# Presentation: 433625

## Update on HIV Testing

**[A Very Fast]**

## Message

**There are numbers of tests**
**They should be used in combination (strategies)**
**Combinations must be consistent**

## Laboratory Tests

**Laboratory Tests**

**diagnosis of infection**
***acute, recent, established or late stage disease***
**prognostic markers**
**monitoring of ARV therapies**
***immunological and virological markers***
***toxicities***
**diagnosis of opportunistic infections**
**drug resistance testing**

## ‘typical’ primary HIV-1 infection

**‘****typical’ primary HIV-1 infection**

**symptoms**

**HIV-1 p24 antigen**

**0****	****1****	****2****	****3****	****4****	****5****	****6****	****/****	****2****	****4****	****6****	****8****	****10****	**

**weeks**

**years**

**HIV antibodies**

**Time following infection**

**HIV viral load**

**HIV proviral DNA**

**symptoms**

**‘****window’**
**period**

**1° infection**

## HIV Assays: Methodologies FOR THE DIAGNOSIS (DETECTION) & MANAGEMENT OF HIV

**DIAGNOSIS**

**Virus Detection & Quantification**

**Antibody**
**Antigen**
**Detection**

**RNA****modified Ag**
**Viral Culture, ****	****phenotyping**

**CD4+**

**ARV Resistance – genotyping****ARV Sensitivity **

**HIV Assays: Methodologies**** ****FOR THE DIAGNOSIS (DETECTION) & MANAGEMENT OF HIV**

**EIA****Simple, rapid ****	**** tests Immunoblots **
**Incident assays**

**MANAGEMENT**

**DNA****(RNA)**

**Notes:**
HIV Assays - Methodologies and Utilisation:
The diagnosis of HIV infection usually rests on the identification of specific antibody to HIV in plasma or serum.  To identify positively the presence of anti-HIV,  two assays  using different technology and different antigen preparations, should be used.
The detection of HIV infection by antibody tests is an indirect confirmation of infection. 
There has been a change in test usage with the introduction of molecular-based tests.
The emphasis in using anti-HIV tests has been somewhat eclipsed by the news around tests that quantify viral load.  Viral load estimations are used to gauge prognosis and to monitor therapy.
Other tests monitor the status of immune function indirectly but these are used  widely in HIV management.

## Spectrum 

**DNA PCR**

**RNA PCR**

**p24 Ag**

**3rd gen ELISA**

**1st gen ELISA**

**Detuned ELISA**

**1wk        2wk        3wk      2mo   6mo     1yr   2yr    3yr    ****	****      +8yr**

- early

- recent / established

- advanced

**Spectrum **
**of anti-HIV **
**testing **

## HIV Testing -Direct Detection of Virus

**HIV p24 antigen– serology**
**- *****In isolation or Ab/Ag Combo test ***
***- Diagnosis of primary infection viraemia***
**Virus culture / isolation**
**Nucleic acid detection - (NAT)**
***	****** ******Clinical uses ******Proviral DNA vs. plasma RNA(viral load)***
***	****** *****resolution of inconclusive serology /     neonatal**
**  *****subtyping ***
***	******drug resistance monitoring***

## Principle of Immunoassays

**ANTIGEN**

**SAMPLE ANTIBODY**

**ANTI-HUMAN IMMUNOGLOBULIN WITH DETECTOR**

**Notes:**
Principles of Immunoassay:
There is a general basic construction format of immunoassays which include:
An antigen which is used to capture any sample antibody, attached to a solid phase, be it a well on a microtitre plate or a gelatin particle, bead or membrane.
The anchored antigen is incubated with the sample so that an antigen-antibody reaction occurs.
In PA assays bound antibody causes particle agglutination.
In EIAs a wash step removes the serum or plasma and leaves only bound antibody.  A conjugate which is usually anti-human immunoglobulin labelled with enzymes or fluorochromes which will subsequently bring about a reaction that will result in a colour change.  After a wash step to remove excessive conjugate a substrate on which the enzyme reacts or by which the fluorochrome is activated, the colour change is read in a spectrophotometer.
Newer machine-based assays may use variations on the system diagrammed above.

