sEMINAR ON LAB DIAGNOSIS OF HIV

1. LABORATORY DIAGNOSIS AND
TRANSMISSION OF HIV INFECTION.

2. OUTLINE
 INTRODUCTION
 LITERATURE REVIEW
 MORPHOLOGY
 EPIDEMIOLOGY
 MODE OF TRANSMISSION
 PATHOGENESIS
 SIGNS AND SYMPTOMS
 LABORATORY DIAGNOSIS
 PREVENTION AND CONTROL
 MANAGEMENT
 CONCLUSION
 RECOMMENDATION
 REFFERENCES

3. INTRODUCTION
HIV stands for Human Immuno Virus is a type of virus that
attacks the immune system, which is the body’s defense
against infections and disease.
HIV specifically attacks CD4 (Cluster of Differeciation ) cells
which are type white blood cell that plays crucial role in
helping the body fight infections and disease.
Upon entering into the system HIV target and infiltrates the
CD4 cells and produces more copies of itself, thereby
reducing the immune system’s ability to combat infection.
Delaney KP et al., 2017

4. INTRODUCTION CONT’D
If left untreated HIV infection leads to AIDS (Acquired Immune-
deficiency syndrome) which is an advanced stage of the
infection.
At this stage the patient would have a CD4 count <200 cells
per cubic millimeter, thereby making the body vulnerable to
other opportunistic infected as a result of the depleted CD4
count.
Delaney KP et al., 2017

5. INTRODCTION CONT’D
HIV has a worldwide distribution and it is transmitted via two
route:
 Vertical route (from mother to child)
 Horizontal route (sexual intercourse, blood transfusion Etc)
HIV can be diagnosed in the Laboratory qualitatively or
quantitatively and early detection is key in the
management of the virus.
They is no specific drug used in the treatment of the virus but
the virus can be managed by the use of some Antirectro
viral drugs
Douek DC et al., 2019

6. LITERATURE REVIEW
•HIV infection came from a type of chimpanzee in central
Africa as far back as 1980s. The chimpanzee version of the
virus is called simian immunodeficiency virus. Over decades
the HIV slowly spread from Africa to other part of the world
(CDC,2017)
•The human immunodeficiency virus (HIV) is a lentivirus
(family of retroviruses) that targets the helper T cells (CD4
lymphocytes) (Cunningham et al., 2022).
•HIV has two subtypes: HIV-1 and HIV-2. Among these strains,
HIV-1 is the most virulent and pathogenic.
• AIDS was first recognized in the United States in 1981
following a sudden outbreak of opportunistic infections,
including Pneumocystis carinii pneumonia and Kaposi’s
sarcoma (KS) among homosexual men (Durack, 1981;
Gottlieb et al., 1981; Masur et al., 1981)

7. LITERATURE REVIEW CONT’D
• HIV is distributed worldwide and it is transmitted via two
route: Vertical route (from mother to child) Horizontal
route (sexual intercourse, blood transfusion, seminal fluid
breast milk, vaginal fluid, pre-ejaculate etc.) (Masciotra et
al., 2016)
• They is no specific drug used in the treatment of the virus
but the virus can be managed by combination of two or
more Antirectro viral drugs
• An estimated 39.0 million people are infected with HIV
globally. Approximately 86% of people with HIV knew their
HIV status in 2022.
• The remaining 14% (about 5.5 million people) did not know
they had HIV and still needed access to HIV testing services.
HIV testing is an essential gateway to HIV prevention,
treatment, care, and support services.

