Testing Related to HIV                in Adults & Children                and ART MonitoringTesting Related to HIV
Session Objectives   At the end of the session, the participant should be   able to:   • understand the general principles...
Objectives of Testing   • Diagnosis       – Adults       – Children   • Test blood/tissue/organ for transplantation safety...
General Principles of Testing   • Voluntary and part of overall comprehensive     prevention and treatment programme   • U...
Uses of Laboratory Testing of HIV/AIDS   • Establishing diagnosis       – Adults       – Children            • <18 months ...
Types of HIV Diagnostic Tests     • HIV antibody test: Serology     • Viral antigen test: p24     • Viral isolation and cu...
Typical Course of Untreated HIV Infection    800     Acute HIV                                                      10^6  ...
Typical’ HIV-1 infection: Lab Markers                     symptoms                                      symptoms          ...
HIV Structure                   p66 & 31                              Image Courtesy GHTM , Tambaram, HIV Fellowship Progr...
Tests for Diagnosing HIV   • Antibody Tests   • Screening Tests       – Rapid tests       – Enzyme linked immunosorbent as...
Antibody Tests: HIV EIA/ELISA               (Microwell Format) Advantages:                       Disadvantages: • Used as ...
ELISA Test: Microwell Format After several                                                An automated reader incubation a...
ELISA Screening Tests         Generation               Antigen / Antibody   First Generation      Viral Lysates as Ag (Not...
Concept of Window Period   • Window Period (WP) is present between time of     infection and the point at which evidence o...
HIV Rapid Tests  • Rapid tests to detect antibodies to HIV-1 and HIV-2    are in-vitro qualitative tests  • Can be perform...
Overview of workflow at ICTC                     Step                      Personnel          Time           Pre test coun...
HIV Rapid Tests                                             Positive Result          Immunological Principles             ...
Rapid Test: Line Test               (Immunochromatography)                         Lateral Flow Devices                   ...
Rapid Test: Dot Test                  (Immunoconcentration)   HIV antibody links to bound HIV peptide antigens   forming t...
False Positive Test   Reasons for False Positive Test        Antibody based tests:            • Lab error            • Cr...
False Negative Test   Reasons for False Negative Test        Antibody based test:            • Window period            •...
HIV Testing Strategies   • Strategy I:       – All samples tested with one ELISA/Rapid   • Strategy II:       – All sample...
HIV Testing Strategies         Testing Objective of                              Place of                                 ...
HIV Testing Strategy III                                         A1                                                       ...
Tests of Choice for Infants &            Children <18 Months of Age    Name the tests used for the diagnosis of HIV in inf...
Tests of Choice for Infants &            Children <18 Months of Age    Name the tests used for the diagnosis of HIV in inf...
Diagnosis of HIV Exposed                  Infants & Children   • Positive HIV antibody (Ab) alone indicates there     has ...
Diagnosis of HIV Exposed                  Infants & Children                         Schedule of visits at ICTC    6 weeks...
Diagnosis of HIV Exposed Infants &                    Children    National Guidelines: General Principles•    Follow two d...
Diagnosis of HIV Exposed Infants &                    Children    National Guidelines: General Principles   • Sample for D...
Children <18 months     with unknown HIV status of Mother   • In Children (<18months) with signs and symptoms     of HIV, ...
Tests for Monitoring   • Tests are required to monitor            - Disease progression            - Staging of disease   ...
Standard Technologies                   for Monitoring ART   • CD4 by Flow Cytometry advised by NACO       – Variations ac...
FACS Machines for CD4 Estimation        Available Through NACOFACS Count                 FACS Calibur       Partec   Image...
When to Perform CD4 Test   • All HIV patients accessing Hospitals - immediately     after their HIV status known   • Pre-A...
CD4 counts and CD4 %   • Used to assess immunological status of the     HIV-infected child   • CD4 counts are higher in in...
Key Points   • Encourage voluntary testing for HIV with pre-test     and post-test counselling   • HIV testing should foll...
Upcoming SlideShare
Loading in …5
×

HIV tests in adults and children

931 views
843 views

Published on

Published in: Health & Medicine
0 Comments
2 Likes
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
931
On SlideShare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
34
Comments
0
Likes
2
Embeds 0
No embeds

No notes for slide
  • Step 1- Presentation of Session Objectives (Slides 1-2) - 3 minutes Trainer Notes : This session should take approximately 60 minutes to implement. Step 1: Presentation of Session Objectives (Slides 1–2) - 3 minutes. Step 2: Principles of Testing (Slide 3-6) - 7 minutes. Step 3: Types of HIV Diagnostic Tests (7-21) - 20minutes. Step 4: HIV Testing Strategies (Slide 22-24) - 10 minutes. Step 5: HIV Testing in Children (Slides 25-31) – 20 minutes. Step 6: Tests for Monitoring (Slides 32-36) – 8 minutes. Step 7: Summary (Slide 37) - 2 minutes.