## Available Assays

**EIAs  including **
- rapid, simple
- particle agglutination,
- dot/blot
- Western blot
- Antigen & Ab/Ag
- Incidence assays
**Direct Virus Detection**

**Notes:**
Available Assays:
There is a large range of assays available, especially for anti-HIV screening and HIV-diagnosis.
In considering the most suitable assay for a given testing strategy there will be a number of considerations including:
suitability of assay format
antigens used
characteristics of the assay
skills of the technical staff, etc.

## Particle Agglutination

## Western Blot

**Western Blot**

**Expensive – $ 80 - 100**
**technically more difficult**
**visual interpretation**
**lack standardisation**
**- performance**
**- interpretation**
**- indeterminate reactions – resolution of ??**
**‘****Gold Standard’ for confirmation**

## Antibody testing limitations

**Difficulties in interpretation**
**Limitations - ‘window period’**
**antibodies appear within 3-4 weeks**
**Direct detection – HIV p24 antigen or DNA/RNA (NAT) – pre-antibody**
***Combo test****** = earlier detection***
**Primary infection + therapy = delayed antibody response**

## Ag/Ab Combo tests

**Detection of Ag & Ab in a single test**
**utility in primary infection – pre-seroconversion ‘window period’**
**Incident populations – ‘at risk’**
**Blood bank**
**Automated platforms available**

**Ab & Ag**

## Issues with Combo Assays

**Testing strategies**
**False reactivity rates**
**Confirmation strategies**
**Replacement of other assays ****(especially in the USA)**
**Cost**
**Legal issues**

## Conclusions	

**HIV-1/2 Ag/Ab combo assays perform well**
**Differences in limit of detection of Ag **
**(140 - <25 pg/mL)**
**May shorten window period by 3-5 days**
**PHI detection without indication**
**Issues associated with introduction**
**Strategies and confirmatory algorithms**
**ARV therapy effects seroconversion events**
** **

## What about simple assays?

## HIV Determine test

**HIV Determine test**

**Detect HIV-1 & HIV-2**
**Cannot differentiate**
**Procedural control – anti Hu IgG**
**Whole blood or serum/plasma**
**Widely available**
**No additional reagents required**
**Room temperature storage**
**15 minutes to result**

## BioRad HIV-1/2 Multispot

**BioRad HIV-1/2 Multispot**

**Detects HIV-1 and HIV-2**
**Will differentiate 1 and 2**
**Procedural control – anti-Hu IgG**
**Serum / plasma only **
**Additional reagents (included)**
**Requires refrigerated storage**
**‘****Immunoconcentration’ principle**
**15 minutes to result**

## WHO Recommended Strategies

_**Strategy I**_**  Test all samples with one EIA**
_**Strategy II  **_**Strategy I with all reactives retested in a more specific test with different principle and/or antigen.**
_**Strategy III**_**  Strategy II with reactives tested in a third test differing from the first two tests.**

**Notes:**
WHO Recommended Strategies:
Because of the cost of supplemental testing WHO has devised and recommended alternative testing strategies.  These strategies are designed to avoid the use of the costly and poorly standardised Western Blot. 
The strategies rest entirely on assays that may be conducted by screening laboratories.
It is emphasised that tests must be carefully selected.  Each test used in the strategy should employ a different principle and different antigen preparation.
The strategy selected also depends on the prevalence of HIV infection within the population being tested.  The predictive values of the strategies will change with the prevalence.

## WHO Recommended Testing Strategies

- Transfusion safety
- Surveillance
- Diagnosis
-     Risk factors
-     No risk factors

- Strategy  I
- >10%       I
- _<_10%      II
- Strategy II
- >10%      II
- _<_10%     III

**Notes:**
WHO Recommended Testing Strategy 2
The strategies are recommended by their prospective use and depending on the prevalence of the population being tested. Furthermore if the test strategy is being used to achieve diagnoses, risk factors will be taken into account.
These alternative testing strategies have been used to advantage in numbers of countries.