8. MORPHOLOGY
HIV is a spherical, enveloped
virus, which measures up to
120 nm in diameter It has a
unique three-layered
structure:
 The innermost genome
layer
 Middle cone-shaped
nucleocapsid
 An outer membrane of
glyco-protein surrounded by
lipoprotein envelope
Powell MK et al., 2016

9. EPIDEMIOLOGY
Globally,
• An estimated number of 39.0 million people were living with HIV at
the end of 2022.
• 1.5 million children (0–14 years old).
HIV incidence
• 1.3 million people acquired HIV in 2022. Since 2010, the number of
people acquiring HIV has been reduced by 38%, from 2.1 million to
1.3 million people.
• 130 000 children acquired HIV in 2022.
HIV-related mortality
• In 2022, 630 000 people died from HIV-related causes globally.
Since 2010, HIV-related deaths have been reduced by 51%, from 1.3
million. The global HIV epidemic claimed 69% fewer lives in 2022
since the peak in 2004.
• 84 000 children died from HIV-related causes in 2022.
HIV continues to be a major global public health issue, claiming 40.4
million lives so far
(WHO 2022).

10. EPIDEMIOLOGY CONT’D

11. EPIDEMIOLOGY CONT’D
NIGERIA HIV EPIDEMIOLOGY
In 2022, 1.9 million people in Nigeria were living with HIV. Women
were the most affected group, counting 1.1 million individuals.
Also, children up to age 14 who were HIV positive equalled 170
thousand.
1.9 million people with HIV
1.3% adult HIV prevalence
74,000 new HIV infections
51,000 AIDS-related deaths
1.7 million people on antiretroviral treatment
(National Agency for the Control of AIDS (NACA) 2021).

12. Akwa Ibom 5.6%
Benue 4.9%
Rivers 3.8%
Taraba 2.7%
Anambra 2.4%
Enugu 2.1%
Abia 2.1%
Delta 1.9%
Nasarawa 1.9%
Edo 1.8%
Bayelsa 1.8%
Cross River 1.7%
Imo 1.6%
Plateau 1.5%
FCT 1.5%
Lagos 1.3%
Borno 1.3%
Adamawa 1.3%
Ogun 1.2%
Gombe 1.2%
Kaduna 1.0%
Kogi 1.0%
Kwara 1.0%
Ondo 0.9%
Osun 0.9%
Oyo 0.9%
Ebonyi 0.8%
Niger 0.7%
Ekiti 0.7%
Kebbi 0.6%
Kano 0.5%
Zamfara 0.5%
Yobe 0.4%
Bauchi 0.4%
Sokoto 0.4%
Jigawa 0.3%
Katsina 0.3%
HIV PREVALENCE IN DIFFERENT STATES IN NIGERIA
2022/2023

13. NIGERIA HIV EPIDEMIOLOGY
EPIDEMIOLOGY CONT’D

14. EPIDEMIOLOGY CONT’D

15. MODE OF TRANSMISSION
Transmission of HIV infection: HIV infection occurs either by
the transfer of HIV-infected cells or free HIV not associated
with cells.
HIV is transmitted through two route
 Vertical Route: mother-to-child transmission in pregnancy
 Horizontal Route:
• Unprotected sexual contact is the most common means of
transmission.
• blood transfusion
• breast milk
• sharing of needles in IV drug use
•breastfeeding. Etc
Branson BM et al.,

16. TRANSMISSION OF HIV

17. PATHOGENESIS
•HIV is primarily a sexually transmitted pathogen transmitted
by high-risk behaviours, such as unprotected intercourse,
male homosexual intercourse, and also by intravenous (IV)
drug abuse.
•HIV infects the cells of the immune system and destroys
them or makes them ineffective.
• The tropism of the HIV for CD4-expressing T-cells and
macrophages is the principal determinant of the
pathogenicity of HIV
Hurt CB et al., 2021

18. PATHOGENESIS CONT’D
• HIV shows tropism for all the cells expressing CD4 antigens on
their cell surfaces. The CD4 antigens act as receptors for HIV.
The virus infects helper T cells and kills them, resulting in
HIV-induced immunosuppression, leading to full-blown AIDS—a
key feature of the pathogenesis of HIV infection. This makes
the patient most susceptible to opportunistic infections and
certain cancerous conditions, such as Kaposi’s sarcoma and
lymphoma. However, the virus does not directly cause any
tumor, because HIV genes are not found in these tumor cells
Hurt CB et al., 2021

