Guidelines for Appropriate Evaluations of

HIV Testing Technologies in Africa

In-Press

TABLE OF CONTENTS

Acknowledgements

Abbreviations

Definition of Terms

Executive Summary

1.  Background

    1.  Serodiagnosis of HIV

    2.  EIAs

    3.  Rapid/Simple assays

    4.  Importance of Rapid/simple Assays

    5.  Synopsis of HIV Testing Strategies

        1.  WHO/UNAIDS Testing Strategies

2.  Rational for Justifications for Conducting Test Evaluations

    1.  Evaluating HIV Assays in Africa

    2.  Justification for evaluating new HIV test kits

3.  Laboratory Quality Assurance (QA) and Safety

    1.  Importance of QA

    2.  Quality Control

    3.  External Quality Assessment (EQA)

    4.  Safety Precautions

4.  Planning an Evaluation

    1.  Responsibilities of NRL

    2.  Program Coordination

    3.  Funding Considerations

    4.  Test Selection Criteria for Country-level Evaluation

    5.  Planning Activities and Timeline

    6.  Technical Training Requirements

5.  Conducting the Evaluation

    1.  Overview of Evaluation Phases

    2.  Objectives of Evaluation Phases

    3.  Evaluation Scenarios

> Advantages and Disadvantages
>
> 5.4 Phase I: Laboratory Evaluation
>
> 5.4.1 Use of Stored Serum
>
> 5.4.2 Sample Size
>
> 5.4.3 Sample Population
>
> 5.5 Phase II: Field Evaluation/Pilot Testing
>
> 5.5.1 Number of Sites
>
> 5.5.2 Sample Population
>
> 5.5.3 Sample Size
>
> 5.6 Phase III: Implementation and Monitoring of Test Performance
>
> 5.6.1 Implementation
>
> 5.6.2 EQA
>
> 5.6.2.1 Proficiency Testing
>
> 5.6.2.2 Dried Blood Spots (DBS)
>
> 5.6.3 Remediation / Corrective Measures

6.0 Evaluation Materials

> 6.1 Types of Evaluation Materials
>
> 6.2 Specimen Collection and Handling

1.  Specimen Collection

    1.  Plasma

    2.  Serum

    3.  Whole Blood

> 6.2.2 Transfer and Storage of Specimens
>
> 6.2.3 Improving the Quality of Stored Sera
>
> 6.3 Serum Library
>
> 6.3.1 Characterization of Evaluation Panel
>
> 6.4 Trouble-shooting of Problematic Samples
>
> 7.0 Data Analysis

1.  Data Management

2.  Resolving Discordants

3.  Sensitivity, Specificity, PPV, NPV, CI, Delta values,
    Reproducibility,

> Inter-reader variability of rapid tests

8.  Reporting Results, Conclusions, Recommendations

    1.  Developing an Algorithm

    2.  Reporting Results

    3.  Aggregation and Dissemination of Evaluation Data

Appendix

A Parallel and Serial Testing Algorithms

B Summary of WHO Testing Strategies

C Potential Testing Strategies

D Rapid Test Methodologies and Degree of Implementation

E Laboratory Safety Rules

F Sample Evaluation Expenditures

G Sample Outline of an Evaluation Protocol

H Confidence Ranges

References

Use of trade names and commercial sources is for identification only and
does not imply endorsement by the Public Health Service or by the U.S.
Department of Health and Human Services.

Acknowledgements

The Centers for Disease Control and Prevention (CDC) and the African
Regional Office of the World Health Organization (WHO/AFRO) wishes to
express their gratitude to individuals who contributed their time and
expertise by participating in a workgroup meeting November 28 --
December 1, 2001 in Harare, Zimbabwe, to develop these guidelines.

Workgroup Participants

> Ms. Hiwot Berhanu
>
> Ethiopian Health and Nutrition Research Institute
>
> National Referral Laboratory for AIDS
>
> Ethiopia
>
> Ms. Eileen Burke
>
> Centers for Disease Control and Prevention (CDC/ZIM)
>
> Zimbabwe
>
> Dr. Guy-Michel Gershy-Damet

World Health Organization, Regional Office for Africa (WHO/AFRO)

Zimbabwe

> Mr. Henry Feluzi
>
> Lilongwe Central Hospital
>
> Malawi
>
> Ms. Stacy Howard
>
> Centers for Disease Control and Prevention (CDC)
>
> Public Health Practice Program Office (PHPPO)
>
> Atlanta, GA
>
> Mr. Brighden Kakonkanya
>
> Virology Laboratory
>
> University Teaching Hospital
>
> Zambia
>
> Dr. Eligius Lyamuya
>
> Department of Microbiology and Immunology
>
> Muhimbili University College of Health Sciences
>
> Tanzania
>
> Dr. Terry Marshall
>
> National Institute for Virology
>
> South Africa
>
> Mr. Louis Mururi
>
> Harare Central Hospital
>
> Zimbabwe
>
> Dr. Manase Mutingwende
>
> National Microbiology Reference Laboratory (NMRL)
>
> Zimbabwe
>
> Dr. Christina Mwangi
>
> Nyangabgwe Hospital
>
> Botswana
>
> Dr. Patrick Osewe
>
> USAID/Zimbabwe
>
> Dr. Mark Rayfield
>
> CDC
>
> National Center for Infectious Diseases (NCID)
>
> Atlanta, GA
>
> Dr. John Ridderhof
>
> CDC /PHPPO
>
> Atlanta, GA
>
> Mr. Pierre Rugimbanya
>
> Treatment Research AIDS Center (TRAC)
>
> Rwanda
>
> Dr. Michael St. Louis
>
> CDC/Zimbabwe
>
> Mr. Sergio Stakteas

CDC /Maputo

> Mozambique
>
> Mr. Ziyambi Ziyanbi
>
> Population Services International (PSI)
>
> Zimbabwe
>
> Ms. Judith Wethers
>
> Association of Public Health Laboratories (APHL)

Appreciation is also given to Dr. El hadj Belabbes (Algeria), Dr. Robert
Downing (Uganda), Dr. Chantal Maurice (Cote d'Ivoire), Professeur
Souleymane Mboup (Senegal), and Dr. John Nkegasong (Cote d'Ivoire) for
their technical contributions.

**ABBREVIATIONS**

CDC Centers for Disease Control and Prevention

EIA Enzyme Immunoassay

ELISA Enzyme-Linked Immunoassay

EQA External Quality Assessment

HIV Human Immunodeficiency Virus

PMTCT Prevention of Mother to Child Transmission

NAP National AIDS Program

NPV Negative Predictive Value

NRL National Reference Laboratory

POS Point of Service

PPV Positive Predictive Value

PT Proficiency Testing

QA Quality Assurance

QC Quality Control

Se Sensitivity

Sp Specificity

SOP Standard Operating Procedure

VCT Voluntary Counseling and Testing

WB Western Blot

WHO World Health Organization

WHO/AFRO World Health Organization -- Regional Office for Africa

**Definition of Terms**

> **Algorithm** - The sequence in which assays are performed to detect
> HIV antibody in a body fluid.
>
> **Confidence Interval --** An interval estimate of a population
> parameter computed so that the statement "the population parameter
> lies in this interval" will be true at a stated confidence, usually
> 95%.
>
> **Evaluation** -- A process for determining whether a test system
> meets defined needs in the potential user's environment.
>
> **Evaluation Panel --** Specimens that are used during the evaluation
> for which the serostatus has been previously defined by the gold
> standard.
>
> **External Quality Assessment (EQA)** -- A program that allows
> laboratories or testing sites to assess the quality of their
> performance by comparison of their results with other laboratories,
> through analyzing proficiency panels, or blind rechecking. EQA also
> includes on-site evaluation of the laboratory to review the quality of
> test performance and operations.
>
> **Gold Standard** -- A country defined algorithm for determining a
> sample's true serostatus.
>
> **National Reference Laboratory** -- A nationally recognized
> laboratory with appropriate testing capabilities and facilities for
> performing or providing access to confirmatory HIV testing sufficient
> to determine HIV status.

**Negative predictive value -** In HIV testing, the probability that
when a test is non-reactive, the specimen does not have antibody to HIV.

**Positive predictive value -** In HIV testing, the probability that
when a test is reactive, the specimen actually contains antibody to HIV.

**Prevalence -** The percentage of persons in a given population with a
disease or condition at a given point in time.

**Proficiency testing panel** -- A set of approximately 3-5 samples with
known values used to assess the performance capabilities of testing
personnel.

**Quality Assurance** - Planned and systematic activities to provide
adequate confidence that requirements for quality will be met.

**Quality Control --** Operational techniques and activities that are
used to fulfill requirements for quality.

**Reference Panel --** Aliquoted, stable serum or plasma specimens that
have been highly characterized; known cutoff-points, subtype, titer,
etc.

**Sensitivity of a test -** A measure of the probability for correctly
identifying an HIV-infected person.

**Serum Library** -- A source of serum specimens from which a panel is
drawn for evaluation purposes.

**Specificity of a test** - A measure of the probability of correctly
identifying an HIV-uninfected person.

**Testing strategy -** The use of an appropriate HIV test or combination
of HIV tests for identifying positive specimens. The choice of testing
strategy used is based on the objective of the test, the sensitivity and
specificity of the test, and HIV prevalence in the population being
tested.

**Window Period -** The period of time following exposure and infection
with HIV and the generation of detectable antibodies by the infected
person.

1.  **Executive Summary**

Ensuring the quality of HIV testing in support of prevention and care
efforts has been identified as a priority by the U.S. Centers for
Disease Control and Prevention (CDC) and the World Health
Organization/African Regional Office (WHO/AFRO). Rapid/simple HIV tests
are marketed widely, and promoted for use by a variety of HIV/AIDS
prevention strategies such as voluntary counseling and testing (VCT) and
prevention of mother to child transmission (MTCT). It is vitally
important that before these and other HIV assays are utilized, countries
evaluate the performance of each assay to determine its performance
characteristics and suitability for use within a given country setting.
This evaluation is considered a critical aspect of assuring the quality
of test results, and all countries must make this a priority.

This document is intended to provide those involved with planning or
conducting any aspect of test evaluations practical guidance for
developing country-specific protocols for conducting evaluations of HIV
EIA and rapid/simple test methods. As test evaluations require both time
and resources, specific guidance is given on the rationale and
justification for evaluating new tests, issues to consider when planning
an evaluation, and projected timeline for an evaluation. Detailed
descriptions of phases of the evaluation quality assurance, evaluation
materials, e.g., specimens, and laboratory safety precautions are also
presented in this document.

**1.0 Background**

#### 1.1 [Serodiagnosis of HIV]{.underline}

> Africa is the continent most affected with human immunodeficiency
> virus (HIV) epidemic: of the estimated 40 million persons infected
> with HIV in the world by the year 2001, 28 million live in Africa
> \[1\]. HIV antibody testing is critical for controlling the epidemic
> because it is the critical entry point for both prevention and care
> efforts for HIV/AIDS. For instance, short-course regimen of antiviral
> therapeutics administered to HIV-infected pregnant women reduces rates
> of transmission of HIV-1 from infected mothers to infants by 38% to
> 50% \[2,3, 4, 5, 6\]. Also, cotrimoxazole administered together with
> standard tuberculosis therapy reduces mortality and morbidity by 40 -
> 45% among HIV- infected tuberculosis patients \[7\]. For HIV-infected
> persons to benefit from such therapies, they must be diagnosed
> appropriately. Serologic diagnosis of HIV infection is based on a
> multi-test algorithm for detecting antibodies to HIV. Screening tests
> provide presumptive identification of specimens that contain antibody
> to HIV. These enzyme immunosorbent assays (EIAs) or simple/rapid
> immuno-diagnostics are selected for their high sensitivity of
> detecting antibodies to HIV. Supplemental or confirmatory tests, such
> as Western blot (WB), can be used to confirm infection in samples that
> are initially reactive on conventional EIAs. Alternatively, repetitive
> testing incorporating EIAs or rapid tests selected for their
> specificity may be used to confirm whether specimens found to be
> reactive for HIV antibodies with a particular screening test are
> specific to HIV. For practical purposes, resource-poor settings depend
> heavily on EIA and rapid tests for screening and confirmation.

#### 

#### 1.2 [EIAs]{.underline}

> EIAs are the most widely used screening tests because of their
> suitability for analyzing large numbers of specimens, particularly in
> blood screening centers. Since 1985, EIAs have progressed considerably
> from first to fourth generation assays: first generation assays were
> based on purified HIV whole viral lysates, however, sensitivity and
> specificity of these assays were poor; second generation assays used
> HIV-recombinant proteins and/or synthetic peptides, which enabled the
> production of assays capable of detecting HIV-1 and HIV-2. The assays
> had improved specificity, although their overall sensitivity was
> similar to that of first-generation assays. Third-generation assays
> used the solid phase coated with recombinant antigens and /or peptides
> and similar recombinant antigens and peptides conjugated to a
> detection enzyme or hapten that could detect HIV-specific antibodies
> bound to a solid phase. These assays could detect immunoglobulin M,
> early antibodies to HIV, in addition to IgG, thus resulting in a
> reduction of the seroconversion window. Fourth generation assays are
> very similar to third-generations tests but have the ability to detect
> simultaneously HIV antibodies and antigens. Typical fourth-generation
> EIAs incorporate cocktails of HIV-1 group M (HIV-1 p24, HIV-1 gp160),
> HIV-1 group O, and HIV-2 antigens (HIV-2 *env* peptide). Furthermore,
> third and fourth-generation assays are able to detect IgM and IgG
> antibodies to both HIV-1 and HIV-2. These assays may reduce the 2-4
> week time period or "window period" of detecting HIV antibodies.

