1. 1 Lecture 3: Laboratory Diagnostics in ARV Therapy delivered by Dr. Madisa Mine, Botswana Ministry of Health KITSO AIDS Training Program

2. 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. 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. 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. 5 Minimum Time from Infection to First Detection of HIV-1 Markers 5101520253035 ELISA p24 DNA PCR RNA (VL) Detection of HIV: time (in days) after infection KEY: TIME WHEN HIV DETECTION BY TEST IS POSSIBLE TIME PERIOD BEFORE DETECTION Infection

6. 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. 7 ELISA Algorithm for Diagnosing HIV 2 Parallel ELISA Tests Negative Patient uninfected or in window period of infection

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

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

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

11. 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. 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 0 C and NOT frozen. Samples should be sent to the lab within 24 hours.

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

14. 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. 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 in utero and via breast milk. Hence detection of HIV nucleic acid in infant blood is done by DNA PCR.

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

17. 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. 18 Monitoring HIV Disease Progression

19. 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. 20 CD 4 and Viral Load Progression Viral load CD4 TIME Infection High Low

21. 21 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. Viral Load

22. 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 0 C 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 0 C.

23. 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. 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. 25 CD4 Cell Counts

26. 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. 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. 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 0 C (cool box without ice pack).

29. 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. 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. 31 Monitoring for ARV Toxicity

32. 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. 33 Samples required for chemistry and haematology tests TestsType of tubeVolume requiredComments FBCEDTA ( purple top)3 mlMix gently by inverting 5-10 times LFT Creatinine AmylasePlain/no additive Do not mix/ allow time to Proteins, CO 2 ( red top)3-5 mlClot before testing CPK, TBil Lipase Cholesterol Triglycerides HDL/LDL Plain (red) 3 mlFasting sample/clotted LactateHeparin tube on ice 3 mlMix gently by inverting 5-10 times GlucoseSodium Fluoride (Grey Top) 2 mlMix gently by inverting 5-10 times

34. 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. 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. 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 0 C 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 0 C.

37. 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. 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