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New Advances in HIV testing in Vietnam Welcome to the 4th Generation of Rapid Tests

March 20, 20143/9/2013Presentation by Nguyen Nhat QuangThe presentation was supported and advised from the C&T team in HCM city1The contentsReview the knowledge of basic HIV testing And New Advances in HIV testing in VietnamThe results of HIV testing study in San FranciscoCurrent Vietnam HIV testing Algorithm.Mountainous Model Pilot ApproachRecommendations 4th Generation Test Use in Vietnam

3/9/2013HIV: Serotypes, Groups, and Subtypes3/9/2013

The subtype CRF01_AE is most prevalence in Vietnam HIV is a highly variable virus which mutates very readily. This means there are many different strains of HIV, even within the body of a single infected person.Based on genetic similarities, the numerous virus strains may be classified into types, groups and subtypes

Within group M there are known to be at least nine genetically distinct subtypes (or clades) of HIV-1. These are subtypes A, B, C, D, F, G, H, J and K.2Occasionally, two viruses of different subtypes can meet in the cell of an infected person and mix together their genetic material to create a new hybrid virus

3Global distribution of HIV-1 by subtypes3/9/2013RegionSubtypesN. America, Caribbean, latin America and West EuropeBEast Europe, Central AsiaA, BN. America, Mid EastB, CSoutheast AsiaB, AESub-Saharan Africa

A, C, D, F, G, H, J, K, CRF

East AsiaB, C, BC, CRF 01Source: Lancet 2007; 368:4894Transmission and Mechanism of Immunological Response3/9/2013

IgM

IgGAntigenTime of Immune responseAnti body

53/9/2013

Potential Antigen Targets for HIV Testing3/9/2013What do HIV Tests Detect?EIA (enzyme immunoassay) and ELSIA can be used interchangeably. Rapid tests use other principles instead: immunochromatography, immunoconcentration and particle agglutination.

ELISA (Enzyme- Linked Immunosorbent Assay)

EIA (Enzyme-linked immunoassay) Rapid Tests

Western blot

IFA Immuno- Fluorescence Assay

RIPA Radio - Immuno - Precipiation Assay

DBS Dried Blood Spot

P24 Ag TEST AVAILABLEARCHITECT HIV Ag/Ab Combo Alere Determine HIV-1/2 Ag/Ab Combo

7Classification of Rapid HIV tests3/9/2013FirstGeneration testSecondGeneration testThirdGeneration testFourthGeneration testDetect IgG antibody

Unacceptable rate of false positives

Contamination issues

Detect /IgG antibody

Improved sensitivity and specificity

False positives and contamination continued to be an issue Detect IgM/IgG antibody

Sandwich technique employing conjugated antigen

Improved sensitivity and specificity

Detect IgM/ IgG antibody and P24 antigen

10-14 day window to detect Acute HIV infection (AHI)

Sensitivity of p24 testing is still questionable in AHI

May increase indeterminate resultsSource: Future Microbiol.2009;4(8):963-982.Most tests can clarify prevalence but not incidence as difficult to know when infection occurred.

Refer from : What Is New in HIV Infection?KEVIN SHERIN, MD, MPH, MBA, Florida State University College of Medicine, Orlando, FloridaBENJAMIN G. KLEKAMP, MSPH, Florida Department of Health, Orlando, FloridaJEFFREY BEAL, MD, University of South Florida, Tampa, FloridaNICOLLE MARTIN, MD, MPH, Morehouse School of Medicine, Atlanta, Georgia

The generation Biologicals: first generation is using virus antigens were fully lysed and purified from infected cells with HIV . The biologicals have sensitivity and specificity is limitations . Second Generation Biologicals antigens using recombinant proteins or protein synthesis . Biologicals sensitivity and higher specificity . Third Generation antigens using recombinant proteins and synthetic antibodies detected but on the principle of sandwich ELISA . Sensitivity and specificity were significantly improved . Fourth Generation Biologicals simultaneous detection of antigens and antibodies in the sample . Born This product allows to detect HIV infection prior to sero-conversion and biologicals are advised to check the option for safe blood transfusion .

Busch MP, Satten GA: Time course of viremia and antibody seroconversion following human immunodeficiency virus exposure. Am. J. Med. 102,117124 (1997).The first-generation immunoassay tests employed HIV lysate as the source of antigen to capture antibody present in the sample. This generation of tests suffered from many problems associated with the antigen preparation. These included a high number of false-positive results caused by contamination of the antigen with proteins from the cells used to culture the virus, and batch-to-batch variation in the lysates employed. April 1987 saw the introduction of second-generation anti-HIV-1 immunoassays into blood-bank screening. This led to a reduction of the mean infectious window period from 56 to 42 days.8Evolution of Rapid HIV tests3/9/2013