##  Testing Strategies

**AIM:****	****	****To develop the logic used in  establishing the use of HIV tests (testing strategies)**

**Notes:**
Developing Testing Strategies:
To develop testing strategies effectively you should know:-
the prevalence of the infection being detected
the incidence of the infection
the characteristics of tests used in testing your population
that a supply of the tests to be used will be constant over time
the interactive properties of the tests
the requirements of the clinical staff that use the laboratory

All these parameters need to be available and understood for a testing strategy to be developed so that the predictive values of the strategies will be understood.

## Objectives of Testing Strategies

- To achieve the correct diagnosis in the most efficient manner
- To maintain consistency in testing
- To know the predictive value of the testing process
- To develop baseline data for assessing changes
- To deliver useful results

**Notes:**
Objectives Of Testing Strategies:
Achieving the correct diagnosis
In a fully developed testing strategy, the limitations of any tests and of the testing process are understood.
Tests are used appropriately.
There are supporting data against which ongoing performance may be measured.
Consistency
Laboratory performance can only be tracked if the testing process is consistent
Laboratory staff and doctors alike understand results reported with consistent test usage.
 Laboratory records can be maintained with consistency.
Strategies are based on data therefore the predictive value of the testing process is known.
The limitations of the testing process may be defined.
If any changes in tests or testing occur, the changes will be understood in the context of the strategy.

## Aims in Developing HIV Testing Strategies

- To arrive at the correct sero-diagnosis
- To minimise total testing; thus cost
- Minimise samples classed as indeterminate or dual reactors
- Detect HIV-1 negative but HIV-2 positive
- Follow likely seroconverters (HIV-1 or -2)

**Notes:**
Aims in Developing HIV Testing Strategies:
To arrive at a correct sero-diagnosis
Characteristics of the tests need to be understood.
The sequence in which the tests are to be used have to be understood by laboratory personnel and health care workers alike.  
The deficiencies of the strategies must be understood.
Minimising total testing
Developing strategies generates data that will enable laboratories to achieve the correct diagnosis in the most efficient manner. Therefore, total testing is minimised. 
 If different tests are continually introduced into testing there is confusion about what aberrant test results might mean, and no consistent means of resolving these. Therefore further testing and confusion is generated.  This can be costly.

CONTINUED ON NEXT PAGE

## Screening Assays

**Are used to detect antibody-- specific or nonspecific **
** ****Are designed to handle large numbers of samples with rapid throughput**
** ****Must be high performance**
** ****Should include a full range of HIV antigens **

**Notes:**
Screening or First Line Tests:
Screening assays usually detect antibody.
Because the assays are geared to detecting any antibody that resembles the target antibody they demonstrate false reactivity that cannot be ignored.
The most commonly used screening assays are enzyme immunoassays (EIAs) and particle agglutination(PA) assays. In many countries the blood service laboratories are using fully automated micro-particle immunoassays.
The assays are designed so that large sample numbers can be processed in short time frames.
The use of screening assays has almost eliminated the transmission of HIV, HCV and HBV through blood transfusion when the assays are used correctly. 
This has been possible because most screening assays are highly sensitive (designed to detect all positive sera).
Screening assays are the assays used initially in diagnostic testing strategies.

## Ab + Ag          AbAg       

**Notes:**
The Basis of Immunoassay reactions:

All immunoassays are based on an equilibrium between the binding of an antibody with an antigen.