  • Trainer Notes:   Present the session learning objectives. This presentation will cover details relevant to adolescent and adult HIV. There will be interactive case discussions in between the slide presentation. The time allotment for the session is 90 minutes, so discussions should be relevant to the themes in this session.
  • Step 2: Principles of Testing (Slides 3-6) - 7 minutes Trainer Notes:   The above slide illustrates the objectives of testing. HIV testing forms the basis of epidemiological mapping, population studies and other trends in the community. These are mostly done as sentinel surveillance. Diagnosis includes those with symptoms, those with history of high risk behaviour, as well as testing in antenatal clinics to monitor and prevent mother to child transmission.
  • Trainer Notes: Highlight the importance of ‘voluntary’ testing as opposed to ‘mandatory’ testing. Participants need to understand that use of good quality kits as well as reliable laboratory methods form the basis of a successful programme. Reader Notes: The concept of voluntary testing for HIV is very important. Mandatory testing performed in certain private sector institutions should be condemned. These services include pre-employment checks and health check profiles, without pre-test counselling or consent of the individual. Quality Assurance (QA) and Quality Control (QC) procedures are very important in lab testing protocols to avoid erroneous test results. NACO and SACS have procedures built-in to ensure procurement and use of kits of acceptable quality. However the importance of the external and internal quality assurance must be stressed for ideal standardisation of the techniques and accuracy of the results. Appropriate to the field take into account the situation where testing is offered and the type of testing offered. For example, for a woman in advanced labour, it would be inappropriate to test with a procedure that may take 3 hours to generate the result. Instead, a rapid test which offers a result in 15 minutes or less would be more appropriate. Cost effectiveness must be an important consideration in resource limited or constrained areas. In the governmental sector, resources are not generated from the patient population. It is important to maintain a balance through use of the most appropriate tests without compromising quality
  • Trainer Notes: Emphasise to participants the basis of various diagnostic approaches in adult and paediatric HIV. Reader Notes: Laboratory testing of HIV/AIDS is an organised process that involves first establishing the HIV status of an individual. Tests for diagnosing HIV differ between adolescents and adults as compared to infants &lt;18 months of age. Infant testing will be discussed briefly in subsequent slides. Tests for monitoring HIV involve studying the progress of the disease in 2 groups: Progress of disease in ART naïve patients. Response to therapy in patients on ART.
  • Step 3: Types of HIV Diagnostic Tests (Slides 7-21) - 20 minutes Trainer Notes: Explain these three HIV diagnostic tests. Reader Notes: HIV antibody tests are the most commonly used test for the diagnosis of HIV infection. These tests are economical, rapid, and can be performed easily in most laboratories. HIV antibody assays are now commercially available in various formats. With improvement in the quality of kits, antibody tests have become the back-bone of HIV testing in diagnosis. Viral antigen tests are more specific for monitoring disease/response to ARV therapy and in infants &lt;18 months. Being very expensive, requiring expertise and expensive infrastructure, they are not easily available. Viral culture is done in a reference lab due to the cumbersome nature of the tests.
  • Trainer Notes: Explain the details of how virological load of HIV is inversely proportional to the immunity of the host (CD4 count) Discuss the occurrence of opportunistic infections is related to increased viral load and reduced CD4 count Virological set point, after the Acute retroviral syndrome, decides the progression of HIV disease; higher the set point earlier the progression to AIDS and vice versa Emphasise the development of detectable antibodies to HIV only a few weeks after the entry of HIV (window period) Reader Notes: Pink color depicts the viral load. It reaches the maximum level during acute retroviral syndrome, then it settles down to a “viral set point” and this set point is maintained over a period of years. After 5-8 years the viral load starts increasing again and the patient progresses to the stage of AIDS. The blue line depicts the CD4 count. The CD4 count is depleted initially and then reaches a set point which is maintained over a period of 5-8 years. When the patient reaches the stage of AIDS, CD4 count starts declining very fast.
  • Trainer Notes: Discuss the window period once again. Emphasise the window period is there for HIV (RNA &amp; DNA) as well as for the antibodies RNA can be detected in lab tests ahead of DNA &amp; antibodies to HIV DNA can be detected in lab tests ahead of antibodies to HIV Tests related to detection of p24 antigen will be useful only during initial weeks of HIV infection
  • Trainer Notes: Three types of antigens present on the HIV virus: Core antigens – GAG (P17), (P24), (P55). Polymerase antigens POL (P31), (P51), and (P66). Envelope antigens ENV - GP (41), (120), (160). Denatured antigens are impregnated into the test kits that are used for antibody-based testing.