19. PATHOGENESIS CONT’D
.•Without antiretroviral treatment the disease typically advances
through several phases:
•First stage: rapid and transient decline in the number of CD4
lymphocytes;
•Progression over anywhere from a few months to more than 10
years: CD4+ T cell numbers slowly decline to below a critical
level and the immune system can no longer fight opportunistic
infections.
•Final stage: Onset of full-blown AIDS (Acquired Immune
Deficiency Syndrome), marked by infection with one or more
HIV-associated opportunistic infections or cancers and
ultimately death.
Delaney KP et al ., 2019

21. SIGNS AND SYMPTOMS

22. LABORATORY DIAGNOSIS
The laboratory diagnosis of HIV infection can be categorized into
the following;
 Qualitative assay: These help to determine whether HIV
antibodies or antigen is present in a blood specimen. It involves
the use of several test kit (Determine, Uni-Gold, stat pack),
ELISA (Enzyme linked immunosorbent assay)
 Quantitative assay: These involves measuring the amount of
the virus present (viral load) in the blood specimen. It involves
the use of PCR (Polymerase Chain Reaction)

23. CDC,2016
LABORATORY DIAGNOSIS CONT’D
Qualitative assay
Serial algorithm

24. LABORATORY DIAGNOSIS CONT’D
• PRINCIPLE FOR DETERMINE
The test check for the presence of HIV antibodies. During
testing the whole blood, serum, or plasma specimen reacts
with HIV antigen coated particles in the test strip. The
mixture then migrates upward on the membrane
chromatographically by capillary action and reacts with
recombinant HIV antigen on the membrane in the test line
region.
MATERIALS REQUIRED
• Blood sample (capillary blood or venous blood)
• Test kit
• Timer
• Marker pen
• Buffer

25. LABORATORY DIAGNOSIS CONT’D
PROCEDURE FOR DETERMINE
• Label the patient identification number on the test strip
• Pull off the protective foil cover
• Collect 50ul of blood sample using Pasteur pipette
• Apply the specimen to the absorbent part of the strip
• Add 1 drop of buffer to the specimen
• Set the timer to 15 minutes
• Read and record the result

26. LABORATORY DIAGNOSIS CONT’D
CWI,
2019

27. LABORATORY DIAGNOSIS CONT’D
PROCEDURE FOR UNI GOLD
• Label the patient identification number on the test strip
• Collect specimen using disposable pipette
• Add 2 drops (approx 60ul) of the buffer in the sample port
• Wait for 15 minutes (no longer than 20 minutes)
• Read and record the result

28. LABORATORY DIAGNOSIS CONT’D

29. LABORATORY DIAGNOSIS CONT’D
PROCEDURE FOR STAT PAK
• Label the patient identification number on the test
strip
• Collect approximately 5ul 0f the specimen using a new
disposable loop
• Dispense the sample in the center of sample well
• Add 3 drops of buffer, holding vertically over the
sample well
• Wait for 15 minutes before reading the result
• Read and record the result

30. LABORATORY DIAGNOSIS CONT’D

31. LABORATORY DIAGNOSIS CONT’D
ELISA
• Elisa principle; The ELISA HIV test detects HIV antibodies in a
person's blood. It involves coating a solid surface with HIV
antigens, adding the blood sample to allow binding of
antibodies, washing to remove unbound substances, adding a
secondary antibody linked to an enzyme that binds to HIV
antibodies, adding a substrate for the enzyme, and measuring
the resulting signal to determine if HIV antibodies are present
(positive) or not (negative).