1.3 [Rapid/Simple assays]{.underline}

> Simple, instrument-free assays are also available and are now widely
> used in Africa. They include agglutination, immunofiltration, and
> immunochromatographic assays. The appearance of a colored dot or line,
> or an agglutination pattern indicates a positive result. Most of these
> tests can be performed in less than 20 minutes, and are therefore
> called simple/rapid assays. Some simple tests, such as agglutination
> assays, are less rapid and may require about 30 minutes to 2 hours to
> be completed. In general, these rapid/simple tests are most suitable
> for use in settings that have limited facilities and process fewer
> than 100 samples per day.

####### 1.4 [Importance of rapid/simple assays]{.underline}

#######  Although EIA--based serodiagnostic algorithms are highly cost effective, their application in resource-poor settings is limited by several factors. They require well-trained personnel, need a consistent supply of electricity, and maintenance and cost of most equipment. Rapid assays have high sensitivity and specificity and perform as well as EIAs on specimens from persons seroconverting for non-B HIV-1 subtypes \[8\]. Rapid enzyme assays circumvent the issue of low rates of return for serologic results associated with EIA-based testing algorithms because results can be delivered on the same day. In addition, their performance has improved considerably, and some do not require reconstitution of reagents or refrigeration; thus, making them very suitable for use in resource limited settings and hard to reach populations. Practical applications for the use of simple/rapid assays are in settings such as Voluntary Counseling and Testing (VCT) and Prevention of Mother to Child Transmission (PMTCT) programs. Studies have shown that using rapid assay testing algorithms result in remarkable increase in the number of HIV-positive women identified as eligible to receive the short-course therapy that reduces mother-to-child transmission of HIV \[9\].

1.5 [Synopsis of HIV Testing]{.underline}

> A testing algorithm for serologic diagnosis of HIV-infection is the
> sequence in which assays are performed to detect HIV antibody in a
> body fluid. The most common referenced testing algorithm employs an
> EIA to screen specimens with those found to be positive then confirmed
> by WB testing. This so-called conventional algorithm has several
> limitations:
>
> \- WB is expensive and requires technical expertise

-   WB often yields indeterminate results with certain types of
    specimens with uncertain diagnostic significance, e.g.,
    hyperimmunoglobulinemia specimens

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-   Both ELISA and WB are time consuming and require a well-equipped
    laboratory infrastructure

> Several alternative testing algorithms exist for the serologic
> diagnosis of HIV infection that are based on a combination of
> screening assays, without using WB. In a parallel testing algorithm,
> sera are simultaneously tested by two assays. In the serial algorithm
> all specimens are tested by a first test that is highly sensitive.
> Specimens are considered as true negative if they react negatively in
> the first test. Specimens that are reactive in this assay are retested
> by a second EIA that has a high specificity. Parallel testing
> algorithms are often used in the clinic setting, such as with rapid
> assays using whole blood fingerstick specimens, to avoid requesting a
> second specimen from the client when the first test is HIV reactive.
> Serial algorithms may be more cost effective and convenient when
> sufficient specimen, such as with a venipuncture, is available to
> perform additional tests when the initial test is HIV reactive.
>
> These algorithms maintain accuracy and minimize cost. Most of these
> algorithms have been evaluated in field conditions in Africa and found
> to be highly effective. Regardless of the testing algorithm (Appendix
> A[),]{.underline} the first test must be highly sensitive and the
> second should be highly specific.

1.5.1 [WHO/UNAIDS testing strategies]{.underline}

> In considering both serial and parallel testing algorithms, WHO and
> UNAIDS have recommended three testing strategies (figure 1). Criteria
> for choosing the appropriate HIV testing strategy (Appendix B)
> include:

1.  Objective of the test (diagnosis, surveillance, blood safety, or
    research),

2.  Sensitivity and specificity of the test(s) being used, and

> 3\. HIV prevalence in the population being tested

######## Potential testing strategies based on data from several countries can be found in 

> Appendix C. Information on the manufacturers of rapid test kits can be
> found in Appendix D.
>
> Figure 1: Schematic representation of the WHO/UNAIDS HIV testing
> strategies

**[Strategy I]{.underline}: [Strategy II:]{.underline} [Strategy
III:]{.underline}**

Transfusion/transplant safety Surveillance- Diagnosis - Prevalence \<10%

Surveillance - Prevalence \>10% Diagnosis -

Prevalence \>10%, asymptomatic

Prevalence \< 30%, symptomatic

######## 

**2.0 Rational and Justifications for Conducting Test Evaluations**

2.1 [Rationale for evaluating assays in Africa]{.underline}

> HIV testing algorithms involving the use of supplemental assays such
> as Western blot (WB) or line immunoassay (LIAs) to confirm infection
> in samples that are initially reactive on EIA conventional algorithms
> are still impractical in most African countries due to the high cost
> of the supplemental assays, long turnaround time, and difficulties
> related to interpreting WB and EIA strips. To circumvent these
> limitations, reliable and less expensive HIV serodiagnostic algorithms
> have been evaluated and shown to be as sensitive and specific as the
> conventional algorithm \[10, 11, 12, 13, 14, 15\]. For the testing
> algorithms to be effective, assays employed in them must be highly
> sensitive and specific within the context of the HIV situation in each
> country.
>
> A high degree of genetic diversity exists in several countries in
> Africa \[16\]. For instance, HIV-1 circulating recombinant form
> (CRF_02), and HIV-2 predominate the epidemic in West Africa. In
> Central Africa, a mixture of subtypes, CRFs, group O and N exists. In
> East Africa, subtypes A, C, and D predominate; and in Southern Africa,
> subtype C is most frequent. Although rapid tests continue to improve
> like EIAs, antigens used for these assays were originally derived from
> HIV-1 subtype B viruses. Thus, the existence of newly identified
> aberrant HIV variants in Africa coupled with the high degree of
> genetic diversity of HIV has historically posed a challenge,
> especially for persons during early seroconversion. Indeed, some
> studies have shown a significantly lower sensitivity of some screening
> assays to detect non-B subtypes antibodies during seroconversion
> \[17\]. Moreover, several EIAs were withdrawn from circulation when it
> was shown that some variants of HIV-1 group O viruses were missed by
> these assays.
>
> 2.2 [Justification for evaluating new HIV tests kits]{.underline}

There are many reasons to perform evaluations of HIV tests. Many
countries are performing evaluations to determine an algorithm of simple
rapid tests that can be used at the point-of-service for VCT, PMTCT, and
surveillance. If a country has previously conducted evaluations and has
selected an algorithm of rapid tests that performs adequately, then
there must be compelling reasons for considering evaluating additional
tests. There is often much demand from manufacturers or donors to
evaluate specific tests for use within a country. Due to the number of
kits appearing on the market, a preliminary review of available
performance data cannot be over emphasized. Data are often available
regionally that permit a presumptive determination of the assay's
sensitivity and specificity, reducing the need to evaluate numerous
tests. As a consequence of available data, the decision may be made to
tailor an evaluation to focus solely on the potential implications of
integrating the product into an existing algorithm. An evaluation of
testing algorithms requires time and resources, and each country must
determine the potential advantages of a test (s) before deciding to
perform a formal evaluation.

-   Is there evidence from published studies that indicate the test has
    greatly improved performance characteristics?

-   Is the test(s) much simpler to perform?

-   Is the test(s) more stable to ship and store?

-   Is there a significantly reduced cost with evidence that the
    proposed cost will not increase significantly after implementation?

In many cases there may be no demonstrable improvement gained in a
full-scale evaluation of a new product, either because evidence is
already sufficient to determine its efficacy or there is no demonstrable
need. For example, if a test or algorithm has proven efficacy (Se and
Sp) within the immediate region, then a country may decide to start the
evaluation in the point of service (POS) setting rather than an initial
full scale laboratory-based evaluation. Other circumstances requiring a
limited evaluation at the POS include revising the order of tests within
an approved algorithm or replacing a single test within the algorithm.

Countries should resist pressures to evaluate products solely for in
country marketing concerns. For tests that will be evaluated in-country,
every effort should be made to allow manufacturers or marketers to bear
the costs of evaluating new tests, as evaluations consume a considerable
amount of time and precious resources. Adopting new tests without
adequate evaluation should NOT be considered an option. Doing so will
compromise the integrity of the testing facility, personnel, and quality
of reported results to the patient and/or client.

**3.0 Laboratory Quality Assurance (QA) and Safety**

3.1 [Importance of Quality Assurance]{.underline}

> Laboratory Quality Assurance (QA) is defined as planned and systematic
> activities to provide adequate confidence that requirements for
> quality will be met. It is critical that each facility performing
> laboratory testing establishes and implements a QA program to monitor
> and evaluate laboratory functions and services throughout the total
> testing process. The total testing process is comprised of the
> pre-analytical, analytical, and post-analytical phases of laboratory
> testing. Specific activities (although not all inclusive) of the total
> testing process related to evaluations are outlined below.

[Pre-Analytical phase]{.underline} encompasses the following components:

-   Test request

-   Test selection

-   Trained testing personnel

-   Patient/client preparation

-   Specimen collection, labeling, and transport

[Analytical Phase]{.underline}

-   Specimen processing and storage

-   Reagent preparation

-   Preventative maintenance / Equipment checks

-   [Quality control]{.underline}

-   Test performance

-   [Proficiency Testing / External Quality Assessment]{.underline}

-   Specimen storage

    [Post-analytical Phase]{.underline}

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-   Reviewing quality control

-   Transcribing results

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-   Reporting results

-   Interpreting results

-   Maintaining records

> Written polices and procedures for each activity will assist in
> continually assessing the total testing process for areas needing
> improvement, in identifying problems, and in having defined mechanisms
> to prevent the reoccurrence of problems. A successful QA program will
> need the support of the National Reference Lab and requirements should
> be rigorously complied with to ensure the accuracy of the results from
> the evaluation and all other assays. Comprehensive QA program guidance
> is beyond the scope of this document and can be found in an
> internationally accepted quality management document, e.g., ISO 15189
> --Quality Laboratory Management.

# 3.2 [Quality control (QC)]{.underline} 

> Quality control (QC) refers to those measures that are taken to
> monitor the quality of the assay itself. QC may include the assay of
> samples/materials with known test results to verify the procedure
> itself is working properly. When QC materials analyzed daily produce
> acceptable results, and all other testing conditions have been met,
> then the results of the samples being analyzed may be considered
> acceptable.

3.3 [External Quality Assessment (EQA) / Proficiency
Testing]{.underline}

> Every testing facility must at any time be ready to demonstrate and
> document its competence in performing HIV serology that is carried out
> as part of its routine services. External Quality Assessment (EQA) is
> one component of a laboratory QA program. The focus of EQA is on
> identifying laboratories or testing sites and technicians exhibiting
> poor performance. There are three methods that can be used as part of
> a program to evaluate laboratory performance:

-   On-site Evaluation

-   Proficiency Testing

-   Blinded Rechecking

> The choices for which type of EQA program to implement will depend on
> both the available resources and the ability to obtain additional
> resources as needed to support the EQA program.

Additional information on proficiency testing and the use of Dried Blood
Spots (DBS) as a form of EQA are highlighted in section 5.6.2 (Phase
III: Implementation and Monitoring of Test Performance - EQA).

3.4 [Safety Precautions]{.underline}

Each laboratory or testing site must follow Universal (Standard)
Precautions designed to prevent transmission of HIV, hepatitis B virus
(HBV), and other bloodborne pathogens. When laboratories adhere to
universal precautions, blood and certain body fluids of all patients are
considered potentially infectious for HIV, HBV and other bloodborne
pathogens. Refer to Appendix E for safety rules \[18\] that should be
followed when working in the laboratory.

**4.0 Planning an Evaluation**

4.1 The Ministry of Health (MOH) and the national authority responsible
for HIV/AIDS control, e.g., National AIDS Control Program (NACP), should
designate a National Reference Laboratory (NRL) or other recognized
laboratory in the country that is assigned overall responsibility for
coordinating and conducting evaluations of HIV tests. The NRL should
work closely with the National AIDS control authorities in each country
to ensure coordination of efforts and activities. Each country will need
to evaluate its support structure and available resources in order to
determine the most effective way to conduct the evaluations.

[Responsibilities of the NRL]{.underline}

> The NRL should:

-   Be mandated by the government to either coordinate or perform test
    evaluations

-   Have sufficient resources to conduct or oversee country test
    evaluations

-   Strive to adhere to internationally recognized quality standards,
    e.g., ISO 15189; Quality management in the medical laboratory, UK
    Standards for the Medical Laboratory, etc.

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-   Advise the government about making recommendations and setting
    policy

-   Maintain existing reference methods, such as EIA, and perform or
    provide access to additional reference methods, e.g., WB, PCR, etc.