  • Trainer Notes: Emphasise that it is possible that an antibody based test will not detect the HIV infection during the “ window period ”. During this period the infection can be detected using antigen detection methods like NAT (Nucleic Acid Tests), P24 Ag and virus culture (these two are not commonly done). Reader Notes: The Microwell ELISA test can take up to 3 hours to complete, while rapid test readings should be available in less than 30 minutes from start of test (per NACO requirement). ELISA / Rapid - both have the possibility of false positives (as in any serological test). The result of HIV has a serious impact , hence all of the tests that result in a positive result, must be re-checked or confirmed by use of a 2 nd/ 3rd set of tests. Currently the confirmation can be done by 2 nd /3 rd E/R (ELISA/Rapid) tests which narrows down the chance of a false positive. In the past, the western blot was used as the confirmation test, but due to the high cost per test, this is no longer the main stay of confirmation. It is now available only for isolated cases in reference labs. All such re-tests using 2 nd /3 rd tests should use the same serum/plasma sample as the 1 st test. Explain that the tests listed on this slide are more appropriate in adolescents and adults. Window period denotes the time lag between actual infection with HIV and the appearance of detectable antibody in serum/plasma using the currently available test kits. Interpretation of NEGATIVE TEST (For Ab) Not infected Infected- but Antibody not measurable (True –ve) (False –ve)
  • Trainer Notes: This slide illustrates the advantages and disadvantages of the EIA/ELISA Test. Reader Notes: ELISA Test is the screening test used when &gt;30 samples are tested in a batch. Cost per test is much less than rapid tests but requires expensive infrastructure as well as trained technicians. The QA/QC is more stringent. These tests also require a 2 nd /3 rd test for confirmation. The test reading is based on colour development in the positive samples. It is objective as it uses a reader for measuring the colour development.
  • Trainer Notes:   List equipment used for ELISA testing. Briefly explain the function of the equipment shown in this slide. Inform participants that they will observe the equipment and their function during the visit to the laboratory. Reader Notes:   The ELISA Washer – is a ‘multi-channel’ automated washer, to assist in washing out the contents of the microwells between the various steps. The end result is a colour development which is quantified using an ELISA reader. The ELISA reader measures the colour developed in the wells as the ‘absorbance ‘ or optical density (OD). This measurable result is then compared with the cut-off value set for that test run. The wells showing a reading above the cut-off value (COV) are reported as reactive (positive) and those below the COV are reported as non-reactive (negative). The readings by a Microwell ELISA test are thus objective &amp; not subjective. The whole process of testing takes 2-3 hours. The Microwell ELISA as seen here requires trained technicians, good standardisation, QA/QC, and supervision
  • Trainer Notes: Review the 4 generations of ELISA screening tests. Reader Notes: The first generation tests used viral lysates as antigens and false positives were a major concern. The second generation tests use recombinant HIV proteins and/or synthetic peptides as antigens. The third generation tests use double antigen binding and enable IgM detection also. The fourth generation tests are based on simultaneous detection of HIV antibodies, P24 antigen and immune complexes and have very high sensitivity and specificity. Their use narrows the window period to as close as the NAT, but the cost prevent its availability in the national programme.
  • Trainer Notes: Refer participants to the graphical representation of the narrowing window period and ensure understanding of how the window period affects the test results. Reader Notes: A graphic representation of the narrowing ‘Window Period’.
  • Trainer Notes: Explain that Rapid tests form an important limb of HIV testing in the programme and kit quality has improved considerably over time. It is important to ensure that the sensitivity of kits is 99.7% and specificity more than 98% as per NACO specifications. The advantages of rapid tests: Ease of testing No expertise required Can be used in field testing No accessories or equipment required One to multiple people can be tested Reader Notes: Advantages of Rapid HIV Antibody Tests: More flexible (1- multiple tests at a time) Requires minimal equipment and reagents Does not require highly skilled staff On-site clinic or field testing possible Very easy to interpret test results (naked eye) Samples can obtained less invasively (finger-prick) - safer for lab technician Same-day screening and confirmation of results
  • Trainer Notes: Explain that work flow of counselling at ICTC and testing procedures at the lab and the post test counselling and handing over the report to the concerned person Also give the details of the time taken for the procedure Emphasise that the test result will be made available on the same day
  • Trainer Notes: Explain the different principles and methods of Rapid testing. Explain how to make an interpretation of Rapid tests based on their principles and methods.
  • Trainer Notes: Explain the procure of the rapid line test This procedure involves immunochromatography
  • Trainer Notes: Explain the procure of the rapid Dot test; Refer to Tridot test This procedure involves immunoconcentration)
  • Trainer Notes: Ask the participants to brainstorm reasons for the FALSE POSITIVE REPORT– Ab based test and review the answers. Time permitting, record participants’ answers on flipchart paper labeled with the same title as the slide ‘Reasons for False Positive Report-Ab based Test’. Highlight the importance of correct lab techniques and QA/QC. Reader Notes: There are many causes of false positives. This is considerably reduced if quality kits are available. 2 nd /3 rd tests for confirmation of the 1 st positive test are performed to overcome the possibility of a false positive. Other clinical causes of false positive are similar to those in other serological tests and are rare in practice.
