1. Unit 6 Diagnosis & Follow-up of HIV Infection
Laboratory Testing
Unit 6
Diagnosis & Follow-up
of HIV Infection
This unit should take 1 hour and 10 minutes to complete.
Step 1: Overview of Unit Learning Objectives (Slide 2) – 5 minutes
Step 2: Laboratory Monitoring for HIV/AIDS (Slides 3-42) – 60 minutes
Step 3: Key Points, Questions (Slide 43) – 5 minutes

2. Learning Objectives Describe the role of laboratory tests in:
The diagnosis of HIV infection
Ongoing monitoring of HIV’s effect on the body
Initiating ART and prophylaxis of OIs
Monitoring response to treatment
Monitoring toxicity of treatments
Step 1: Overview of Unit Learning Objectives (Slide 2) – 5 minutes

3. Patient Management Tasks That Depend on Laboratory Support
Diagnosis of HIV
Staging of HIV’s effects
Baseline lab assessment
ARV regimen selection
Monitoring response to ART
Monitoring adverse drug effects
Detection of treatment failure
Diagnosis of OIs
Resistance profile
Step 2: Laboratory Monitoring for HIV/AIDS (Slides 3-42) – 60 minutes
Use this slide to elaborate on the objectives described in the previous slide, and to create an interactive environment with the participants.
Select a few of the items on the slide and ask the audience to describe how lab tests are needed for that particular task.
For example, “How do we use laboratory support in the diagnosis of OIs?” Correct responses might be: 1) staining of BAL specimen for Pneumocystis 2) detection of cryptococcal antigen in a patient’s serum or CSF, etc.
Don’t make the group go through this exercise for all the items on this page – that would be tedious. Just do this enough to “activate” and engage the group, e.g., 3-4 items

4. Diagnostic Tests for HIV Infection
Serological methods for detection of antibody
Rapid tests
ELISA
Western blot
Antigen detection methods
P24 antigen capture test
Polymerase Chain Reaction (also known as PCR or viral load)
HIV infection is in almost all instances diagnosed and confirmed by detecting anti-HIV antibodies
Diagnostic Methods:
Serological assays for HIV infection have been refined over the past years to unprecedented levels of sensitivity and specificity.
However, diagnostic problems, which cannot be resolved with serological methods, are usually cleared using methods for direct detection of HIV infection.
In addition, molecular amplification methods are currently the methods of choice for prediction of HIV progression and for monitoring antiretroviral treatment.

5. HIV Serological Diagnosis
Tests are based on a reaction between HIV antigens and antibodies in the patient’s serum
Tests use viral antigen extracts as a testing material
Both a screening test and a confirmatory test should be used for diagnosis
Serological methods detect antibodies to HIV.
These methods are used solely for diagnosis of HIV infection, not for treatment monitoring.
Tests are more than 99% sensitive and 99% effective
Because antibodies develop within 2-8 weeks after infection, testing shortly after a potential exposure may not be reliable.

6. “Window Period” Following HIV Infection
Acute HIV syndrome
Antibody
Primary HIV infection
Asymptomatic
Viremia
WINDOW
PCR
P24 a.g
ELISA
Standard HIV antibody tests do not detect HIV in the “window period.”
PCR and p24 antigen tests can detect HIV during the latter part of the “window period” but there is not presently a test that reliably identifies early HIV infection within the first two weeks following acute infection.
Source: S Conway and J.G Bartlett, 2003 a b
Years 2 3 4
(Weeks since infection)
Source: S Conway and J.G Bartlett, 2003

7. False Negatives and Positives
False negative results may result from:
Window period
Agammaglobulinemia
Seroreversion (loss of HIV antibodies due to extreme immune system destruction) in late disease
Technical error
Type N or O strains, or HIV-2
False positive results may result from:
Autoantibodies e.g., SLE
HIV vaccines

8. Rapid Test Use two consecutive kits
Initial test kit is very sensitive
Repeat test is very specific
Visual tests that do not require the ELISA Reader
Technically simpler to perform
Most have sensitivity and specificity comparable to ELISA
Based on the principles of dot immunoassay, or particle agglutination (e.g. gelatin or latex)
Requires a second test only if the first test is positive:
A single negative result is valid and need not be repeated, due to the very high sensitivity (> 99.7%) of the test.
First test is relatively less specific (may have false positives) but the second test is very specific and differentiates the true positives from false positives.
These tests enable health providers to supply definitive negative and preliminary positive results to clients at the time of testing.
The method of determination is projected in subsequent slides.

