# Presentation: 175700

## Overview of HIV testing

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**Overview of HIV testing**
**Evolution of technologies **

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**Chantal Maurice & Projet RETRO-CI team, Côte d'Ivoire**
**Global AIDS Program**
**Centers for Disease Control and Prevention, USA**

**Notes:**
Good afternoom,
I am presenting here a rapid overview of HIV testing technologies. I’d like to focus on the testing strategies for the developing word and share with you our experience of evaluating and using Elisa and rapid tests as part of programs at Projet RETRO-CI in Côte d'Ivoire. 

## HIV particle and genome

**envelope**

**   ****gp120**
**gp41 **

**matrix**

**p17**

**core**

**p24**

**RT**

**RNA**

**MHC**

**lipid bilayer**

**Notes:**
Just some generalities to remind you that the HIVs are complex Retroviruses belonging to the lentivirus subfamily.

These  RNA viruses are  characterized by an extensive variability which has an impact on, diagnosis 
In the classification system There are 2 types of viruses the HIV- 1 and HIV- 2
HIV 1 strains are divided in 3 groups M (for main or major), groups N and O (for outlier)
Multiple subtypes have been documented. 
Group M is  widespread. Subtype B is found in the US and Europe.
Groups O and N have limited distribution primarily in Central Africa.
The epidemic of HIV- 2 is focused in west Africa.
Principal antigenes of HIV-1 viruses from envelope,  gp120 and gp 41 and from core p24 are listed on this slide. 
They are the main antigens used in HIV tests for the detection of HIV-1  antibodies.  

## Clinical course of HIV infection Typical HIV infection profile 

**Notes:**
HIV infection has various stages from primary infection  (or acute phase)  to asymptomatic phase (or chronic phase) and late stage disease (or AIDS)

The first weeks after primary infection, seroconversion occurs and is associated with a rapid increase in circulating viral titers and a signifant drop in the number of CD4+ cells. 
Then  . Adaptive host immunity develops including neutralizing antibodies to HIV antigens (envelope, core).
The patient enters the stage of clinical latency so called  the asymptomatic period for several years up to 12 years.
This stage is characterized by gradual deterioration of immune response and depletion of CD4 cells..

 The terminal stage of HIV infection is marked  by a complete collapse of the immune system and AIDS  

## Immune response and evolution of antibodies

-   **Window period**

**Immune response and evolution of antibodies**

**Notes:**
The serologic profile of HIV infection is as follows:
After initial exposure, the time frame between exposure to HIV and the ability to detect HIV antibody is known as the window period.
Median length of the window period is 40 days and the viremic phase of the window period is 22 days.

During the seroconversion period the p24 antigene is detectable, mean time for its  detection is 5 to 10 days.  

Most of the antibodies  assays became reactive during the first week after onset of symptoms of primary HIV-1infection. 
The first HIV antibodies detected are Ig M then IgG . 
HIV tests which can detect Ag and IgM improve the performance of the diagnosis.Anti HIV envelopegp160 appear first, anti HIV core p24 gp120 and gp41 follow.

.    

## HIV testing objectives

** ****Voluntary**
** ****Informed consent**
** ****Surveillance**
** ****Unlinked anonymous**
** ****Without informed consent**
** ****Samples collected for several purposes**
** ****Linked (confidential or anonymous)**
** ****Informed consent**
** ****Samples collected for HIV testing only**
**  ****Blood transfusion safety**
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**HIV testing objectives**

**Notes:**
What are the objectives of HIV testing
UNAIDS and WHO have defined that HIV testing could be conducted for three main objectives:

Diagnosis , epidemiological surveillance, and blood transfusion safety.

HIV diagnosis is an voluntary testing with informed consent for asymptomatic and  symptomatic of HIV/AIDS patients.

HIV surveillance, performed with unlinked anonymous testing strategy without informed consent, is the most used testing strategy worldwide. In this case, samples are collected for several purposes by example for syphilis testing of pregnant women.
 But the preferred approach is the linked strategy with inform consent.     

To assure the safety of the blood transfusion it is mandatory to screen the blood or blood products, including screening of donors of tissues and organs.

