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Assuring the Quality of Laboratory Testing in Countries Fighting the HIV/AIDS Epidemic. CDC November 29-30, 2000. Test Verification & Test Validation. Niel T. Constantine, Ph. D. Professor of Pathology Director Clinical Immunology. University of Maryland School of Medicine And

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Assuring the quality of laboratory testing in countries fighting the hiv aids epidemic l.jpg

Assuring the Quality of Laboratory Testing in Countries Fighting the HIV/AIDS Epidemic

CDC

November 29-30, 2000


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Test Verification Fighting the HIV/AIDS Epidemic&Test Validation

Niel T. Constantine, Ph. D.

Professor of Pathology

Director Clinical Immunology

University of Maryland

School of Medicine

And

Institute of Human Virology


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Test Verification &Test Validation Fighting the HIV/AIDS Epidemic

  • Considerations when determining the utility of tests

    A. Protocols for Evaluation of Tests

    B. Reference Tests

    C. Algorithms

    D. Choice and Number of Samples

    E. Testing Conditions

    F. Resolution of Discordant Results

    G. Indicators of test performance


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Considerations When Determining the Utility of Tests Fighting the HIV/AIDS Epidemic

Protocols for Evaluations of Tests


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Protocols for Evaluation of Tests Fighting the HIV/AIDS Epidemic

  • Essential to set guidelines.

  • Must be followed exactly.

  • Must outline all characteristics of samples and procedures.

  • Must describe detailed algorithm to follow for discordant results.

  • Must include QA/QC section.


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Considerations When Determining the Utility of Tests Fighting the HIV/AIDS Epidemic

Reference Tests


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Reference Tests Fighting the HIV/AIDS Epidemic

  • Needed to fully characterize samples.

  • Choice depends on purpose of testing.

    • Concordance – against reference screening test.

    • Accuracy – against confirmatory test.

  • Must be careful about “pre-selected samples” to evaluate false positives.

  • Should be tests that are recognized by the scientific community.


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Considerations When Determining the Utility of Tests Fighting the HIV/AIDS Epidemic

Algorithms


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UNAIDS and WHO Fighting the HIV/AIDS EpidemicRecommended Alternative Algorithms

  • To maximize accuracy while minimizing cost

  • Depends on objectives of the test and the prevalence of infection


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Table 2 UNAIDs and SHO reccommendations for HIV testing strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

according to test objective and prevalence of infection in the de depistage du VIH, en fonction de l’obectif du test et de la

sample population prevalence de l’infection dans la population

Objective of testing Prevalences of infections Testing strategy

Objectif du dépistage Prévalences de l’infection Stratégie de dépistage

Transfustion/transplant safety All Prevalences

Sécurité des transfusions/transplantations Toutes prévalences 

Surveillance >10% 

10% 

Clinical signs/symptoms of >30% 

HIV Infection- Signnes

Cliniques/symptôms de 30% 

l’infection à VIHa

Asymptomatic >10% 

Asymptomatique

10% 

aWorld Health Organizaion, Intenm proposal for a WHO staging system for HIV infection and desease (WER no.29, 1990, pp 221-228)- Organisation mondiale de la sante. Echelle provisoire OMS proposee pour la determinationdes strades de l’infecrtiono VIH et de la malodie (REN no 29, 1990. P.221-228)


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Considerations When Determining the Utility of Tests strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Choice and Number of Samples


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Choice and Number of Samples strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Samples:

  • Should represent population where test will be performed.

  • Same matrix of sample (e.g. plasma).

  • Must meet guidelines stated by manufacturer (e.g. not lipemic).

  • Avoid multiple freeze/thaw, etc.

  • Use “clean”samples.

  • Multiple aliquots if possible.

  • Must be well categorized.


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Choice and Number of Samples strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Samples:

  • Should represent population where test will be performed.

  • Same matrix of sample (e.g. plasma).

  • Must meet guidelines stated by manufacturer (e.g. not lipemic).

  • Avoid multiple freeze/thaw, etc.

  • Use “clean”samples.

  • Multiple aliquots if possible.

    Numbers of Samples:

  • The more the better (min. 30 positives, 200 negatives).