Perfect Immunoassays:

To create a perfect immunoassay there would be a single, totally specific interaction, which was 100% sensitive in detecting the particular antibody and 100% specific to the analyte in question

## Ab +Ag

**Ab +Ag**

**AbAg**

**Ab +Ag**

**Ab +Ag**

**Ab +Ag**

**Ab +Ag**

**Ab +Ag**

**Ab +Ag**

**AbAg**

**AbAg**

**AbAg**

**AbAg**

**AbAg**

**AbAg**

**AbAg**

**Notes:**
There Is No Perfect Assay!

Because humans generate a repertoire of antibodies to many proteins there is always some degree of overlap and false reactivity can occurs in 0.1 to 2 or 3% of the population for any given assay.
On the other hand sometimes antibody is of low level or low concentration and therefore is not detected.
When 100% sensitivity and specificity are quoted for an assay - the reported evaluation has not incorporated sufficient samples (always look for Confidence Intervals).
Therefore where a result is sensitive such as for the diagnosis of HIV, the result must be as close to a100% accurate as possible.  Because this is not possible using a single test multiple tests must be used in sequence to arrive at a diagnosis that is as close to 100% as possible.  The careful development of testing strategy will enable this.

## Serological Testing Strategy

**Notes:**
A Reference Laboratory Testing Strategy: 
In a reference laboratory tests additional to the normal screening and first supplemental test used in diagnostic laboratories, may be performed to decipher complex reactivity in samples that may be referred from other laboratories.
“Supplemental tests” used could include other EIA’s, p24, immunoassays, commercial Western Blots and molecular methods.
As a reference laboratory we aim to minimise the need for follow up of samples.
It should be realised that most samples will never enter this complex type of strategy.  Most samples will not go beyond the first screen.  

## HIV  Testing Strategy

**NEG**

**HIV1/2 SCREEN**

**POS**

**HIV-1 WB**

**ADDITIONAL**
**TESTS**

***REACTIVE***

***NEG***

***NEG***

***IND***

**POS**

***IND***

**HIV  Testing Strategy**

_**SCREENING**_

_**SUPPLEMENTAL**_

**Notes:**
Principles involved in a Testing Strategy: 
Testing strategies are set up according to the characteristics of the tests.
The screening test is a highly sensitive test which identifies anything that resembles the target.
Supplemental testing sorts out true from false reactivity - tests should therefore be:

highly sensitive
of different configuration
use different capture targets  

## Supplemental Assays

**Range of assays that  further define sero-status**
**High Performance  (higher specificity)**

**Notes:**
Supplemental assays - Definition:

Supplemental assays are those that further define the sero-status.  

To distinguish true from falser reactivity supplemental assays are necessarily of high specificity.

## The Use of Screening Assays

**Define samples as negative for a given analyte**
**Enable high throughput**

**Notes:**
The Use Of Screening Assays:
Screening assays are used in both blood bank and diagnostic programs to exclude negative samples from any further testing in  the testing strategy.
Because of the very high negative predictive value of screening tests, the first test in screening programs can use the specimen as a single sample and if it is negative no further testing need occur.

## Assay Selection depends on:

**laboratory infrastructure**
**access to reference laboratory**
**desired characteristics of the test**
**equipment**
**performance time**
**shelf life and stability of reagents**
** ****price**
** ****technical skills of personnel**
**support (technical, kit supply, etc)**

**Notes:**
Assay Selection:
The assay selected in strategy development will depend on a large number of factors.
These factors will be different for every country or region.
An advantage of collaborations between regions is that each may utilise aspects of others testing strategies for unusual situations.

## Predictive Values

_**Positive Predictive Values**_**:**
**The likelihood of a sample identified as a reactive by a test being truly POSITIVE for the analyte used as the basis of the test.**

**PPV**

**=**

**True Positives**

**True Positives + False Reactives**

**X 100%**

**Notes:**
Predictive Values:
Understanding predictive values is the key to understanding the predictability of a strategy’s effectiveness.
The predictive values of the same strategy in different areas will be different because of factors such as 
the prevalence of HIV in the community
the characteristics of  the tests being used
the rate of false reactivity.

Low Prevalence :	True Positives < False Reactives
High Prevalence:	True Positives > False Reactives

Therefore the same tests and strategies used in different areas will not give the same predictive values.  Thus it is important that each testing network understand what its individual predictive values are.

