The document discusses the role of clinical laboratories in diagnosing and monitoring HIV infection. It describes that initial diagnosis involves screening tests like ELISA or rapid tests, followed by confirmatory tests like Western Blot or nucleic acid amplification tests. Monitoring involves regular CD4 counts to track immune status and viral load tests to monitor response to antiretroviral treatment. It also discusses additional tests like drug resistance and co-receptor tropism tests to help guide treatment decisions.

1. The role of the clinical laboratory
in diagnosis of HIV infection
By
Dr Ayman A Allam
Prof of Medical Microbiology and Immunology
Faculty of Medicine
Zagazig University

2. Human Immunodeficiency Virus
•Acquired Immunodeficiency syndrome
first described in 1981
•HIV-1 isolated in 1984 by Luc Montanier
(Pasteur Institute) and HIV-2 in 1986
•Belong to the lentivirus subfamily of the
retroviridae
•Enveloped RNA virus, 120nm in
diameter

3. Human Immunodeficiency Virus
• RNA retrovirus
• HIV-2 shares 40% nucleotide
homology with HIV-1
• three sub-groups of HIV-1, M (main
or major), N (new) and O (outlier).
• Within Group M: A to J subtypes

4. HIV

5. HIV
KEY VIRAL ENZYMES
• Reverse transcriptase
• Integrase
• Protease

6. 6
The Genome of HIVThe Genome of HIV
Three structural genes
LTRs
Extra open reading frames are clue to latency
These ORFs code for small proteins - antibodies in AIDS
patients

7. HIV

8. The role of Laboratory
• Initial diagnosis of HIV
1) Screening tests 2) confirmatory tests
• Follow up of patients
1.CD4 count (immune reconstitution )
2.Viral load (viral replication)
3.Virus drug resistance tests
4.HLA-B*5701 testing before use of abacavir in tt
5.viral tropism testing in the case of maraviroc.
6.Other tests to detect the complications of the
disease

9. Initial diagnosis of HIV
• Screening tests
1.Rapid tests (point of care tests)
2.ELISA
• Confirmatory tests
1.Western blot
2.Nucleic acid amplification tests
Qualitative TMA (FDA approved)
Quantitative tests as Real Time- PCR

10. The window period
• The time between when a person may have been
exposed to HIV and when a test can tell for sure
whether He has HIV infection
• The window period varies :
(1) the genetics of the virus
(2) the genetics and immunocompetence of the host
(3) The assay (detects antigen, antibodies , RNA)
(4)the specimen type, such as oral fluid, venous or
capillary whole blood and serum/plasma.
• For a period of about 10 days following HIV
infection, no currently available serological or
virological assay can detect any marker of HIV
infection.

11. The window period
• A nucleic acid test (NAT):10 to 33 days after
an exposure.
• Antibody tests: 23 to 90 days to reliably
detect HIV infection
• Antigen/antibody test: 10 to 45 days after an
exposure
• Rapid Antigen/antibody test : 18 to 90 days
after an exposure

12. Window period

13. Screening tests
1- Premarital examination
2- Blood Donors.
3-Antenatal Care
4- Pre-employment screening
5- Before Surgical operations (Medico-legal)
6- Follow up for hemodialysis patients.
7- IV Drug abuse
8-prisoners
9-homosexuals

14. Rapid HIV-
1/2 antibody-
based tests
performed at the
point of care
(POC) are
becoming the
global standard
for HIV testing,
particularly in
the developing
world

15. Rapid tests (point of care tests)
• Rapid (point-of-care) give results in less than 20-30
minutes.
• Rapid testing is the method of choice when
immediate information is necessary to determine
the need for prophylaxis:
in the labor/delivery
post-exposure settings
When the person who is being tested is unlikely
to return for a follow-up visit.

