dr.biol.tatjana kolupajeva head of hiv

1. Dr.biol.Tatjana Kolupajeva Head of HIV / AIDS Reference Laboratory Infectology Center of Latvia Dear colleagues! The task of my presentation is to observe the laboratory methods and algorithms being used in HIV diagnostics and monitoring of HIV patients and demonstrate how we apply them here in Latvia as well. I hope these materials may be the basis for your comments and questions. Especially welcome is information about your experience in these fields. Dear colleagues! The task of my presentation is to observe the laboratory methods and algorithms being used in HIV diagnostics and monitoring of HIV patients and demonstrate how we apply them here in Latvia as well. I hope these materials may be the basis for your comments and questions. Especially welcome is information about your experience in these fields.

2. Dr.Tatjana Kolupajeva 11.09.2004 2 Agenda: HIV situation in Baltic States HIV Diagnostics in Latvia Traditional methods Early diagnostics Diagnostics in infants till 18 months HIV disease management testing First of all I would like to tell you about organization of HIV diagnostics in Latvia. Then we will analyze the problems concerning traditional methods of diagnostics, possibilities and current trends in early diagnostics, approaches to diagnostics in infants born to the infected mothers and recent methods in HIV disease management. First of all I would like to tell you about organization of HIV diagnostics in Latvia. Then we will analyze the problems concerning traditional methods of diagnostics, possibilities and current trends in early diagnostics, approaches to diagnostics in infants born to the infected mothers and recent methods in HIV disease management.

3. Dr.Tatjana Kolupajeva 11.09.2004 3 HIV / AIDS Surveillance in Baltic States Just some remarks about the testing of infants born to the infected mothers. You know that traditionally the tests like Ag detection by ELISA, NA detection by PCR or other molecular biological techniques are used. Detection of seroreversion is a significant marker of the infection status of the child. Very useful may be HIV IgA ELISA test because IgA Ab don�t cross the placenta. Commercial tests are recently under development. Just some remarks about the testing of infants born to the infected mothers. You know that traditionally the tests like Ag detection by ELISA, NA detection by PCR or other molecular biological techniques are used. Detection of seroreversion is a significant marker of the infection status of the child. Very useful may be HIV IgA ELISA test because IgA Ab don�t cross the placenta. Commercial tests are recently under development.

4. Dr.Tatjana Kolupajeva 11.09.2004 4 HIV Testing Laboratories Let me present you the system of HIV testing in Latvia. On this map you see the net of laboratories dealing with HIV testing. Most of them are concentrated in Riga. There are two big labs: one of them Blood Bank laboratory, the other HIV Reference laboratory in Infectology Center of Latvia, and 12 smaller laboratories. There are labs in all big towns of Latvia: in two ports - Ventspils and Liepaja, on the north � in Valmiera, and in east � Blood Bank and diagnostic laboratories in Rezekne and one in Daugavpils. Let me present you the system of HIV testing in Latvia. On this map you see the net of laboratories dealing with HIV testing. Most of them are concentrated in Riga. There are two big labs: one of them Blood Bank laboratory, the other HIV Reference laboratory in Infectology Center of Latvia, and 12 smaller laboratories. There are labs in all big towns of Latvia: in two ports - Ventspils and Liepaja, on the north � in Valmiera, and in east � Blood Bank and diagnostic laboratories in Rezekne and one in Daugavpils.

5. Dr.Tatjana Kolupajeva 11.09.2004 5 HIV testing laboratories network in Latvia 2 Blood Bank laboratories 19 Primary testing laboratories: 3 laboratories are accreditated according to LVS EN ISO/IEC 17025 Standards 16 laboratories are certificated testing for diagnostic and surveillance purposes by 3rd and 4th generation ELISA and rapid tests 1 Reference laboratory � HIV/AIDS Laboratory Department of Infectology Center of Latvia: is accreditated according to LVS EN ISO/IEC 17025 Standards confirmatory testing of all primary reactive samples testing for diagnostic and surveillance purposes by ELISA and rapid tests more than 35000 blood samples /year monitoring HIV patients and treatment efficacy 19 primary laboratories test samples for diagnostic and surveillance purposes using 3rd and 4th generation ELISA and rapid tests. 3 of them are accreditated according to ISO 17025 standards, 16 are certificated. Reference laboratory is accreditated � 19 primary laboratories test samples for diagnostic and surveillance purposes using 3rd and 4th generation ELISA and rapid tests. 3 of them are accreditated according to ISO 17025 standards, 16 are certificated. Reference laboratory is accreditated �

6. Dr.Tatjana Kolupajeva 11.09.2004 6 Prevalence of HIV Infection in Latvia, 2003 Here you can see groups of population which are tested and number of tests performed in each group in 2002. Total number of tests this year is more then 161 000. About half tests were performed for blood donations and other half for diagnostics. Largest group for testing is pregnant women (group for recommended systematic testing). Other important groups are I/V drug users � with HIV prevalence in group 13.1 , prisoners �. During my further presentation you will hear about other specifics of HIV testing in Latvia as well. Here you can see groups of population which are tested and number of tests performed in each group in 2002. Total number of tests this year is more then 161 000. About half tests were performed for blood donations and other half for diagnostics. Largest group for testing is pregnant women (group for recommended systematic testing). Other important groups are I/V drug users � with HIV prevalence in group 13.1 , prisoners �. During my further presentation you will hear about other specifics of HIV testing in Latvia as well.

7. Dr.Tatjana Kolupajeva 11.09.2004 7 HIV Testing Strategies HIV testing strategy can be selected to maximize sensitivity and specificity while minimizing cost Criteria for choosing HIV testing strategy*: HIV prevalence in the population being tested Objective of the test (surveillance, blood screening, or diagnosis) Sensitivity and specificity of the tests *(Guidelines for Using HIV Testing Technologies in Surveillance: Selection, Evaluation, and Implementation; UNAIDS/ WHO 2001) To maximize sensitivity and specificity of HIV diagnostics, testing strategy must be selected. Criteria for choosing����.depend on: 1. 2. 3. In general, the higher is infection prevalence in the population we are testing, the higher is the prognostic value of positive result, that means the greater possibility, that person testing positive is truly infected. Conversely, the probability, that a person with a negative result is truly uninfected, decreases. To maximize sensitivity and specificity of HIV diagnostics, testing strategy must be selected. Criteria for choosing����.depend on: 1. 2. 3. In general, the higher is infection prevalence in the population we are testing, the higher is the prognostic value of positive result, that means the greater possibility, that person testing positive is truly infected. Conversely, the probability, that a person with a negative result is truly uninfected, decreases.

8. Dr.Tatjana Kolupajeva 11.09.2004 8 UNAIDS and WHO HIV Testing Strategies Taking in account all these problems experts of WHO have developed three HIV testing strategies. 1. 2. 3. Main idea of these strategies is that the combinations of ELISA and / or rapid assays can provide results as reliable as ELISA / WB combination and more cheap. They recommend to use confirmatory assays only to resolve indeterminate results in diagnostic purposes. Here in Latvia we don�t use such strategies. It would be interesting to hear about experience of using them in other states. Taking in account all these problems experts of WHO have developed three HIV testing strategies. 1. 2. 3. Main idea of these strategies is that the combinations of ELISA and / or rapid assays can provide results as reliable as ELISA / WB combination and more cheap. They recommend to use confirmatory assays only to resolve indeterminate results in diagnostic purposes. Here in Latvia we don�t use such strategies. It would be interesting to hear about experience of using them in other states.

