Lecture 3: Laboratory Diagnostics in ARV Therapy delivered by Dr. Madisa Mine, Botswana Ministry of Health

1. KITSO AIDS Training Program Lecture 3:Laboratory Diagnostics in ARV Therapydelivered byDr. Madisa Mine, Botswana Ministry of Health

2. Main Lab Tests in ARV Therapy • HIV diagnosis: • Adults: ELISA test • Infants under 18 months: DNA PCR test • Monitor viral suppression: Viral load • Monitor recovery of immune system: CD4 cell count • Monitor drug toxicity: chemistry and haematology • ARV Resistance Assay

3. Detection of HIV Infection • Antibody methods for diagnosing HIV - (ELISA) Enzyme Linked Immunosorbent Assay - Western Blot - Rapid tests • DNA PCR tests • p24 antigen (used to screen blood donations) • HIV culture (used for research)

4. Window Period • Period between infection and first reliable detection of HIV by lab test. • Window period varies by test and by individual. • The majority of infected individuals are positive by ELISA, antigen, and/or DNA/RNA tests by 6-8 weeks after infection.

5. Minimum Time from Infection to First Detection of HIV-1 Markers KEY: TIME PERIOD BEFORE DETECTION TIME WHEN HIV DETECTION BY TEST IS POSSIBLE ELISA p24 DNA PCR RNA (VL) 5 10 15 20 25 30 35 Infection Detection of HIV: time (in days) after infection

6. Diagnosis in Adults • In adults, diagnosis of HIV infection is best determined by the detection of antibodies (markers) to the virus in blood. • The antibodies are specific for particular virus proteins and are unique to HIV.

7. ELISA Algorithm for Diagnosing HIV 2 Parallel ELISA Tests Negative Patient uninfected or in window period of infection

8. 2 Parallel ELISA Test Positive Patient infected Discordant Parallel ELISA Tests (positive/negative) Discordant Western Blot Repeat ELISAs on same sample Positive Positive Indeterminate Patient infected Re-draw blood and repeat ELISA Negative Negative Patient uninfected or in window period of infection

9. Western Blot • Most commonly used confirmatory test. • Detects antibodies directed at specific HIV envelope and core proteins.

10. Alternative Methods of Sero-diagnosis of HIV Infection • Rapid tests • Non-invasive testing methods • Saliva or urine

11. Rapid Tests • Quick, cheap, easy to use. • Easy to store. • A laboratory is not required. • Combinations of rapid tests are highly sensitive and specific. • However, interpretation does require trained personnel.

12. Sample Required for ELISA • Five (5) milliliters of whole blood in plain or purple-top EDTA tubes. • Samples should be stored in the fridge at 4 0C and NOT frozen. • Samples should be sent to the lab within 24 hours.

13. Labeling of Lab Test Tubes (all assays) • Tubes should be labeled with: • PATIENT IDENTIFICATION NUMBER • (ID/Omang) • Date of collection

14. Filling out Lab Test Forms (all assays) • Fill in patient information: • ID / Omang • Patient initials (as a cross-check for errors) • Patient gender (M or F) • Patient date of birth (day / month / year) • Fill in sample information: • Date specimen drawn (day / month / year) • Time specimen drawn (24 hour clock) • Site information: • Initials and signature or stamp of clinician

15. Diagnosis in Infants • Diagnosis of HIV by serology in infants under 18 months is complicated by the presence of maternal antibodies passed along both inutero and via breast milk. • Hence detection of HIV nucleic acid in infant blood is done by DNA PCR.

16. DNA PCR Assay • PCR based on cellular proviral HIV DNA provides a qualitative result: Positive or Negative

17. Sample Required for DNA PCR • .5-1.0 ml of whole blood in purple-top EDTA tubes w/ anticoagulant. • Tube should be mixed well by inverting slowly 5-10 times IMMEDIATELY after collection to prevent clotting. • Sample should be stored in the fridge and NOT frozen. • The sample should be sent to the lab immediately. • Turn-around-time is 2 weeks.

18. Monitoring HIV Disease Progression

19. HIV Disease Progression • Progression can be monitored by: Clinical markers: ▪ HIV/AIDS-related conditions and mortality. Laboratory markers: • Increase in blood virus levels (viral load) • Decrease in CD4 cell count

20. CD 4 and Viral Load Progression High Viral load CD4 Low TIME Infection

21. Viral Load • Measures the number of virus particles per ml of blood by quantifying HIV RNA. • With the standard test used in Botswana, 400 – 750,000 HIV copies per ml of blood can be detected. • Measure viral load at start of therapy, after 3 months and every 3 months thereafter.

