Laboratory Tests and Resistance Testing

1. Laboratory Monitoring of Progression of
Human Immunodeficiency Virus infection
By- Dr Ankita Mohanty
Resident
Department of Microbiology
LTMMC , Sion

2. Contents
A. Epidemiology – Global and India
B. Overview of HIV infection & Immunity
C. Laboratory Tests for Monitoring of HIV progression
D. Treatment Monitoring
E. Treatment Failure

3. A. Epidemiology-Global
0.6
(70%)
(9%) (9%)
(6%)
(4%)
(2%)

4. 0.4
(60%)
(65%) (49%)
(52%) (67%)
(60%)

5. Source: NACO

7. B. Overview of HIV life cycle
• HIV life cycle:
1. Binding and Fusion
2. Entry
3. Reverse transcription
4. Integration
5. Viral RNA and protein
expression
6. Assembly and budding
7. Maturation
• HIV target cells:
1. CD4T cells,
2. Macrophages,
3. Dendritic cells

9. HIV disease progression
–Acute infection

10. Stem Cell
Mature
T-Cells
HIV Infected Cells
HIV-Specific
T Cell
Expansion of
HIV-Specific
Cells
Killing of HIV
Infected Cells
Incomplete Clearance
of HIV Infected Cells
and Exhaustion
HIV Specific T Cell Responses
Thymus Periphery
R
T
R
T
R
T
R
T
R
T
R
T
R
T
R
T
R
T
R
T
R
T
R
T
R
T
R
T

11. HIV disease progression --
Clinical Latency
• During this period of the disease, the immune
systems remains competent at handling most
infections with opportunistic microbes
• Few or no clinical manifestation.
• Steady destruction of CD4+ T cells and steady
decline of circulating blood CD4+ T cells

12. HIV disease progression -- AIDS
• Acquired Immune Deficiency Syndrome:
• Catastrophic breakdown of host defenses,
marked increase in viremia and clinical disease.
• CD4+ cell count less than or equal to 200 per
microliter
• Clinical Features:
• Opportunistic infection
• Neoplasms
• CNS involvement

13. HIV disease progression
Levels
(Separate
Scales)
CD4+ T cell
HIV viral load
CD8+ T cell
Neutralizing Antibodies
Years
AIDS and
Death
Acute Asymptomatic
(clinical latency)
4 – 8
weeks
Primary
infection

14. C. Markers of Disease Progression
Non-specific
1. CD4 Count
2. CBC
3. Serum Neopterin
4. Beta 2 microglobulin
Specific
• Viral Load

15. Non-specific - 1. CD4 Count
• As HIV disease progresses, CD4 cell counts decline,
typically by about 30-100 cells/μl per year
(depending on viral load)
• it provides information about how well anti-HIV
therapy is working

16. Mechanism of CD4 T cell depletion in HIV
infection

17. Methods for detection of CD4 counts
Flow cytometric
• Dual platform approach
• Single Platform
Non flow cytometric
• Coulter manual CD4
count assay
• Dynal T4-T8
quantitative assay
• ELISA based counting
systems (Capcellia ELISA
and TRAx and
Zymmune)

18. Flow Cytometry-
Principle

22. 2.Complete Blood count (CBC)
• People with HIV may have low blood cell counts
(cytopaenias) due to chronic HIV infection or as a
side effect of medications, particularly drugs that
damage the bone marrow, where all blood cells are
produced.
• Blood cell counts are typically reported as the
number of cells per μl of blood (cells/μl) or as a
percentage of all blood cells.
• People with HIV infection should receive a CBC
about every six months, and more often if they are
experiencing symptoms or taking drugs associated
with low blood cell counts

23. Red and white blood cells, Platelets
• Anemia - common in HIV positives. HIV itself and various
OIs such as Mycobacterium avium complex (MAC) can
affect red blood cells and their oxygen-carrying capacity
• Neutropenia - Various anti-HIV drugs, OI medications
[including ganciclovir (Cytovene), used to treat
cytomegalovirus], and cancer chemotherapies that
suppress the bone marrow may lead to low neutrophil
levels
• Thrombocytopenia - may be caused by certain drugs,
autoimmune reactions, accelerated destruction by the
spleen, or HIV disease itself.