Source: Future Microbiol.2009;4(8):963-982.Refer from : What Is New in HIV Infection?KEVIN SHERIN, MD, MPH, MBA, Florida State University College of Medicine, Orlando, FloridaBENJAMIN G. KLEKAMP, MSPH, Florida Department of Health, Orlando, FloridaJEFFREY BEAL, MD, University of South Florida, Tampa, FloridaNICOLLE MARTIN, MD, MPH, Morehouse School of Medicine, Atlanta, Georgia

The generation Biologicals: first generation is using virus antigens were fully lysed and purified from infected cells with HIV . The biologicals have sensitivity and specificity is limitations . Second Generation Biologicals antigens using recombinant proteins or protein synthesis . Biologicals sensitivity and higher specificity . Third Generation antigens using recombinant proteins and synthetic antibodies detected but on the principle of sandwich ELISA . Sensitivity and specificity were significantly improved . Fourth Generation Biologicals simultaneous detection of antigens and antibodies in the sample . Born This product allows to detect HIV infection prior to sero-conversion and biologicals are advised to check the option for safe blood transfusion .

Busch MP, Satten GA: Time course of viremia and antibody seroconversion following human immunodeficiency virus exposure. Am. J. Med. 102,117124 (1997).The first-generation immunoassay tests employed HIV lysate as the source of antigen to capture antibody present in the sample. This generation of tests suffered from many problems associated with the antigen preparation. These included a high number of false-positive results caused by contamination of the antigen with proteins from the cells used to culture the virus, and batch-to-batch variation in the lysates employed. April 1987 saw the introduction of second-generation anti-HIV-1 immunoassays into blood-bank screening. This led to a reduction of the mean infectious window period from 56 to 42 days.9HIV Transmission, Eclipse, and Window Period3/9/2013Early HIV infection

1st generation EIA 2nd generation EIA 3rdgeneration EIA 4th-generation Ag/AbIgMIgGWindow periodEclipse Phase Horizontal is days post infectionColumn is amount of antigen and Antibody The chart describe the time can detect Antigen and Antibody after HIV nfection- 10 days after infection can detect RNA- 02 weeks after infection can detect HIV P24- 20 days after infection can detect IgM soon then IgG later

Eclipse period: time after HIV infection when HIV RNA may be present in very small quantities but is undetectable. Acute HIV infection: phase of early HIV infection when HIV RNA and p24 antigen are detectable but HIV antibodies are not. Early HIV infection: stage of infection prior to HIV seroconversion or Western blot positivity.

Fourth-generation assay: detects p24 antigen and IgM/IgG HIV antibodies; Third-generation assay detects IgM/IgG HIV antibodies; second-generation and first-generation assays detect IgG HIV antibodies. (Ab = antibody; Ag = antigen. AHI = acute HIV infection; Ig = immunoglobulin.) The generation Biologicals: first generation is using virus antigens were fully lysed and purified from infected cells with HIV . The biologicals have sensitivity and specificity limitations . Second Generation Biologicals antigens using recombinant proteins or protein synthesis . Biologicals sensitivity and higher specificity . Third Generation antigens using recombinant proteins and synthetic antibodies detected but on the principle of sandwich ELISA . Sensitivity and specificity were significantly improved . Fourth Generation Biologicals simultaneous detection of antigens and antibodies in the sample . This product allows to detect HIV infection prior to sero-conversion and biologicals are advised to check the option for safe blood transfusion Refer from : What Is New in HIV Infection?KEVIN SHERIN, MD, MPH, MBA, Florida State University College of Medicine, Orlando, FloridaBENJAMIN G. KLEKAMP, MSPH, Florida Department of Health, Orlando, FloridaJEFFREY BEAL, MD, University of South Florida, Tampa, FloridaNICOLLE MARTIN, MD, MPH, Morehouse School of Medicine, Atlanta, Georgia10Rapid tests: short timing of detectionResults in ten minutes to 2 hours using blood, urine or saliva.

Extremely useful in immediate situations.

Many different tests are now available, all with excellent sensitivity and specificity.

Negative test can be reported as negative

Positive results must be confirmed with additional testing

3/9/2013RESULT OF DETERMINE HIV 1/23/9/2013

+-Test is dameged 02 Red bands : confirm the test response positive with HIV antibodies. 01 red band : test is negative.(verifiable band)(3) No red lines : no comment, the tests was failed12Criteria for WB confirmationWestern blot negative: there is no specific color band. Western blot positive: there are at least 2 color bands correspond to the envelop gene. Western blot suspect (in determinate): one or more color bands does not fix with the standard Western blot positive.