9. HIV Serial Rapid Test Algorithm
Neg
Pos
SAMPLE T1 T3 T2
T1: Screening test
If (+) go to T2
T2: Confirmatory test
If (-) go to T3
T3: Tie breaker
This testing algorithm and method of interpretation is used in rapid tests, because a single positive result is not reliable due to the high sensitivity of T1.
It is a cost effective algorithm, because tests are only done when needed, i.e., when indicated based on the previous test’s results.
Results that appear in bold text on the flow diagram represent final results.

10. HIV Parallel Rapid Test Algorithm
T1 and T2: Screening and Confirmatory tests
If T1 and T2 do not agree go to T3
T3: Tie breaker
Neg
SAMPLE T1
Pos T3 T2
This mode of testing is more expensive than the serial method, because two test kits are used for even a negative test. However, it provides results more quickly because it requires only two rounds of testing
Results that appear in bold text on the flow diagram represent final results.

11. ELISA Test Very sensitive Results:
Negative means patient does not have evidence of HIV
Repeatedly reactive indicates need for confirmatory test (by Western blot)
Ideally, all tests performed would screen for HIV 1 and 2
Uses the same principles of interpretation of results as in the Rapid test
Many of the current HIV ELISA assays also contain HIV-2 antigens. Specimens failing to demonstrate HIV-1 should be examined for the presence of HIV-2 specific proteins in patients where the suspicion of HIV infection is high.

12. Western Blot Test Confirmatory test for all positive ELISA assays
Two or more “key” bands indicate a positive test
Should not be used alone for HIV diagnosis
The Western Blot (WB)
Detects antibodies to HIV-1 proteins, including core (p17, p24, p55), polymerase (p31, p51, p66) and envelope (gp41, gp 120, gp160).
Should not be used alone; should always be coupled with a screening test due to a 2% rate of false positives.

13. Western Blot Result Interpretation
Results are interpreted as follows:
Negative: no bands
Positive: reactivity to gp120/160, plus either gp41 or p24
Indeterminate: one reactive band (or anything other than a positive test) should be repeated at a later time, e.g., 1-3 months later
Repeatedly indeterminate: no HIV infection
Western blot techniques involves placing specimen at one end of a gel strip and subjecting the specimen to electrophoresis, where an electrical current causes migration of antibodies in the specimen.
Antibodies to HIV proteins of specific molecular weights can be detected, and these correlate to particular HIV proteins.

14. Case Study: Tewabech
Tewabech is a 27 year-old female who began a new relationship with an older man last month
Yesterday she learned that this man had been married previously and that his wife died from AIDS
Tewabech is concerned that she might have HIV and wants to be tested
SUGGESTED DISCUSSION
Discuss the following questions regarding the case study with the participants. Read each question and ask participants to volunteer answers
Should she be tested today? If not, when?
If she tests positive, does that mean her new partner infected her?
If she tests negative, does that mean she does not have HIV?
What tests are available to diagnose HIV infection?