22. Sample Collection for Viral Load • Collect blood in 5-7 ml EDTA anticoagulant tube for adults, 3ml tube for infants. • Tube should be mixed well by inverting slowly 5-10 times IMMEDIATELY after collection to prevent clotting. • Store in the 4 0C fridge while awaiting transportation to lab. • Sample should be transported to lab within 4-6 hours of collection in cool box with ice pack at 4 0C.

23. Sample Collection for Viral Load • If sample cannot reach testing labs within 6 hours, collection viral load sample in PPT tube. • Local lab should centrifuge PPT tube samples to separate plasma and then ship to testing lab within 24 hours.

24. Viral Load Results • Turn-around time is 2 weeks. • Detection limit of assay = 400 copies/ml. • Undetectable result indicates viral load below 400 copies/ml. • Recent study in Botswana: • median viral load in asymptomatic patients: 36,000 copies/ml. • median viral load in AIDS patients: 296,000 copies/ml. • Potential 0.2-0.3 log inherent variability in viral load assay (60-80% due to biologic variation).

25. CD4 Cell Counts

26. CD4 Cell Counts • CD4 cell count measures the number of CD4 cells per cubic milliliter of blood. • The CD4 count is a measure of the degree of immuno-compromise and stage of HIV disease progression. • The CD4 count is an important test for deciding whether ARV therapy is required and for monitoring the recovery of the immune system under treatment.

27. CD4 Counts in Botswana • Uninfected : 750 cells/ul (IQR: 560-900) • Asymptomatic HIV-1 positive: 350 cells/ul (IQR: 268-574) • Patients with AIDS : 121 cells/ul (IQR: 50-250)

28. Sample Collection for CD4 Count • Three (3) ml of whole blood in EDTA anticoagulant (purple top tube). • Tube should be mixed well by inverting slowly 5-10 times IMMEDIATELY after collection to prevent clotting. • Sample should be transported to the lab within 24 hours. • Store and transport sample at room temperature: 20-30 0C (cool box without ice pack).

29. CD4 cell counts • Turn-around time is 72 hours. • Measure CD4 cell count: • As an eligibility screen for therapy. • At baseline and every 3 months on therapy. • Off-therapy: • If CD4 count > 350: every 6 months. • If CD4 between 201 and 349: every 3 months.

30. Interpretation of CD4 Counts • Use absolute CD4 cell count in adults. • Use CD4% in infants and children. • Ignore CD8 count and CD4:CD8 ratio. • 10-25 % variability in CD4 cell count due to: • biologic variation. • sampling/measurement error. • Variation usually not clinically relevant.

31. Monitoring for ARV Toxicity

32. Chemistry and Haematology • Before start of therapy: full blood count, liver function, kidney function, blood sugar. • At start of therapy, 1 month, 3 months, then 3-monthly: • FBC • liver and kidney function • Glucose • NVP-containing regimens: liver function tests are drawn two weeks after HAART initiation.

33. Samples required for chemistry and haematology tests

34. Other Tests • Syphilis serology (5 ml plain red-top tube). • Hepatitis B antigen (5 ml plain red-top tube). • No longer part of baseline laboratory tests under Revised 2005 Guidelines.

35. Resistance Assays • A genotypic resistance assay is performed for patients failing second-line therapy to determine which drugs to which a patient may no longer be susceptible. • The resistance assay, combined with expert opinion, will guide treatment decisions for third line therapy. • The blood sample for resistance assays should be drawn while a patient is still on the failing regimen. Or no more than four weeks after discontinuation.

36. Sample Collection for Resistance Assay • Collect blood in 5-7 ml EDTA anticoagulant tube for adults, 3ml tube for infants. • Tube should be mixed well by inverting slowly 5-10 times IMMEDIATELY after collection to prevent clotting. • Store in the 4 0C fridge while awaiting transportation to lab. • Sample should be transported to lab within 4-6 hours of collection in cool box with ice pack at 4 0C.

37. Sample Collection for Resistance Assay • If sample cannot reach testing labs within 6 hours, collection viral load sample in PPT tube. • Local lab should centrifuge PPT tube samples to separate plasma and then ship to testing lab within 24 hours.

38. Summary • HIV diagnosis: • Adults: ELISA test • Infants under 18 months: DNA PCR test • Monitor viral suppression: Viral load • Monitor recovery of immune system: CD4 cell count • Monitor drug toxicity: chemistry and haematology • ARV Resistance Assay