  • Trainer Notes: Ask the participants to brainstorm reasons for the FALSE NEGATIVE REPORT-Ab based test; review the answers. Time permitting, record participants’ answers on the corresponding flipchart paper labeled with the same title as the slide ‘Reasons for False Negative Report – Ab based test’. Emphasise the importance of the window period, especially in patients engaging in high risk behaviour. The window period has narrowed with quality improvement of tests. Just as ‘sero-conversion’ takes place in the early part of the disease, ‘sero-reversion’ takes place at the end of the spectrum of disease progression, as a result of immense immuno-suppression. This is not seen very often. Laboratory error and poor quality kits are issues that can be addressed by programme managers. Inform participants that further clarifications on the lab tests, equipment and other allied issues will discussed during the visit to the laboratory.
  • Step 4: HIV Testing Strategy (Slides 22-24) - 10 minutes Trainer Notes: The three strategies of testing for HIV are based on different situations. In strategy I, only a single test is performed. In strategy II all samples tested positive by the 1 st test are tested on another kit. In strategy III, all samples found positive by sequential testing on two different kits are re-tested on a third kit. The 3 different kits used must use different antigens and/or different principles of testing. The choice of kits are such that the 1 st test must use kits with the highest sensitivity, the 2 nd and 3 rd test kits must use kits with highest specificity. The 1 st kit thus detects maximum infected individuals, while the 2 nd /3 rd kits weed out the false positives and detect the truly infected persons. Refer the participants’ to the Handout 1: HIV Testing Strategies as per NACO Guidelines after explaining these three testing strategies Reader Notes: The three strategies of testing for HIV are based on different situations. In strategy I, only a single test is performed. In strategy II all samples tested positive by the 1 st test are tested on another kit. In strategy III, all samples found positive by sequential testing on two different kits are re-tested on a third kit. The 3 different kits used must use different antigens and/or different principles of testing. The choice of kits are such that the 1 st test must use kits with the highest sensitivity, the 2 nd and 3 rd test kits must use kits with highest specificity. The 1 st kit thus detects maximum infected individuals, while the 2 nd /3 rd kits weed out the false positives and detect the truly infected persons.
  • Trainer Notes: Emphasise that the testing strategy should be appropriate to the objective of testing and the population being tested. The prevalence of HIV in the population is an important denominator. This table sums up the choice of testing strategies. Highlight the importance of ‘confidentiality’ to improve effectiveness of ICTCs. Stress voluntary and confidential HIV testing methods, with appropriate pre- and post-test counselling. Reader Notes: Three methods to label blood samples to ensure confidentiality: Unlinked anonymous - all identifiers are removed from blood and it is HIV antibody tested- as in sentinel surveillance. Linked testing –the blood sample sent has an identifier on it, such as a name, PID or a centre number, which links the sample to the individual client. Ideally samples sent for HIV testing should not be identified with a name, but with some other identifier. Linked-anonymous testing - no names or other identifiers from the client are recorded. The client receives a unique number (in no way linked to any medical records) that matches the number placed on the blood sample sent to the laboratory. “ Unlinked anonymous” forms the basis of the population based surveillance method. This is also called sentinel surveillance. Mandatory testing in the general population should be condemned. The only situations where mandatory tests are appropriate are in blood banks and organ/tissue transplantation services.
  • Trainer Notes: Explain the HIV testing strategy III with the help of this flow chart. Use the pointer and follow the arrow marks and explain them how to interpret the results in HIV testing strategy III Explain them about Positive, Negative and Indeterminate.
  • Step 5: HIV Testing in Children (Slides 25-31) – 20 minutes. Trainer Notes: Explain The trainer may request the participants to recollect the tests used for the diagnosis of HIV They may be requested to answer the following question; Question 1: “Name the tests that are used for the diagnosis of HIV in children? The trainer can write down the answers on the flip chart” Answer: Antibody based tests, antigen based tests like HIV DNA PCR, P24 antigen test and viral culture.
  • Trainer Notes: Ask the participants: recommending or not of HIV DNA PCR and reason for it. Explain the merits and demerits of HIV DNA PCR. It is very sensitive - 98% at 4 weeks of age.