15. Direct Detection Methods
Uses:
Window period
Indeterminate WB results
Monitor HIV infected individuals
Two methods
P24 antigen capture test
Polymerase Chain Reaction
In certain situations serologic tests are not reliable and direct detection of HIV is required. Such as:
Newborns born to HIV-infected mothers, individuals with indeterminate WB results, to determine the HIV status during “window period”, and to monitor HIV infected individuals

16. P24 Antigen Capture Test
Detected during acute phase of primary HIV infection
Sensitivity in adults: 50-75%
Sensitivity in children: 20%
Specificity: 95%
Following seroconversion, antigen is less detectable (sensitivity declines)
p24 antigen
Can be detected in serum or plasma
Associated with intact or disrupted virions or released by infected cells.
Detected using EIA, utilizing immobilized antibodies to p24, which bind to p24 antigen present in the patient's serum.
Test
Used during primary HIV infection (“window period”), during very late symptomatic stages of infection, and in newborns to HIV-infected mothers.
Negative result does not rule out HIV
For confirmation of a reactive result, the sample must be subjected to an additional confirmatory neutralization assay
Estimated average time from detection of p24 antigen to detection of HIV antibody by routine screening examinations is approximately six days (not all recently infected individuals exhibit p24 antigenemia)

17. Polymerase Chain Reaction
PCR or “viral load”
Very expensive
Not routinely used for diagnosis except in children <18 months
Also used for:
Diagnosis in special situations
Evaluation of response to treatment (success)
Prognosis (Viral set point)
Detection of virological treatment failure
A significant drop in viral load is expected three to four weeks after initiation of treatment.
Diagnosis in special situations
Confusing serological test
Examples: indeterminate WB or serologic positive result in children less than 18 months (and particularly less than 12 months of age) due to maternal antibodies
Neonatal HIV infections
Window period
Evaluation of response to treatment (success)
Drop of >1 log within 4-8 weeks of starting ART
Goal: undetectable viral level at 4-6 months
Undetectable viral load is defined as < 50 copies virus per mcl of serum
Prognosis (Viral set point)
Viral load at 6-12 month after acquisition of HIV
Detection of virological treatment failure
Failure to achieve complete viral suppression
Detectable virus after complete suppression

18. Case Study: Tewabech (cont.)
Tewabech requests an HIV test
The test is performed and comes back negative
Tewabech remains concerned
Although she is counseled to return in one month for repeat testing, she returns one week later with fever, rash, pharyngitis and adenopathy
HIV PCR is positive
DISCUSSION SUGGESTIONS
What do her symptoms represent? (Acute HIV Syndrome)
What other symptoms are associated with this syndrome? (see other unit)
Was the test defective?

19. PCR for Viral Load Commercial methods/kits in use: Amplicor B-DNA
Amplification: RT-PCR detection of DNA
Detection: labeled probe
B-DNA
Detection: labeled antibodies
EasyQ
Amplification: NASBA detection of RNA
Methodologies used to determine viral load.

20. Viral Load Monitoring
Where available, PCR or NASBA monitoring provide valuable information about ART effectiveness
Viral load should be checked:
Every 3-6 months when not on ART
One month after starting or changing ART
Every 3-4 months in stable ART patients

21. CD4 Determination CD4+ cells CD4 receptor serves as a target for HIV
Subset of T lymphocytes
CD4 receptor serves as a target for HIV
Normal Values
T helper (CD4+) cell count =
T suppressor (CD8+) cell count =
CD4+ cells
Identified by functional proteins (receptors) on the cell surface
Preferentially depleted during the course of the disease
HIV binds to the receptor in the process of entering/infecting the cell

22. Uses of CD4 Cell Count Decision to initiate ARV
Guide in initiating OI prophylaxis
Assess progression of disease
Measure response to treatment (prognostication)
Detect immunologic treatment failure
Accurate and reliable enumeration of CD4 T cell counts is crucial for monitoring the rate of progression to AIDS, both for initiating prophylaxis for opportunistic infections as well as monitoring the impact of antiretroviral therapy (ART).

