LABORATORY INVESTIGATION AND MANAGEMENT OF HIV

2. LABORATORY DIAGNOSIS Of HIV

3. • PURPOSE OF TESTING FOR HIV
• • A matter of great importance as in Blood donors to prevent
infected blood being transfused.
• • To diagnose the patients infected with HIV virus.
• • For surveillance purpose.
• • Persons with high risk behavior.
• • In Pregnant women to prevent Mother to Child transmission.
• • Patient's presenting with opportunistic infections.
• • For persons who need to undergo a surgery

4. SPECIFIC TESTS FOR H.I.V INFECTION
Antigen detection: p24 antigen
Virus isolation
Detection of viral nucleic acid
Antibody detection
HIV Diagnostic Confirmatory tests
1- Differential Antibody tests
(Western Blotting, WB).
2- Indirect Immunofluorescene Assay.
3- Nucleic acid Amplification tests
(NAT, PCR) done in most hospitals for
screening blood donors.
4- Virus Isolation (not done for
clinical diagnosis only in research
purposes).

5. NON SPECIFIC TESTS
Total leucocyte and lymphocyte count to demonstrate leucopenia
and a lymphocyte count usually below 2000/mm3.
T cell subset assays. Absolute CD4+T cell count will be usually less
then 200/mm3.
Platelet count will show thrombocytopenia.
Raised IgA & IgG levels.
Diminished CMI as indicated by skin test.
Lymph node biopsy showing profound abnormalities

6. Specific Tests For Hiv Infection
Antigen Detection:
• Following a contact, as by blood transfusion, the viral antigen may be
detectable in blood after about 2 weeks
• Note: The p24 antigen is the earliest viral marker to appear in the
blood.
• IgM antibodies appear in about 4-6 weeks to be followed by IgG
antibodies.

7. • If the infecting dose is small example in needle-stick injury the
process maybe considerably delayed.
• Free p24 antigen disappears from circulation and remains absent duri
ng the long asymptomatic phase to reappear only when severe clinic
al disease sets in..
• The p24 antigen capture assay (ELISA) which uses anti-p24 antibodies
as the solid phase and can be used for this.
• The test is positive in about 30% cases.

8. Virus isolation
• The viruses present in circulation and body fluids, within lymphocytes
or free cells.
• Virus titres parallel p24 titres, being high soon after infection, low and
antibodie-bound during the asymptomatic period, and again high
towards the end.
• The infectivity being highest in “the early phase and when the person
becomes terminally ill

9. • The virus is present in many parts of the body and can be isolated
from the peripheral lymphocytes by the technique of co-cultivation of
the patient’s lymphocytes with uninfected lymphocytes in the
presence of interleukin-2.
• Viral replication can be detected by RT.
• Note: Because of the risk involved, virus isolation is to be attempted
only in laboratory with adequate containment facilities.

10. Detection of Viral Nucleic Acid
• As the most sensitive and specific test, PCR has become the gold
standard for diagnosis in all stages of HIV.
DNA PCR
PCR
RNA PCR
A related test, HIV RNA PCR can be
used for diagnosis as well as for
monitoring the level of viremia.

11. Antibody detection
• It takes 2-8 weeks to months for the antibodies to appear in
circulation
• Infection can be detected during the window period by the p24 assay.
• Antibody testing will have to be done after 2- 6 months to ascertain
whether infection has occurred or not.
• IgM antibodies dissappear in 8-10 weeks while IgG antibodies remain
throughout.
• IgM antibodies appear first, to be followed by IgG antibodies

12. Detection of antibodies
• ELISA
Screening
test
• Western blot test
• Indirect
immunofluorescence
test
• Radio immuno-
precipitation assay
Supplement
tests
• Dot Blot assay
• Particle Agglutination test
• HIV spot and comb test
• Fluorimetric micro
particle technologies
Rapid test

13. • Screening test performed in
• 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- Others

14. Screening Tests
ELISA tests:
• ELISA is the most frequently used method for screening of blood sam
ples for HIV antibody
• Direct solid phase antiglobulin ELISA is the method most commonly
used.
.
• The antigen is coated on the microtitre wells or other suitable solid
surface.
• The test serum is added. And if the antibody is present, it binds to the
antigen

15. Significance of ELISA
• • Antibody can be detected in a majority of individuals within 6-12
weeks after infection using the earlier generation of assays.
• • But it can be detected within 3-4 weeks when using the newer third
generation ELISA.
• • Due to their ability to detect p24 antigen, the fourth-generation
ELISA can be of value in detecting early infection.
• • The window period can be shortened to two weeks using p24-
antigen assay.
Do not forget ELISA is Screening not Confirmatory

16. Supplement Tests
• Western Blot test:
The confirmatory test commonly employed is the Western
Blot test.
The sensitivity of the test systems are currently being improved by the
use of recombinant antigens.