  • Depends on purpose of testing (e.g. blood donors).

  • Include appropriate percent of variants.

  • Perform precision and reproducibility studies (lg. Volumes).


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HIV Classification strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

HIV

Types

HIV-1

HIV-2

M

O

Groups

N

ROD NIH2

ANT 70,

MVP5180,

VAU

A, B, C, D,

E, F, G, H, I, J

Guidelines for Classification

Types: HIV-1 and HIV-2

50% homology

Subtypes/Groups: HIV-1 group M, N and O

60-70% homology

Clades: HIV-1 Clades A-J

>70% homology

Clades


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Considerations When Determining the Utility of Tests strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Testing Conditions


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Testing Conditions strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Must test under identical conditions.

    (e.g. same lab, equipment, day, tech).

  • Use non-expired kits that have been properly stored.

  • Follow manufacturer’s recommendations.

  • Sample integrity.

  • Test in a blinded fashion.


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Considerations When Determining the Utility of Tests strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Resolution of Discordant Results


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Resolution of Discordant Results strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Check sample integrity, labeling, paperwork, and procedures.

  • Repeat by same technologist.

  • Repeat blindly by another technologist.

  • Repeat reference test blindly.

  • Repeat at different laboratory.

  • Determine true status by other means.

  • What parameters would these investigate?


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Resolution of Discordant Results strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategiesPossible Variants

  • Synthetic peptide tests

  • Specific Western blots

  • Specific IFAs

  • Combination tests

    • Dot blots

    • Immunoconcentration tests

    • Augmented blots and LIA

  • PCR - specific


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Rapid Assay Evaluation Algorithm strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Rapid Assay +

ELISA -

Rapid Assay -

ELISA +

Discordant Results

Repeat Rapid & ELISA

Western Blot Assay (FDA Licensed)

Negative

Indeterminate

Positive

IFA (FDA Licensed)

Resolved

Negative

Indeterminate

Positive

Sample Volume

> 1 mL

Sample Volume

(<1 mL & >0.2 mL)

Resolved

Resolved

P24 Ag Assay (FDA Licensed)

RT-PCR Assay

Negative

Positive

Inconclusive

Ag Neutralization

Negative

Positive

Positive

Negative

Inconclusive

Resolved

Resolved


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Considerations When Determining the Utility of Tests strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Indicators of Test Performance


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Indicators of the Value of a Diagnostic Assay strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Sensitivity

  • Specificity

  • Test efficiency

  • Delta values

  • Predictive values


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Sensitivity of Tests strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Sensitivity (epidemiologic)

  • Sensitivity (analytical)

    • Low titer

    • Seroconversion

    • Dilutions


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Indicators of the Value of a Diagnostic Assay strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Sensitivity = True Positives

True Positives + False Negatives

X 100%

Specificity = True Negatives

True Negatives + False Positives

X 100%


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Indicators of the Value of a Diagnostic Assay strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Positive Predictive = True Positives

Value True Positives + False Positives

X 100%

Negative Predictive = True Negatives

Value True Negatives +False Negatives

X 100%


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Predictive Values strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Assume: Test Sensitivity = 100% / Specificity = 99.5%

Population #1, where the prevalence of infection is high (5%)

  • Population: 1000 sera tested

    50 sera from infected individuals

    950 sera from non-infected individuals

  • Test Results: 50 positives: 45 from the infected group

    5 false pos from the non-infected group

  • Therefore, the positive predictive value is:

    PPV = 45 = 90%

    45+5

  • 9 out of 10 positive results will be from infected persons


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Predictive Values strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Assume: Test Sensitivity = 100% / Specificity = 99.5%

Population #2, where the prevalence of infection is low (0.7%)

  • Population: 1000 sera tested

    7 sera from infected individuals

    993 sera from non-infected individuals

  • Test Results: 7 positives: 2 from the infected group

    5 false pos from the non-infected group

  • Therefore, the positive predictive value is:

    PPV = 2 = 28.6%

    2+5


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Predictive Values strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Therefore, the same test that yields the same number of false-positives produces a different positive predictive value when testing two different populations


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Predictive Values strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Therefore, the same test that yields the same number of false-positives produces a different positive predictive value when testing two different populations.