## Predictive Values

_**Negative Predictive Values**_**:**
**The likelihood that a sample identified as a non-reactive by a test is truly NEGATIVE for the analyte used as the basis of the test.**

**Notes:**
Predictive Values Continued:
Negative predictive values are much higher  than positive predictive values because of the way of tests are constructed. 

Antibody tests are geared to recognise antibody.  (The tests can be thought of as hypersensitive therefore, the number of false negatives is usually very low.)

## Negative Predictive Value

**Non-reactive test, prevalence 2%**

** ****99.17  99.38 99.59 99.79  99.90  99.96  99.98**
** ****99.17  99.38 99.59 99.79  99.90  99.96  99.98**
** ****99.17  99.38 99.59 99.79  99.90  99.96  99.98**
**  **

**Specificity**

**Sensitivity**

**60**
**70**
**90**

**60      70      80      90       95       98       99   **

**%**

**Notes:**
Negative Predictive Values:
Because assays are constructed to pick up antibody that even vaguely resembles anti-HIV antibody, the negative predictive value of  tests is very high, even for tests with relatively low sensitivity and specificity.
This explains why it is possible to screen using a single sample.
It is notable that most samples screened will fall into the negative category.  This means that if samples are screened appropriately with a high sensitivity test the chances of a false negative are very low.

## Positive Predictive Value

**Reactive test, prevalence 2%**

**    ****4.95     4.95**
**    ****6.49      6.49**
**    ****9.43      9.43    9.43**
**  ****17.24    17.24  17.24   17.24   17.24   17.24    17.24 **
**  ****29.41   29.41  29.41    29.41   29.41   29.41     29.41**
**  ****51.02   51.02  51.02    51.02   51.02   51.02     51.02 **
** ****67.57    67.57  67.57    67.57   67.57    67.57     67.57**** **

**Sensitivity**

**Notes:**
Positive Predictive Value:
The positive predictive value depends on the prevalence of the measured analyte in the community being tested as well as the false reactivity of the tests i.e. test specificity.
The positive predictive value is the ratio of true reactivity to the sum of and true false reactivity and therefore predicts the likelihood of a reactive test being a true positive.
It is notable that for tests with high sensitivity that if the specificity is low the predictive value falls precipitously.  This is particularly true of populations where the true prevalence is low.
In developing a testing strategy, laboratories need to have a clear understanding of what the positive predictive value of tests are.  Furthermore it is extremely important to be able to communicate to the public and to the health care workers and the public the importance of predictive values.  How many times have we all heard that the test is 100% sensitive therefore the performance is perfect!

## Cross reactivity

**Cut-off value**

**    ****1 **

**Frequency**

**3SD**

**2SD**

**1SD**

**    ****2 **

**    ****3 **

**Cross reactivity**

**Notes:**
The Cutoff Value:

Cutoff values are set by manufacturers by examining wide populations - both negative and positive for the analyte.
If the cutoff value is move the predictive value of the test will change.
Cutoff values should never be altered unless a full evaluation has been undertaken.

## Slide 41

**Notes:**
Positive Predictive Values Of Strategies:
If an assay of high sensitivity and specificity is used in a low prevalence population its predictive value will be low  but in a high prevalence population it will be much higher.
Even if the sensitivity and specificity of an assay approach 100% (as is the case in assay 2 above) the predictive value may still be quite low for low prevalence populations.

## Slide 42

**Notes:**
Positive Predictive Values (2):
If assays are used in sequence as in assay 1 and assay 2 in the example above, the predictive value, even at low prevalence, is enhanced considerably.
It is the use of assays in sequence that makes strategy development essential  in populations of low prevalence.  