-   Support the NACP and other laboratories meet the increased need for
    simple/rapid tests in an environment of decentralization

-   Establish and oversee implementation of a national QA program for
    HIV testing

-   Write standard operating procedures for distribution to all testing
    sites

-   Characterize and maintain evaluation and reference panels

    4.2 [Program coordination]{.underline}

    Evaluation of HIV test kits should always be coordinated with the
    NACP and any other organizations that will be using the tests and/or
    results. Program staff should help pre-select test methods,
    especially if rapid tests are being evaluated for use at POS
    locations and non-laboratory staff might perform tests. Shared
    decisions in the planning stages might include the costs of tests,
    test result reporting, ease of use, storage, data-sharing
    mechanisms, in addition to test performance characteristics.

> 4.3 [Funding considerations]{.underline}

Evaluation of tests will require funding over and above the normal
operating costs of performing diagnostic testing. One component of
planning involves developing an itemized budget for each additional cost
and ensuring that funds are available before initiating an evaluation.
The itemized budget should include estimates for the additional test
kits, supplies, any necessary equipment for testing or storage,
transport of specimens during field-testing, and any additional staff
costs (Appendix - F).

> 4.4 [Test Selection Criteria for Country-level
> Evaluations]{.underline}
>
> Having appropriate justification for conducting test evaluations,
> guidelines for selecting assays for evaluation include:

-   Assays that have been previously reviewed by WHO, CDC or other
    independent international organizations with relevant expertise

### 

### Published regional test performance data from:

######  Journal Publications 

WHO/UNAIDS

Manufacturer-provided data

Websites: WHO/AFRO -- [www.AFRO.WHO.INT](http://www.AFRO.WHO.INT/)

> CDC -- <http://www.phppo.cdc.gov/DLS/default.asp>

-   Documented ability of the test to detect HIV-1 (group M and O) and
    HIV-2

-   Documented ability to detect IgG and IgM antibodies

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-   Cost per test and possibility for bulk purchase

-   Storage requirements

-   Equipment and maintenance requirement

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-   Required technical skill

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-   Ease of use; Simplicity of test procedure

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-   Experience with the assay(s)

-   Availability

-   Shelf-life and robustness

-   Service and trouble-shooting provided locally by manufacturers

-   Laboratory infrastructure

> 4.5 [Overview of Planning Activities and Timeline]{.underline}

The following list of activities and timeline (figure \# 2) represents a
typical process for conducting laboratory test evaluations. Details of
each phase of the evaluation are explained in detail in section 5.0
(Conducting the Evaluation). Sample contents of and evaluation protocol
can be found in Appendix G.

-   Determine capacity to conduct evaluations

-   List kits available in country and/or kits approached to evaluate

-   Conduct literature and data review

-   Conduct situation analysis

-   Conduct needs analysis

> Select kits worth assessing
>
> Conduct consensus meetings to gain cooperation of stakeholders

-   Develop Evaluation Protocol

-   Obtain ethics clearance

-   Procure kits, supplies, etc.

-   Conduct Training

Clinic and lab staff

-   Pilot test logistics of plan

-   Implement phase I

-   Evaluate phase I

-   Analyze phase I data

-   Decide which kits to use in phase II / Determine algorithm

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-   Publish phase I findings

-   Select sites for phase II

-   Implement phase II

-   Evaluate phase II

-   Analyze phase II findings

-   Decide which kits/ algorithm to use in the country/setting

-   Publish phase II findings

-   Implement phase III = ongoing monitoring

Build capacity for this during phase I and II trials

FIGURE 2:

Project Development Test Evaluation / Algorithm Development Monitoring

#  Planning Period Phase I Phase II Phase III

(\~ 3 months) (3-6 months) (6 months) (\> 3 months)

Determine Capacity

Literature Review

Situation Analysis

Needs Analysis Proposal

Ethical Review

Procurement Establish Panels

Training Evaluation

Analysis of Data

Algorithm Decision Site Selection

Publish Findings Training of Staff

On Site Evaluation

Data Analysis

Algorithm Approval Pilot Manuals

Monitor Performance

Publish Algorithm

> 4.6 [Technical Training Requirements:]{.underline}

Training should be provided for laboratory and POS testing staff,
ideally at the site in which testing will occur, rather than at a
centralized venue. Training should also be provided for assessors
responsible for monitoring EQA activities of testing sites.

Every effort should be made to ensure continuity of training throughout
the evaluation process through the use of documented processes and
procedures. In addition to performance of assays, the training should
include QA, QC, data management, and laboratory safety. Organizers of
the training should ensure availability of training venue, test kits,
supplies and samples.

Expansion of training activities is further addressed in section 5.6.1
(Phase III -- Training Requirements).

**5.0 CONDUCTING THE EVALUATION**

> 5.1 [Overview evaluation phases]{.underline}

Evaluation of HIV testing performance is an ongoing process that begins
prior to implementation of testing and continues after tests have been
implemented in the field. The evaluation process is divided into three
phases. Although these phases can apply to evaluation of any HIV tests
using serum, plasma, saliva, or whole blood, for the purposes of this
document, emphasis is focused on evaluating rapid test methods that can
be used in the POS setting with whole blood specimens. Evaluation of
rapid tests for use in the POS setting is usually more complex than
evaluations of standard EIA formats that can be tested in parallel with
the existing EIA in a laboratory setting.

Phase I is a laboratory-based evaluation to provide preliminary results
on test performance characteristics (Se, Sp) on the same set of samples.
Having evaluated the same sample set that may consist of 4-7 rapid
tests, an algorithm of 2-3 tests may then be proposed based on the
performance of the combination of test methods.

Phase II involves evaluation of the selected algorithm under field
conditions that may include test performance and interpretation by
non-laboratory clinic staff. Phase II is often referred to as the field
trials, and typically is conducted in at least 2-3 POS sites. Tests
under evaluation in this phase should be performed in the same manner in
which it is to be used, e.g., finger stick specimens.

Phase III represents ongoing evaluation of performance through EQA
programs that not only monitor the performance of individual clinic
and/or staff, but also provide aggregate data for ongoing assessment of
test performance.

5.2 [Objectives of Evaluation Phases]{.underline}

> Objectives of Phase I:

-   Provide preliminary performance characteristics on tests under
    evaluation

-   Develop a panel of well-characterized serum for future use

-   Review performance of each test combination to develop 2-3 test
    algorithm

> Objectives of Phase II:

-   Evaluate the performance of the 2-3 test algorithm in the POS
    setting

-   Perform a demonstration study in selected sites and conditions that
    will provide a reasonable/reliable indication of how the testing
    methods and algorithm will perform when implementation is expanded
    to multiple sites through the country

Objectives of Phase III:

-   Ensure each new testing site has appropriate training and
    preliminary observation of performance prior to reporting results

-   Assess clinic/ staff performance through EQA

-   Monitor aggregate test performance

> 5.3 [Evaluation Scenarios]{.underline}

![](media/image1.wmf)

> Diagram 1 is a scenario in which the NRL is preparing for the
> evaluation by collecting, characterizing and storing serum specimens
> for later evaluation. This allows the NRL to collect and store
> approximately 500 specimens over a period of weeks to months and then
> separately evaluate several new tests in a few days.
>
> **[Advantages]{.underline}**

-   The NRL can pick and choose the appropriate number of positive and
    negative specimens for evaluating tests from all the specimens
    received over time.

-   This scenario avoids unnecessary testing of excess negatives or
    specimens that cannot be characterized with the tests that are under
    evaluation.

-   The evaluation panel can be collected without making major changes
    to the laboratory workload.

-   Multiple tests can be evaluated with stored sera in a short time
    (e.g., \<1 week).

> **[Disadvantages]{.underline}**

-   Evaluation with stored serum may be sub-optimal, as additional
    requirements must be met for sample preparation and storage, and
    different performance characteristics may result in testing fresh
    sera compared with testing stored sera.

-   Different performance characteristics may be observed with whole
    blood after initial evaluation with serum.

-   For whole blood-based rapid tests, an additional step using whole
    blood to provide preliminary validation data of performance
    characteristics is required before implementing phase II.

-   The laboratory must have sufficient resources to meet quality
    standards for storing specimens. At a minimum, a subset of stored
    specimens should be retested to ensure validity of earlier results.
    If any deviation is found in the subset of re-tested specimens, then
    all stored samples must be retested.

![](media/image1.wmf)

> Diagram 2 is a scenario in which the tests being evaluated are
> performed concurrently with standard test methods. This scenario still
> represents the use of serum due to limited availability and logistical
> difficulty in transporting whole blood to the NRL. Since the tests are
> performed concurrently, less is required for managing the storage and
> retrieval of specimens.
>
> **Advantages**

-   By testing fresh sera, the NRL avoids the necessity of previous
    rigid requirements for aliquoting and storing specimens prior to
    beginning the evaluation.

-   Since evaluation tests are performed concurrently, there will be
    earlier indications of unacceptable performance. Given these early
    indicators, one may stop evaluation of tests as soon as a
    statistically significant sample size is reached.

**Disadvantages**

-   In a lower prevalence setting, the laboratory may have to perform
    preliminary tests on excess negatives; leading to an increase in the
    length of time before phase I is completed.

-   Evaluation with stored sera may be sub-optimal for whole blood-based
    rapid tests. There is the possibility of observing different
    performance characteristics when used with whole blood in phase II.

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-   For whole blood-based rapid tests, an additional step is required to
    provide preliminary validation of performance characteristics data
    before implementing phase II.

![](media/image1.wmf)

> Diagram 3 is a scenario in which the laboratory can perform a
> concurrent prospective evaluation using whole blood prior to
> characterizing the serum with the gold standard methods. This type of
> evaluation is possible when there are laboratory resources to perform
> 3-5 tests in a clinic setting, such as blood center, where whole blood
> is immediately available.

**Advantages**

-   Using whole blood to evaluate rapid tests that will be used in POS
    settings with whole blood is the best method in directly determining
    the performance characteristics and selecting an algorithm

-   This scenario negates the need for an additional step of validating
    using whole blood specimens

**Disadvantages**

-   The whole blood is often retrieved from a venous sample and may not
    mimic all the aspects of test performance when used with a
    fingerstick specimen

-   Performing a concurrent evaluation of several tests with whole blood
    may be logistically difficult due to requiring a laboratory in the
    POS setting

-   Performing 3-4 tests concurrently directly from fingersticks may be
    logistically difficult, particularly if non-lab staff are performing
    tests

> 5.4 [Phase I: Laboratory Evaluation]{.underline}
>
> 5.4.1 [Use of Stored Serum]{.underline}
>
> Fresh sera are the preferred specimens for evaluation of serum-based
> tests and preliminary evaluation of whole blood tests when whole blood
> is not immediately available. If sera are frozen before the
> evaluation, there should be some standards and practices to ensure
> that the quality of the thawed serum has not been impaired by
> freeze/thawing, contamination, excess particulate matter, etc. The
> sera should be aliquoted in separate vials to avoid multiple
> freezing/thawing. For monitoring the quality of frozen storage, a
> percentage of specimens should be retested with standard tests prior
> to performing the evaluation to ensure that test results do not
> change.

######## 5.4.2 [Sample Size]{.underline}

> A test evaluation should include a minimum of approximately 200
> HIV-positive and 200 HIV-negative specimens to provide 95% confidence
> intervals of less than ± 2% for both the estimated sensitivity and
> specificity. Lower numbers of HIV-positive and HIV negatives specimens
> may be used, but this will increase the confidence interval for
> sensitivity and specificity. The total number of specimens included in
> the evaluation will depend on whether the HIV reactivity of the
> specimens is known prior to evaluating the test. In a prospective
> evaluation, such as using whole blood in the clinic setting where the
> HIV reactivity is unknown, the evaluation would be performed until a
> minimum of 200 positives are obtained. For instance, in a setting with
> 20% prevalence this might require testing upwards of 1000 specimens
> until 200 positives are obtained (Appendix H). In a laboratory-based
> evaluation where the HIV reactivity of specimens is known, such as
> with previously tested and stored serum or plasma specimens, it is
> preferable from a cost perspective to select 200 HIV-positives and 200
> HIV- negatives.
>
> When a whole blood or serum-based rapid/simple test is initially
> evaluated in phase 1 with serum, then an additional validation step is
> required to provide some reassurance that the performance in whole
> blood is similar to that obtained in serum before initiating a more
> extensive evaluation in Phase 2. This assessment does not need to be
> as extensive as the serum-based evaluation. The test methods
> representing a 2-3 test algorithm should be validated with 50-100
> whole blood specimens (containing a minimum of 20 positives).

######## 5.4.3 [Sample Population]{.underline}

Selecting the sample population for a test evaluation will include
several considerations. Although there are considerations of having a
sample that is representative of the various areas of the country, this
may not be feasible in phase I when the NRL is limited to available
specimens. If specific concerns exist about how HIV-1 and HIV-2 or
specific subtypes are distributed, these might be addressed by selected
specimens in a panel. In most instances the primary goal should be
selecting a population with a high prevalence of infection to obtain a
sufficient number of positives.