## Challenges of HIV testing

**Sensitivity- Early diagnostic ****( window period)**
**Specificity- Cross reactivity **
**Detection of HIV-1 and HIV-2 and discrimination between the two viruses**
**Easy to perform, low cost **
**==> One test can not fulfill these requirements **
**    ****Need to perform a combination of HIV tests for screening and confirmation **

**Notes:**
HIV testing is challenging: it should be highly sensitive and specific.
The Sensitivity of a test  is a measure of the probability of correctly identifying an HIV infected  person 
The  specificity of a test  is a measure of the probability of correctly identifying an HIV uninfected person
The variablity of HIV viruses impacts on the sensitivity the specificity of HIV tests.Some examples.  In the nineties some  licensed diagnostic tests could not detect antibodies produced against strains of group O. Assays show significantly lower specificity for testing African specimens which are non- B subtypes.
In addition, the window period improves the challenge for the detection of new infections. 
Cross reactivities are possible particulary during pregnancy and in primary infections with Cytomegalovirus and Epstein-Barr virus.  
In some areas the cross reactivity between HIV 1 and HIV 2 causes problems. Most of the HIV infections occur in the developping world so there is a need for easy and low cost tests
 One test can not fulfill these requirements 
We need to perform a combination of HIV tests for screening and confirmation 

##     Current HIV technologies 

-  **Screening tests**
** ****Enzyme immunosorbent assays (EIAs)**
** ****Simple/rapid immuno-diagnostics  assays**
-  **Confirmatory or supplemental tests**
-  **Western blot (WB) **
** ****Line immunoassays (LlAs)**
-  **Alternatives to confirmatory tests**
** ****Repetitive EIA or rapid assays**

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**    ****Current HIV technologies **

**Notes:**
Serodiagnosis 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 are enzyme immunosorbent assays (EIAs) or simple/rapid immuno-diagnostics assays selected for their high sensitivity of detection. 

 Supplemental or confirmatory tests, such as western blot (WB), or line immunoassays (LIAs) are used to confirm infection in samples that are initially reactive.,

Repetitive testing incorporating EIAs or rapid tests selected for their specificity of detection may be used to confirm whether specimens found to be reactive for HIV antibodies by the screening test.

 For practical purposes, resource poor settings depend heavily on EIA and rapid tests for the screening and confirmation 

**	**** ****ELISAs**** ****(Enzyme Linked Immunosorbent Assays) **
** ****Four immunologic principles**
** ****Indirect**
** ****Competition**
** ****Sandwich**
**Immuno-capture**
** ****Suitable for large numbers of specimens****	**
**   **

**HIV Screening  tests**
**EIAs (Enzyme Immuno Assays)**
** **

**Notes:**
ELISA Enzyme linked Immunosorbent Assays are the most widely tests used since 1985 when the HIV antibody tests became available

They are based on  Four principles: Indirect, Competition, Sandwich,Immuno-capture and suitable for large numbers of specimens

**	**
** ****First generation**
** ****Ag : Purified lysates of HIV**
** ****Poor sensitivity and specificity**
** ****Second generation**
** ****Ag : HIV-recombinant proteins and/ or peptides**
** ****Detection of HIV-1 and HIV-2**
** ****Poor sensitivity, improved specificity **
** ****Third generation **
**Ag : HIV-recombinant proteins and/ or peptides**
**Detection of IgM and Ig G, improved sensitivity **
**Detection of HIV group O **
** ****Fourth generation ****	**
** ****Capacity to detect P24 Ag and antibodies **
**   **

**         ****Evolution of HIV ELlSAs**
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**Notes:**
HIV immunoassays have become more sensitive and specific over the years with the use of recombinant and or synthetic peptide antigens as the basis of the tests. First generation of HIV assays relied on the detection of antibody to HIV viral proteins. They had  poor sensitivty and specificityensitivity and 
The second generation assays were more specific and sensitive , they used HIV-recombinant proteins and/ or peptides as antigens.  
in addition they were able to detect HIV-2
The Third generation assays use the same antigens as the sencond generation. They are able to detect IgM in addidition to Ig G and have improved their sensitivity in reducing the seroconversion window

As the same time assays were developped to detect HIV-1 antigen using p24 antibodies.

Then Fourth generation assays which have the capacity to detect P24 Ag and antibodies have been developed .They have good sensitivities and specificities for the detection of antibodies.The HIV antigen sensitivity varies  among the assays specially for group O . But the overall performance is acceptable

## 		Detection of HIV p24 Ag 

** ****Objectives **
**Blood bank safety (Primary infections)**
** ****Standard ****ELISAs**
** ****Ag assays**
** ****Ag/Ab assays**
**Diagnosis of pediatric HIV-1 infections**
**Signal-amplification-boosted ELISA**
**Heat-denatured plasma**
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**	****	****Detection of HIV p24 Ag **

**Notes:**
Let me talk more about Tests which can detect p24 antigens
They were developed to shorten the window period and decrease the risk of  transmission of HIV through blood transfusion.
They are ELISAs based assays on  an  immunocapture format. The first step is to detect p24 Antigen with antibodies to p24 antigene. Positive results are confirmed by a neutralization assay.
These asays  have some problems of sensitivity. Underdetection is due to the biding of p24 antigen  in immune complex with p24 specific antibodies. 
Rheumatoid factor like antibodies may cause false positive results and affect the specificity of the test.
Compared to nuleic acid based methods (RNA PCR) they are less sensitive

In the mean time , Ag assays have been modified . A new HIV-1 antigen signal amplification boosted ELISA using tyramid has been developed. This assay combines the use of heat denatured plasma instead of sera which eliminates the intefering antibodies. 
This assay has an excellent sensitivity comparable to RNA PCR and can be used for the diagnosis of pediatric HIV-1 infecions for children above 10 days.
This assay is still under evaluation but a commercial version of this assay will be available soon. 