17. Indirect Immunofluorescence Test:
• HIV infected cells are fixed onto glass slides and then reacted with
serum followed by fluorescein conjugated antihuman gamma
globulin. In a positive test, apple-green fluorescence appears when
examined under fluorescent microscope.

18. Advantages
 Easy to use immediate and fast
results (10-20 minutes).
 Can be used at home or clinic.
 Inexpensive (1-2 $/test)
 Recently approved
 Done upon whole blood or saliva
 No equipment, refrigeration, No
electricity, No multiple timing
steps required.
 Built in controls
Disadvantages
 Only detects Antibodies to HIV ½
 Not detecting Antigens
 Positive late after infection; after
seroconversion.
 Need confirmation for reactive
tests.
 Subjective variability in result
reading.
R
A
P
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D
T
E
S
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19. What is CD4 test ?
• What is it mean ?
• What’s its indicate and usefullness ?
CD4 indicates immunity level
• Usefulness
1. Current Status of Immunity
2. Prognosis
3. To monitor effect of Therapy
4. Identify Treatment Faillure

20. Specific tests for the laboratory diagnosis of HIV
infection
Test
Window
period
Acute
infection
Asymptomatic
infection
ARC & AIDS
p24,RT
antigensp24,RT
antigens
+
+ - +
Virus isolation ++
± - ++
ELISA -
+ + +
Western blot -
+ + +

22. How to carry out pre-test counselling
• Discuss meaning of positive and negative test results
• Realise importance of maintaining confidentiality
• Identify person to whom positive result could be disclosed
• Explore knowledge and explain natural history of HIV
• Discuss transmission and risk reduction
• Assess coping strategy
• Explain test procedure
• Obtain informed consent

23. How to carry out post-test counselling
• Test result negative
• • Discuss transmission and
need for behaviour
modification
• • Advise second test 3
months after last exposure
Test result positive
• Explain meaning of result
• Organise medical follow-up
• Stress importance of disclosure
• Explain value of antiretroviral therapy
• Provide written information and useful
Internet resources
• Discuss confidentiality issues
• Organise emotional and practical support
• Facilitate notification of sexual partners

24. Baseline investigations
 CD4 count
 Viralload
• Hepatitis B surface antigen
• Hepatitis C antibody
 Syphilis serology
 Cervical smear in women
• Liver function tests
• Full blood count
• Urinalysis, serum creatinine
Serum cryptococcal antigen (if
CD4 < 100)
Tuberculin skin test
Sexually transmitted infection
screen

25. Viral transmission
Acute retroviral
syndrome: 2-3 weeks
Seroconversion: 2-20
weeks
Asymptomatic chronic
HIV infection: 8 yrs
Symptomatic HIV
infection/AIDS 1.3 yr
Stages of
Untreated HIV
Infection

26. Type of HIV Progression
Proportion
among PLHIV
CD4 cells drop Characteristic features
Typical progression 50-70% 35-50 CD4
cells/year
Develop end-stage disease within 8-10
years after seroconversion
Rapid progression 10% 50 CD4
cells/month
Develop symptoms of AIDS or end-stage
HIV disease within 2-3 years after
infection & also in children
Slow progression 5-15% Very slow Remain free of symptoms of AIDS for
more than 10-15 years
Long term Non-
progression
5-10% Stable CD4 Living with HIV for >15 years and have
stable CD4+ counts of ≥ 500 cells/mm³
blood. No HIV related diseases and no
previous ART

27. Antiretroviral Therapy (ART)
To achieve maximal
and most durable
suppression of viral
replication
improve the
quantity and
quality of life
improve the CD4
count to over 200
cells/mm3
To prevent
emergence of drug
resistant mutants
To reduce HIV
transmission
The
goals of
ART

28. Goals Principle
Clinical To prolong life & improve quality of life
Virological Greatest possible sustained reduction in viral load
Immunological Immune reconstitution is both: 1.Quantitative (CD4 within normal
range) 2.Qualitative (pathogen specific immune response
Therapeutic Rational sequencing of drugs to achieve previous 3 goals while:
1.Maintaining future therapeutic options
2.Minimising drug toxicities & side effects
3.Maximising treatment adherence
Epidemiological Reduce HIV transmission