  • Trainer Notes: Discuss the transfer of HIV antibodies to a child (passive transfer) from an infected mother. Maternal antibodies take 18-20 months to disappear if the child is uninfected. Hence a positive antibody test may be misleading in diagnosing HIV in children &lt;18 months. Reader Notes: Positive HIV antibody (Ab) alone indicates there has been exposure to HIV and need to perform HIV DNA PCR for confirmation. It requires to be followed up with antibody tests after 18 months of age even the child is positive for HIV DNA PCR One positive virological test should be confirmed by a repeat positive test on a separate specimen
  • Trainer Notes:   Trainer has to summarise the entire testing strategies using the flow diagram in the slide. Discuss the following questions about testing at 18 Months and Discordant results in testing. All three rapid tests are to be used at 18 months irrespective of the first test result, i.e. even if the first screening rapid test is negative, then proceed for the test 2 and 3. Ask the following question to the participants.   Question 1: What do you do when you observe a discordant result during testing? Answer: In case discordance is observed between the test results obtained from the two specimens (DBS and whole blood) by the testing laboratory, a fresh whole blood specimen will be requested by the testing lab from the respective ART centre (test requisition cum result form) TRRF. The baby may be recalled for collection of fresh whole blood specimen for retesting on an urgent basis. The repeat specimen must be accompanied by a fresh TRRF that must specify that the specimen is a repeat specimen in the space provided and the reason why the repeat specimen was needed.
  • Trainer Notes: Discuss the National Guidelines on Early Infant diagnosis in this and in the next slide Inform the trainees that there are two diagnostic algorithms for the infants and children &lt;18months of age; one for those aged 6 weeks and 6 months and the second one for the children aged 6-18 months born to HIV positive mother Describe the basic principles of the sample collection and testing procedures involved in the diagnostic algorithms
  • Trainer Notes: Continue to describe the basic principles of the testing procedures involved in the diagnostic algorithms
  • Trainer Notes: Discuss the procedure one has to resort when encountered with a child, whose mother’s HIV status remained “unknown”, using the contents of the slide.
  • Step 6: Tests for Monitoring (Slides 32-36) – 8 minutes. Trainer Notes: Mention the objectives of HIV disease monitoring. Explain the direct marker –Viral Load Assay and Indirect marker – CD4 assay. Explain the definition of Viral load – Number of RNA copies per milliliter of blood or plasma Reader Notes: Tests are required to monitor to know about the disease progression, staging of disease and response to ART Tests available to monitor HIV are CD4 T-cell Assay and Viral load assay
  • Trainer Notes: Explain the two concepts used to obtain CD4 cell count. Reader Notes: Immunofluorescence assays by Flow Cytometry is the gold standard for CD4 cell measurements. CD4 – Methods of enumeration: The following two concepts are used to obtain the absolute CD4 cell count: 1. Dual platform approach: Uses two instruments to generate absolute CD4 cell counts: a FCM for generating a % of CD4 cells among lymphocytes and a haematology analyser to enumerate the absolute lymphocyte count. An absolute CD4 count is then derived by multiplying the %CD4 by the absolute lymphocyte count. Instruments include BD FACS Calibur/ FACS Scan/ FACSort or Beckman-Coulter Epics XL. Some of these combine the concept of a dual platform into one machine –e.g. a FACS Calibur. The results directly obtained include an absolute CD4 count and CD4%. The process involves ‘gating’ of CD45 bright cells (Lymphocytes). 2. Single platform approach: Enables absolute CD4 cell counts to be derived directly without the need for a haematological analyser, e.g., the use of volumetric counting (Partec CyFlow), microfluorometry (Guava) and most commonly, the addition of a known density of reference fluorescent beads to the sample (FACS Count).
  • Trainer Notes: NACO has currently installed FACS Count, FACS Calibur and Partec machines in various ART Centres. For Paediatric HIV- CD4% are more important than the absolute CD4 counts as counts are inherently high in children especially those under 5 years of age. FACS Calibur as compared to FACS Count and Partec requires more training of technicians and greater commitment to equipment care &amp; maintenance. The differences between the FACS Calibur and FACS Count and Partec Machine need to be explained to the participants. Partec cyflow A simple and portable equipment for flow cytometric assay. Single Platform technology - Absolute CD4 count, CD4:CD8 ratio only It can be used as a mobile system that can run on car batteries It works on simple no-lyse and no-wash protocol However, its robustness and reproducibility is under evaluation especially on stabilised cells. (Source : Laboratory guidelines for enumerating CD4 T lymphocytes in the context of HIV/AIDS -http://www.searo.who.int/LinkFiles/Publications_BLT-SEA-HLM-392.pdf on 30.6.2010) Reader Notes: Keep in mind the following when requesting CD4 assays: Repeat in same lab and by same method. Blood should be processed within 48 hours of collection for FACS Count. Freezing blood - false low counts. Ensure no clots (micro-clots) in sample. False values- inter-current illness, acute infections, steroids, major surgery. Normal variation up to 30%. Children &lt;6 years have high CD4 counts. Diurnal variation. FACS Count gives results of absolute CD4, CD8, CD3 as well as CD4/CD8 ratio. It does not give CD4 percentage. One can do maximum of 30 tests per working day. It is easier to use and requires less training than the FACS Calibur. FACS Calibur is a much more expensive equipment, has many more functions other than CD4 assay. This machine gives absolute and percentage of CD4 hence useful in Peadiatric HIV monitoring. Large number of CD4 estimations per day can be done (More than 100). However it requires much more training and equipment maintenance than the FACS Count. Source: Guidelines for HIV Diagnosis and Monitoring of ART- Dec, 2005, WHO .