##     Rapid HIV tests

**  ****Simple **
**  ****Provide same day results**
** ****Four immunologic principles**
**  ****Particle agglutination**
**  ****Immunodot (dipstick)**
**  ****Immunofiltration (flow-through device)**
**  ****Immunochromatography (lateral flow)**
** ****Suitable for low volumes and limited ressources                   **
**   ****testing****	****sites **

**  **
**    ****Rapid HIV tests**

**Notes:**
The rapid HIV tests are simpler than ELISAs tests and can provide same day results.

Several types of tests exist based on their principles. The  most known principles are the agglutination, the immunodot,  the immunofiltration, or (flow-through device) and the  Immunochromatography (lateral flow)

These tests are suitable for low volumes and limited ressources testing sites 

## Confirmatory tests 

**  ****Western Blot (WB)**
**    ****Purified antigenes from lysates of HIV on **
**    ****nitrocellulose bands**
**  ****Line immuno assays  (LIAS)**
-     **HIV synthetic peptides on bands**

**Confirmatory tests **
** **

**Notes:**
The Western Blot is the most common test use for confirmation of HIV infection. 
However wb tests have several Disadvantages  such Lack of standardization in performance and interpretation by example  CDC and WHO have not the same criteria for interpretation. They give a high range of indeterminate results, they are complex.in addition they are very expensive.

Line immunoassays as well are considere as supplemental tests  

## Complexity of  HIV  tests

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**Complexity of  HIV  tests**

**  ****Level 1 : No additionnal equipment and little or **
** ****	****        ****no laboratory experience needed**
**  ****Level 2 : Reagent preparation or a multistep process is**
**        ****required; Centrifugation or optimal equipment **
**  ****Level 3 : Specific skills such as diluting are required**
**  ****Level 4 : Equipment and trained laboratory technician**
**                   ****are required**

**Notes:**
4 levels of complexity for HIV tests have been described 

Level 1 : No additionnal equipment and little or 
 	        no laboratory experience are  needed

  Level 2 : Reagent preparation or a multistep process is
        required; Centrifugation or optimal equipment may be required

  Level 3 : Specific skills such as diluting are required

  Level 4 : Equipment and trained laboratory technician
                   are required

##       Rapid tests and ELISAs advantages Rapid tests                          ELISAs

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**	****	****       **

| * Flexible  | * Batch capability                                        good for ≥ 100 specimens at same time |
| --- | --- |
| * Time to result(< 30 min) | *Can be automated |
|  * Skilled staff not required  | *Centralized  (QA/QC) |
| * Very easy to interpret  results | * May be highly  sensitive for
seroconverters |
| * On site testing  | * Cost per test less than cost                                         per rapid test |
| * Minimal equipment and  reagents required |  |
| * Tests can be stored at room temperature |  |
| * Complexity 1-3 |  |

**Notes:**
On this slide you can see the advantages of the rapid tests and the ELISAS.
Rapid tests are flexible, may be used for a small number , time to get  results is less than 30 minutes. Skilled staff is not required. Results are very esay to interpret and permit on site testing. 

ELISAs are good for high volumes of specimen. And good to test samples in batch. The testing could be automated and the Quality assurance program centralized and easy to implement. Cost per  test less expensive than rapid test. 

##    Rapid tests and ELISAs disadvantages          Rapid tests                                    ELISAs 

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**	****	****       **

| *Small numbers for each test run  | * Less flexible(need a minimum numbers for maximum  |
| --- | --- |
| *QA/QC at multiple sites | * Time to result(>1.5h)
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| *Some tests less sensitive (for   seroconverters)  | * Complexity 4 |
| *May cost more per individual test 
than EIA  | *Skilled technician required

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| * Interreader variability may provide  inconsistent results (e.g., particle                agglutination) | *Refrigerated reagents

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|  | * requires sophisticated equipments |
|  | * High equipment maintenance
 |

**Notes:**
The disavantages of rapid tests and ELISAs are opposite to their advantages. 
Quality control program are difficult to implement for HIV rapid testing.  

ELISAs need skilled technicians and equipment and electricity 

The choice of  an HIV test should be based on the objective of the testing and the capacities of the testing site. 