> 5.5 [Phase II -- Field Evaluation / Pilot testing]{.underline}
>
> 5.5.1 [Number of Sites]{.underline}

In Phase II, the selecting testing sites from different areas of the
country should be balanced with the need and logistics of monitoring
on-site testing and transport of specimens to the NRL for
characterization by the gold standard method. At a minimum, 2-3 sites
should be considered for inclusion in Phase II of the evaluation. Some
larger countries may need to consider up to 4-5 sites that are
implemented sequentially to allow for training at each site. Managing
the logistics of transporting specimens and reporting may be difficult
with more than 3 sites.

5.5.2 [Sample Size]{.underline}

> The same sample size for Phase II evaluation should be used as in
> phase I. This will require finding a sufficient number of field test
> sites with high prevalence to obtain the minimum of 200 positives
> distributed across all sites.
>
> 5.5.3 [Sample Population]{.underline}
>
> If a country has specific concerns about having a representative
> population for test evaluation, these should be addressed through the
> selection of testing sites in Phase II. The primary concern should be
> about representative testing conditions.

5.6 [Phase III -- Implementation and Monitoring]{.underline}

> 5.6.1 [Training Requirements]{.underline}
>
> When tests and algorithms have been evaluated in phase II and
> considered acceptable, there is continued need to provide training and
> support for systematic implementation in additional sites. The NRL and
> NAP must develop a plan than involves training and evaluation of staff
> at new sites prior to reporting results to patients. In many cases,
> implementation will involve merging testing practices, evaluation, and
> quality assessment into counseling programs and settings.
>
> Training all staff, laboratory or non-laboratory, who will perform the
> test(s) is a necessary and important prerequisite to expanding the
> testing sites. Training topics should include at a minimum, test
> performance, quality control, safety, and also include some measure of
> test performance with standard competency proficiency panels
> established by the NRL. Successful participants should receive a
> certificate acknowledging their competency. The certificate, however,
> should recognize that the training and competency are limited to
> specific tests performed during the training.
>
> Every new testing site should receive a laboratory visit that combines
> training and evaluation by observation. This visit should be a
> standard component of implementation and occur before any patient test
> results are reported. Each site should be provided with SOPs for
> testing either during training or as part of the initial visit. When
> appropriate, the NRL should provide control materials for the specific
> tests.
>
> *Initial evaluation of the performance of testing personnel*
>
> The performance of individuals at each site should be evaluated before
> results are reported. For rapid tests, this should involve taking an
> additional venous sample on the first 50-100 patients and comparing
> the rapid test results obtained in the POS with the standard EIA
> results. The results reported to the patient/client should be based on
> results from the standard EIA.
>
> 5.6.2 [EQA]{.underline}
>
> There should be at least one or more methods available to assess the
> quality of testing within a country. This should include every NRL
> establishing a program for monitoring different manufactured lots of
> tests kits that are received/purchased by the country. This will
> require using a standard reference panel to assess lot-to-lot
> performance for each individual test. Special consideration should be
> given to including weak positives for adequately assessing any
> lot-to-lot variations in test sensitivity.
>
> 5.6.2.1 Onsite Evaluation
>
> External quality assessment programs should provide onsite evaluation
> of each testing site in addition to methods that will assess testing
> performance. Having onsite evaluation is necessary to review QC,
> record keeping, and observation of test performance. Additionally,
> this evaluation is an opportunity to directly administer a proficiency
> test to each individual performing testing during the visit. A program
> of onsite evaluation should include a standard checklist of laboratory
> indicators and evaluators should be trained to perform consistent
> reviews of laboratories and other POS sites. Standard checklists and
> evaluation methods allow for collecting and comparing consistent
> information from multiple sites.

5.6.2.2 [Proficiency Testing]{.underline}

> Proficiency testing (PT) is the most common form of EQA and involves
> development of specimen panels by the NRL for distributing to POS
> sites. Laboratories administering PT panels should strive to adhere to
> international guidelines, e.g., ISO Guide 43. There are standard
> methods available to develop PT samples and might be the easiest type
> of program for implementation at sites where serum-based tests are
> performed. The limitations of PT are that it usually involves only a
> few specimens and the test results may not represent the routine test
> performance. This may be due in part to the greater care in handling
> PT specimens.

5.6.2.3 [Blinded Rechecking]{.underline}

> Retesting selected specimens in a reference/referral laboratory may
> also assess the quality of testing. This can be accomplished by
> forwarding all positive and 10% of negative specimens for standard
> EIAs when a venous specimen is available. Another systematic sampling
> method may be considered to reduce the potential bias of selecting
> test specimens for referral.

5.6.2.4 [Dried Blood Spots (DBS)]{.underline}

> The use of dried blood spots (DBS) is one method that is being
> developed as EQA for whole blood tests where it may be impractical to
> refer specimens for additional testing or where there is limited or no
> access to serum PT specimens for monitoring test performance. The DBS
> are collected at the time of patient testing (e.g. fingerstick) on
> filter paper and easily transported to a reference laboratory. The use
> of DBS will require a reference laboratory that has demonstrated
> proficiency with eluting the DBS specimens and performing standard EIA
> methods. Additional concerns include the logistics and methods of
> collecting DBS in the testing protocol. Although a statistical sample
> of specimens re-tested by DBS based on testing volume may be
> desirable, this may be difficult to implement in the flow of testing
> and counseling of patients. Additionally, testing a percentage of
> specimens, such as 10% may be problematic. Countries may consider
> random sampling of DBS such as bimonthly, or at a given time or day.
> Further development of DBS protocols, proficiency testing and EQA
> guidelines are necessary to assist with the expansion and monitoring
> of rapid testing.
>
> 5.6.3 [Remediation / Corrective Measures]{.underline}
>
> When deficiencies are noted during on-site visits, corrective measures
> should be taken to ensure the quality of results. This may include
> additional training and discontinuation of services.

**6.0 EVALUATION MATERIALS**

### 6.1 [Types of evaluation materials]{.underline} 

> These guidelines describe several types of evaluation panels that may
> differ by the composition of negatives and positives, and by degree of
> characterization. Specimen library is a term given to the source or
> collection of all specimens that may be selected and retrieved for
> evaluation purposes. In some instances, this might represent a large
> collection of stored sera from which a set of positives and negatives
> is selected and retested for inclusion in the evaluation. The specimen
> library could also represent all fresh specimens tested in the
> laboratory where only a subset of specimens is selected for
> evaluation.
>
> The evaluation panel consists of those specimens that are tested by
> the gold standard method and evaluation test methods and included in
> calculating the sensitivity and specificity for individual tests and
> algorithms. The evaluation panel should usually consist at a minimum
> of 400-500 total specimens including at least 200 positives.
>
> A laboratory may also have available several special reference panels.
> These panels may represent a collection of difficult or unusual
> specimens that provide a unique challenge to the tests being
> evaluated. Samples from uninfected and infected persons, which
> represent unusual screening results and have been further tested to
> resolve serostatus, may be used in the panel as challenges to the
> sensitivity and specificity of an assay under evaluation. Because the
> sensitivity of some antibody tests is less for sera collected early in
> HIV infection for persons infected with non-B subtypes, it is
> important to evaluate the assays on panels containing specimens from
> persons recently infected with the HIV-1 or HIV-2 subtypes circulating
> in the country.
>
> Each specimen in the reference panel should be tested with multiple
> EIAs and positives confirmed with Western Blot and when possible,
> additional tests including p24, PCR, genotype, etc.
>
> Because of repetitive use of reference panels during Phase II and
> Phase III, stability and storage of samples are critical. Samples
> should be aliquoted into storage vials and preferably frozen at --70ºC
> (minimum standard is --20ºC when molecular procedures are not used).

### 

### 6.2 [Specimen Collection and Handling]{.underline}

1.  [Specimen Collection]{.underline}

> [Plasma]{.underline}
>
> Collect up to 10 ml of blood from the patient's vein into a sterile
> anticoagulated tube. Choice of anticoagulant should be appropriate to
> the test being evaluated according to the manufacturer's insert. Using
> an evacuated blood collection system is recommended for safety. The
> blood drawn is immediately mixed by gently inverting the tube 10
> times. Shaking should be avoided to prevent hemolysis.
>
> The specimen should be centrifuged at 300-400g for 10 minutes to
> separate the plasma. After centrifugation, the separated plasma should
> be withdrawn using a clean pipette and transferred to a storage tube.
> Ideally, specimens are prepared for storage in 0.5ml aliquots.

## 

##  [Serum]{.underline}

> Collect up to 10 ml of blood from the patient's vein into a sterile
> serum separation tube, preferably an evacuated blood collection tube
> without anticoagulants. Again, shaking should be avoided to prevent
> hemolysis. Let the blood stand for 20-30 minutes at room temperature
> to allow for clot formation. Serum can be separated from the clot by
> centrifugation at 300-400 g for 10 minutes. Alternatively, gently draw
> the serum off the clot using a sterile pipette. The serum can be
> subsequently clarified further by centrifugation at a remote site.
> Specimens should be prepared for storage in 0.5 ml aliquots.

##  [Whole Blood]{.underline}

> Collect up to 10 ml of blood from the patient's vein into a sterile
> tube containing an anticoagulant. Again, choice of anticoagulant
> should follow test manufacturer's recommendations. Immediately draw
> off sufficient quantities of whole blood to run the tests under
> evaluation. The remaining blood should be used for preparing of plasma
> as described above.

## 6.2.2 [Transfer and Storage of Specimens]{.underline}

> Ideally, aliquoted serum or plasma specimens should be stored
> immediately at --20ºC. If specimens are to be transferred to a central
> facility they should be maintained at 4ºC and shipped on cold packs to
> the storage site. If cold packs are not available, serum specimens can
> remain at room temperature for up to 3 days, whereas whole blood
> hemolyzes over time. Signed specimen transfer sheets should accompany
> specimens during shipment. Upon receipt at the central facility,
> specimens should be immediately transferred to a non-self-defrosting
> freezer for storage. Specimens should be stored uniformly aliquoted
> and stored in a polypropylene tube. Specimen identifiers should be
> labeled directly on the tube, and not on the screw-cap top. Specimen
> inventories should be maintained for storage freezers that are
> specifically reserved for reposited specimens. Every effort should be
> made to limit the number of freeze-thaw cycles, since repetitive thaws
> may result in loss of antibody titer and formation of serum
> flocculates. For long-term storage, specimens should be frozen --70ºC.

### It is important to store these specimens with all pertinent detailed information concerning specimens in a computer database or bound logbook, which is periodically updated to reflect specimen use or transfer. Maintaining this database and deposited specimens will facilitate additional evaluations at a later date.

> 6.2.3 [To improve quality of sera for storage, the following steps may
> be followed:]{.underline}

-   Centrifuge

-   Pipette serum from clot rather than pouring the serum

-   Filter the serum

-   Make aliquots of serum to avoid multiple cycles of freezing and
    thawing

-   Store at --70 degrees centigrade in non-self defrosting freezer

-   Keep good daily freezer logs

-   Exclude specimens that are:

    -   Particulate

    -   Lipemic

    -   Hemolyzed

    -   Contaminated with bacteria

        6.3 [Serum Library: Collection and use of stored
        serum]{.underline}

### 

###  

###  

> 6.3.1 [Characterization of Evaluation Panel]{.underline}

### Characterization of the library of specimens used in the evaluation should be based on a multi-test algorithm that allows for establishing a gold standard to determine serostatus. Consideration should be given for confirmation of [only the positive]{.underline} samples by the Western blot for the following reasons:

### Use of WB allows for characterization of sera to develop a panel for repeated use; 

### Use of the WB is recommended to allow countries to share evaluation data that represent standard confirmation methods and a more complete and accurate characterization of specimens for evaluation. 

### Although some countries may currently evaluate tests using only an EIA algorithm, countries should strive to adopt the WB for standardization and to increase data sharing. 

## 6.4 [Trouble-shooting of problematic specimens]{.underline}

> Occasionally, assays produce results that are difficult to interpret
> and are erroneous, which may be due to factors inherent with the
> specimens or clerical errors. If such results occur, consider the
> following:

-   Check specimen integrity for evidence of bacteria contamination,
    hemolysis, and lipidic substances

-   Verify labeling, paper-work, and procedures

-   Re-check equipment and reagents

-   Have the same technologist re-test the specimen

-   Repeat testing blindly by another technologist

-   Repeat on reference test blindly

-   Repeat at different laboratory or reference laboratory

-   Determine true status by other assays (PCR testing, p24)

    **7.0 DATA ANALYSIS**

    7.1 [Data Management]{.underline}

> Before collecting blood specimens, it is important to design a simple
> questionnaire and tracking records for specimen management, which
> should include a unique specimen number, date, and site of draw. They
> may also include limited demographic information such as age, sex,
> profession, and home district. Tracking documents should include an
> inventory of specimens being shipped, their origin, destination, and
> time and date of transfer. Also create a database that will allow the
> variables to be entered and linked with the associated specimen. Such
> variables will include the unique specimen identification number,
> relevant tracking information, the name of tests used, test results
> (positive or negative), optical density values, optical density ratios
> (OD ratio), any additional confirmatory information such as WB
> pattern, and final determination of sero-status (positive or
> negative).