23. Cellular Blood Elements & CD4 Count
WBCs
RBCs
platelets
PMNs
Eos
Lymphs
Grans
Macroph.
This slide puts “CD4 cells” in context of all cellular blood elements.
CD4 cells are identified in the lab by a process called “flow cytometry.”
CD4 (T helper)
CD8 (T helper)
others

24. ART Baseline Labs HIV antibody test Full blood count and differential
AST or ALT
Serum creatinine or blood urea nitrogen
Serum glucose
Pregnancy tests for women
Urine dipstick
Hep B surface antigen
Serum RPR
Serum lipids (for pts. with other cardiac risk factors or to receive PIs or EFV)
CD4 lymphocyte count
Viral load
In the absence of resource constraints, this list provides a thorough baseline assessment which can help you to determine baseline health status, and assist in deciding which medications the patient can use and when they should be started.
These tests might not be available in all Ethiopian settings.

25. ART Baseline Labs (2) Minimum required in Ethiopian context
HIV antibody test
CBC and differential
Liver function test – ALT
Pregnancy test
BUN and creatinine

26. Laboratory Indicators for Initiating ART
Total lymphocyte count <1200
CD4 count <200
Viral load
Ethiopian guidelines for initiation of ART also include clinical indicators, such as Stage IV regardless of CD4 count, and Stage III with CD4 <350.

27. Case Study: Tewabech (cont.)
Tewabech is referred to her local hospital clinic for ongoing care
Baseline laboratory tests performed all return with normal results, except:
White blood cell count is 4200 cells/mm3
CD4 count is 380 and 23%
DISCUSSION SUGGESTIONS
What ART is needed presently? (nothing; ART is not initiated until CD4<200)
What OI prophylaxis is needed? (nothing; no significant risk for serious OIs. Generally, none started until CD4<200)

28. Total Lymphocyte Count (TLC)
May be used as a substitute marker for CD4 count
TLC value <1200 cells/ul corresponds roughly to CD4 cell count <200 cells/ml
TLC used to initiate ART but not for monitoring response to therapy
While the total lymphocyte count correlates poorly with the CD4 T cell count in asymptomatic patients, in combination with clinical staging, it is still a useful marker of prognosis and survival.
Reference Laboratory Support to HIV Diagnosis and Monitoring of Antiretroviral Therapy Report of a Regional WorkshopPune, India, July 2004

29. CD4 Count and TLC Thresholds for OI Prophylaxis
Pneumocystis pneumonia
CD4 <200
TLC <1200
Toxoplasmosis
CD4 <100 and positive Toxoplasma serology
Cryptococcal meningitis
CD4 <100

30. Case Study: Tewabech (cont.)
Tewabech establishes ongoing care in the HIV clinic, with repeat CD4 counts done every 6 months
Ultimately, her CD4 count declines to 180 and 13%; she begins zidovudine, lamivudine and nevirapine
DISCUSSION SUGGESTIONS
Ask the group if they agree with her care and treatment (they should, since it is in accord with national guidelines).
How will they follow-up with (what laboratory tests will they order for) Tewabech now that she is on ART? How often will they be repeated?

31. Recovery of CD4 Cells Continues Years After Starting HAART
The CD4 count typically increases > 50 cells/mm3 at 4-8 weeks after viral suppression with HAART and then increases an additional 50 – 100 cells / mm3 per year thereafter. (Ref. GID 2000;185:471 JAMA 2002;288:222)
Image source: Binquet C, et al. Modeling changes in CD4-positive T-lymphocyte counts after the start of highly active antiretroviral therapy and the relation with risk of opportunistic infections: the Aquitaine Cohort, Am J Epidem Feb 15;153(4):
Source: Binquet C, et al. Am J Epidem, 2000.

32. Interpretation of CD4 Cell Count
CD4 count and interpretation are affected by many factors
Patient age
Time blood is drawn (diurnal variation )
Seasonal variation
Presence of intercurrent infections, e.g., HTLV-1, flu
Steroid administration
Stress (psychological, physical, pregnancy)
Splenectomy
Results may fluctuate up to 30% in same individual

33. Interpreting CD4 Counts in Pediatric Patients
Level varies with age
Absolute CD4 cell count is high in children <6 years of age
CD4 percentage varies less than CD4 number
Because of this, CD4 percentage (rather than count) is often used in pediatric ART initiation and follow-up

34. Pediatric Age-Specific HIV Classification: CD4+ Immunologic Categories
Modified from: CDC Revised classification system for human immunodeficiency virus infection in children less than 13 years of age. MMWR, 1994; 43 (No. RR-12): p. 1–10.