3/9/2013

+_WB is gold standard for confirmation testing, but it is high intensive and high level of External quality assurance system,So it has not been used in developing country ( include Viet Nam)

WB testing detect the specific antibodies to HIV core antigen as P24, envelop antigen : gp41, gp120, gp160 ...- These antigens were prepared to arranged in bands- IgG antibodies combined with antigens to creating the bands.13Example of Confirmatory testWestern blot test (WB)

Used as a confirmatory test.Very specific for HIV antibodiesIndeterminate tests must be repeatedSamples that give a negative result are reported as negativeResource intensive and require high level of external quality assurance

3/9/201314Sensitivity and specificity and predictive value3/9/2013Test resultHIV statusHIV infectionsNo HIV infectionsPositiveTrue PositiveaFalse Positiveb(a + b)NegativeFalse NegativecTrue Negatived(c + d)(a + c)(b + d)Sensitivity= a/(a+c)Specificity= d/(b+d)Positive predictive value (PPV)= a/(a+b)Negative predictive value (NPV)

= d/(c+d)Sensitivitiy : how many per cent of HIV infection cases will get positive result ( true positive) by the HIV testing?So the HIV testing has high sensitivity will be use for screening test.Sensitivity = a over/ a plus c

Specificity : how many per cent of no HIV infection cases will get negative result ( True Negative) by the HIV Testing? So the HIV test has high specificity will be use for determine testing.Specificity = d over/ d plus b

Predictive values may be defined as the probability that a result is correct, Predictive values depend on prevalence of the target population.NPV: Negative predictive value; PPV: Positive predictive value.

NPVs of HIV tests and testing strategies are always high even if the sensitivity and/or specificity of tests is suboptimal (see text). However, the PPVs change markedly, particularly in low prevalence populations where the proportion of false results may approximate or even exceed the true results. 15The Sensitivity and Specificity of Different Tests 3/9/2013HIV testSensitivitySpecificityDetermine HIV-1/2 99.999.7Elisa Genscreen ultra HIV Ag/Ab10099.87Elisa Murex HIV Ag/Ab combination10099.80ARCHITECT HIV Ag/Ab Combo10098WB9899.9Objectives of the San Francisco StudyTo assess how new tests might influence the performance of HIV testing programs in San Francisco.

To determine how use of these new tests might perform given their variable ability to detect acute infections.

3/9/2013Source: Pilcher et al. PLOS, Volume 8, Issue 12, e80629. December 2013MethodsAnalyzed 21,234 consecutive HIV tests with venous blood obtained from 2003 to 2008.

Using an HIV antibody-plus-RNA test algorithm.

New tests and re-testing all specimens that had negative/discrepant antibody results on initial screening

Separate analysis of oral HIV test on 127 oral samples from HIV+ patients

3/9/2013Process of study3/9/2013

Ability of Tests to Detect Acute HIV Cases, and All HIV Cases San Francisco Targeted Testing Programs, 20032008.

Test NameTypeFormatDetection of HIV infection among Ab-/RNA+specimens (n = 66 cases)Estimate of potential for case finding by each testAb Screening Assay-, WB-/ind Acute Specimens (n = 58)Ab Screening Assay- but WB+Specimens (n = 8)Numerator includes 695 Ab screening test+, WB+specimensTest+%Test+%Test+%ARCHITECT HIV Ag/Ab Combo(4th-generation)Ag-AbRapid lab IA48/5587.36/6100.0754/76199.1Determine HIV-1/2 Ag/Ab ComboAg-AbPOC RT31/5754.48/8100.0735/76196.6Genetic Systems HIV-1/2 Plus O (3rdgeneration )AbLab IA20/5834.58/8100.0723/76195.0Oraquick Advance (on blood plasma)*AbPOC RT3/585.28/8100.0706/76192.8Genetic Systems HIV WB (any bands positive)AbWestern blot16/5827.68/8100.0719/76194.4.20

20Detecting Acute HIV Infection3/9/2013

Findings and Conclusions of the studySome rapid antibody blood tests will give similar case detection to laboratory antibody tests.

Oral fluid testing has limited ability to detect Acute HIV infection.

New 4th-generation combo tests can increase detection of acute infections detectable by HIV RNA and could be used for high risk population groups with active transmission.

3/9/2013HIV cascade in Vietnam3/9/2013

A model that links prevention, HIV testing and treatment services across the continuum of care (CoPC). A means to:- Reduce HIV transmission, extend lives, and improve health outcomes;- Increase demand for HTC and diagnosis; - Enroll PLHIV into care and initiate treatment as soon as possible; - Promote adherence and sustain life-long ensure life-long ART. - Reducing viral load of individuals and within the communities.

Number of infected persons enroll in care and treatment is small compare with the total number of infected persons

To increase in PLHIV enroll in ART early

23The HIV testing in VietnamAdults and Children>18 months: HIV tests (screening rapid and confirmation with 2 ELISA + 1 Rapid) used popularly to confirm HIV positive.Delays from time of first positive rapid test to confirmation (3-5 days). PCR HIV tests are used to detect HIV DNA applied in HIV exposed children