# Presentation: 746121

## Proposed International Guidelines for External Quality Assessment (EQA) of AFB Smear microscopy

**World Health Organization (WHO)**
**International Union Against TB and Lung Disease (IUATLD)**
**Royal Netherlands Tuberculosis Association (KNCV)**
**Association of Public Health Laboratories (APHL)**
**Centers for Disease Control and Prevention (CDC)24**

**Proposed International Guidelines for External Quality Assessment (EQA) of AFB Smear microscopy**

## Slide 2

**Notes:**
Staining Procedure

Since tubercle bacilli retain the primary stain even after
exposure to strong acid solutions, they are called acid-fast.
In the Ziehl Neelsen staining procedure, using carbol fuschsin
and methylene blue, the acid-fast organisms appear red.

## Proposed Guidelines EQA AFB Microscopy Co-sponsors - WHO, IUATLD, KNCV, APHL, and CDC

- Karin Weyer (WHO-South Africa)
- Adalbert Laszlo (IUATLD)
- Armand Van Deun (IUATLD)
- Maks Rahman (Canada)
- Mike Iademarco (CDC-USA)
- Fatoumata Ba (Senegal)
- Mohamed Aziz (WHO-Uganda)
- Masashi Suchi (JATA)

- C N Paramisivam (India)
- Sang Jae Kim (Korea)
- Frantz Lamothe (Haiti)
- Marijke Becx-Bleumink (KNCV-Netherlands)
- Gisela Bretzel (Germany)
- Alexander Sloutsky (USA)
- Kajari Shah (APHL-USA)
- Rosemary Humes (APHL-USA)
- John Ridderhof (CDC-USA)

## Proposed International GuidelinesEQA – AFB Microscopy

**External Quality Assessment (EQA) Components**
**onsite evaluation**
**rechecking**
**proficiency testing**

## EQA  --  AFB MicroscopyOn-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**

## EQA - AFB MicroscopyProficiency 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**

## PT Implementation in Mexico

**Inspected 587 of 637 laboratories**
**604 microscopists given a 2 hour, 10 slide test**
**52% had score **_**>**_**80**
**33% had score 60-79**
**15% had score <60**
**536/604 (88.7%) finished all 10 slides**
**216 persons with score <80 received training followed by second PT:  average scores improved from 61 to 90 (P-value < 0.0001).**

**Ref: Balandrano et al, National QC of AFB Microscopy **
**in Mexico 1999 IUATLD, Budapest**

## EQA - AFB MicroscopyRechecking - Background

**Recommended by IUATLD and WHO**
**Usually 100% of positive and 10% of negative smears**
**Usually un-blinded — 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**

## 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**
**Ref: Balandrano et al, National QC of AFB Microscopy in Mexico 1999 IUATLD, Budapest **

## Proposed International GuidelinesEQA - 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**

## Proposed EQA - AFB Microscopy Guidelines Key FeaturesResource Analysis

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

## Proposed EQA – AFB Microscopy Guidelines Key FeaturesOn-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**

## 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?**

## Slide 14

**Notes:**
Quality Control in the TB Laboratory

Quality Control (QC) should be routinely
performed in the laboratory to ensure
reliability as well as reproducibility of results.

Perform QC on each new batch of stain. Observe these results before reading patient slides. This important step will verify correct staining procedure and microscope function.

When controls are not demonstrating proper staining,
determine the cause and correct it.

If the negative control appears red after the staining procedure, this is the result of incomplete decolorization. 

## 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?**

## Proposed EQA – AFB Microscopy Guidelines Key FeaturesProficiency 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**

## Semi-quantitative ReportingZiehl 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+**

**Notes:**
The following semi-quantitative reporting method is
recommended for the Ziehl Neelsen technique.

If No AFB are found in 100 fields, report:
No acid-fast bacilli observed.
If 1-9 are found in 100 fields:
record the exact figure.
If 10-99 AFB are found in 100 fields:
report as 1+.
If 1-10AFB are found per field, report as 2+.
f greater than 10 AFB per field, report as 3+.

## Slide 18

## Slide 19

| Correct | No errors |  |
| --- | --- | --- |
| QE | Quantification Error | Minor error |
| LFN | Low false negative | Minor error
 |
| LFP | Low false positive | Minor error
 |
| HFN | High false negative
 | Major error |
| HFP | High false positive | Major error |

## Proposed EQA – AFB Microscopy Guidelines Key FeaturesRechecking

**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 sensitivity**
**Positives and negatives sampled**
**Minor errors (FP or FN with 1-9 AFB/ 100 f) are used as a surrogate  **

## Rechecking LQAS example -Mexico

| Annual Volume | Positive | Negative | Current
Rechecking | LQAS
Rechecking |
| --- | --- | --- | --- | --- |
| 501 | 76 | 425 | 119 | 61 |
| 2219 | 44 | 2175 | 262 | 194 |
| 6650 | 138 | 6512 | 789 | 214 |

## Prevalence of positive slides

** **

## Sample size example

- 104 = 94  +  10

**N = 4500 negatives (5000 – 500 positives)**
**Sensitivity = 80%**
**Corresponding CV = 2.20**

**Increased proportional to prevalence **
**of positives - 10%**

## Interpretation of rechecking errors

**If there are no errors then lab is meeting sensitivity of 80% (95% confidence level)**
**Many/most labs will have at least one error and each laboratory should be evaluated based on the number and types of errors**
**Some low false negatives (LFN) are to be expected, but HFN may signal a problem**
**Any false positives may indicate a systematic problem**

## Proposed EQA – AFB Microscopy GuidelinesResource AnalysisPhased 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**

## Proposed EQA – AFB Microscopy GuidelinesResource AnalysisPhased Approach (cont)

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

## Slide 27

**Notes:**
“An Ancient disease is killing more people today
than ever before. Tuberculosis, which many of us
believed would disappear in our lifetime, has staged
a frightening comeback.
We are at the crossroads.
We can permit the TB epidemic to become more
deadly, or we can act now to end the suffering and
deaths.
We are at the crossroads.”
G. Harlem Brundtland, M.D., Director, WHO 1998
As we enter the twenty first century, many diseases
continue to challenge the health of people around the
world. Tuberculosis, both an ancient and a modern
disease, looms in the foreground.

Over 8 million cases of TB continue to occur annually.
Close to 2 million people still die of TB each year.

Poverty and malnutrition fuel the fire of disease in
developing countries, while migrations, wars, and natural
catastrophes set the scene for mass infection.

The global escalation in TB,the rise in HIV and the
emergence of multiple drug resistant strains of TB have
caused urgency and concern.

Several international agencies are dedicated to the control and eradication of tuberculosis worldwide.

Practical and affordable diagnosis is a key link in the chain.