24. 3. Serum Neopterin
• Neopterin (6-D- erythrohydroxy-propylpterin) is a
low molecular weight compound derived from an
intermediate product of the de novo biosynthesis of
tetrahydrobiopterin from guanosine triphosphate
(GTP)
• It is an early marker of HIV infection
• The levels rise further on progression from pre AIDS
to clinical AIDS.

25. Application of Neopterin measurements

26. HPLC Method
• Neopterin is measured
by its native
fluorescence (350nm
excitation, 440nm
emission).

27. Shimadzu LC-VP
• HPLC System Available: Shimadzu LC-VP
• Sensitivity: High sensitivity analysis possible
– As little as 1 ppb of F-can be analyzed without any
preconcentration procedures.

28. 4.Beta 2 microglobulin (β-2M)
• 11kD protein which forms a heterodimer with class I MHC
molecules present on the surface of most nucleated cells
• Free β-2M can be measured in both serum and urine
• It correlates with the degree of progression of HIV
disease. It spikes in acute infection, declines and then
rises during the infection.

29. Determination of β-2M levels
• The β-2M levels can be determined by enzyme
immunoassay (EIA) using the in vitro diagnostic test
kit (Immunotech, France).
• The EIA test used is a competition assay. The
intensity of the colour developed and the measured
optical density (OD) is inversely proportional to the
concentration of β-2M in the samples

30. Specific Marker: Viral load
• The number of copies of HIV-1 RNA per milliliter of
plasma.
• Viral load is an accurate reflection of the burden of
infection and the magnitude of viral replication.
• It is critical in monitoring virologic response to ART.

31. Viral Load Progression

32. Viral Load -
determined by
PCR

34. Method COBAS
AmpliPrep/
COBAS
TaqMan HIV-1
Real
Time
HIV-1
NucliSENSEa
sy
Q HIV-1 v2.0
VERSANT
HIV-1
RNA 1.5
(kPCR)
Xpert HIV-1
Manufacturer Roche Abbott BioMerieux Siemens Cepheid
Advantages •High throughput
•Can be used for
other diseases
•Large
range
•High
throughput
•Compatible
with dried blood
spots as well as
body fluids
•Can be used for
other diseases
•Low risk of
contaminatio
n
•Can be used
for other
diseases
•Easy
•Rapid
•Flexible
Disadvantages •High Cost
•Requires trained
personnel and
support
•High Cost
•Requires
trained
personnel
and support
•High Cost
•Prone to
contamination
•High Cost
•Specific
space and
infrastructur
e demand
•High Cost
•Specific space
and
infrastructure
demand

35. Recommendations for Viral Load and CD4
Count Monitoring
Clinical Scenario Viral Load Monitoring CD4 Monitoring
Before initiating ART At entry into care
If ART initiation is deferred,
repeat before initiating ART
At entry into care
If ART is deferred, every 3-
6 months
After initiating ART Within 2 to 4 weeks, after
that every 4 to 8 weeks
6 months after initiation
After ART modification(
drug toxicity or regimen
simplification with viral
suppression)
4 to 8 weeks after
modification
According to prior CD4
count
ART modification due to
virological failure
Within 2 to 4 weeks, after
that every 4 to 8 weeks
Till viral load is suppressed
Every 3 to 6 months

36. E. Treatment Monitoring tests
1. Resistance testing
a. Genotypic tests
b. Phenotypic tests
c. Virtual phenotype
2. Therapeutic drug monitoring

37. 1. Resistance Testing
• HIV can develop drug resistance by mutating in such a
way that the virus can continue to replicate despite the
drug.
• Usually occurs when there is inadequate treatment
adherence or when drug is not completely effective,
allowing HIV to multiply and mutate in the presence of
the drug
• Prevention of the emergence of HIV drug resistance (HIV
DR), is the key for success of ART programme and
prevention of transmission of resistant virus

38. a. Genotypic Tests
• These tests examine the genetic sequence of HIV's
reverse transcriptase and/or protease enzymes to
look for mutations that are known to be associated
with resistance to particular drugs.
• These assays require a plasma viral load of at least
500 to 1,000 copies/mL.

