

• Recommended by IUATLD and WHO
• Usually 100 of positive and 10 of negative
  smears
• Usually unblinded adds bias
• Reviews patient testing including smear
  preparation, staining, and interpretation
• Lan N.T.N. et al, 1999 Int J Tuberc Lung Dis
  3(1) 55-61

28
Rechecking Slides in Mexico 1998

• States provided data for 438 of 637 laboratories
• Only 303 laboratories had complete and consistent
  data
• Only 109/303 (36) had any FN or FP error
• 194 (64) laboratories with no errors had 55 of
  total test volume so presence of errors was not
  dependent on volume

29
EQA - AFB MicroscopyProficiency Testing -
Background

• Uncommon in resource-limited countries
• Prepared smears (South Africa) or patient slides
  (Senegal) sent from central laboratory
• Consistent challenge of laboratory test
  performance
• PT test performance may be different from testing
  routine patient specimens

30
PT Implementation in Mexico

• Inspected 587 of 637 laboratories
• 604 microscopists given a 2 hour, 10 slide test
• 52 had score gt80
• 33 had score 60-79
• 15 had score lt60
• 536/604 (88.7) finished all 10 slides
• 216 persons with score lt80 received training
  followed by second PT average scores improved
  from 61 to 90 (P-value lt 0.0001).

31
Proposed International GuidelinesEQA - AFB
Microscopy Components

• Resource analysis to determine appropriate EQA
• Checklists for onsite evaluation by
  non-laboratory district supervisor or supervising
  laboratory staff
• Blinded rechecking using a random statistical
  sample from each laboratory
• Procedures to develop PT slides
• Sample forms

32
Proposed EQA - AFB Microscopy Guidelines Key
FeaturesResource Analysis

• Inventory available resources (actual/projected)
• Manpower, supplies, communication,
  administrative, financial
• Examine effectiveness of current EQA activities
• Gather laboratory service information
• Planningoptions for the evolution of EQA
• Pilot test and document changes
• Expansion based on availability of resources

33
Proposed EQA AFB Microscopy GuidelinesResource
AnalysisPhased Approach

• Assure the five elements of DOTS
• Develop a central reference and intermediate
  laboratories to carry out EQA
• Determine the existing capacity for EQA
• Train district health officials to evaluate the
  minimal functions of microscopy laboratories

34
Proposed EQA AFB Microscopy GuidelinesResource
AnalysisPhased Approach (cont)

• Proficiency testing to evaluate performance
• Pilot rechecking program
• Determine resources additional PT or phased
  implementation of rechecking

35
Proposed EQA AFB Microscopy Guidelines Key
FeaturesOn-site Evaluation

• Develop a standard checklist of questions and
  indicators
• Include minimal evaluation that can be performed
  by non-laboratory trained personnel (e.g.,
  inventory supplies, reagents, equipment)
• Include detailed evaluation that can be performed
  by supervisory laboratory staff
• Train laboratory and non laboratory staff to
  assure consistent application

36
On-site Evaluation Performed by Non-laboratory
Staff Examples

• Are all staining reagents available and within
  expiration dates?
• How are wire loops cleaned?
• Is the laboratory register present and all
  columns completed properly?
• How is maintenance on the microscope performed?

37
(No Transcript)
38
On-site Evaluation Performed by Laboratory Staff
Examples

• Does the technician verify that the container is
  properly labeled?
• How are slides labeled?
• How often is the carbol fuchsin filtered?
• How many fields are examined to report a negative
  smear?

39
Proposed EQA AFB Microscopy Guidelines Key
FeaturesProficiency Testing

• Laboratory may re-use patient slides but a
  procedure is provided to produce test slides for
  consistent slide sets
• Recommended slide set is 10 slides 5 stained and
  5 unstained
• Simple forms for slide production and collection
  of test results

40
Proposed EQA AFB Microscopy Guidelines Key
FeaturesRechecking

• Emphasizes blinding and random sample using the
  laboratory register
• Sample size is based on Lot Quality Assurance
  Sampling (LQAS) with parameters selected for test
  volume and desired specificity
• Positives and negatives sampled
• Minor errors (FP or FN with 1-9 AFB/ 100 f) are
  used as a surrogate

41
Semi-quantitative ReportingZiehl Neelsen

• No AFB are found in 100 fields
• No acid-fast bacilli observed.
• 1-9 /100 fields Report the exact figure.
• 10-99 AFB /100 fields, 1
• 1-10AFB /field, 2
• Greater than 10 AFB/ field, 3

42
(No Transcript)
43
Rechecking LQAS example -Mexico
44
Some Reasons for False Positive Results
45
Some Reasons for False Positive Results

• Errors in specimen handling or recording
  information
• Re-use of containers or positive slides
• Unfiltered fuchsin
• Contaminated immersion oil
• Inadequate decolorization

46
Some Reasons for False Negative Results
47
Some Reasons for False Negative Results

• Errors in specimen handling or recording
  information
• Poor quality sputum
• Excessive decolorization
• Reading less than 100 microscopic fields

48
(No Transcript)