  • Trainer Notes: All ART sites have access to CD4 testing. This may be done either within the campus, or the samples may need to be transported to a nearby location for testing. The choice for patients for CD4 testing is crucial to ensure that the kits are properly utilised. Reader Notes: All ART sites have access to CD4 testing. This may be done either within the campus, or the samples may need to be transported to a nearby location for testing. The choice for patients for CD4 testing is crucial to ensure that the kits are properly utilised.
  • Trainer Notes: The trainer may inform the participants about the CD4 counting methods. FACS Calibur/ FACS count/ Partec cyflow / Guava/EPICS XL(automated machines) Calculating percentage of CD4 cells using the formula when only the CD4 absolute count is available. Reader Notes: CD4 is the best measurement for assessing immune deficiency. CD4 should be used in conjunction with clinical assessment; however, CD4 allows an earlier detection of worsening of HIV disease as CD4 decline usually occurs prior to clinical progression. CD4 monitoring can aid in the decision to initiate or switch ART. Younger children normally have higher CD4 than older children and adults. %CD4 cells vary less in children &lt;6 years old and are the preferred measurement. Reference: Table-9, Page 16; Reference: Guidelines for HIV care and treatment in Infants and Children, November 2006,developed by Indian Academy of Paediatrics and National AIDS Control Organisation with support from Clinton Foundation, UNICEF and WHO)
  • Step 7: Summary (Slide 37) - 2 minutes. Trainer Notes: Review key points and clarify any remaining questions. Reader Notes: Further reading: NACO Guidelines on HIV testing. WHO Antiretroviral Therapy of HIV infection in Adults and Adolescents in resource-limited settings: Towards universal access – 2006. WHO Antiretroviral Therapy of HIV infection in infants and children in resource-limited settings: Towards universal access – 2006.
  • HIV tests in adults and children

    1. 1. Testing Related to HIV in Adults & Children and ART MonitoringTesting Related to HIV
    2. 2. Session Objectives At the end of the session, the participant should be able to: • understand the general principles of HIV testing • classify the testing procedures • elaborate on the policy of three strategies of testing and its applications • learn the tests for diagnosis of HIV-infection in children • discuss the tests for monitoring disease progression in adults and childrenTesting Related to HIV 2
    3. 3. Objectives of Testing • Diagnosis – Adults – Children • Test blood/tissue/organ for transplantation safety • Monitor trend of HIV infection and surveillance • ResearchTesting Related to HIV 3
    4. 4. General Principles of Testing • Voluntary and part of overall comprehensive prevention and treatment programme • Use of quality testing kits, standardised techniques, and QA/QC procedures • Test kit and procedure must be appropriate to the field situation • Cost-effectiveTesting Related to HIV 4
    5. 5. Uses of Laboratory Testing of HIV/AIDS • Establishing diagnosis – Adults – Children • <18 months • >18 months • Monitoring progression of HIV disease and response to ART – CD4 T-cell estimation (Indirect Marker) – Viral load estimation (Direct Marker)Testing Related to HIV 5
    6. 6. Types of HIV Diagnostic Tests • HIV antibody test: Serology • Viral antigen test: p24 • Viral isolation and culture • Viral DNA PCRTesting Related to HIV 6
    7. 7. Typical Course of Untreated HIV Infection 800 Acute HIV 10^6 HIV antibodiesCD4 Asymptomaticcount HIVcells/µl RNA Minor HIV-related Copies /ml symptoms Virologic set-point 200 Varies from patient to Opportunistic patient infections 10^2 Death 1 3 Wk about 6mths // 5yrs 10 yrs Time DurationTesting Related to HIV 7
    8. 8. Typical’ HIV-1 infection: Lab Markers symptoms symptoms HIV proviral DNA HIV antibodies ‘window’ period HIV viral load HIV-1 p24 antigen 0 1 2 3 4 5 6 / 2 4 6 8 101° infection weeks years Time following infectionTesting Related to HIV 8
    9. 9. HIV Structure p66 & 31 Image Courtesy GHTM , Tambaram, HIV Fellowship ProgrammeTesting Related to HIV 9
    10. 10. Tests for Diagnosing HIV • Antibody Tests • Screening Tests – Rapid tests – Enzyme linked immunosorbent assays (ELISA) • Confirmatory/Supplemental Tests – 2nd/3 rd R/E to confirm 1st R/E – Western blot assay • Same blood sample is utilised for performing all the tests for identifying HIV antibodiesTesting Related to HIV 10