35. Case Study: Tewabech (cont.)
Viral load monitoring becomes available after Tewabech has started on ART, and her first level checked 8 months after starting ART is undetectable (<50 copies/mcl)
One year after starting ART, her viral load is 255; the next viral load is undetectable
Eighteen months later, her viral load again rises to 330, though the next viral load is undetectable
BACKGROUND INFORMATION
These transient, low-level rises in her viral load have been termed “blips.”
Blips have not been shown to be associated with ART failure, and ART should not be changed when blips occur.
Consecutive, persistent elevations is viral load are clinically significant, and may indicate a need to change ART. At minimum, they should prompt a meticulous review of adherence.

36. Drug Resistance
Definition: growth of HIV despite presence of therapeutic levels of drug concentration
Arises from genetic mutations of HIV
As the number of mutations increases, this leads to an increasing degree of resistance

37. Treatment Failure
Detected by laboratory evaluation before it becomes obvious clinically
Detected by a few different methods
Treatment failure due to resistance is detected early by rise in virological means
Viral load testing is the most sensitive, earliest means to detect ART failure.
CD4 cell decline is intermittent in its ability to detect ART failure (between viral load, and clinical signs of failure, e.g., new OI).
Where available, resistance testing is useful to help guide changes in ART when the current regimen is failing.

38. Detection of Treatment Failure
CD4 cell count
Viral load testing
Viral resistance testing
Genotype
Phenotype
Changes in CD4 count that would be evidence of treatment failure include:
Decline after previous increase; return to pre-ART baseline; failure to rise when ART is started
Changes in viral load that would be evidence of treatment failure include:
Possibilities: failure to achieve 1 log drop in viral load within first 4 weeks treatment; failure to achieve undetectable viral load within first 4-6 months of ART; persistent detectable virus after becoming undetectable; rise after achieving a stable, low level of persistent virus
Resistance testing should be ordered when:
available, a change in ART is being considered, and while patient is still taking the failing regimen

39. Indications for Resistance Testing
Recently acquired infection (primary HIV infection)
Chronically infected treatment-naïve patients
Patients receiving ART who show evidence of virologic failure (rising viral load)
The rationale to have resistance testing in primary HIV infection is that recently infected persons show high level of resistance to ARVs probably due to a wider dissemination of resistant viruses in the community.
In patients receiving ARVs resistance testing guides therapy changes.

40. Types of Resistance Testing
Genotype
Phenotype
Virtual phenotype
Genotype – analyzes genetic sequence of virus to detect presence of mutations
Phenotype – measures ability of the virus to grow in vitro in presence of varying drug concentrations.
Virtual phenotype – a proprietary process that compares genotype results to a database of hundreds of comparable patients, in an attempt to predict phenotypic response

41. Toxicity Monitoring Goals
Assure safety of ARVs
Detect specific organ dysfunctions:
Known to be associated with specific ARVs
That can be detected before they become clinically apparent
To prevent or limit adverse health effects

42. Ethiopian Guidelines for Laboratory Monitoring of Patients Taking ART
These are the laboratory guidelines from the “Guidelines for use of antiretroviral drugs in Ethiopia, Jan 2005”

43. Key Points Laboratory tests determine: Laboratory tests measure:
The diagnosis of HIV infection
Timing for initiating ART and OI prophylaxis
Laboratory tests measure:
HIV’s effect on the immune and other body systems
Response to and toxicity of treatments
Laboratory testing is key to determine HIV diagnosis, optimize ART effect, and minimize ART toxicity
Step 3: Key Points, Questions (Slide 43) – 5 minutes