39. Drug Resistance Testing- Panel recommendations
• For Anti retroviral therapy- Naïve Persons
- Entry into care
- In persons with acute or recent(early) HIV Infection
- In Pregnant people with HIV
- In People who will initiate ART on day of or soon
after HIV Diagnosis

40. Drug Resistance Testing- Panel recommendations
• For Anti retroviral therapy- Experienced Persons
- When changing ART regimen in following:
1) Virologic failure and RNA copies > 1000/ml
2) Virologic failure and RNA copies 500- 1000/ml
3) Sub optimal viral load reduction

41. Systems Available
Manufacturer ViroLogic VIRCO Visible
Genetics
Applied
Biosystems
System
Available
GeneSeq HIV GENChec TruGene ViroSeq
Principle RT-PCR RT-PCR RT-PCR RT-PCR
Minimum
Detectable
Viral Load
500 copies/ml 500 copies/ml 500 copies per
ml
1000 copies/ml
TAT 10 days 7 to 14 days 7 to 14 days 7 to 14 days

42. • The highly sensitive parallel allele-specific
sequencing (PASS) assay can simultaneously look at a
large number of viral genomes and detect minor (0.1
to 0.01%) drug-resistant populations.
• A dried serum spot method of HIV genotypic testing
is also being developed. This method is relatively
simple and it is a potential means of making
resistance testing available throughout the world.

43. PASS
• After isolation of the genetic material in blood
samples, tiny fluorescent tags designed to stick to
HIV genes in particular ways are added. Tags
designed to stick to mutated gene locations known
to produce drug resistance are labelled to appear
green, while tags designed to stick to the same gene
locations but where the genes had not mutated are
labelled to appear red.

44. • A sophisticated computer program is used to count
the number of molecules with green or red
fluorescent tags in each sample.
• The test proves sensitive enough to detect a single
mutated virus out of 10,000 non mutated viruses in
the patient samples
• The test also can detect when a virus molecule has
more than one mutation, a capability that no
commercially available test has achieved.

45. b.Phenotypic tests
• done by adding a medication to an HIV culture in the
laboratory to determine how much drug is needed to
inhibit viral replication
• Resistance levels are usually reported in terms of
how much drug is needed to inhibit viral replication
by 50 or 90 per cent (the IC50 or IC90, respectively),
compared with wild-type virus.
• more direct measure of resistance, but are more
difficult, time-consuming, and expensive.

46. Systems Available
Manufacturer ViroLogic Viralliance Virgo
System Available Phenosense Phenoscript Antivirogram
Principle PCR and direct
ligation
PCR fully replication-
competent
recombinant virus
Minimum
detectable load
500 copies/ml 200-1000 copies/ml 500 copies/ml
TAT 2-3 weeks 7 to 14 days 2 weeks

47. c.Virtual phenotype
• Estimation of the viral phenotype using a large
database of more than 18,000 pairs of genotypic and
phenotypic data.
• HIV with a similar genotype is identified in the
database, and the corresponding phenotypic
information is used to estimate resistance
• The reliability of virtual phenotyping has been
investigated in several studies

48. VIRA 3001
• The first was an analysis of VIRA 3001, where the
actual phenotype established during the study was
compared with a virtual phenotype derived from
genotypic data and the Virco database.
• There was an 86% agreement between actual and
virtual phenotypes, with major discrepancies in only
3% of cases.

49. Virco TYPE HIV-1
• The Virco TYPE HIV-1 virtual phenotype assay uses
one of the world's largest reference databases of
viruses isolated from people with HIV.
• As of January 2008, the database contained 314,000
genotype samples and 86,000 phenotype samples.
• Samples of each assay for an individual were
available for 53,000 and this is the basis of
calculation for the correlative database.

