1. Human Immunodeficiency Virus - HIV Laboratory Testing
CLS 552 Application of Clinical Medical Microbiology & Immunology
Karen Honeycutt, MEd, MLS(ASCP)CMSMCM

2. Human: infecting human beings
What is HIV?
Human: infecting human beings
Immunodefficiency: decrease or weakness in the body’s ability to fight off infections
Virus: a pathogen that only reproduces inside a living cell
RNA, single stranded, enveloped virus
Retrovirus: contains reverse transcriptase enzyme that converts RNA to DNA

3. Types of HIV HIV 1 Most common throughout the world HIV 2
Found in West Central Africa, parts of Europe and India
Both produce the same patterns of illness
HIV 2 disease progress slower than HIV 1

4. HIV Entry Into Cells
Viral envelope protein (gp 120) binds to target cells with CD4 receptor
CD4 T lymphocytes primary target cells
Other cells with CD4 receptor:
Macrophages
Peripheral blood monocytes
B lymphocytes (≈ 5%)
HIV turns host cell into HIV replication factory

5. How is HIV Transmitted
Unprotected sexual contact with infected partner
Exposure of broken skin to infected blood or body fluids
Transfusion with HIV infected blood products
Tissue transplantation
Injection with contaminated object
Mother to child during pregnancy, birth or breastfeeding
NOT by: saliva, respiratory droplets, insect vectors or close personal contact

6. Early Disease Progression ≈ 2-4 Weeks
HIV localizes in lymphoid organs
Viremia ensues after infection
Rapid spread within first few weeks after infection
≈ 30 billion virus particles produced in first weeks of infection
Acute retroviral syndrome: fever, fatigue, rash, headache, lymphadenopathy, pharyngitis, myalgias, nausea, vomiting, diarrhea, night sweats
Resolves in a few days to a few weeks

7. HIV Antibody Development
Detectable levels usually at 3 to 8 weeks after infection
Time between infection and detectable antibody levels = ‘window period’
Serologic tests (looking for patient antibody) will be negative during window period
Viremia greatly decreased due to antibody
Patient usually asymptomatic
Clinical latency (average 10 years)
HIV continues to replicate in lymphoid tissue

8. Disease Progression Severity of illness determined by:
Amount of virus in body
Degree of immune suppression: CD4 lymphocyte counts decrease
CD4 counts <500 usually become symptomatic, develop opportunistic infections

9. Acquired: come into possession of something new
What is AIDS?
Acquired: come into possession of something new
Immune Deficiency: decrease or weakness in the body’s ability to fight off infections
Syndrome: signs and symptoms occurring together characterizing a particular abnormality
HIV infection leads to a weakened immune system. This makes a person with HIV vulnerable to a group of illness, e.g., opportunistic infections, that would not as easily affect a healthy person
AIDS results when HIV infection progresses to an advanced stage, damaging the immune system to a point at which the body can no longer fight illness.
AIDS is a syndrome because it is characterized by a group of illnesses
Drugs are available which can treat HIV and AIDS.
These drugs are called antiretrovirals (ARVs). They prevent the virus from replicating and slow the progress of the disease, but there is still no cure for AIDS or vaccine to prevent HIV transmission.
AIDS is the final stage of the disease caused by infection with HIV.

11. Reasons for Testing for HIV
Identify those with infection so antiviral therapy can be initiated
Identify carriers who may transmit infection to others (blood & organ donors, pregnant women, sex partners)
Monitor disease progression
Evaluate treatment efficacy

12. Types of Testing Most common Less common
Serology to detect patient antibody production to HIV components
Nucleic acid testing (NAT) to detect HIV viral nucleic acid or characterize nucleic acid (resistance to antiviral drugs)
Less common
Detect HIV antigen (viral components) – usually used to screen blood products
Culture: very difficult and dangerous to perform

13. CDC Recommendation: Opt-Out Testing
Testing all persons aged 13 to 64 years in all health care settings
Why? 250,000 in US unaware of HIV infection
Informed consent: inform patient HIV testing will be part of routine testing
Consent is inferred unless patient declines