9. Dr.Tatjana Kolupajeva 11.09.2004 9 Predictive value of the first positive test depending on HIV prevalence Latvia, 2003 On this slide you can see, how the PPV increases with the increasing of prevalence in the tested group. These are results of testing in three groups with different HIV infection prevalence (data of AIDS Prevention Center) in the AIDS Reference laboratory in Latvia in 2003: ��������������������������. Unfortunately, we do not have the data about the NPV, but risk of a false-negative result now is higher because of increase of HIV infection level. That is why the choose of optimal testing algorithm is of critical importance for the correct diagnostics. Testing algorithm means both selection of assays and the order in which the assays are used. On this slide you can see, how the PPV increases with the increasing of prevalence in the tested group. These are results of testing in three groups with different HIV infection prevalence (data of AIDS Prevention Center) in the AIDS Reference laboratory in Latvia in 2003: ��������������������������. Unfortunately, we do not have the data about the NPV, but risk of a false-negative result now is higher because of increase of HIV infection level. That is why the choose of optimal testing algorithm is of critical importance for the correct diagnostics. Testing algorithm means both selection of assays and the order in which the assays are used.

10. Dr.Tatjana Kolupajeva 11.09.2004 10 Traditional technologies for HIV testing 2nd and 3rd generation ELISA (EIA, MEIA) � Enzyme Linked ImmunoSorbent Assay � for HIV � antibody detection Very high sensitivity ( > 99%) and specificity ( > 95%) 3rd generation assays have sensitivity higher than confirmatory tests ! are able to detect both IgM and IgG specific antibodies Good results on both HIV 1 Major (M) and Outlier (O) groups viruses Rapid tests for HIV � antibody: particle agglutination, immunofiltration , immunochromatography, etc. Western Blot and Immunoblot (LIA, RIBA) � for confirmation of HIV 1 or/and HIV 2 antibodies Nucleic acid detection: highly sensitive and specific tests HIV-DNA-PCR: detect the integrated DNA form of the virus. HIV-RNA-PCR: qualitative or quantitative (in plasma) Traditional technologies recently used for HIV testing are the following: 2nd and 3rd �. Both assays are very �. But 3rd generation assays have � Highly sensitive and specific tests such as nucleic acid detection by PCR are often used in routine practice. Traditional technologies recently used for HIV testing are the following: 2nd and 3rd �. Both assays are very �. But 3rd generation assays have � Highly sensitive and specific tests such as nucleic acid detection by PCR are often used in routine practice.

11. Dr.Tatjana Kolupajeva 11.09.2004 11 ELISA tests principle

12. Dr.Tatjana Kolupajeva 11.09.2004 12 Traditional algorithm for HIV diagnostics Traditional algorithm for HIV diagnostic using these technologies is presented on this slide. All ELISA negative results are reported as negative, but repeatedly positive need confirmation by Western Blot. Positive WB results are reported as positive, all indeterminate ones usually require call for the 2nd sample, but negative ones are reported as negative. The problem is that this algorithm was developed when 1st and 2nd generation ELISA, which were as sensitive as WB, were used. With the improvement of ELISA sensitivities in 2nd and especially in 3rd generation assays, applying of such algorithm may result in false negative results. The other problem is variety of WB criteria. Traditional algorithm for HIV diagnostic using these technologies is presented on this slide. All ELISA negative results are reported as negative, but repeatedly positive need confirmation by Western Blot. Positive WB results are reported as positive, all indeterminate ones usually require call for the 2nd sample, but negative ones are reported as negative. The problem is that this algorithm was developed when 1st and 2nd generation ELISA, which were as sensitive as WB, were used. With the improvement of ELISA sensitivities in 2nd and especially in 3rd generation assays, applying of such algorithm may result in false negative results. The other problem is variety of WB criteria.

13. Dr.Tatjana Kolupajeva 11.09.2004 13 Criteria for interpretation of positive Western Blot results As you see in different regions criteria for interpretation of positive WB results differ. Sometimes in one country two and more criteria are used. That was the reason for speculation about the usefulness of WB applying in practical medicine. WHO proposed criteria when antibodies to at least 2 envelope glycoprotein are presented in sample. Here in Latvia we apply WHO criteria. Higher sensitivity of 1st line assays combined with these criteria PROVOKE significant number of indeterminate results. That is why the schema of confirmatory diagnostic algorithm became more complicated and expensive. As you see in different regions criteria for interpretation of positive WB results differ. Sometimes in one country two and more criteria are used. That was the reason for speculation about the usefulness of WB applying in practical medicine. WHO proposed criteria when antibodies to at least 2 envelope glycoprotein are presented in sample. Here in Latvia we apply WHO criteria. Higher sensitivity of 1st line assays combined with these criteria PROVOKE significant number of indeterminate results. That is why the schema of confirmatory diagnostic algorithm became more complicated and expensive.