50. Action of
Anti-
Retroviral
Drugs

51. 2. Therapeutic Drug Monitoring
• used to help individualize anti-HIV therapy by
measuring the amount of drug in an individual's
blood.
• different people absorb, process, and eliminate drugs
at different rates, and blood levels vary .
• Ideally, the lowest plasma drug concentration
between doses (the trough level, or Cmin) should still
be high enough to inhibit HIV, but the highest
concentration (the peak level, or Cmax) should not
cause intolerable side effects.

52. Normal Response to ART
• Virological response to ART: The viral load done 6
months after initiation of ART should be less than
1000 copies/ml.
• Immunological response to ART: CD4 count of the
patients should increase after initiation of ART. (at
least 50 CD4 cells/cubic mm at 6 months of the
initiation of the ART in ARV naïve patients, who are
adherent to their treatment)

53. Evidences of suspected treatment failure
• New OIs/ recurrence/ clinical events after 6 months
on ART (after ruling out IRIS)
• Progressive decline in CD4 counts
• Slow/ no clinical improvement over 6- 12 months,
associated with stationary CD4 count, despite good
adherence.
• In cases of sub-optimal rise in CD4 count at 6
months, despite good adherence and absence of
clinical symptoms, one may repeat CD4 count at 3
months.

54. Chronology of Treatment Failure

55. Definitions of ART Failure

56. SOP for Determining Failure
• Treatment Adherence: A detailed assessment of
reasons for non-adherence needs to be made. Unless
these reasons are identified, a patient will find it
difficult to adhere to the second-line regimen.
• Drug- drug interactions: Assessing whether the
patient is concomitantly taking medications that
interfere with ARV activity. For example, many
patients may not reveal that they take herbal
treatments along with the prescribed ART regimen.
• Certain Opportunistic infections may lead to decline
in the CD4 count, which may revert after treating
those infections.

57. Viral Load Testing

58. Classification of Anti-HIV Drugs
1. Nucleoside reverse transcriptase inhibitors
(NRTIs): Zidovudine, Stavudine, Lamivudine,
Abacavir, Zalcitabine, Emtricitabine, Didanosine.
2. Non nucleoside reverse transcriptase inhibitors
(NNRTIs): Efavirenz, Nevirapine, Delaviridine.
3. Nucleotide reverse transcriptase inhibitors
(NTRTIs): Tenofovir
4. Protease inhibitors (PIs): Saquinavir, Indinavir,
Nelfinavir, Amprenavir, Fosamprenavir, Ritonavir,
Lopinavir, Atazanavir.
5. Entry/Fusion inhibitors: Enfuvirtide, Maraviroc

59. NACO guidelines on ART
•First line anti- retroviral therapy
•Second line ART , for HIV-1 patients failing to first line
NNRTI based regimen
•Second line ART , for HIV-1 patients failing to first line
PI based regimen(not exposed to NNRTI)
•Second line ART , for HIV-1 patients failing to first line
PI based regimen(intolerant to NNRTI or where NNRTI
cannot be used)
•Second line ART , for HIV-2 patients failing to first line
PI based regimen

60. First line anti- retroviral therapy

61. Steps in initiating Second-line ART

66. Summary
Monitoring of HIV Disease progression must include
CD4 count, CBC, Viral load
 Reaching an undetectable viral load is a key goal of
ART.
Treatment Monitoring :
 Resistance tests-
 Phenotypic
 Genotypic
 Virtual phenotype
 Therapeutic Drug Monitoring

67. References
1. Harrison’s Principles of Internal Medicine, 20th
Edition
2. C. M. Tsoukas And N. F. Bernard ,Markers Predicting
Progression of Human Immunodeficiency Virus-
Related Disease, CLINICAL MICROBIOLOGY
REVIEWS, Jan. 1994, p. 14-28
3. Madhu Vajpayee and Teena Mohan, Current
practices in laboratory monitoring of HIV infection
Indian J Med Res. 2011 Dec; 134(6): 801–822.

68. References-continued
4. www.naco.gov.in (Accessed on 1/6/2019)
5.https://www.unaids.org/en (Accessed on
1/6/2019)
6.https://www.who.int/ (Accessed on 1/6/2019)