    11. 11. Antibody Tests: HIV EIA/ELISA (Microwell Format) Advantages: Disadvantages: • Used as a screening test – Technical expertise since 1985 – Special equipment • Easy for mass screening – More stringent • Easy to automate standardisation • Accurate • Less costly than other testsTesting Related to HIV 11
    12. 12. ELISA Test: Microwell Format After several An automated reader incubation and wash gives a measurement of steps, a colour optical density reaction occurs if (presence of colour) HIV antibody is for each well present Images Courtesy: GHTM, Tambaram, ChennaiTesting Related to HIV 12
    13. 13. ELISA Screening Tests Generation Antigen / Antibody First Generation Viral Lysates as Ag (Not in use anywhere) Second Generation Synthetic peptides/Recombinant Ag Synthetic peptides/Recombinant Ag, Third Generation Double Ag binding, Enables IgM, IgA Ab detection Fourth Generation Detects Ag and Ab to HIVTesting Related to HIV 13
    14. 14. Concept of Window Period • Window Period (WP) is present between time of infection and the point at which evidence of infection is detected. Relative shortening of Window Period with different ELISA Kits 4 GENERATIONS OF HIV Ab TESTS Image Courtesy Abbott PublicationTesting Related to HIV 14
    15. 15. HIV Rapid Tests • Rapid tests to detect antibodies to HIV-1 and HIV-2 are in-vitro qualitative tests • Can be performed on whole blood, plasma, serum and saliva. In India whole blood and serum are used • Most rapid test kits come with all materials required to perform the testTesting Related to HIV 15
    16. 16. Overview of workflow at ICTC Step Personnel Time Pre test counselling and 1. Counsellor 15-20 min informed consent Blood (2-5ml) collection in 2. Lab Technician 5-10 min sterile vial 3. Serum separation Lab Technician 30-60 min 4. HIV testing on serum sample Lab Technician 30-60 min 5. Report preparation Lab technician 30-60 min Report dispensing with Post 6. Counsellor 15-20 min test counselling All times excluding waiting times; Waiting time depends on client load; Reports will be available on the same dayTesting Related to HIV 16
    17. 17. HIV Rapid Tests Positive Result Immunological Principles (Visual) Particle agglutination Clumping Immunofiltration/dot Dot Immunochromatography Line ELISA based rapid tests Dot Most rapid tests detect Antibodies to both HIV-1 & HIV-2Testing Related to HIV 17
    18. 18. Rapid Test: Line Test (Immunochromatography) Lateral Flow Devices Non- Reactive Control HIV Antigen Reactive Specimen Flow Sample pad Sample Pad Test line Control lineTesting Related to HIV 18
    19. 19. Rapid Test: Dot Test (Immunoconcentration) HIV antibody links to bound HIV peptide antigens forming the colour spot Internal Control HIV-1 peptide HIV-2 peptideTesting Related to HIV 19
    20. 20. False Positive Test Reasons for False Positive Test  Antibody based tests: • Lab error • Cross reacting antibodies (Autoimmunity) • IV drug abuse • Multiple pregnancies • Recent immunisation • Chronic alcoholics • Cirrhosis of LiverTesting Related to HIV 20
    21. 21. False Negative Test Reasons for False Negative Test  Antibody based test: • Window period • Lab/Clerical error • Other immunodeficiency statesTesting Related to HIV 21
    22. 22. HIV Testing Strategies • Strategy I: – All samples tested with one ELISA/Rapid • Strategy II: – All samples tested with one ELISA/Rapid (E/R) – Reactive samples tested again on different system (different antigen or principle) • Strategy III: – All samples tested with one ELISA/Rapid (E/R) – Reactive samples from the first test tested with different antigen or preparation – Reactive samples from the second test again tested with third system of different antigen or principleTesting Related to HIV 22
    23. 23. HIV Testing Strategies Testing Objective of Place of Type of Testing Strategy Testing Testing Transfusion/ I Donation Mandatory Blood Bank safety Unlinked Designated II Surveillance Anonymous laboratories Voluntary Diagnosis of  Counselling III patients at  Informed ICTC ICTC Consent  ConfidentialTesting Related to HIV 23
    24. 24. HIV Testing Strategy III A1 Report: A2 A1+ A1- Non-Reactive A2+ A2- Report: A3- Non-Reactive A3 A3 A1+, A2-,A3+ A3+ (indeterminate) A3+ A3- A1+, A2+, A3- (indeterminate) A1+, A2+, A3+ Report: ReactiveTesting Related to HIV 24
    25. 25. Tests of Choice for Infants & Children <18 Months of Age Name the tests used for the diagnosis of HIV in infants and children aged <18 Months and give their relevance Test Recommendation ReasonHIV antibodyHIV p 24 AntigenHIV viral cultureHIV DNA PCRTesting Related to HIV 25