14. Dr.Tatjana Kolupajeva 11.09.2004 14 Tests for early diagnostics of HIV infection 4th generation ELISA � Antigen-Antibody Combination Assay Simultaneous detection of HIV � Antibodies and HIV Antigen Reduces diagnostic window by 4 � 9 days ( as demonstrated on seroconversion panels) Sensitivities: from 99,5% to 100% (in several studies from 98,00% to 99,87%) Specificities: from 98% to 100% (88,2% in one study on potentially cross-reactive samples) ELISA HIV Ag and ELISA HIV Ag neutralization for confirmation Nucleic acids detection assays: HIV-DNA-PCR, HIV-RNA-PCR highly sensitive � detection limit till 50 cop/ml Current improvement of diagnostic technologies concerns mostly possibilities of early diagnostics to reduce window period. Development of ELISA assay results in creation of 4th generation ELISA. 4th generation ELISA � Some authors write about lower sensitivity of combination assays on Ab comparing to the ELISA tests for Ab, and lower sensitivity on Ag comparing to the ELISA tests for Ag ????? At the same time in other articles there are data that sensitivity� Applying the 4th g. ELISA more often became the necessity of using ELISA HIV antigen tests for the confirmation of primary reactive results. Certainly NA detection assays such as ���. still show the better results in the case of early infection but using of them is limited by high cost. Current improvement of diagnostic technologies concerns mostly possibilities of early diagnostics to reduce window period. Development of ELISA assay results in creation of 4th generation ELISA. 4th generation ELISA � Some authors write about lower sensitivity of combination assays on Ab comparing to the ELISA tests for Ab, and lower sensitivity on Ag comparing to the ELISA tests for Ag ????? At the same time in other articles there are data that sensitivity� Applying the 4th g. ELISA more often became the necessity of using ELISA HIV antigen tests for the confirmation of primary reactive results. Certainly NA detection assays such as ���. still show the better results in the case of early infection but using of them is limited by high cost.

15. Dr.Tatjana Kolupajeva 11.09.2004 15 Principle of ELISA tests

16. Dr.Tatjana Kolupajeva 11.09.2004 16 Detection of recent HIV Seroconversion

17. Dr.Tatjana Kolupajeva 11.09.2004 17 Early Diagnostics of HIV Infection

18. Dr.Tatjana Kolupajeva 11.09.2004 18 Project study : Improving the early diagnostics in Latvia (2000-2001) Patients: blood- born infections STD ARS-like syndrome drug addicts pregnant women Total number : 1524 individuals Number of confirmed HIV infection : 119 Number of early HIV infection (when primary antibodies tests or Western Blot were negative or indeterminate): 19 Proportion of early infection in all confirmed HIV infection : 16.1%

19. Dr.Tatjana Kolupajeva 11.09.2004 19 Actualities of early HIV infection AIEDRP (Acute HIV Infection and Early Disease Research Program): study of mechanisms and course of HIV disease, outcomes of interventions with HAART therapy Proposed staging system: Pre-seroconversion, Early post-seroconversion, Fully HIV positive Methods and finding used for the staging of the early HIV infection: 2nd and 3rd generation ELISA: registration of last negative result ( if possible) ELISA HIV Ag (p24) positive result Western Blot: negative or indeterminate result p31 band (endonuclease) � discussed as possible part of earlier criteria for positive WB result HIV-RNA-PCR quantitative � viral load detection LS EIA (less sensitive enzyme immunoassay)