## Body Fluids used for HIV Testing

**                                         ****Technologies**
**      ****ELISA                         Rapid tests              Western Blot**
**     **Serum                              Serum                              Serum
-       Plasma                             Plasma                            Plasma
-       Dried Blood Spots           Whole blood                     Dried Blood Spots
-       Oral Fluids                       Oral Fluids                       Oral Fluids
-       Urine

**Notes:**
HIV tests could be performed on a wild range of body fluids. Sera Plasma and whole blood are the most used and may give the opportunity to do supplemental testing as biomolecular testing and quality assurance

## HIV testing strategies

** ****Definition**
**   ****Combination of tests**
** ****First test:  Highly sensitive**
** ****Second test: Highly specific**
** ****Parallel testing algorithm**
** ****Samples are tested simultaneously by two **
**    ****different tests**
** ****Serial testing algorithm**
** ****Samples tested by a first test****	****	****	****	****	**
** ****Negative samples are not retested**
** ****Positive samples are retested by one or **
**  ****several tests upon results**

**HIV testing strategies**
** **

**Notes:**
Alternative strategies or algorithms for confirmation instead of convetionnal WB testing have been evaluated extensively the last past years.
A strategy is a combination of tests with different antigens preparations and or different test principles . First HIV test should be highly sensitive (sensitivity should be  more than 99%) 
Second tests should be Highly specific.

They are several types of HIV testing algorithms
 Parallel testing algorithm
 Samples are tested simultaneously by two different tests

 Serial testing algorithm
 Samples tested by a first test.			 Negative samples are not retested
 Positive samples are retested by one or multiple tests

## 	HIV Parallel testing algorithm  

** **

**Assay 1**

**Assay 2**

**Assay 1 ****(-)**

**Assay 2 ****(-)**

**Assay 1 ****(+)**

**Assay 2 ****(+)**

**Discordant:**

**Assay 1**

**Assay 2**

**Assay 3**** **
**Tie-breaker**

**HIV status**

**Negative**

**HIV status**

**Positive**

**-**

**+**

**-**

**+**

**Notes:**
Here is an example of a parralel algorithm 
Samples are tested by an assay 1 et 2 simultaneously. 
Concordant results are reported negative or positive
Discordant samples are tested with a third test called tie breaker. The result of the tie braker is reported.  

## HIV Serial testing algorithm

** **

**Assay 1**** **

**HIV Positive**** **

**HIV Negative **

**     ****( - )                                        ( + )**

**Assay 2**

**( + )****                       ****( - )**

**     ****Assay 3 ****Tie-breaker**

**( + )**

**( - )**

**Notes:**
In ithe serial  testing algorithm : 
Samples are tested by an assay 1. 
If assay 1 is negative the sample is reported as negative while positive samples are confirmed by assay 2
Same as for the parallel strategy discordant samples are tested with an third assay as a tiebreaker.   

## Characteristic of WHO /UNAIDS strategies 

**Strategies**

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**Numbers of  EIAs**

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**or rapid tests for **
**screening**

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**Objectives / (HIV  Prevalences)**

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**WHO I**

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**1 test**

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**-**

** ****Surveillance/ (>10%)**

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**-**

** ****Blood safety/ ( All rates)                                                        **

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**-**

** ****Diagnosis/**

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** ****Symptomatic ( >30%)**

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**    **

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**WHO II**

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**2 tests/ serial testing**

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**-**

** ****Surveillance**

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** ****/ ( < or =10%)**

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**-**

**Diagnosis**

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**Asymptomatic (> 10%)**

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**Symptomatic (< or = 30%)**

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**WHO III**

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**3 tests/ serial testing**

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**-**

** ****Diagnosis**

**  ****Asymptomatic (<10%) **

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**        **

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**Notes:**
WHO and UNAIDS have set up three strategies based on the objective of the test, the  HIV prevalence in the population tested and the presence of symptoms or not. 

## WHO/UNAIDS HIV testing strategy II

**Assay 1 (A1)**

**Sample**

**Assay 2 (A2)**

** ****Negative**

**-**

**+**

**-**

**+**

** ****Positive**

**WHO/UNAIDS HIV testing strategy II**

**Repeat**
**A1 and A2**

**A1 -/A2-**
**Negative**

**A1+/A2-; A2+/A1-**
**Indeterminate**

**A1 +/ A2+**
**Positive**

**Notes:**
Here is presented the strategy II from WHO and UNAIDS This is a serial algorithm

## HIV testing Projet RETRO-CI’s experience

**Notes:**
After this rapid oveview let me share with you the experience of PROJET RETRO-CI  in HIV testing

## Projet RETRO-CI clinical laboratory

-  **Capacity building and programmatic activities  **
** ****lab support for HIV care and VCT services  **
** ****sentinel surveillance **
** ****validation of HIV testing algorithms (HIV-1/2) **
** ****ELISA based **
** ****rapid tests **
** ****staff training   **
** ****quality assurance  **

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**Projet RETRO-CI clinical laboratory**

**Notes:**

The clinical laboratory  of Projet RETRO-CI-performs multiple functions. 
- It supports all the research studies as well as the expanded programmatic activities
Including lab monitoring for all patients receiving HAART in Cote d’Ivoire, support for VCT, sentinel surveillance and other activities
Another major work and the topic of this talk has been the validation of HIV-1 and 2 testing algorithms for both ELISA and rapid tests along with quality assurance, staff training and implementation in the field .          