29. MANAGEMENT OF HIV FALLS UNDER THREE
MAJOR CATEGORIES
1) TREATMENT/MANAGEMENT OF HIV-AIDS
2) POST EXPOSURE PROPHYLAXIS(P.E.P)
3) TREATMENT OF ADJOINING CONDITIONS
- Fungal Infections
-Bacterial infections -Viral infections
-NEOPLASIAS -misc.( recurrent apthos ulcers, xerostomia, salivary G.
enlargement)

30. MANAGEMENT OF HIV-AIDS
• The management of HIV/AIDS normally includes the use of multiple
antiretroviral drugs in an attempt to control HIV infection.
• •There are several classes of antiretroviral agents that act on different
stages of the HIV life-cycle. The use of multiple drugs that act on
different viral targets is known as highly active antiretroviral therapy
(HAART).
HAART decreases the patient's total burden of HIV, maintains function of
the immune system, and prevents opportunistic infections that often
lead to death.
• •Treatment has been so successful that in many parts of the world, HIV
has become a chronic condition in which progression to AIDS is
increasingly rare.

31. Classes of drugs
• There are five classes of drugs, which are usually used in combination,
to treat HIV infection.
• Use of these drugs in combination can be termed anti-retroviral
therapy (ART), combination anti-retroviral therapy (cART) or highly
active anti-retroviral therapy (HAART).
Anti-retroviral (ARV) drugs are broadly classified by the phase of the
retrovirus life-cycle that the drug inhibits.

32. Entry
inhibitors
• Block binding of HIV to the target cell
Reverse
Transcriptase
Inhibitors
• Block the viral RNA cleavage and one that inhibits
reverse transcriptase
Integrase
Inhibitors
• Block the enzyme integrase, which helps in the provirus
DNA being incorporated into the host cell chromosome
• Block the RNA to prevent viral protein production
Protease
Inhibitors
• Block enzyme protease
• Inhibit the budding of virus from host cells
The ARV drugs act on
the viral replication in
the following steps
and are labelled
according to site of
their action

33. • Initiate ART
• If CD4 cell count < 500 Cells/mm3
• If WHO Clinical Stage 3 or 4 Initiate ART Regardless of WHO clinical
stage and CD4 cell count
• Active TB Disease or Past History of TB in life time
• HBV or HCV Co-infection with sever chronic liver disease
• Pregnant and lactating woman with HIV
• HIV positive individual (to reduce HIV tranmission risk)

34. Commonly used antiretroviral drugs
classes drugs
Nucleoside reverse transcriptase
inhibitors (NRTIs)
Abacavir, emtricitabine, lamivudine,
tenofovir, zidovudine*
Non-nucleoside reverse transcriptase
inhibitors (NNRTIs
Nevirapine, Delavirdine, Efavirenz,
rilpivirine
Protease inhibitors (PIs) Indinavir, Ritonavir,Atazanavir, darunavir,
lopinavir
Integrase inhibitors Raltegravir, dolutegravir, elvitegravir
Chemokine receptor inhibitor Maraviroc

35. Generic Name Dose Adverse effects
Tenofovir (TDF) 300 mg once daily Renal toxicity, bone
demineralization
Lamivudine (3TC) 150 mg twice daily
or 300 mg once
daily
Minimal toxicity, rash (though very
rare)
Abacavir (ABC) 300 mg twice daily
or 600 mg once
daily
Hypersensitivity reaction in 3 to 5%
(can be fatal), fever,
rash, fatigue, nausea, vomiting,
anorexia, RS- (sore throat, cough,
shortness of breath)
Nevirapine 200 mg once
daily for 14 days,
followed by 200
mg twice daily
Hepatitis (usually within 12 weeks);
Stevens Johnson syndrome (SJS)
and Toxic Epidermal Necrolysis
(TEN).
Ritonavir 100 mg twice daily diarrhoea, nausea, vomiting,
abdominal pain

36. Selecting antiretroviral regimens
• The standard combination antiretroviral regimens are two NRTIs together
with an NNRTI, protease inhibitor (PI) or integrase inhibitor.
• Dual NRTI combinations are usually emtricitabine or lamivudine (they have
the same mechanism of action and so are never combined), together with
one of abacavir, tenofovir or zidovudine.
• It is possible to construct effective regimens without NRTIs if there is
intolerance or resistance to the NRTIs.
• Currently used PIs should always be administered with ritonavir.

37. POST EXPOSURE PROPHYLAXIS (THE EMERGENCY
PILL)
• • If an individual believes they have been exposed to the virus within
the last 72 hours (three days), anti-HIV medication, called PEP (post-
exposure prophylaxis) may stop infection.
The treatment should be taken as soon as possible after contact
with the virus.
• • PEP is a very demanding treatment lasting four weeks. It is also
associated with unpleasant side effects (diarrhea, malaise, nausea,
weakness and fatigue).
• • After a positive HIV diagnosis, regular blood tests are necessary to
monitor the progress of the virus before starting treatment.
The therapy is designed to reduce the level of HIV in the blood.