7.2 [Resolving Discordants]{.underline}

There are two types of discordant results in an evaluation: One such
discordant is a specimen that does not meet the criteria of positive or
negative using the gold standard method/definition. Before the
evaluation, the laboratory should determine the gold standard for
positives and negatives. In the case of an evaluation this may differ
from normal testing practices, such as the use of WB to confirm positive
obtained in an evaluation setting. An example of a discordant result may
be a specimen that is positive by EIA(s), but indeterminate on WB. In
the case of prospective evaluation, the laboratory must ensure that the
reason for the discordant is not sample mix-up or transcription error
before deciding to perform additional testing to resolve these types of
discordants, such as p24 antigen testing or PCR. Only the specimens that
are positive or negative by the gold standard method should be used in
calculating the sensitivity and specificity of test performance. The
results of further testing may be listed in the evaluation summary to
provide further information on the performance of tests used in the
evaluation.

> The second type of discordant result occurs when the result of the
> test(s) being evaluated differs from the result of the gold standard.
> An example might be a specimen that is negative with the gold standard
> algorithm of EIA(s), but positive on one or more of the tests being
> evaluated. Once again the laboratory may decide to perform additional
> tests to provide further information on the patient specimen; however,
> these results should not be included in calculating the sensitivity
> and specificity.

##### 7.3 [Sensitivity, Specificity, PPV, NPV, Confidence Interval, Delta value, Reproducibility, Inter-reader variability]{.underline} 

> Several key parameters need to be evaluated for each assay:
> sensitivity, specificity, positive and negative predictive values, and
> delta values. The sensitivity and specificity of each assay are
> calculated using the gold standard.
>
> *[Sensitivity]{.underline}* is defined as the ability of an assay
> being evaluated to correctly detect specimens containing antibody to
> HIV. In other words, sensitivity is the percentage of true positive
> HIV specimens identified by the assay under evaluation as positive
> (A), divided by the number of specimens identified by the reference
> assays as positive (A+C).
>
> *[Specificity]{.underline}* is defined as the ability of an assay
> being evaluated to correctly detect specimens that do not contain
> antibody to HIV. In other words, specificity is the percentage of true
> negative specimens identified by the assay being evaluated as negative
> (D), divided by the number of specimens identified by the reference
> assays as negative (B+D).

Example:

Evaluation of a rapid test on a panel of specimens that have been tested
by the gold standard is shown to contain HIV antibodies to 300 serum
samples and no HIV antibodies to 200 samples (Figure 3). Of the 300
serum samples that were antibody positive, the rapid test classified 275
of the samples as positive. Of the 200 samples that were HIV antibody
negative by the gold standard, 125 were classified by the rapid test as
not containing HIV antibodies

Figure 3: Results of Evaluation panel using Gold Standard

**Gold Standard Results**

**+ -**

+-----------------------+----------------+--------------+------------+
| **Results of assay    | A              | B            | A + B      |
| +**                   |                |              |            |
|                       | True-positives | False        | 350        |
| **under evaluation    |                | positives    |            |
| -**                   | 275            |              |            |
|                       |                | 75           |            |
+-----------------------+----------------+--------------+------------+
|                       | C              | D            | C + D      |
|                       |                |              |            |
|                       | F              | Tr           | 150        |
|                       | alse-negatives | ue-negatives |            |
|                       |                |              |            |
|                       | 25             | 125          |            |
+-----------------------+----------------+--------------+------------+
|                       | A + C = 300    | B+D = 200    | 500        |
+-----------------------+----------------+--------------+------------+

Sensitivity=A/(A+C), 275/(275 + 25) = 91.67%

Specificity=D/(B+D), (125/(75 + 125) = 62.5%

> Positive Predictive value=A/(A+B), 275/(275 + 75) = 78.57%
>
> Negative Predictive value=D/(C+D), 125/(25 + 125) = 83.33%
>
> *[Positive Predictive Value (PPV):]{.underline}* is the probability
> that when the test is reactive, the specimen actually contains
> antibody to HIV. PPV is calculated as follows: A/(A+B). PPV can also
> be calculated as follows:
>
> (prevalence) (sensitivity)
>
> PPV=
> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
>
> (prevalence) (sensitivity) + (1-prevalence)(1-specificity)
>
> *[Negative Predictive Value (NPV):]{.underline}* is the probability
> that when a test is negative, a specimen does not have antibody to
> HIV. NPV is calculated as follows: D/(C+D) or as:
>
> (1-prevalence)(specificity)
>
> NPV=\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
>
> (1-prevalence)(specificity) +(prevalence)(1-sensitivity)
>
> The proportion of false positives and false negatives varies with the
> prevalence of HIV infection in various segments of the population. In
> general, the higher the prevalence of HIV infection in the population,
> the greater the probability that a person testing positive is truly
> infected, i.e., the greater the positive predictive value (PPV). Thus,
> with increasing prevalence, the proportion of positive results that
> are false-positive decreases. Conversely, the likelihood that a person
> having a negative test result is truly uninfected (i.e., the negative
> predictive value \[NPV\]), decreases as prevalence increases.
> Therefore, as prevalence increases, so does the proportion of samples
> testing false-negative.
>
> *[Confidence Interval (CI):]{.underline}* The 95% confidence interval
> is an estimate of a population parameter computed so that the
> statement "the population parameter lies in this interval" will be
> true at a stated confidence, e.g., 95%.
>
> 95% CI of the calculated sensitivity and specificity are calculated
> using the formula:

P ± 1.96√

where P is the sensitivity or specificity

where N is the number of sera analyzed.

> *[Delta value(δ )]{.underline}*
>
> Delta values are used to determine the ability of EIAs to separate the
> negative and positive anti-HIV serum populations from the cut-off.
> Delta (δ) values of the anti-HIV positive and negative sample
> populations are calculated by dividing the mean Optical Density (OD)
> ratio (log~10~) by the standard deviation of each population. OD
> ratios are calculated by dividing by the relevant cut-off:
>
> OD sample
>
> OD ratio = \-\-\-\-\-\-\-\-\-\-\-\-\--
>
> OD cutoff
>
> In case of overflow, usually denoted as "\*\*\*\*" in the print out,
> an OD of 3.000 is attributed to the specimen. The higher the positive
> (δ+) and negative (δ -) values, the higher the probability that the
> test will clearly distinguish antibody positive and negative
> specimens.
>
> *[Reproducibility]{.underline}*
>
> To determine reproducibility, retest approximately 10% of the
> initially reactive and non-reactive samples. Reproducibility,
> expressed as a percentage, is calculated by dividing the number of
> concordant results by the total number of samples retested.
>
> *[Inter-reader variability of rapid test]{.underline}*
>
> It is important to determine the inter-reader variability of rapid
> tests. Three persons independently interpret each test result, and the
> reader variability is expressed as percentage of sera for which
> different readers interpret test results differently.

**8.0 REPORTING RESULTS, CONCLUSIONS, RECOMMENDATIONS**

8.1 [Developing an Algorithm]{.underline}

> Evaluation data should be analyzed to determine the performance of
> individual tests and the combination of tests used in a proposed
> algorithm. In phase I, this will involve determining the performance
> of various test combinations in addition to the individual test
> performance. An important point to consider in the analyzing potential
> algorithms is whether the tests will be performed in a parallel or
> serial testing algorithm. Most standard EIAs will be used in a serial
> algorithm in which the use of the second test is dependent on a
> reactive result in the first test. Many rapid tests that are used in
> POS, however, may be tested in parallel logistical reasons. A typical
> example might involve determining the concordance of 2 tests performed
> in combination and then evaluating the results when both tests agree
> (concordance) and when a 3^rd^ test is required as a tiebreaker
> because the first 2 tests have discordant results (Figure 4).
>
> Figure 4:
>
> ![](media/image2.wmf)
>
> Samples in panel \# 660 and #506 would have completely different
> interpretations in the algorithm based on whether the tests were
> performed sequentially (Fig. 4) or in parallel. This is also true if
> the algorithm is a two test only or three tests with tiebreaker. The
> probable cause of the difference in results in the EIA status vs. the
> rapid tests results is sample mix-up.

8.2 [Reporting Results]{.underline}

> Analysis of evaluation data should be completed and reported to the
> NAP, MOH, and other partners immediately following the phase of
> evaluation in which it was performed, and before beginning the next
> phase.
>
> The report for Phase I evaluation typically includes the data
> presented in a table that itemizes the test methods, and the Se, Sp,
> PPV, NPV for each method and combination of methods evaluated (Figure
> 5). Phase II reports should include the on-site performance data in
> addition to the subjective input of the client/patient flow. Having
> completed Phase III of the evaluation, countries should consider
> including the following recommendations in the final report.

-   The names and manufacturers of all EIA or rapid tests evaluated with
    documented test performance

-   The name and required specimen type for each test approved for use
    in POS settings

-   The name of the test to be used as the tiebreaker for resolving
    discordant specimens and justification for use

-   The names and manufacturers of each test with demonstrable testing
    performance, but excluded for use in POS setting. Justifications for
    excluding tests should be noted.

-   Summary of individual test data

Figure 5:

  ---------------- ---------------- -------------------- -------- ----------
  Test method      Sensitivity      Specificity          PPV      NPV

  A                95% (190/200)    98% (294/300)                 

  B                97% (194/200)    98.5% (295/300)               

  C                96% (192/200)    99% (297/300)                 
  ---------------- ---------------- -------------------- -------- ----------

Example of evaluation of algorithm of tests performed in parallel

+----------+---------+----------+-------------------+-----------------+
| Test     | Tie     | Dis      | Combined          | Combined        |
| com      | breaker | cordants | Sensitivity of    | Specificity of  |
| bination |         |          | concordant (2     | concordant (2   |
|          |         |          | tests) and        | tests) and      |
|          |         |          | discordant (3     | discordant      |
|          |         |          | tests)            |                 |
|          |         |          |                   | (3 tests)       |
+----------+---------+----------+-------------------+-----------------+
| A and B  | C       | 35       |                   |                 |
+----------+---------+----------+-------------------+-----------------+
| B and C  | A       | 40       |                   |                 |
+----------+---------+----------+-------------------+-----------------+
| A and C  | B       |          |                   |                 |
+----------+---------+----------+-------------------+-----------------+

Test method combination with tiebreaker test for discordant results

3.  [Aggregation and dissemination of evaluation data]{.underline}

> Conclusions and recommendations from evaluation of tests should be
> submitted to WHO for access and dissemination to other countries
> within the region. This compilation of test performance will allow
> countries to review data from neighboring countries which should limit
> the need for full-scale evaluations.
>
> The following should be included in the report to WHO:

-   Protocol for evaluating tests, including designation of gold
    standard

-   Discordant results as tested by WB, if part of country's gold
    standard

**[Phase I (n = \# of samples)]{.underline}** **[Phase II
(n)]{.underline}** **[Phase III (n)]{.underline}**

  ----------- ---------- --------- --------- ----------- --------- ---------
  **Tests**   **Se**     **Sp**    **Se**    **Sp**      **Se**    **Sp**

  A                                                                

  B                                                                

  C                                                                

  D                                                                
  ----------- ---------- --------- --------- ----------- --------- ---------

> Summary reports should be submitted to:
>
> Dr. Guy-Michel Gershy-Damet or Designate\
> Regional Advisor For Laboratory\
> Regional Program on AIDS\
> WHO Regional Office For Africa\
> PO BOX BE 773\
> Harare -Zimbabwe\
> Tel: 263-4- 746342/827/323/359\
> Fax: 263-4-746867\
> Email: gershyg@whoafr.org

APPENDIX -- A

> **Testing Algorithms**

### *Parallel testing algorithm*

In a parallel testing algorithm, sera are simultaneously tested by two
assays. True-positive sera are concordantly reactive by two different
initial assays. A true-negative specimen in the algorithm is defined as
being concordantly negative in the two initial assays. Sera yielding
discordant results between the two assays are tested in a third assay,
and the outcome of the latter assay is considered definitive.

*Serial testing algorithm*

> The serial testing algorithm is most consistent with the proposed
> testing strategies of WHO/UNAIDS. In the serial algorithm, all
> specimens are tested by a first test that is highly sensitive.
> Specimens are considered as true negative if they react negatively in
> the first test. Specimens reactive in this assay are retested by a
> second assay that has a high specificity (this second assay must be
> one which possesses dissimilar antigen presentations than that of the
> first assay. If specimens are concordantly positive by the two assays,
> they are considered as true-positives. Discordantly reactive sera are
> further tested by a third assay, whose outcome is considered as
> definitive. This algorithm is recommended for identification of
> asymptomatic seropositive persons in areas with an HIV seroprevalence
> of more than 10% \[19\].

APPENDIX -- B

> **Summary of WHO Testing Strategies**

[WHO Strategy I:]{.underline}

-   Requires one test.

```{=html}
<!-- -->
```
-   For use in diagnostic testing in populations with an HIV prevalence
    \>30% among persons with clinical signs or symptoms of HIV
    infection.

-   For use in blood screening, for all prevalence rates.

-   For use in surveillance testing in populations with an HIV
    prevalence \>10% (e.g., unlinked anonymous testing for surveillance
    among pregnant women at antenatal clinics). No results are provided.

    [WHO Strategy II]{.underline}:

```{=html}
<!-- -->
```
-   Requires up to two tests.

-   For use in diagnostic testing in populations with an HIV prevalence
    [\<]{.underline}30% among persons with clinical signs or symptoms of
    HIV infection or \>10% among asymptomatic persons.