## Annual HIV tests performed at RETRO-CI’s clinical laboratory N>25,000

**Notes:**
More than twenty five thousand HIV tests are performed at the RETRO-CI laboratory each year among various populations. 

Pregnant women make up more than a third of these tests.   

## HIV positive test resultsDistribution of HIV-1 and HIV-2 

**Notes:**
An added complexity in West Africa is the circulation of both HIV-1 and HIV-2 viruses. 

Of the more than 25,000 samples collected from these various patient populations, 26% tested HIV-positive. 

If we just take the positive test results shown here, 96% were HIV-1 positive, including 6% dually infected with HIV-1 and HIV-2. 
A further 4% were infected with HIV-2 alone. 

## HIV testing among pregnant women

**         ****Short course AZT trial**

**PMTCT program and scale-up**

**Antenatal sentinel surveillance**

- Rapid
- tests

**HIV testing among pregnant women**

**Notes:**
Since 1995, more than 5000 ELISA based HIV tests have been performed among pregnant women each year.   

Early on these were tests done as part of research studies. 
Later antenatal sentinel surveillance was introduced
And mother-child HIV prevention programs introduced and expanded.
Now the introduction of on-site rapid tests is facilitating the scaling up and decentralizing of the PMTCT program.    

## HIV ELISA-based Algorithms in Projet Retro-CI I

**1988  **
** ****Screening **
**1****st ****Generation ELISAs **
** ****Confirmation of reactive samples**
**Western Blots HIV-1 and HIV-2**
**1990   **
** ****Confirmation of reactive samples**** **
**LIA (Peptilav1/2) **
**Western Blot HIV-1 and HIV-2 for discrepant results ELISAs/ LIAs**
** ****1993 **
** ****Screening 2****nd ****Generation ELISA in duplicate  **
**	**

**Parallel Algorithms**

**Notes:**
Since the beginning in 1988 Projet RETRO-CI has used ELISA  parallel algorithms.

With first then second, third and fourth generation ELISAs.
Confirmation was based on western blot, then on LIAs and now with repetitive ELISAs  

##  HIV ELISA-based Algorithms in Projet Retro-CI II

**1997 **
**Screening and confirmation **
**Three 3****rd ****Generation ELISAs**
**3****rd ****ELISA is used as Tie-breaker  **
**2000**
**Screening and confirmation **
**One 4****th**** and two 3****rd ****Generation ELISAs**
**Tie-breaker is a 3****rd ****ELISA**
**	**

**Parallel Algorithms**

## 	 ELISA Algorithm of  Projet RETRO-CI

**Negative**

**ELISA 1  (-)**

**ELISA 2 (-)**

**ELISA 3**

**Discordant**

**HIV-1**

**HIV-2**

**HIV-D**

**Positive**

**Positive **

**P24 Ag**

**Indeterminate**

**Negative **

**WB VIH-1 / VIH-2**

**Negative**

**  ****Monospecific**
**     ****ELISAs**

**Positive**

**ELISA 1   (+)**

**ELISA 2  (+)**

**ELISA 1**

**ELISA 2**

**Sera**

**PCR**

- PARAL
- LEL

- CME

**Notes:**
As shown here the testing strategy is complex with 3different  tests for the screening In addition 2 monospecific ELISAs are use to do the serogical typing of HIV-1 and 2 .
But Wb is used   only on .5% of the samples tested 
P24 A g assays are used on inderterminate results with WBs
RNA PCR testing is performed in some cases for research purposes    

## Rationale for rapid test strategy 

**   ****based algorithms **
** ****Reference laboratory strategy**
-  **Disadvantages of ELISA:**
** ****Expensive equipment **
** ****Electricity supply**
** ****Testing off-site away from program site **
** ****Laboratory expertise        **
** ****Batch processing **
** ****Slow turnaround time**

** **

**Rationale for rapid test strategy **

**Notes:**

Until 1999, all HIV tests were performed with previously validated ELISA based algorithms and these are the standard Project RETRO-CI reference
 But there was a rationale for use of rapid HIV tests
 because ELISAs  strategies have disadvantages from both the laboratory and clinical service perspectives.
 Testing requires expensive equipment, supply of electricity,
 is usually  performed off site in central laboratories as they require laboratory expertise. Samples are tested in large batches and turnaround time for testing is slow.
In the PMTCT programs we found a substantial proportion of the women accepted HIV testing but did not return to receive their results and therefore could not access effective PMTCT interventions. 