##      PPV vs Prevalence

**                                                    **

**     ****PPV vs Prevalence**

**(in sequence)**

**Sensitivity ****	****        99.0%      99.0%             **

**       ****Specificity         99.5%      99.9%**

**Prevalence**

**0.2%**
**2.0%**
**20.0%**

**28.4% **

**80.2% **

**66.4% **

**99.75% **

**98.0% **

**80.2% **

**99.97% **

**99.6% **

**99.99% **

**PPV**

**PPV**

**PPV**

## Why Follow  a Strategy?

## The Importance of Maintaining a Strategy

**Consistency of laboratory records**
**Consistency of results**
**Clarity of results to doctors**
**Maintaining data base to assess performances**
**Avoiding common false reactivity**
**Avoiding technical errors**
**Reducing costs**

**Notes:**
The Importance of Maintaining a Strategy:.
Consistency of Laboratory Records
The records can be set up consistently and therefore data will be in better order 
If a strategy is carefully followed, laboratory records will be more easily maintained.  Log books or computer records can be consistent. 
Consistency of results 
If a strategy is followed the results will be more consistent because technical staff will be more familiar with assays and procedures.  
The staff will become more familiar with interpretation procedures.
Similarly, the clarity of reported results will be consistent and easier to understand.  Therefore better diagnoses will be given to patients.
Databases will be more useful if tests are used consistently.  Any difficulties with the strategy may be assessed from data that are carefully maintained.
If assays are changed over the course of time it may become evident that there is common false reactivity among samples.  This will only be detected if a strategy is followed and a database maintained.
Consistency of data and results will lead ultimately to the reduction in costs as will a reduction or avoidance of technical errors.  In the previous example of developing HTLV strategies the reduction in the number of referrals and indeterminate results would only have been possible if the strategy had been maintained for a sufficient period of time to analyse the results.

## WHO Recommended Strategies

_**Strategy I**_**  Test all samples with one EIA**
_**Strategy II  **_**Strategy I with all reactives retested in a more specific test with different principle and/or antigen.**
_**Strategy III**_**  Strategy II with reactives tested in a third test differing from the first two tests.**

**Notes:**
WHO Recommended Strategies:
Because of the cost of supplemental testing WHO has devised and recommended alternative testing strategies.  These strategies are designed to avoid the use of the costly and poorly standardised Western Blot. 
The strategies rest entirely on assays that may be conducted by screening laboratories.
It is emphasised that tests must be carefully selected.  Each test used in the strategy should employ a different principle and different antigen preparation.
The strategy selected also depends on the prevalence of HIV infection within the population being tested.  The predictive values of the strategies will change with the prevalence.

## WHO Recommended Testing Strategies

**Transfusion safety**
**Surveillance**
**Diagnosis**
**    ****Risk factors**
**    ****No risk factors**

**Strategy  I**
**>10%       I**
_**<**_**10%      II**
**Strategy II**
**>10%      II**
_**<**_**10%     III**

**Notes:**
WHO Recommended Testing Strategy 2
The strategies are recommended by their prospective use and depending on the prevalence of the population being tested. Furthermore if the test strategy is being used to achieve diagnoses, risk factors will be taken into account.
These alternative testing strategies have been used to advantage in numbers of countries.

## WHO Guidelines

***Other possibilities***
**strategy for confirmation**
**combination of affordable & simple assays**
**different test principles**
**different antigen preparations**
**two or three ELISAs or rapid tests**
**diagnosis confirmed by second sample**
**detection of virus (PCR)**
**antigen detection (limited lab.facilities)**
_**Always use a QC sample**_

## Cost of HIV Testing

***comparative costs (AUD$)***
**ELISA (Ab only) - $2 per test**
**EIA (Ab/Ag combo) - $3.50**
**rapid test - $10-20 per test**
**Western blot $80 - 100**
**p24 antigen $30**
**PCR - qualitative $80 - 100**
**PCR - quantitative (viral load) $90 – 150***
**DNA sequencing (resistance) $400 – 700**

## Summary of Testing Strategies

## Message

**There are numbers of tests**
**They should be used in combination (strategies)**
**Combinations must be consistent**