-   For use in surveillance testing in populations with an HIV
    prevalence [\<]{.underline}10% (e.g., unlinked anonymous testing for
    surveillance among patients at antenatal clinics or sexually
    transmitted infection clinics). No results are provided.

    [WHO Strategy III]{.underline}:

```{=html}
<!-- -->
```
-   Requires up to three tests.

-   For use in diagnostic testing in populations with an HIV prevalence
    ≤ 10% among asymptomatic persons.

    Alternative approaches that address limitations to these strategies
    are addressed in WHO/UNAIDS and surveillance documents.

APPENDIX -- C

> **Potential Testing Strategies**

+----------------+-----------------------+-----------------------------+
|                | **Screening**         | **Confirmation**            |
+----------------+-----------------------+-----------------------------+
| ## Whole Blood | Determine HIV ½       | HemaStrip HIV ½             |
+----------------+-----------------------+-----------------------------+
|                |                       | UniGold HIV Recombinant     |
+----------------+-----------------------+-----------------------------+
|                |                       | OraQuick HIV --1/2          |
+----------------+-----------------------+-----------------------------+
|                | HemaStrip HIV ½       | UniGold HIV Recombinant     |
+----------------+-----------------------+-----------------------------+
|                |                       | OraQuick HIV -- ½           |
+----------------+-----------------------+-----------------------------+
| ##             | OraQuick HIV1/2       | HemaStrip HIV1/2            |
+----------------+-----------------------+-----------------------------+
| ##             |                       | UniGold HIV Recombinant     |
+----------------+-----------------------+-----------------------------+
| ##             | Capillus HIV ½        | SeroCard HIV                |
| Serum / Plasma |                       |                             |
+----------------+-----------------------+-----------------------------+
|                |                       | MultiSpot HIV ½             |
+----------------+-----------------------+-----------------------------+
|                |                       | HIVChek System 3            |
+----------------+-----------------------+-----------------------------+
|                |                       | SeroStrip HIV ½             |
+----------------+-----------------------+-----------------------------+
|                |                       | HIVSav 1&2                  |
+----------------+-----------------------+-----------------------------+
|                |                       | DoubleCheck HIV 1&2         |
+----------------+-----------------------+-----------------------------+
|                |                       | Genie II HIV1/2             |
+----------------+-----------------------+-----------------------------+
|                |                       | HIVSpot HIV                 |
+----------------+-----------------------+-----------------------------+
|                | HIVSpot HIV           | SeroCard HIV                |
+----------------+-----------------------+-----------------------------+
|                |                       | SeroStrip HIV ½             |
+----------------+-----------------------+-----------------------------+
|                |                       | DoubleCheck HIV ½           |
+----------------+-----------------------+-----------------------------+
|                |                       | Genie II HIV ½              |
+----------------+-----------------------+-----------------------------+
|                |                       | HIVSav 1&2                  |
+----------------+-----------------------+-----------------------------+
|                | Determine HIV ½       | SeroCard HIV                |
+----------------+-----------------------+-----------------------------+
|                |                       | SeroStrip HIV ½             |
+----------------+-----------------------+-----------------------------+
|                |                       | DoubleCheck HIV ½           |
+----------------+-----------------------+-----------------------------+
|                |                       | Genie II HIV ½              |
+----------------+-----------------------+-----------------------------+
|                |                       | HIVSpot HIV                 |
+----------------+-----------------------+-----------------------------+
|                |                       | MultiSpot HIV ½             |
+----------------+-----------------------+-----------------------------+
| ## Oral Fluids | OraQuick HIV ½        | Saliva-Strip HIV1/2         |
+----------------+-----------------------+-----------------------------+
|                |                       | SalivaCard HIV              |
+----------------+-----------------------+-----------------------------+
|                |                       |                             |
+----------------+-----------------------+-----------------------------+

NOTE:

1\. This is a very limited review based on experiences of CDC
investigators and collaborators.

2\. Choice of screening / confirmation order should be based on review
of sensitivity and specificity in country. Examples above are starting
points based on work in several countries. (Tests like Determine and
Capillus have high sensitivity and are designed as screening tests but
consistently give some false positives, therefore, are not recommended
as confirmatory tests).

APPENDIX - D

+----------------+------------+------+------+----+------+----+------+
| ##             | # **P      | #    | #    | #  | #    | #  | #    |
| **Manufacturer | roduct Des |  **S |  **S |  * |  **C | ** | **Co |
| /Distributor** | cription** | ensi | peci | *F | omme | St | st/T |
|                |            | tivi | fici | ie | nts^ | at | est^ |
|                |            | ty** | ty** | ld | 5^** | us | 6^** |
|                |            |      |      |  U |      | ** |      |
|                |            |      |      | se |      |    |      |
|                |            |      |      | ** |      |    |      |
+================+============+======+======+====+======+====+======+
| # Abbot        | # Determi  | 97.9 | 100  | L  | Com  | F  | \$   |
| t Laboratories | ne HIV-1/2 | -100 | %^1^ | im | plex | or | 3.80 |
|                |            | %^1^ |      | it | ity: | sa |      |
| Abbott Park,   | I          |      |      | ed | 1    | le | Ne   |
| IL, USA        | mmunochrom |      |      |    |      |    | goti |
|                | atographic |      |      |    | S    |    | able |
| [www.ab        |            |      |      |    | tore |    |      |
| bottdiagnostic | R          |      |      |    | at   |    |      |
| s.com](http:// | ecombinant |      |      |    | 2-   |    |      |
| www.abbottdiag | antigens   |      |      |    | 30°C |    |      |
| nostics.com/)\ | and        |      |      |    |      |    |      |
| Manufacturer:\ | synthetic  |      |      |    |      |    |      |
| Dainabot Co.,  | peptides   |      |      |    |      |    |      |
| Ltd.\          | for        |      |      |    |      |    |      |
| Tokyo, Japan   | HIV-1/2    |      |      |    |      |    |      |
|                |            |      |      |    |      |    |      |
|                | Single     |      |      |    |      |    |      |
|                | reagent    |      |      |    |      |    |      |
|                |            |      |      |    |      |    |      |
|                | Uses whole |      |      |    |      |    |      |
|                | blood,     |      |      |    |      |    |      |
|                | plasma, or |      |      |    |      |    |      |
|                | serum      |      |      |    |      |    |      |
+----------------+------------+------+------+----+------+----+------+
| # BIONOR A/S   | # Bion     | 99   | 95   | Mo | Com  | F  | Ne   |
|                | or HIV-1&2 | .8-1 | .6-1 | de | plex | or | goti |
| Skien,         |            | 00%^ | 00%^ | ra | ity: | s  | able |
| Norway6g       | Magnetic   | 1,2^ | 1,2^ | te | 3    | al |      |
|                | part       |      |      |    |      | e\ |      |
| [www.biono     | icle-bound |      |      |    | S    | WH |      |
| r.no](http://w | EIA        |      |      |    | tore | O- |      |
| ww.bionor.no/) |            |      |      |    | at   | e  |      |
|                | Synthetic  |      |      |    | 2    | va |      |
|                | peptides   |      |      |    | -8°C | lu |      |
|                | for        |      |      |    |      | at |      |
|                | HIV-1/2    |      |      |    | Requ | ed |      |
|                |            |      |      |    | ires |    |      |
|                | Multiple   |      |      |    | Bi   |    |      |
|                | reagents   |      |      |    | onor |    |      |
|                |            |      |      |    | e    |    |      |
|                | Uses whole |      |      |    | quip |    |      |
|                | blood,     |      |      |    | ment |    |      |
|                | plasma, or |      |      |    |      |    |      |
|                | serum      |      |      |    |      |    |      |
+----------------+------------+------+------+----+------+----+------+
| # Sayvon Di    | #          | 95-  | 9    | E  | Com  | F  | Ne   |
| agnostics Ltd. | HIVSav 1&2 | 99%^ | 6-99 | xt | plex | or | goti |
|                |            | 1,3^ | .9%^ | en | ity: | s  | able |
| Ashdod, Isreal | M          |      | 1,3^ | si | 2\   | al |      |
|                | icrofiltra |      |      | ve | S    | e\ |      |
| [www.hctech    | tion-bound |      |      |    | tore | WH |      |
| .com/savyon](h | EIA        |      |      |    | at   | O- |      |
| ttp://www.hcte |            |      |      |    | 2    | e  |      |
| ch.com/savyon) | R          |      |      |    | -25° | va |      |
|                | ecombinant |      |      |    | C^4^ | lu |      |
|                | antigens   |      |      |    |      | at |      |
|                | and        |      |      |    | Op   | ed |      |
|                | synthetic  |      |      |    | tion |    |      |
|                | peptides   |      |      |    | al:\ |    |      |
|                | for        |      |      |    | ce   |    |      |
|                | HIV-1/2    |      |      |    | ntri |    |      |
|                |            |      |      |    | fuge |    |      |
|                | Multiple   |      |      |    |      |    |      |
|                | reagents   |      |      |    |      |    |      |
|                |            |      |      |    |      |    |      |
|                | Uses       |      |      |    |      |    |      |
|                | plasma or  |      |      |    |      |    |      |
|                | serum      |      |      |    |      |    |      |
+----------------+------------+------+------+----+------+----+------+
| #              | ###        | 1    | 1    | L  | Com  | E  | Ne   |
|  Orasure Techn | # *OraQuic | 00%^ | 00%^ | im | plex | va | goti |
| ologies, Inc.  | k HIV-1/2* | 1,3^ | 1,3^ | it | ity: | lu | able |
| Bethlehem, Pen |            |      |      | ed | 1    | at |      |
| nsylvania, USA | I          |      |      |    |      | io |      |
|                | mmunochrom |      |      |    | S    | ns |      |
| [www.orasure.c | atographic |      |      |    | tore | o  |      |
| om](http://www |            |      |      |    | at   | ng |      |
| .orasure.com/) | Synthetic  |      |      |    | 18-  | oi |      |
|                | peptides   |      |      |    | 30°C | ng |      |
| #              | for        |      |      |    |      |    |      |
|                | HIV-1/2    |      |      |    |      |    |      |
|                |            |      |      |    |      |    |      |
|                | Single     |      |      |    |      |    |      |
|                | reagent    |      |      |    |      |    |      |
|                |            |      |      |    |      |    |      |
|                | #          |      |      |    |      |    |      |
|                | **Uses ora |      |      |    |      |    |      |
|                | l fluid, w |      |      |    |      |    |      |
|                | hole blood |      |      |    |      |    |      |
|                | , plasma,  |      |      |    |      |    |      |
|                | or serum** |      |      |    |      |    |      |
+----------------+------------+------+------+----+------+----+------+
| # Gen          | # HIV SPOT | 97-  | 96-  | E  | Com  | F  | \$1  |
| elabs Diagnost |            | 99%^ | 99%^ | xt | plex | or | .20- |
| ics, Pte, Ltd. | M          | 1,3^ | 1,3^ | en | ity: | s  | 1.80 |
|                | icrofiltra |      |      | si | 2\   | al |      |
| Singapore      | tion-bound |      |      | ve | S    | e\ |      |
|                | EIA        |      |      |    | tore | WH |      |
| Parent         |            |      |      |    | at   | O- |      |
| Company:\      | R          |      |      |    | 2    | e  |      |
| Genelabs       | ecombinant |      |      |    | -25° | va |      |
| Technologies,  | antigens   |      |      |    | C^4^ | lu |      |
| Inc.\          | and        |      |      |    |      | at |      |
| Redwood City,  | synthetic  |      |      |    | Op   | ed |      |
| CA, USA        | peptides   |      |      |    | tion |    |      |
|                | for        |      |      |    | al:\ |    |      |
| [www.gen       | HIV-1/2    |      |      |    | ce   |    |      |
| elabs.com.sg]( |            |      |      |    | ntri |    |      |
| http://www.gen | Multiple   |      |      |    | fuge |    |      |
| elabs.com.sg/) | reagents   |      |      |    |      |    |      |
|                |            |      |      |    |      |    |      |
|                | Uses       |      |      |    |      |    |      |
|                | plasma or  |      |      |    |      |    |      |
|                | serum      |      |      |    |      |    |      |
+----------------+------------+------+------+----+------+----+------+
| #              | #          | 1    | 99   | Mo | Com  | F  | ne   |
| Orgenics, Ltd. |  DoubleChe | 00%^ | .5-1 | de | plex | or | goti |
|  Yavne, Isreal | ck HIV 1&2 | 1,3^ | 00^1 | ra | ity: | s  | able |
|                |            |      | ,3^% | te | 2\   | al |      |
| [w             | I          |      |      |    | S    | e\ |      |
| ww.orgenics.co | mmunochrom |      |      |    | tore | WH |      |
| m](http://www. | atographic |      |      |    | at   | O- |      |
| orgenics.com/) |            |      |      |    | 2    | e  |      |
|                | R          |      |      |    | -30° | va |      |
|                | ecombinant |      |      |    | C^4^ | lu |      |
|                | antigens   |      |      |    |      | at |      |
|                | for        |      |      |    | Op   | ed |      |
|                | HIV-1/2    |      |      |    | tion |    |      |
|                |            |      |      |    | al:\ |    |      |
|                | Single     |      |      |    | ce   |    |      |
|                | reagent    |      |      |    | ntri |    |      |
|                |            |      |      |    | fuge |    |      |
|                | Use with   |      |      |    |      |    |      |
|                | plasma, or |      |      |    |      |    |      |
|                | serum      |      |      |    |      |    |      |
+----------------+------------+------+------+----+------+----+------+