## Phase 1 – Lab based evaluation of rapid HIV test algorithms 

**1.**** **Determine the sensitivity and specificity
-     of candidate rapid HIV tests
- 2. Evaluate test performance on a recent
-     infection panel
- 3. Evaluate the performance of parallel and
-     serial rapid HIV test algorithms

**Notes:**
Thus in 1998 we decided to evaluate some of the newly available rapid HIV tests to see if a combination of these tests could be used in place of the ELISA testing strategy.

The 1st phase consisted of laboratory based evaluations of rapid test algorithms. 
This required 3 steps: 
First -  to select individual candidate rapid HIV tests which were available in country and determine the sensitivity and specificity against the HIV viruses circulating in the region. 
2nd to evaluate  the performance of the rapid assays among a small panel of samples from recently infected persons
And 3rd to use a combination of the best performing individual rapid tests and evaluate the performance of both parallel and serial HIV testing algorithms 

## Step  3. Evaluation of algorithms

** ****Selection of tests based on:  **
** ****ability to detect HIV-1 and HIV-2**
** ****sensitivity and specificity for prevalent  samples**
**  ****and sensitivity to detect recent infections**
** ****Determine 100% sensitive**
** ****Genie II and Capillus high specificity**
** ****Evaluation: serial and parallel HIV testing**
**   ****algorithm against gold standard   **

** **

** **

**Notes:**
THE STEP 3 is detailed on this slide
 Based on the results of the sensitivity and specificty of each test evaluated We  selected the best performing tests

 Remember the principle in sequential testing is a test with high sensitivity followed by a test with high specificity. 

Determine was selected as the 1st test due to its 100% sensitivity on both new and old infections. Genie II and Capillus were selected due to their high sensitivity and specificity.   

Serial and  parallel HIV testing algorithms were then evaluated against our gold standard ELISA algorithms. 

## 3: Parallel rapid testing algorithm

**n=1179**

** **

**Determine and Genie II**** on serum **

**Both negative**** **

**Both positive                   Discordant **

**     ****Tie-breaker: ****Capillus**

**HIV positive**

** **

**HIV negative **

**Sensitivity =  100%**
**Specificity =  100%**

** ****- **

**+ **

**Notes:**
In the parallel  testing algorithm :  more than 1100 sera were tested by both Determine and genie 2  
Concordant negative samples were reported as negative and concordant positive samples as positive. 
Discordant samples were then tested with Capillus as a tiebreaker and classified according to the Capillus result.  

This algorithm was found to be 100% sensitive and specific when compared to our ELISA reference algorithm. 

## 3: Serial rapid testing algorithm                                       n=1179

** **

**Determine**** on serum **

**HIV positive**** **

**HIV negative **

**     ****( - )                                        ( + )**

**Genie II**

**( + )****                       ****( - )**

**Sensitivity  =  100%**
**Specificity  =  100%**

**     ****Tie-breaker: ****Capillus**

**( + )**

**( - )**

**Notes:**
In the serial  testing algorithm :  we tested the same samples. 
If Determine was negative the sample was labeled as negative while positive samples were confirmed by Genie-2. 
As for the parallel strategy discordant samples were tested with Capillus as a tiebreaker.   

Due to the 100% sensitivity of Determine this algorithm was also found to accurately classify all samples correctly. 

## Phase 2 :  To introduce rapid test algorithm

**  ****At PMTCT site (site of short course AZT trial)**
**  ****Highly trained and experienced lab technicians **
**        ****from Projet RETRO-CI performed on-site testing**
**  ****Tests on sera **
**  ****Quality Control Program**
**  ****Parallel testing strategy **

** **

**Phase 2 :  To introduce rapid test algorithm**
** ****in a highly controlled field setting**

**Notes:**
So after validating our rapid test algorithm on prevalent strains we were ready for the next step.    

In May 1999 – we introduced on-site rapid tests in a highly controlled field setting. 

This was at the PMTCT program implemented at our previous research site.    
All tests were performed by highly trained lab technicians from the project on –site 
Tests were done on sera not whole blood
A quality control program  was put in place
And the HIV rapid parallel testing strategy was used with 2 modifications.  

## Parallel  testing algorithm

** **

**Determine and Genie II**** on serum **

**Both negative**** **

**Both positive                   Discordant **

**     ****Capillus**

**RETRO-CI reference laboratory**

**HIV dual**

** ****HIV-1       HIV-2**

**HIV negative **

**HIV indeterminate**

- Final classification

**Notes:**
The 1st modification included the discrimination of HIV-1 and HIV-2 by Genie –2 as the implications for PMTCT interventions are different between the 2 viruses. 
The 2nd included the referral to the RETRO-CI reference lab of all samples identified as HIV dual by Genie 2 or those that are discordant for final classification. 