  • The chance of a positive result being from a truly infected individual in the low prevalence population is only 28.6% (2 true positive detected by the test and 5 false-positives).


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Predictive Values strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Therefore, the same test that yields the same number of false-positives produces a different positive predictive value when testing two different populations.

  • The chance of a positive result being from a truly infected individual in the low prevalence population is only 28.6% (2 true positive detected by the test and 5 false-positives).

  • This indicates that a positive result by the test will be from an infectd individual in only one of four cases (a guess could yield better chance!).


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Test Verification &Test Validation strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Quality Assurance and Errors

  • A. Common Errors

  • B. Quality Assurance Needs

  • 1. Fundamentals of QA

  • 2. Quality Control

  • 3. Quality Assessment

  • 4. Equipment Issues

  • 5. 10 Key Issues for QA


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Most Common Errors strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Transcription

  • Carelessness

    • Procedures

    • Specimens

  • Environmental conditions

  • Pipettes and pipetting


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Clerical Errors strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Logging specimens

  • Aliquoting

  • Worksheets

  • Result printouts

  • Translating results

  • Computer entering

  • Reports

  • Supervisory Review


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Specimen Problems strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Insufficient volume for repeating

  • Hemolysis, lipemia, and bacterial contamination

  • Insufficient and inadequate labeling

  • Misidentified specimens

  • Frozen / Thawed (multiple)


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Other Types of Errors strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Kit Dependent Problems.

  • Technologist – dependent errors.

  • Inter-lot variations and Intra-lot variations.

  • Environmental problems.

  • Non repeatable results.

  • Inter-laboratory and Intra-laboratory variations.

  • Equipment problems.


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Quality Assurance strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategiesFundamental for Quality Test Results

  • Record keeping

  • Monitoring laboratory staff

  • Vigilance in the laboratory

  • Verification of true positive and true negatives

  • Parallel testing of resubmitted samples

  • Reporting of results

  • Confidentiality

  • Interaction with physicians

  • Storage of specimens for follow-up testing

  • Laboratory efficiency

  • Total quality management


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Components of Quality Control strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategiesRecord Keeping

  • Kit lot numbers (expiration and open dates).

  • Clearly label reagents with date opened or prepared (include open and expiration date) on each label.

  • Daily temperature monitoring and recording i.e. Incubators water baths, ambient.

  • Performance of controls and action taken when out-of-range.

  • Photograph or clear photocopies of Western blots.

  • Ratios of in-house controls to cut-off values.


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Components of Quality Control strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategiesControls

  • Kit controls: Use as directed by the manufacturer.

  • In-house controls: preferably three levels to monitor variability between runs and lot numbers of kits.

    • Low positive – absorbance enough above cut-off that it should not be misclassified because of expected run-to-run variability.

    • High positive – well above the cut-off.

    • Negative – well below cut-off.

  • Storage of in-house control sera:

    • Dispense in aliquots sufficient for one week of use.

    • Freeze at -20°C in a non-self-defrosting freezer.

    • Thaw each aliquot once, store at 4 °C when not in use, do not refreeze and discard after 1 week.


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Monitoring by External Controls strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Trend

Shift


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Quality Assessment strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Internal Quality Assessment

  • Known Reactors

  • Unknown Reactors

  • Blind Testing

    External Quality Assessment

  • Proficiency Panels

  • Blind Proficiency Panels


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Equipment Issues strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Pipette Calibrations

ESSENTIAL FOR ACCURACY

  • Frequency

    • At least every 6 months

  • Reasoning

    • 1l inaccuracy = 10% error (total volume of 10 l)

    • Controls – o.k., borderline specimens – loss of sensitivity


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Quality Assurance: What Must Be Done? strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies10 Key Issues

  • Detailed SOP with total compliance.

  • Supervising review of all paperwork.

  • Develop checklists for monitoring all activities.

  • Dev. Organizational schemes for processing, documentation, and assessment.

  • Monitor staff – blind proficiencies.

  • Neat and complete documentation of all results.