20. Dr.Tatjana Kolupajeva 11.09.2004 20 Tests for HIV Infection Statusin Infants <18 months More than 95% of infected infants can be diagnosed by 3-6 months HIV-P24 Antigen Assay:� Standard Assay: Primary HIV Ag detection by ELISA ELISA Ag neitralisation: Confirmation of positive results HIV nucleic acid detection: Highly sensitive and specific test for early detection of HIV infection in infants. HIV-DNA-PCR: detects the integrated DNA form of the virus HIV-RNA-PCR: qualitative or quantitative (in plasma) HIV-IgA Antibody Test: Very sensitive assay in infants after 6 months. 94% detected at 6 months, 100% detected at 9 months. (IgA antibodies do not cross the placenta) Detection of serorevertion by anti-HIV � ELISA: repeatedly negative by 18-months of age is considered as serorevertion

21. Dr.Tatjana Kolupajeva 11.09.2004 21 HIV Desease Management Testing Lymphocyte phenotyping- �measure of damage� CD4/CD8 absolute counts, ratios and percentages Flow cytometry � most common method Plasma HIV-RNA quantitative test � Viral Load � �measure of pathogen�: Target amplification: PCR, NASBA (nucleic acid sequence based amplification) Signal amplification: bDNA (branched DNA)

22. Dr.Tatjana Kolupajeva 11.09.2004 22 HIV Desease Management Testing HIV drug resistance testing � predicts pathogen response to drugs Importance of resistance testing: high proportion of patients on HAART (till 50%) has one or more mutations Genotyping: detection of point mutation associated with definite resistance Phenotyping: measuring by growing the virus in cell culture in the presence of different drugs

23. Dr.Tatjana Kolupajeva 11.09.2004 23 HIV Drug Resistance: SPREAD-NAS What is SPREAD? Strategy to Control SPREAD of HIV Drug Resistance SPREAD - official European Commission supported programme 18 European countries including NAS (Newly Associated States)

24. Dr.Tatjana Kolupajeva 11.09.2004 24 HIV Drug Resistance: SPREAD-NAS Focus of the programme Prevalence of HIV-drug resistance among recently identified HIV-infected individuals: Collection of viruses from 4000 newly diagnosed individuals (2002 � 2005 ) Characterisation on mutational and phenotypic level Final issues of the SPREAD-project Establishment of an European Surveillance Program for collection of comparable and reliable data on the incidence of transmission of drug-resistant HIV: Development and implementation of a European strategy to control the further spread of drug resistant HIV Just some remarks about the testing of infants born to the infected mothers. You know that traditionally the tests like Ag detection by ELISA, NA detection by PCR or other molecular biological techniques are used. Detection of seroreversion is a significant marker of the infection status of the child. Very useful may be HIV IgA ELISA test because IgA Ab don�t cross the placenta. Commercial tests are recently under development. Just some remarks about the testing of infants born to the infected mothers. You know that traditionally the tests like Ag detection by ELISA, NA detection by PCR or other molecular biological techniques are used. Detection of seroreversion is a significant marker of the infection status of the child. Very useful may be HIV IgA ELISA test because IgA Ab don�t cross the placenta. Commercial tests are recently under development.

25. Dr.Tatjana Kolupajeva 11.09.2004 25 Conclusion Basic achievements during last years: laboratory diagnostics - the shortening of the � window� period by introduction of 4th generation ELISA for simultaneous detection of HIV 1/2 Ab and HIV Ag HIV Desease Management Testing � developing of HIV Drug resistance testing methods Main tasks: introduction of HIV Drug Resistance testing methods in the clinical laboratory practice improving of laboratory diagnostics and monitoring of AIDS opportunistic infections Just some remarks about the testing of infants born to the infected mothers. You know that traditionally the tests like Ag detection by ELISA, NA detection by PCR or other molecular biological techniques are used. Detection of seroreversion is a significant marker of the infection status of the child. Very useful may be HIV IgA ELISA test because IgA Ab don�t cross the placenta. Commercial tests are recently under development. Just some remarks about the testing of infants born to the infected mothers. You know that traditionally the tests like Ag detection by ELISA, NA detection by PCR or other molecular biological techniques are used. Detection of seroreversion is a significant marker of the infection status of the child. Very useful may be HIV IgA ELISA test because IgA Ab don�t cross the placenta. Commercial tests are recently under development.