## Quality control program

** ****reference = ELISA-based testing algorithm**
**   ****100% dual and indeterminate samples retested**
**   ****HIV-1, HIV-2 positive and HIV negative samples ****	****stored and a random 25% re-tested**
**   ****Parallel testing strategy evaluated and **
**    ****serial testing strategy modeled**

** **

**Quality control program**

**Notes:**
A quality control program was implemented at Projet RETRO-CI with the standard reference tests

As I showed on the previous slide, 100% of the dual and indeterminate  samples were retested, other samples were stored and a random 25% retested. 

  We tested 1114 randomly selected samples and found 100% concordance between the parallel algorithm and the gold standard for HIV negative, HIV-1 and HIV-2 infected samples. 

 We found a perfect concordance between the parallel and the serial algorithms.too. 

## Phase 3 : Expert workshop recommendations & expansion

** ****11/2001 national expert workshop convened:**
** ****review of data: sens/spec, test properties & cost  **
** ****serial HIV testing algorithm recommended**
**    **
** ****Projet RETRO-CI activities:**
**   ****HIV serial testing algorithm defined as “standard” **
**   ****Training for external lab staff (3 weeks)  **
**   ****Ongoing program evaluation: **
** ****Quality control on a smaller proportion of samples**
** ****Evaluation of new modifications (serial/staff etc)**

** **

**Notes:**
The next phase in implementation involved sharing the data. 
In November 2001 a national expert workshop was convened to review the evaluation and consider the sensitivity and specificity of the test algorithms, the  properties of the tests and their costs. 
The workshop recommended use of the serial HIV testing algorithm with Determine followed by Genie-2. 

At projet RETRO-CI, 
We incorporated the serial algorithm as our new standard on-site testing strategy for all
   RETRO-CI supported programs. 
We also began training for external lab staff. 
And there were ongoing program evaluation activities including quality control and the evaluation of new program modifications with the use of the serial algorithm and changed staff training. 

## “Standard” serial testing algorithm

** **

**Determine ****on serum **

**RETRO-CI reference laboratory**

**HIV dual**

** ****HIV-1      HIV-2**

**HIV negative **

**     ****( - )                                     ( + )**

**Genie II**

**( + )****                            ****( - )**

**HIV indeterminate**

**Notes:**
Our new standard algorithm for new PMTCT sites was Determine on serum followed by Genie-2 if Determine was positive. 
If the results were discordant or if Genie-2 showed dual infection these samples were sent to 
the reference laboratory for evaluation and final classification. 

## HIV results at two PMTCT clinics

- 			   **n = 4172****                           ****n = 535****	**
** ****	****	**Parallel & trained          Serial & 3 week training **                               **
**	****	****	****      ****   ****%****	****	****	****  %**
**HIV-negative****	****	****      87.1****	****	****	****             87.1**
**HIV-1****	****	****	****      10.9 ****	**		** 11.8     ****	****	**
**HIV-2****	****	****	****        0.9****	****	****	****	**** 0.0**
**Dual****	****	****	****        ****0.5**** ****	****	****             ****	**** ****0.4**
**Indeterminate****	****	****        ****0.6**** ****	****             ****	****              ****0.7**
**Total ****	****	****	****    100.0 ****	****                        100.0**

-  }      1.1%      {
-        referred

**HIV results at two PMTCT clinics**

- 98.9%
- on-site

**Notes:**
This slide shows  the results of 2 different testing strategies at 2 PMTCT sites. 
The column on the left shows results from the former research site with the parallel algorithm performed by trained RETRO-CI lab techs. 
While the column on the right shows results from a MOH clinic with a new PMTCT program. Tests were done with a serial algorithm and MOH staff who had received 3 weeks training.

With either strategy almost 99% of women could be given their results immediately of which around 11% were HIV-positive. 
Just over 1% of women had to return for their results after confirmation at the reference laboratory.           

## Evaluation of tie-breaker Capillus for discordant results

**	****                                **
**Determine (+) / Genie (-)     compared to ELISA referent **
**Tiebreaker: Capillus (-)**** ****	****   **
**	****	****	****	****   ****N      (%)**** **
**	****             ****	**
** ****HIV negative****	****   ****	****   ****31****	****(100)**
** ****HIV-1****	****	****     ****	****	****    0****	****             **
**  **
** ****HIV Dual ****	****                          0****	****              **
** ****HIV-2****	****	****       ****	****               0****	****             **
** **
** ****Total                                      ****31 (< 0.7% of 4707)**

- * 99.4%
- Specificity
- of Determine
- => False pos

**Notes:**
We also found that the tiebreaker test Capillus correctly classified all the discordant results – with all 31 samples being correctly classified as HIV negative.  