16. Rapid tests
CLIA-waived tests: performed on whole blood
or oral fluid
• The OraQuick Advance
• Uni-Gold Recombigen assays
Moderate complexity: performed on serum or
plasma
• The Reveal G2
• Multispot assays

17. Rapid tests (point of care tests)

18. OraQuick rapid test
• OraQuick is a home
HIV test approved by
the United States
Food and Drug Administration
in 2012
• Sample is oral mucosa
transudate
• It can detect reactive
patients after 14 days of
infection

19. oral fluid sample
Step 1 – Collect sample.
Step 2 - Insert the device
into the buffer.
Step 3 - Read between 20
and 40 minutes.
Non-
Reactive
Preliminary Positive

20. OraQuick rapid test
Step 1 - Collect
sample.
Step 2- Mix sample
in buffer.
Step 3: Insert the device
into the buffer.
Step 4 - Read between
20 and 40 minutes.
Preliminary Positive

21. Alere Determine HIV-1/2 Ag/Ab
Combo test
U.S. Food and Drug Administration approved the
Alere Determine HIV-1/2 Ag/Ab Combo test on
August 9, 2013,
• Add sample---buffer---read result in 20 min.

22. Multispot rapid assays

26. Rapid tests: False positives
False positives are possible:
• hepatitis A virus (HAV)
• hepatitis B virus (HBV) infections
• rheumatoid factor
• Epstein-Barr virus infection
• in multiparous women.

27. Rapid tests: False negative
• Before seroconversion
• The adaptive immune response may decline after HIV
acquisition, leading to lower levels of specific gp41 HIV
antibodies.
• false-negative test results may be more common for oral
fluid specimens
• Infection with HIV-1 group O or HIV-2.
• Concurrent acute hepatitis C
• Delayed antibody synthesis in infants and persons receiving
post-exposure prophylaxis
• Diminished immune response in individuals receiving
intensive or long-term immunosuppressive therapy

28. ELISA

29. ELISA for HIV antibody
Microplate ELISA for HIV antibody: coloured wells indicate
reactivity

30. ELISA

31. ELISA fully automated
• Fully Automated ELISA machines (dilution,
pipetting, washing, incubation and reading)

32. fourth generation serological
assays
• fourth generation serological assays are the
first-line assay in blood screening.
• Antibody tests are typically positive no sooner
than 2 weeks after exposure, but
seroconversion may take as long as several
months.
• Thus, testing algorithms for exposed patients
include baseline serology with repeat testing at
1, 3, and 6 months post exposure.

33. ELISA
False positive:
• Epstein-Barr virus
• Influenza vaccination
• Pregnancy
• Receipt of immune globulin
• Autoimmune disease
False negative:
• Before seroconversion
• Using kit not detectiong HIV2 or group O HIV1

34. Testing algorithm

35. Confirmatory test
Western blot / line probe assay

36. Western blot
It detects specific antibodies to specific viral antigens:
1.gp 160 - precursor of ENV glycoprotein
2.gp 120 - outer ENV glycoprotein
3.p66 - reverse transcriptase component of POL
translate
4.p55 - precursor of GAG proteins
5.gp 41 - transmembrane ENV glycoprotein
6.p31 - endonuclease component of POL translate
7.p24 - GAG protein
8.p17 - GAG protein

37. Western blot
Positive : Any two or more of the bands : p24, gp41 and
gp120/160.
Negative: No bands present
Indeterminate: Any bands present but pattern does not meet
criteria for positive

38. Indeterminate western blot
Probable True Positive (HIV Infection)
• Sero-converting
• HIV-2 infection
• Technical errors
Probable True Negative (No HIV Infection)
• Recipients of HIV-1 trial vaccine
o Multiparous women
o Polytransfused patients
o Patients receiving chronic hemodialysis
o Patients with autoimmune disease
• Recipients of influenza and hepatitis B virus vaccines
• Persons with non-HIV acute viral infections
• Congenital bleeding disorders
• Alcoholic hepatitis and other chronic liver diseases
• Hematologic malignancies, lymphomas

39. Nucleic acid amplification tests for
HIV
1) Qualitative
• HIV-1 RNA TMA
• FDA-approved as a confirmation test
• 3-5 days for results
2) Quantitative (viral load test): Not FDA-approved
as a confirmation test
I. HIV-1 RNA, Real-time PCR
II.Quantiplex bDNA or branched DNA test
III.NASBA

40. • Gen-Probe's (Aptima) HIV-1 RNA qualitative assay is the only
molecular assay that has been approved by the FDA for diagnosis of
acute infection and as a confirmatory test for detection of HIV-1 in
samples that test reactive for HIV-1 antibodies

41. Real time PCR
•Viral load is a quantitative
measure of viral RNA genomes in
the peripheral circulation.
•Standard assays have a
lower limit of detection of
400 copies/mL
•ultrasensitive assays have
lower limit of detection of 50
copies/mL
•An optimal response to
treatment would be a reduction in
the viral load to undetectable
levels within 6 months
•false-positive quantitative
RNA viral load tests can
occur at counts under
1000/mL.