38. Recommended PEP regimens
Dosages of the drugs for PEP
for adults
Recommendation for PEP Recommendation for PEP
Tenofovir (TDF) 300 mg +
Lamivudine(3TC) 300 mg(1-
OD)
One tab Immediately within
2 hours of accidental
exposure, either at day time
or at night time
Next day one tab once OD,
continue for 4 weeks
Lopinavir (200 mg) +
Ritonavir (50 mg) Two Tab
(FDC) twice daily
(2-BD)
Two Tab Immediately within
2 hours of accidental
exposure, either at day time
or at night time
Next day two-tab BD,
continue
for 4 weeks
PEP must be initiated as soon as possible, preferably
within 2 hours

39. • CLINICAL MONITORING
Monthly clinical evaluation
o Body weight, overall well-being, any new symptoms / signs, four symptom
screening for TB at every visit
o Monthly Treatment Adherence-Evaluation--pill count, self-reported adherence
o Adherence to ART must be assessed at each visit and adherence must be
reinforced through counselling at each visit
o Adverse reactions of ART / OI drugs
o Drug-drug interactions, look for all concomitant drug use (prescribed and over
the counter)
o IRIS (Immune Reconstitution Inflammatory Syndrome)

40. • Immunological monitoring
CD4 testing should be done every 6 months
Virological monitoring: At 6 months and 12 months after ART initiation
and then every 12months

41. Adverse effects
• Each class and individual antiretroviral carries unique risks of adverse side effects.
NRTIs
The NRTIs can interfere with mitochondrial DNA synthesis and lead to
high levels of lactate and lactic acidosis, liver steatosis, peripheral
neuropathy, myopathy andlipoatrophy.
Current first line NRTIs such as lamivudine/emtrictabine, tenofovir,
and abacavir are less likely to cause mitochondrial dysfunction.
NNRTIs
NNRTIs are generally safe and well tolerated. The main reason for
discontinuation of drugs is neuro-psychiatric effects including suicidal
ideation. Nevirapine can cause severe hepatotoxicity, especially in
women with high CD4 counts.

42. Protease inhibitors
Protease inhibitors (PIs) are often given with ritonavir, a strong inhibitor
of cytochrome P450 enzymes, leading to numerous drug-drug
interactions. They are also associated with lipodystrophy, elevated
triglycerides and elevated risk of heart attack.
Integrase inhibitors
Integrase inhibitors (INSTIs) are among the best tolerated of the
antiretrovirals with excellent short and medium term outcomes. Given
their relatively new development there is less long term safety data.
They are associated with an increase in creatinine kinase levels and
rarely myopathy.

43. Other adverse effects
The NNRTI efavirenz causes insomnia, agitation, euphoria or
dysphoria in many patients but tolerance to its neuropsychiatric effects
develops in a few weeks in most patients.
The NRTI zidovudine can cause anaemia and neutropenia, and
tenofovir may cause nephrotoxicity and loss of bone mineral density.
Some PIs are associated with dyslipidaemias and may increase the risk
of myocardial infarction.

44. Prevention measures for HIV transmission
sexual
• • Sex education programmes in
schools
• • Easily accessible voluntary
counselling and testing centres
• • Promotion of safer sex practices
• • Effective ART for HIV-infected
individuals
• • Pre-exposure prophylaxis for high-
risk groups
• • Male circumcision
• • Post-exposure prophylaxis
Parenteral
• • Blood product transmission: donor
questionnaire, routine screening of
donated blood
• • Injection drug use: education,
needle/syringe exchange, avoidance
of ‘shooting galleries.,
Perinatal
• • Routine ‘opt-out’ antenatal HIV
antibody testing
• • Measures to reduce vertical
transmission (see text)
 Occupational
• Education/training: universal precautions, needle stick injury avoidance & Post-
exposure prophylaxis

45. ART complications
• Immune reconstitution inflammatory syndrome
• Lipodystrophy
• Hypersensitivity rashes

46. Issues related to ARV treatment
• Treatment is life long
• Complete adherence is essential
• Lack of adherence
• – Drug resistance / treatment failure
• – Client may have many difficulties
• – Client based strategies are important

47. Components of Palliative Care
The main components include:
1. Pain management
2. Symptomatic management
3. Nutritional support
4. Psycho-social support
5. Spiritual support
6. End of life care
7. counselling

48. THANK YOU...!