Note that all the discordant results were with Determine being positive and Genie 2 being negative which is consistent with Determine’s specificity of 99.4% and Genie and Capillus’ high specificity.  

This suggests that Capillus could be used in the field as a tiebreaker without requiring referral to the reference lab. However as Determine has a higher sensitivity for recent infections it might be prudent to recommend repeat testing to exclude recent infection.    

## Added complexity due to HIV-2.Evaluation of Genie II “dual results” 

**	****                                **
**Genie (dual) compared to monospecific ELISA referent **
**	****   **
**	****	****	****	****   ****N      ****	****(%)**** **
**	****             ****	**
** ****HIV negative****	****   ****	****    0****	****	****(0)**
** ****HIV Dual ****	****                         12****	****	****(55) **
**            **
** ****HIV-1****	****	****     ****	****	****    5****	****	****(23)  **
** **
** ****HIV-2****	****	****       ****	****               5****	**** ****	****(23)            **
** **
** ****Total                                      22 (< 0.5% of 4707)**

**Notes:**
We also evaluated dual infections which ocurred in < 1⁄2% of women and found that Genie-2 had no false HIV negative results but  was not able to discriminate well between HIV-1 and HIV-2. 
Around 1⁄2 of the samples were monospecific ELISA positive to both viruses, 1⁄4 were positive for HIV-1 only and 1⁄4 for HIV-2.      

## Lessons learned

** ****Reliable; highly sensitive and specific **
** ****Easy to perform in field by trained lab staff**
** ****Adapted for developing countries and PMTCT **
**    ****programs**
**	****	****	****AND**
** ****Do NOT change HIV test acceptance**
** ****Do INCREASE % of women receiving their results**
**    ****and having access to PMTCT interventions****    **

**Lessons learned**

**Notes:**
So what have we learned through our experience.

That rapid testing algorithms for HIV diagnosis on sera are:
Reliable, highly sensitive and specific 
Easy to perform in field by lab staff with minimal training. 
Adapted for resource poor settings and for PMTCT programs and can be integrated with other antenatal tests
AND that
Voluntary testing uptake does not decrease
While the proportion of women who receive their test results 
and thus have access to PMTCT interventions does increase substantially

## The dilemma of HIV-2

-  current tests do not appear to reliably discriminate
-     between HIV-1 and HIV-2
-  discriminatory tests add expense and complexity
-  implications for staff training – lab/counselors
-  implications for the individual – PMTCT/natural history
-  potential barrier to expanding access to PMTCT
-  Is there a need to differentiate between HIV-1 and HIV-2 ?
- In Côte d’Ivoire:
-     HIV-positive result will be provided to ~12% of pregnant   	women: ~ 11.5% HIV-1/dual and ~ 1⁄2 % HIV-2

**The dilemma of HIV-2**

**Notes:**
I wanted to close with a few thoughts about HIV-2 which creates a small programmatic dilemma
This is because currently available rapid tests cannot always discriminate between HIV-1 and HIV-2. 
Discriminatory tests like Genie-2 add an extra level of complexity and expense and are not available for whole blood
This has implications for staff training for both lab technicians and for counselors as the implications for vertical transmission and PMTCT interventions are different as is the natural history. 
This added complexity may be a potential barrier to expanding access to PMTCT services          

I think this begs the question:
Is there a need to differentiate between HIV-1 and HIV-2?
In Cote d’Ivoire an HIV-positive result will be given to approximately 12% of women of which around 11 and a 1/2 % will carry HIV-1 and around 0.5% will have HIV-2 alone.  

## Conclusion

**  ****Use of rapid HIV tests can dramatically increase access**
**    ****to HIV test results and PMTCT interventions in Africa**
** ****Tool to promote access to test results **
** ****Decreases  waiting time between test and result**
**  ****Ongoing need to simplify and improve testing strategy**
**  ****whole blood rather than sera**
**  ****consider whether counselors can perform tests **
**  ****monitor and evaluate => improve testing  **
** ****ELISA testing is a reference tool for quality control **
**   ****and evaluation for standard laboratories **

**Notes:**
Finally in conclusion:
It appears that: 
Use of rapid HIV test algorithms can dramatically increase access to HIV test results and to PMTCT interventions  in Africa. 
There is an ongoing need to continue to simplify and improve the testing strategy. This may include use of whole  blood rather than sera which requires centrifugation, ongoing staff training, consideration as to whether counselors can perform the tests and, of course, ongoing monitoring and evaluation to continue to improve the program.    
We plan to  evaluate Rapid HIV tests on whole blood and saliva in order  to further simplify testing procedures. 
 and we will explore the use of dried blood spots for quality control purposes .
 But we still need the ELISA testing for large  scale testing,  quality control and is a standard tool for reference laboratories performing evaluation of HIV kits.