    26. 26. Tests of Choice for Infants & Children <18 Months of Age Name the tests used for the diagnosis of HIV in infants and children aged <18 Months and give their relevance Test Recommendation Reason False +ve due to persistentHIV antibody No maternal antibodies Lower sensitivity than PCRHIV p 24 Antigen Yes, but (27% at 6 weeks) Costly, result takes 2-4 wks,HIV viral culture Yes, but not readily available 98% sensitive from 6 weeksHIV DNA PCR Yes of ageTesting Related to HIV 26
    27. 27. Diagnosis of HIV Exposed Infants & Children • Positive HIV antibody (Ab) alone indicates there has been exposure to HIV: DOES NOT mean child is infected, can be circulating maternal antibodies • Test to diagnose HIV in this population: HIV DNA PCR (Qualitative) • Ideal: First positive virological test (HIV DNA PCR) should be confirmed by a repeat positive test on a separate specimenTesting Related to HIV 27
    28. 28. Diagnosis of HIV Exposed Infants & Children Schedule of visits at ICTC 6 weeks 14 weeks 9 months 18 months 10 weeks 6 months 12 months Birth DNA PCR HIV Antibody test followed by DNA PCR if HIV+ DNA PCR for all HIV exposed infants Final confirmatory Antibody Test for all HIV exposed infants irrespective of earlier testing results / treatment status All HIV infected and/ or symptomatic infants/children are to be referred to ART centreTesting Related to HIV 28
    29. 29. Diagnosis of HIV Exposed Infants & Children National Guidelines: General Principles• Follow two different diagnostic algorithms A. Infants <6 months old and born to HIV positive mother B. Child of age 6-18 months born to HIV positive mother• First HIV DNA PCR test sample at ICTC (Dry blood spot)• If DBS is positive, Second sample for DNA PCR test (whole blood) will be collected at linked ART Centre – A positive DNA PCR test reveals the infant/child as HIV-1 infected – A negative DNA PCR test reveals discordance between the first and second tests; needs a tie breaking third DNA PCR test from the whole blood sample to be taken at ART centreTesting Related to HIV 29
    30. 30. Diagnosis of HIV Exposed Infants & Children National Guidelines: General Principles • Sample for DNA PCR test (whole blood) taken at ART Centre for the tie-break – A positive DNA PCR test reveals the infant/child as HIV-1 infected – A negative DNA PCR test reveals the infant/child as HIV-1 uninfected • Final confirmatory Antibody Test for all HIV exposed infants irrespective of earlier testing results / treatment status at 18 monthsTesting Related to HIV 30
    31. 31. Children <18 months with unknown HIV status of Mother • In Children (<18months) with signs and symptoms of HIV, whose exposure status is unknown, perform rapid test for HIV antibodies • If negative, label child as uninfected • If positive, follow algorithm A or B, depending on age of the childTesting Related to HIV 31
    32. 32. Tests for Monitoring • Tests are required to monitor - Disease progression - Staging of disease - Response to ART • Include - CD4 T-cell Assay - Viral load assayTesting Related to HIV 32
    33. 33. Standard Technologies for Monitoring ART • CD4 by Flow Cytometry advised by NACO – Variations across methods can occur • Nucleic acid Amplification Technologies (NAT) – Cannot be widely used in countries with limited resources because: • Lack of adequate infrastructure • Requires highly skilled lab personnel • NAT testing is too expensiveTesting Related to HIV 33
    34. 34. FACS Machines for CD4 Estimation Available Through NACOFACS Count FACS Calibur Partec Images Courtesy: GHTM, Tambaram, ChennaiTesting Related to HIV 34
    35. 35. When to Perform CD4 Test • All HIV patients accessing Hospitals - immediately after their HIV status known • Pre-ART: once in 6 months till they are being initiated on ART • During ART: – Once in 6 months for monitoring – As and when their clinical conditions demandTesting Related to HIV 35
    36. 36. CD4 counts and CD4 % • Used to assess immunological status of the HIV-infected child • CD4 counts are higher in infants as compared to adults and fall to adult values by age 5 – Varies due to diurnal change, undercurrent illness, steroid treatment, splenectomy, after immunisations – Test variability: repeated measurements are more informative than single value • CD4% varies less than CD4 counts, hence considered more valuable in children <5 years of ageTesting Related to HIV 36
    37. 37. Key Points • Encourage voluntary testing for HIV with pre-test and post-test counselling • HIV testing should follow recommended strategies I/II/III depending on the situation • HIV Ab test significance varies in adults and children <18 months; DNA PCR test is advised • A CD4 test for monitoring must be done with a fresh sample and at the same lab • Paediatric HIV monitoring and staging relies on CD4%Testing Related to HIV 37

    ×