  • No deviation from procedures.

  • Maintain confidentiality.

  • Endorse safety measures.

  • Vigilance.


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Test Verification &Test Validation strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

III. Introduction of a New Test

A. Selection

B. Characteristics

C. Approved versus Non-Approved tests

D. Continual Monitoring


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Selection strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Availability

  • Appropriateness

  • Cost and bulk purchases

  • Shelf life and robustness

  • Storage

  • Publications and WHO evaluations

  • Regulations


Characteristics l.jpg
Characteristics strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Laboratory capabilities

  • Testing Purpose

  • Simplicity

  • Cost Concerns

  • Sample type

  • Test limitations

  • Test principles and antigens

  • Test indices


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Approved Versus strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategiesNon-approved Tests

  • Which can be used?

  • When approved tests are unavailable.

  • Validation of non-approved tests.

  • Documentation necessities and qualifications.


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Continual Monitoring strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Necessity to monitor new tests.

  • How long to monitor.

  • Methods of monitoring.

  • Looking for trends.

  • Changing tests – Parallel testing.

  • Documentation.


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Test Verification &Test Validation strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

IV. Special Considerations for Developing Countries

A. Selection of Tests and algorithms

B. Testing under non-optimal conditions.

C. Best fit Strategies

D. When Systems Fail


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Special Considerations for Developing Countries strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Selection of Tests and Algorithms


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Selection of Tests strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Infrastructure

  • Supportability

  • Expertise

  • Accessibility

  • Cost Concerns

  • Algorithms


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Algorithms strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • What’s effective?

  • What can be used?

  • Established and recommended algorithms.

  • Use of additional strategies.

  • Differences due to geographical origins of samples.

  • Cost effectiveness.

  • Sample pooling.

  • Blood donations vs. diagnostic testing.

  • Different algorithms within the same country.

  • Epidemiological testing.


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Simple, Rapid Test Alternative Algorithm strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Rapid Test #1

Positive

Negative

Repeat in Duplicate

REPORT

P/N OR P/P

N/N

REPORT

Rapid Test #2*

*Different configuration or antigens

Positive

Negative

REPORT indeterminate

Report as Positive

Resolve with other tests


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Special Considerations for Developing Countries strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Testing Under Non-optimal Conditions


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Testing Under Non-optimal Conditions strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Use of expired kits.

  • Unsatisfactory environmental conditions.

  • Limited number of test kits.

  • Limited equipment (e.g. thermometers).

  • Non-calibrated pipettes.

  • Old equipment.

  • Poor integrity of samples.

  • Questionably labeled specimens.


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Special Considerations for Developing Countries strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

Best Fit Strategies


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Best-fit Strategies strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies(to Test or Not to Test?)

  • Consequences and necessities.

  • Cost effective strategies.

  • Pooling of samples.

  • Saving reagents.

  • Parallel testing.

  • Sequential testing.

  • Mixing reagents.

  • Alternate testing areas.

  • Testing when temperatures and conditions fail.


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Pooling of Samples strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • In what situations can pooling be used?

  • How many samples can be pooled?

  • Accuracy.

  • Final sample dilution of pools.

  • Proper sample size for evaluation.

  • Effects of the presence of HIV Antigens.


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Non-approved Testing Strategies strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

  • Re-use of rapid tests.

  • Modification of test kits:

    • Cutting WB strips.

    • Halving reagents.

  • Pooling of samples.


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    Special Considerations for Developing Countries strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

    When Test Systems Fail


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    When Test Systems Fail strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

    • Trouble shooting.

    • Repeat testing.

    • Alternative testing.

    • Other personnel, other laboratories.

      • Getting help.

    • Documentation.

    • Reporting.

    • Consequences.


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    Reasons for the Need for Improved Assays strategies Tableau 2 Recommandations de I’onusida et de I’OMS aux strategies

    • Early diagnosis.

    • Resolution of indeterminate results.

    • Differentiation of retroviral infections.

    • Less expensive tests.

    • Simple and foolproof tests.

    • Detection of viral types and variants.

    • Multiple combination tests.

    • Detection of infection in the newborn.


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