+----------------+------------+---+------+------+----+------+----+------+
| ##             | # **P      | # |      | #    | #  | #    | #  | #    |
| **Manufacturer | roduct Des |   |      |  **S |  * |  **C | ** | **Co |
| /Distributor** | cription** | * |      | peci | *F | omme | St | st/T |
|                |            | * |      | fici | ie | nts^ | at | est^ |
|                |            | S |      | ty** | ld | 5^** | us | 6^** |
|                |            | e |      |      |  U |      | ** |      |
|                |            | n |      |      | se |      |    |      |
|                |            | s |      |      | ** |      |    |      |
|                |            | i |      |      |    |      |    |      |
|                |            | t |      |      |    |      |    |      |
|                |            | i |      |      |    |      |    |      |
|                |            | v |      |      |    |      |    |      |
|                |            | i |      |      |    |      |    |      |
|                |            | t |      |      |    |      |    |      |
|                |            | y |      |      |    |      |    |      |
|                |            | * |      |      |    |      |    |      |
|                |            | * |      |      |    |      |    |      |
+----------------+------------+---+------+------+----+------+----+------+
| # Ortho --     | # HIVCHE   | 9 |      | 98   | E  | Com  | F  | Ne   |
|  Clinical Diag | K System 3 | 8 |      | .8-1 | xt | plex | or | goti |
| nostic Systems |            | . |      | 00%^ | en | ity: | s  | able |
|                | M          | 2 |      | 1,2^ | si | 3    | al |      |
| Parent         | icrofiltra | - |      |      | ve |      | e\ |      |
| Company:\      | tion-bound | 1 |      |      |    | S    | WH |      |
| Johnson &      | EIA        | 0 |      |      |    | tore | O- |      |
| Johnson\       |            | 0 |      |      |    | at   | e  |      |
| New Brunswick, | R          | % |      |      |    | 2    | va |      |
| New Jersey,    | ecombinant | ^ |      |      |    | -25° | lu |      |
| USA            | antigens   | 1 |      |      |    | C^4^ | at |      |
|                | and        | , |      |      |    |      | ed |      |
| [www.orthocl   | synthetic  | 2 |      |      |    | Op   |    |      |
| inical.com](ht | peptides   | ^ |      |      |    | tion |    |      |
| tp://www.ortho | for        |   |      |      |    | al:\ |    |      |
| clinical.com/) | HIV-1/2    |   |      |      |    | ce   |    |      |
|                |            |   |      |      |    | ntri |    |      |
|                | Multiple   |   |      |      |    | fuge |    |      |
|                | reagents   |   |      |      |    |      |    |      |
|                |            |   |      |      |    |      |    |      |
|                | Uses       |   |      |      |    |      |    |      |
|                | plasma or  |   |      |      |    |      |    |      |
|                | serum      |   |      |      |    |      |    |      |
+----------------+------------+---+------+------+----+------+----+------+
| # Sa           | # Sero-Str | 9 |      | 99.6 | Mo | Com  | F  | \$   |
| liva Diagnosti | ip HIV-1/2 | 8 |      | -100 | de | plex | or | 1.50 |
| c Systems, Ltd |            | . |      | %^1, | ra | ity: | s  |      |
|                | I          | 4 |      | 2,3^ | te | 2    | al |      |
| Medford, NY,   | mmunochrom | - |      |      |    |      | e\ |      |
| USA            | atographic | 9 |      |      |    | S    | W  |      |
|                |            | 9 |      |      |    | tore | HO |      |
| [www.sal       | Synthetic  | . |      |      |    | at   | -e |      |
| v.com](http:// | peptides   | 9 |      |      |    | 2    | va |      |
| www.salv.com/) | for        | % |      |      |    | -25° | lu |      |
|                | HIV-1/2    | ^ |      |      |    | C^4^ | at |      |
|                |            | 1 |      |      |    |      | ed |      |
|                | Single     | , |      |      |    | Op   |    |      |
|                | reagent    | 2 |      |      |    | tion |    |      |
|                | and buffer | , |      |      |    | al:\ |    |      |
|                |            | 3 |      |      |    | ce   |    |      |
|                | Uses       | ^ |      |      |    | ntri |    |      |
|                | plasma or  |   |      |      |    | fuge |    |      |
|                | serum      |   |      |      |    |      |    |      |
+----------------+------------+---+------+------+----+------+----+------+
|                | # Hema-Str | 9 |      | 99.9 | L  | Com  | F  | \$   |
|                | ip HIV-1/2 | 9 |      | %^1^ | im | plex | or | 3.00 |
|                |            | . |      |      | it | ity: | sa |      |
|                | I          | 6 |      |      | ed | 1    | le | Ne   |
|                | mmunochrom | % |      |      |    |      |    | goti |
|                | atographic | ^ |      |      |    | S    |    | able |
|                |            | 1 |      |      |    | tore |    |      |
|                | Synthetic  | ^ |      |      |    | at   |    |      |
|                | peptides   |   |      |      |    | 20-  |    |      |
|                | for        |   |      |      |    | 33°C |    |      |
|                | HIV-1/2    |   |      |      |    |      |    |      |
|                |            |   |      |      |    |      |    |      |
|                | Single     |   |      |      |    |      |    |      |
|                | reagent    |   |      |      |    |      |    |      |
|                |            |   |      |      |    |      |    |      |
|                | Uses whole |   |      |      |    |      |    |      |
|                | blood,     |   |      |      |    |      |    |      |
|                | plasma, or |   |      |      |    |      |    |      |
|                | serum      |   |      |      |    |      |    |      |
+----------------+------------+---+------+------+----+------+----+------+
|                | #          | 9 |      | 99.4 | Mo | Com  | F  | Ne   |
|                | Saliva-Str | 9 |      | %^1^ | de | plex | or | goti |
|                | ip HIV-1/2 | . |      |      | ra | ity: | sa | able |
|                |            | 4 |      |      | te | 2    | le |      |
|                | I          | % |      |      |    |      |    |      |
|                | mmunochrom | ^ |      |      |    | S    |    |      |
|                | atographic | 1 |      |      |    | tore |    |      |
|                |            | ^ |      |      |    | at   |    |      |
|                | Synthetic  |   |      |      |    | 2    |    |      |
|                | peptides   |   |      |      |    | -25° |    |      |
|                | for        |   |      |      |    | C^4^ |    |      |
|                | HIV-1/2    |   |      |      |    |      |    |      |
|                |            |   |      |      |    | Requ |    |      |
|                | Single     |   |      |      |    | ires |    |      |
|                | reagent    |   |      |      |    | vo   |    |      |
|                | and buffer |   |      |      |    | rtex |    |      |
|                |            |   |      |      |    |      |    |      |
|                | Uses oral  |   |      |      |    |      |    |      |
|                | fluid      |   |      |      |    |      |    |      |
+----------------+------------+---+------+------+----+------+----+------+
| # Sano         | # M        | 9 |      | 98   | E  | Co   | F  | \$4  |
| fi Diagnostics | ultiSpot H | 9 |      | .5-1 | xt | mple | or | .00- |
|  Pasteur, S.A. | IV-1/HIV-2 | . |      | 00%^ | en | xity | s  | 5.00 |
|                |            | 3 |      | 1,3^ | si | : 3  | al |      |
| 92430 MARNES   | M          | - |      |      | ve |      | e\ |      |
| la COQUETTE,   | icrofiltra | 1 |      |      |    | S    | W  |      |
| FRANCE         | tion-bound | 0 |      |      |    | tore | HO |      |
|                | EIA        | 0 |      |      |    | at   | -e |      |
| Parent         |            | % |      |      |    | 2    | va |      |
| Company:       | R          | ^ |      |      |    | -8°C | lu |      |
| BioRad         | ecombinant | 1 |      |      |    |      | at |      |
|                | antigens   | , |      |      |    | Op   | ed |      |
| Hercules,      | and        | 3 |      |      |    | tion |    |      |
| California,    | synthetic  | ^ |      |      |    | al:\ |    |      |
| USA            | peptides   |   |      |      |    | ce   |    |      |
|                | for        |   |      |      |    | ntri |    |      |
| [www.bio-rad.c | HIV-1/2    |   |      |      |    | fuge |    |      |
| om](http://www |            |   |      |      |    |      |    |      |
| .bio-rad.com/) | Multiple   |   |      |      |    |      |    |      |
|                | reagents   |   |      |      |    |      |    |      |
|                |            |   |      |      |    |      |    |      |
|                | Uses       |   |      |      |    |      |    |      |
|                | plasma or  |   |      |      |    |      |    |      |
|                | serum      |   |      |      |    |      |    |      |
+----------------+------------+---+------+------+----+------+----+------+
|                | # Genie II | 9 |      | 99   | L  | Com  | F  | Ne   |
|                |  HIV1/HIV2 | 7 |      | .7-1 | im | plex | or | goti |
|                |            | . |      | 00%^ | it | ity: | sa | able |
|                | Microf     | 8 |      | 1,2^ | ed | 2    | le |      |
|                | iltration- | - |      |      |    |      |    |      |
|                | bound EIA  | 1 |      |      |    | Op   |    |      |
|                |            | 0 |      |      |    | tion |    |      |
|                | R          | 0 |      |      |    | al:\ |    |      |
|                | ecombinant | % |      |      |    | ce   |    |      |
|                | antigens   | ^ |      |      |    | ntri |    |      |
|                | and        | 1 |      |      |    | fuge |    |      |
|                | synthetic  | , |      |      |    |      |    |      |
|                | peptides   | 2 |      |      |    |      |    |      |
|                | for        | ^ |      |      |    |      |    |      |
|                | HIV-1/2    |   |      |      |    |      |    |      |
|                |            |   |      |      |    |      |    |      |
|                | Multiple   |   |      |      |    |      |    |      |
|                | reagents   |   |      |      |    |      |    |      |
|                |            |   |      |      |    |      |    |      |
|                | Uses       |   |      |      |    |      |    |      |
|                | plasma or  |   |      |      |    |      |    |      |
|                | serum      |   |      |      |    |      |    |      |
+----------------+------------+---+------+------+----+------+----+------+
| # Trini        | #          | 9 |      | 98.  | E  | Com  | F  | US\$ |
| ty Biotech USA | Capillus H | 8 |      | 2-99 | xt | plex | or | 1.50 |
|                | IV-1/HIV-2 | . |      | .6%^ | en | ity: | s  |      |
| Jamestown, NY, |            | 6 |      | 1,2^ | si | 2    | al |      |
| USA            | Latex bead | - |      |      | ve |      | e\ |      |
|                | agg        | 9 |      |      |    | S    | WH |      |
| Parent         | lutination | 9 |      |      |    | tore | O- |      |
| Company:\      |            | . |      |      |    | at   | e  |      |
| Trinity        | R          | 9 |      |      |    | 2    | va |      |
| Biotech, Plc\  | ecombinant | % |      |      |    | -8°C | lu |      |
| Dublin,        | antigens   | ^ |      |      |    |      | at |      |
| Ireland\       | and        | 1 |      |      |    | O    | ed |      |
| [www.trinitybi | synthetic  | , |      |      |    | ptio |    |      |
| otech.com](htt | peptides   | 2 |      |      |    | nal: |    |      |
| p://www.trinit | for        | ^ |      |      |    | re   |    |      |
| ybiotech.com/) | HIV-1/2    |   |      |      |    | ader |    |      |
|                |            |   |      |      |    | &    |    |      |
|                | Multiple   |   |      |      |    | ce   |    |      |
|                | reagents   |   |      |      |    | ntri |    |      |
|                |            |   |      |      |    | fuge |    |      |
|                | Uses       |   |      |      |    |      |    |      |
|                | plasma or  |   |      |      |    |      |    |      |
|                | serum      |   |      |      |    |      |    |      |
+----------------+------------+---+------+------+----+------+----+------+
|                | #          |   |      |      |    |      |    |      |
+----------------+------------+---+------+------+----+------+----+------+
| ##             | # **P      |   | #    | #    | #  | #    | #  | #    |
| **Manufacturer | roduct Des |   |  **S |  **S |  * |  **C | ** | **Co |
| /Distributor** | cription** |   | ensi | peci | *F | omme | St | st/T |
|                |            |   | tivi | fici | ie | nts^ | at | est^ |
|                |            |   | ty** | ty** | ld | 5^** | us | 6^** |
|                |            |   |      |      |  U |      | ** |      |
|                |            |   |      |      | se |      |    |      |
|                |            |   |      |      | ** |      |    |      |
+----------------+------------+---+------+------+----+------+----+------+
| ##             | #          |   | 98.9 | 98.8 | L  | Com  | F  | Ne   |
|                |            |   | %^1^ | %^1^ | im | plex | or | goti |
|                | # Sali     |   |      |      | it | ity: | Sa | able |
|                | vaCard HIV |   |      |      | ed | 2    | le |      |
|                |            |   |      |      |    |      |    |      |
|                | M          |   |      |      |    | S    |    |      |
|                | icrofiltra |   |      |      |    | tore |    |      |
|                | tion-bound |   |      |      |    | at   |    |      |
|                | EIA        |   |      |      |    | 2    |    |      |
|                |            |   |      |      |    | -8°C |    |      |
|                | Synthetic  |   |      |      |    |      |    |      |
|                | peptides   |   |      |      |    | R    |    |      |
|                | for        |   |      |      |    | equi |    |      |
|                | HIV-1/2    |   |      |      |    | res: |    |      |
|                |            |   |      |      |    |      |    |      |
|                | Multiple   |   |      |      |    | Orap |    |      |
|                | reagents   |   |      |      |    | ette |    |      |
|                |            |   |      |      |    | de   |    |      |
|                | #          |   |      |      |    | vice |    |      |
|                |  **Uses or |   |      |      |    |      |    |      |
|                | al fluid** |   |      |      |    |      |    |      |
+----------------+------------+---+------+------+----+------+----+------+
| # Tri          | # Se       |   | 99   | 99   | E  | Com  | F  | \$   |
| nity Biotech U | roCard HIV |   | .8-1 | .5%^ | xt | plex | or | 1.80 |
| SA (continued) |            |   | 00%^ | 1,2^ | en | ity: | sa |      |
|                | M          |   | 1,2^ |      | si | 2    | le |      |
| Jamestown, NY, | icrofiltra |   |      |      | ve |      |    |      |
| USA            | tion-bound |   |      |      |    | S    |    |      |
|                | EIA        |   |      |      |    | tore |    |      |
| Parent         |            |   |      |      |    | at   |    |      |
| Company:\      | Synthetic  |   |      |      |    | 2    |    |      |
| Trinity        | peptides   |   |      |      |    | -8°C |    |      |
| Biotech, Plc\  | for        |   |      |      |    |      |    |      |
| Dublin,        | HIV-1/2    |   |      |      |    |      |    |      |
| Ireland\       |            |   |      |      |    |      |    |      |
| [www.trinitybi | Multiple   |   |      |      |    |      |    |      |
| otech.com](htt | reagents   |   |      |      |    |      |    |      |
| p://www.trinit |            |   |      |      |    |      |    |      |
| ybiotech.com/) | Uses whole |   |      |      |    |      |    |      |
|                | blood,     |   |      |      |    |      |    |      |
|                | plasma, or |   |      |      |    |      |    |      |
|                | serum      |   |      |      |    |      |    |      |
+----------------+------------+---+------+------+----+------+----+------+
|                | # Uni      |   | 99.8 | 100  | L  | Com  | F  | \$2  |
|                | Gold HIV R |   | %^1^ | %^1^ | im | plex | or | .25\ |
|                | ecombinant |   |      |      | it | ity: | Sa | Ne   |
|                |            |   |      |      | ed | 1    | le | goti |
|                | I          |   |      |      |    |      |    | able |
|                | mmunochrom |   |      |      |    | S    |    |      |
|                | atographic |   |      |      |    | tore |    |      |
|                |            |   |      |      |    | at   |    |      |
|                | R          |   |      |      |    | 2-   |    |      |
|                | ecombinant |   |      |      |    | 27°C |    |      |
|                | antigens   |   |      |      |    |      |    |      |
|                | for        |   |      |      |    |      |    |      |
|                | HIV-1/2    |   |      |      |    |      |    |      |
|                |            |   |      |      |    |      |    |      |
|                | Single     |   |      |      |    |      |    |      |
|                | reagent    |   |      |      |    |      |    |      |
|                |            |   |      |      |    |      |    |      |
|                | Uses whole |   |      |      |    |      |    |      |
|                | blood,     |   |      |      |    |      |    |      |
|                | plasma, or |   |      |      |    |      |    |      |
|                | serum      |   |      |      |    |      |    |      |
+----------------+------------+---+------+------+----+------+----+------+