14. Nebraska Law Requirements
Still requires patient signature indicating patient is consenting to HIV testing prior to blood being drawn
Bill in legislature (LB 462) to remove this restriction and follow CDC Opt-Out testing recommendations
Research indicates more patients consent to testing if seen as a routine test instead of a test to target at-risk behavior

15. This is a summary of the revised recommendations for HIV Testing of Adults and Adolescents in Healthcare Settings.
(slide)

16. (bullets from slide)

17. Testing Algorithm – Standard Serology Patient >2 years of age
Perform screening test
Enzyme immunoassay that will detect HIV antibodies in patient serum
Sensitivity and specificity ≈ 99%
Turn-around time = 1 to 2 days
If positive, the EIA test is repeated in duplicate on the same specimen
If 2 of 3 screen EIA tests are positive, confirmatory testing automatically performed

18. Testing Algorithm – Standard Serology Patient >2 years of age
Confirmatory Testing – Western Blot
Viral components are separated via electrophoresis on nitrocellulose strips
Incubate patient serum with strips
If antibody present, antigen-antibody complexes form on strip
Strip is stained to visualize any antigen-antibody complexes
Positive: if 2 of 3 specific antigen-antibody bands present
Sensitivity & Specificity >99%
Turn-around time varies: 1-7 days

19. Testing Algorithm – Standard Serology Patient >2 years of age
Confirmatory Testing – Western Blot Example
+ = positive control
(-) = negative control
pt. = patient
Patient WB = positive p24 and p120/160 bands present
(Positive: if 2 of 3 specific antigen-antibody bands present)
= Specific bands looked for

20. Testing Algorithm – Standard Serology Patient >2 years of age
Patient is confirmed HIV positive if:
2 of 3 screening tests are positive with confirmatory test (Western Blot) also positive
Test combination: >99% sensitive and >99.99% specific
If screen + and confirmatory negative, then patient is not considered positive:
Recommend follow up testing in 4 weeks
Reasons for false positive and false negative HIV serology (see next two slides)

21. Examples That Can Cause False Positive HIV Serology
Positive syphilis serology
Some malignant blood and autoimmune disorders
DNA viral infections
Alcoholic hepatitis
Chronic renal failure
Renal transplantation

22. Examples That Can Cause False Negative HIV Serology
Window period before seroconversion (most common)
Immunosuppressive therapy
Some malignancies
Bone marrow transplantation
Test systems that mainly detect antibodies to p24

23. HIV Point-of-Care Testing (POCT)
Public health needs for rapid HIV Tests
High rates of non-return for test results
Need for immediate information or referral for treatment choices
Perinatal settings
Post-exposure treatment settings
Screening in high-volume, high-prevalence settings

24. HIV Point-of-Care Testing (POCT)
Rapid or POCT is performed at the time the patient is seen clinically
Specimens: whole blood, saliva, urine
Only FDA approved assays used in health care settings
Results in minutes
Sensitivity and specificity ≈ 99%
Considered a screen test
If positive confirmatory testing recommended
If negative usually no further testing recommended

25. HIV + Confirmed: Additional Testing Quantitative Plasma HIV RNA (Viral Load)
Not FDA approved for confirmatory testing as 2-9% false positive rate
Determine viral load ‘set point’ at time of diagnosis to monitor
patient disease progression
therapeutic response

26. HIV + Confirmed: Additional Testing CD4 Lymphocyte Count
Adult normal range = 700 to 1100 cells/mm3
Results used to stage the disease
Make therapeutic decisions
When to start antiviral therapy
When to start prophylaxis for specific opportunistic infections
Indicator of prognosis

27. Correlation of Complications with CD4 Counts
Infectious Complications
/mm3
Bacterial pneumonia, Pulmonary Tuberculosis, Herpes Zoster, Thrush, Cryptosporidiosis, Kaposi’s sarcoma
<200/mm3
Pneumocystis jiroveci (carinii), Disseminated Histoplasmosis and Coccidioidomycosis, Extrapulmonary TB
<100/mm3
Disseminated Herpes Simplex Virus, Toxoplosmosis, Cryptococcosis, Candida esophagitis
<50/mm3
Disseminated: Cytomegalovirus (CMV)
Mycobacterium avium complex