42. Nucleic Acid Sequence-Based-
Amplification (NASBA)

43. Other viral load tests
• ExaVir Load Version 2 : An inexpensive HIV
viral load assay measures viral RT activity
• The test is performed mostly manually
• Was designed primarily for resource-limited
settings.
• The assay has a lower limit of detection of 400
copies/mL
• It is not approved by the FDA for clinical use in
the United States.

44. Dignosis of HIV infection in the
neonate
• The use of proviral DNA PCR
• Initial testing should be performed as soon as
possible after birth, and because transmission
typically occurs at the time of delivery
• Optimally, exclusion of infection requires at
least 2 further negative DNA PCR tests, one
at 1 month of age and a second at 4 months.
• Serology is done after 18 months of age.

45. Viral culture
• Viral culture is possible but is labor intensive,
requires specialist laboratory equipment and
training (biosafety level 3 containment
facilities)
• may take several days to weeks to get results.
• It has no place in routine clinical diagnostics.

46. CD4 count
• CD4+
T cells can be
readily measured by
flow cytometry,
using specific
antibodies that label
CD4 as well as other
T cell markers such
as CD3.

47. CD4 counts
• A count below 200 cells/mL has been
associated in many studies with an increase in
the relative risk of opportunistic infections
• Many recent studies of treatment initiation at
various CD4+
T-cell counts have shown that
patients who start treatment when their count
drops below 350/mL experience less morbidity
and mortality than those who wait until their
count drops below 200/mL.

48. Drug Resistance Tests
1. Genotypic methods
2. Phenotypic methods
3. Virtual phenotype

49. Genotypic resistance assay
• Testing analyzes the genes of the virus to detect
the presence of one or more mutations that are
associated with antiretroviral drug (ART)
resistance.
• Two direct sequencing-based methods have been
approved by the FDA:
The TruGene HIV-1 Genotyping assay (Siemens)
The ViroSeq HIV-1 Genotyping System (Celera
Diagnostics).

50. Phenotyping resistance assay
• A phenotypic assay provides a direct measure
of drug resistance.
• These assays use recombinant DNA methods
to measure the ability of a patient’s virus to
grow in the presence of a drug.
• The results from a phenotypic test include the
net effect of any and all resistance mutations.

51. Phenotypic resistance assay
• PhenoSense
HIV can
predict
emergence of
resistance
• It directly
measures the
susceptibility
of a patient's
virus to reverse
transcriptase
inhibitors and
protease
inhibitors

52. VIRTUAL PHENOTYPE
• This test is really a method of interpreting
genotypic test results.
• First, genotypic testing is done on the sample.
• Phenotypic test results for other virus samples
with a similar genotypic pattern are taken from
a database.
• These matched samples tell you how the virus
is likely to behave.
• The virtual phenotype is faster and less
expensive than a phenotypic test.

53. Co receptor tropism assay
• It determines types of co-receptor (CCR5 OR
CXCR4) tropism of the virus.
• Maraviroc is active only in CCR5 phenotype
I. Phenotypic Assays : as Trofile assay 2 weeks
II.Genotypic Assays : rapid 2-3d
is based on sequencing of the V3-coding
region of HIV-1 env, the principal determinant
of co-receptor usage.

54. summary
• The most recommended alogarithm for
diagnosis of HIV is screening by 4th
generation
ELISA followed by confirmation by Western
blot or Transcription Mediated amplification.
• No alogarithm can fit for every scenario.
Physicians should allow for individual
circumstances and unique situations

55. summary
• Rapid HIV-1/2 antibody-based tests are
becoming the global standard for HIV
testing, particularly in the developing world.
• For a period of about 10 days following HIV
infection, no currently available serological
or virological assay can detect any marker
of HIV infection.

56. THANK YOU