Notes:

1.  Sensitivity and specificity data supplied by manufacturer as
    determined for blood/serum/plasma against panel containing multiple
    HIV-1/2 subtypes.

2.  Published sensitivity and specificity data against multiple HIV-1/2
    subtypes.

3.  Sensitivity and specificity data from CDC evaluation against
    multiple HIV-1/2 subtypes.

4.  Stable at room temperature (20-33°C), but shelf life improved with
    refrigeration.

5.  Although all tests are designed to be simple, some require multiple
    steps, including pipetting, and performance may improve with
    centrifugation. In these instances limited training and laboratory
    experience are useful. Complexity rating: (1) Specimen may be whole
    blood, venipuncture not required, sample manipulation limited to
    application followed by addition of buffer reagent or wash, easily
    read; (2) Specimen limited to plasma or serum, centrifugation or
    optional equipment beneficial; (3) Reagent or sample preparation may
    be required, multi-step assay.

6.  Prices vary dependent on market and availability; they are provided
    solely as a point of reference.

APPENDIX - E

**Laboratory Safety Rules**

Important rules, not necessarily in order of importance, should be
adhered to when working in a laboratory:

1.  Pipetting by mouth should be prohibited.

2.  Eating, drinking, smoking, storing food and applying cosmetics must
    not be permitted in the laboratory/testing work areas.

3.  Labels must not be licked, materials must not be placed in the mouth

4.  The laboratory/testing site should be kept neat, clean, and free of
    materials that are not pertinent to the work.

5.  Work surfaces must be decontaminated immediately after any spill of
    potentially dangerous material and at the end of the working day.

6.  Members of the staff must wash their hands after handling infectious
    materials, and before they leave the laboratory.

7.  All technical procedures should be performed in a way that minimizes
    the formation of aerosols and droplets.

8.  All contaminated materials and specimens must be decontaminated
    before disposal or cleaning for reuse. They should be placed in a
    leak-proof, color- coded plastic bag for autoclaving or incineration
    on the premises. These bags should be supported in rigid containers.
    If it is necessary to move the bags to another site for
    decontamination, they should be placed in leak-proof containers
    e.g., solid-bottomed, that can be closed before they are removed
    from the laboratory.

9.  Laboratory coveralls, gowns or uniforms must be worn for work in the
    laboratory. This clothing should not be worn in non-laboratory areas
    such as offices, libraries, staff rooms and canteens. Contaminated
    clothing must be decontaminated by appropriate methods.

10. Open-toed footwear should not be worn.

11. Protective laboratory clothing should not be stored in the same
    lockers or cupboards as street clothing.

12. Safety glasses face shields (visors) or other protective devices
    must be worn when it is necessary to protect the eyes and face from
    splashes and impacting objects.

13. Only persons who have been advised of the potential hazards and who
    meet specific entry requirements (e.g. immunization) should be
    allowed to enter the laboratory working areas. Laboratory doors
    should be kept closed when work is in progress; children should be
    excluded from laboratory working areas.

14. There should be an insect and rodent control program

15. Gloves appropriate for the work must be worn for all procedures that
    may involve accidental direct contact with blood, and infectious
    materials. After use, gloves should be removed aseptically and
    autoclaved with other laboratory wastes before disposal. Hands must
    then be washed. Do not wash or disinfect surgical or examination
    gloves for reuse.

16. All spills, accidents and overt or potential exposures to infectious
    materials must be reported immediately to the laboratory supervisor.
    A written record of such accidents and incidents should be
    maintained.

17. Appropriate medical evaluation, surveillance and treatment should be
    provided.

18. Baseline serum samples may be collected from laboratory staff and
    other persons at risk. These should be stored as appropriate.

19. The laboratory supervisor should ensure that training in laboratory
    safety is provided. A safety or operations manual that identify
    known and potential hazards and that specifies practices and
    procedures to minimize or eliminate such hazards should be adopted.
    Personnel should be advised of special hazards and required to read
    and follow standard practices and procedures. The supervisor should
    make sure that personnel understand these.

    **APPENDIX - F**

**Sample Evaluation Expenditures**

+-------------------------------------------------+--------------------+
| **Test Kits**                                   | **Cost**           |
|                                                 |                    |
| > Kit --1                                       | \$4,000            |
| >                                               |                    |
| > Kit --2                                       | \$3,000            |
| >                                               |                    |
| > Kit --3                                       | \$6,000            |
+-------------------------------------------------+--------------------+
| HIV Western Blot                                | \$2,500            |
+-------------------------------------------------+--------------------+
| Reagents                                        | \$2,000            |
+-------------------------------------------------+--------------------+
| **Supplies**                                    |                    |
+-------------------------------------------------+--------------------+
| > Fine tips                                     | \$2,500            |
+-------------------------------------------------+--------------------+
| Vaccutainer tubes with needles                  | \$2,250            |
+-------------------------------------------------+--------------------+
| General (expendable) supplies                   | \$2,000            |
+-------------------------------------------------+--------------------+
| **Monitoring and Evaluation**                   | \$1,000            |
|                                                 |                    |
| ### Laboratory personnel                        | \$248              |
|                                                 |                    |
| ### Onsite Checks                               | \$2000             |
|                                                 |                    |
| > Proficiency testing                           |                    |
| >                                               |                    |
| > Travel expenses                               |                    |
+-------------------------------------------------+--------------------+
| HIV Western Blot                                | \$2500             |
+-------------------------------------------------+--------------------+
| HIV Reagents                                    | \$2,000            |
+-------------------------------------------------+--------------------+
| **TOTAL BUDGET FOR 6 Months**                   | **\$27,498 US**    |
+-------------------------------------------------+--------------------+

**APPENDIX - G**

**Sample Contents of an Evaluation Protocol**

Introduction

Purpose

Literature review

Limitations

Methods

Specimens required

Study sites

Study populations

Sampling

Sample size

Budget

Kits (ELISA, rapids, +/- WB, P24 Ag, PCR)

> Bench expenses (non-kit reagents, pipette tips, time,

Technicians, equipment costs.

Transport of specimens and personnel

Use of panels and libraries

Venipuncture and collection equipment

Storage cryotubes

Training of lab and field staff

IQA, IQC, EQA

Data management and storage

Implementation

Time frames

Staff duties

Analysis

Reporting and Publishing results

Results

Statistical calculations

Ethical issues

References

Appendices

APPENDIX -- H

**95% confidence ranges for .98 Sensitivity and .98 Specificity**

**Prevalence of HIV**

. 01 .05 .10 .20 .30

Sens Spec Sens Spec Sens Spec Sens Spec Sens Spec

100 1.7785 0.02767 0.161 0.0295 0.099 0.03054 0.0672 0.0310 0.052 0.0334

200 0.4256 0.01950 0.0987 0.0199 0.0666 0.02045 0.0451 0.0216 0.0361
0.02319

300 0.2570 0.01592 0.0769 0.0162 0.0521 0.01669 0.0361 0.0177 0.0295
0.01893

400 0.1946 0.01378 0.0666 0.0140 0.0451 0.01446 0.0313 0.0153 0.0250
0.01639

500 0.1609 0.01233 0.0596 0.0125 0.0403 0.01293 0.0274 0.0137 0.0224
0.01466

600 0.1400 0.01125 0.0521 0.0114 0.0361 0.01180 0.0250 0.0125 0.0204
0.01338

700 0.1248 0.01042 0.0482 0.0106 0.0334 0.01093 0.0231 0.0115 0.0189
0.01239

800 0.1143 0.00975 0.0451 0.0099 0.0313 0.01022 0.0216 0.0108 0.0177
0.01159

900 0.1054 0.00919 0.0425 0.0093 0.0295 0.00964 0.0204 0.0102 0.0167
0.01093

1000 0.0987 0.00872 0.0403 0.0089 0.0274 0.00914 0.0194 0.0097 0.0158
0.01037

Hence with 500 samples .98 sensitivity and .98 specificity and a
prevalence of .05

Sensitivity= 1.00 to .92 /Specificity= 1.00 to .97

Note: These numbers are derived making distributional and data
collection assumptions that may not be suited to a particular data set.
They are intended to be used as an approximation on the number of
samples one needs to use to reach a desired accuracy.

**References**

1.  Report of the global HIV/AIDS epidemic, June 2000

2.  **Dabis F, Msellati P, Meda N, Welffens-Ekra C, You B, Manigart O,
    Leroy V, Simonon A, Cartoux M, Combe P, Ouangré A, Ramon R, Ky-Zerbo
    O, Montcho C, Salamon R, Rouzioux C, Van de Perre P, Mandelbrot L,
    and for the Ditrame Study group**. 1999. 6-month efficacy, tolerance
    and acceptability of a short regimen of oral zidovudine to reduce
    vertical transmission of HIV in breastfed children in Côte d\'Ivoire
    and Burkina Faso: a double-blind placebo-controlled multicentre
    trial. Lancet **353**:786-792.

3.  **Guay LA, Musoke P, Fleming T, Bagenda D, Allen M, Nakabiito C,
    Sherman J, Bakaki P, Ducar C, Deseyve M, Emel L, Mirochnick M,
    Fowler MG, Mofenson L, Miotti P, Dransfield [K]{.underline}, Bray D,
    Mmiro F, and Jackson JB.** 1999. Intrapartum and neonatal
    single-dose nevirapine compared with zidovudine for prevention of
    mother to child transmission of HIV-1 in Kampala, Uganda: HIVNET 012
    randomised trial. Lancet **354**:795-802.

4.  **Marseille E, Kahn JG., Mmiro F, Guay L, Musoke P, Fowler MG, and
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