29. Perinatal HIV Infection in Infants
Utilize nucleic acid testing (NAT)
Can’t utilize serology as mother’s IgG HIV antibody will cross the placenta
Infant + if two HIV NATs positive at two different times
Early antiviral therapy is recommended in HIV + infants

30. Antiretroviral Therapy (2008)
HAART—highly active anti-retroviral therapy
23 approved antiretroviral agents
Nucleoside Reverse Transcriptase Inhibitors
Non-NRTIs
Protease Inhibitors
Entry & Fusion Inhibitors
Integrase Inhibitors
5 fixed dose combinations
Guidelines
DHHS—Department of Health and Human Services
IAS-USA—International AIDS Society - USA

31. Goals of HAART Clinical: prolong life and improve quality of life
Virologic: undetectable viral load (<20-50 copies/mL)
Immunologic: immune reconstitution (normal CD4 count)
Therapeutic: combination of drugs (3 or 4)
Epidemiologic: reduce HIV transmission

32. Starting Antiretroviral Therapy
Start if:
Patient symptomatic, an infant or pregnant
HIV RNA >30,000 copies/ml
CD4 count <350/mm3
Consider if:
HIV RNA <5000 copies/ml, CD4 count /mm3
HIV RNA copies/ml, CD4 count >500 /mm3
Defer if:
HIV RNA <5000 copies/ml, CD4 count >500 /mm3

33. Definition of Treatment Failure
Virologic failure
Viral load not below detectable levels (> c/mL)
Side effects – patient not taking meds
Immunologic failure
CD4 count fails to increase 100 cells/mm3 per year
Clinical failure
>3 months post HAART and still having symptoms

34. HIV Resistance Testing
Genotypic testing: HIV gene sequencing of the patient’s virus to detect mutations known to confer drug resistance
Report out specific gene sequences with the drugs that the virus will be resistant to
Reasons to perform
When patient is first diagnosed as baseline
At the start of HAART or switching drugs
Determine if patient has been infected with other virus strains
Treatment failures

35. Opportunistic Infections
Pneumocystis jiroveci (carinii) - fungi
Causes pneumonia (PCP)
Detection via stains of BAL fluid, lung tissue
Mycobacterium tuberculosis
Lung and systemic disease
Detection via culture
Mycobacterium avium complex (MAC)
Disseminated disease

36. Opportunistic Infections
Cryptosporidium sp. - parasite
Diarrhea
Detection of organism is stool via microscopy or antigen detection
Toxoplasma gondii - parasite
Encephalitis, brain abscess
Detection via serology (looking for antibody), staining tissue or NAT

37. Opportunistic Infections
Candida sp. - yeast
Thrush, vaginitis, esophagitis
Detection with culture
Cryptococcus neoformans - yeast
Meningitis, pneumonia, disseminated disease
Detection via culture or antigen detection in CSF
Cytomegalovirus (CMV)
Retinitis, pneumonia
Detection via viral culture, NAT

38. Opportunistic Infections Prophylaxis Examples
Drug
Start
Stop
Pneumocystis jiroveci (PCP)
SXT
CD4 < 200
CD4 >200 for 3-6 months
MAC
Azithromycin
CD4 < 50
CD4 >100 for 3-6 months
M. tuberculosis
Isoniazid
TST > 5mm
Opportunistic infections are never cured in HIV+ patients

39. Summary HIV: single-stranded, RNA, enveloped, retrovirus
Infect CD4 positive cells: especially CD4 lymphocytes
Serology: 2 of 3 screen tests positive followed by positive confirmatory test = HIV +
Monitor: CD4 count, viral load, resistance testing
CD4 count <500 = possible opportunistic infections
<350 = probably initiate antiviral therapy