32. LABORATORY DIAGNOSIS CONT’D
ELISA PROCEDURE
• COATING: A micro plate or solid surface is coated with a
specific antibody that is known to bind to the target HIV
antigen. This antibody is referred to as the "capture
antibody."
• SAMPLE ADDITION: The patient's blood sample is added to the
coated micro plate. If HIV antigens or antibodies are present
in the sample, they will bind to the capture antibody, forming
an antigen-antibody complex.
• WASHING: After a short incubation period, the micro plate is
washed to remove any unbound substances and contaminants,
leaving only the captured antigen-antibody complexes
attached to the plate.
Powell MK et al.,2017

33. LABORATORY DIAGNOSIS CONT’D
• DETECTION OF ANTIBODY: A second, different antibody that
is specific to a different epitope of the HIV antigen is added
to the well. This antibody is called the "detection antibody." It
is conjugated with an enzyme, such as horseradish peroxidase
(HRP) or alkaline phosphatase (AP).
• BINDING: The detection antibody binds to the captured
antigen-antibody complexes, forming a "sandwich" structure.
This sandwich complex consists of the capture antibody bound
to the HIV antigen and the detection antibody bound to a
different region of the same HIV antigen.
Powell MK et al.,2017

34. LABORATORY DIAGNOSIS CONT’D
• WASHING: Any unbound detection antibodies are washed
away, leaving only the sandwich complexes attached to the
micro plate.
• SUBSTRATE ADDITION: A substrate specific to the enzyme
conjugated to the detection antibody is added ( TMB
3,3’,5,5’a Tetramethylbenzidine). The enzyme reacts with
the substrate, producing a detectable signal, typically a
colored product.
• SIGNAL MEASUREMENT: The intensity of the generated signal
is measured using a spectrophotometer or other appropriate
detection instrument. The signal's intensity is proportional to
the amount of HIV antigen or antibody present in the patient's
blood sample.
Powell MK et al.,2017

35. LABORATORY DIAGNOSIS CONT’D
ADVANTAGES OF ELISA
• Specific and Sensitive
• Equipment cheap and easily available
• Reagents “Cheap”, long shelf life
• Potential for automation
• No radiation hazards

36. LABORATORY DIAGNOSIS CONT’D
DISADVANTAGES OF ELISA
• Contamination
• Expertise required to label and purify conjugates
• Susceptible to interference from non-specific factors

37. LABORATORY DIAGNOSIS CONT’D
PRINCIPLE OF PCR
• The PCR technique is based on the enzymatic replication of
DNA. In PCR, a short segment of DNA is amplified using primer
mediated enzymes. DNA Polymerase synthesizes new strands
of DNA complementary to the template DNA.
PROCEDURE
• DENATURATION- To amplify DNA, the two strands of the
template DNA first have to be separated. This occurs by
heating the dsDNA template to 95'C at this point the hydrogen
bonds break between the base pairs. This results in the
separation of the two DNA strands.

38. LABORATORY DIAGNOSIS CONT’D
• ANNEALING- The temperature is then dropped to 55'C in
which the forward and reverse primers are stable. At this
temperature the primers can anneal to the single stranded
DNA template strands. DNA polymerase is also stable at this
temperature and can bind to the primers.
• EXTENSION- The temperature is then raised slightly to Taq
polymerase’s ideal temperature (70-75oC). At this
temperature Taq polymerase can synthesize and elongate the
target DNA quickly and accurately

39. LABORATORY DIAGNOSIS CONT’D

40. LABORATORY DIAGNOSIS CONT’D
ADVANTAGES 0f PCR
• Highly specific
• Highly sensitive
• Rapid and efficient
• Versatile
DISADVANTAGES OF PCR
• Contamination
• Errors in amplification
• Cost and complexity

41. PREVENTION AND CONTROL
• Safe Sex Practices: Use condoms consistently and correctly
during sexual activity to reduce the risk of transmission.
• HIV Testing: Get tested regularly, especially if sexually active,
to know your status and seek early treatment if positive.
• Pre-Exposure Prophylaxis (PrEP): For individuals at high risk
of HIV, PrEP medication can be taken daily to reduce the
chances of getting infected.
• Treatment as Prevention (TasP): People living with HIV
should start antiretroviral therapy (ART) as soon as possible,
which not only improves their health but also lowers the viral
load, reducing the risk of transmitting the virus.
Pierce VM et al., 2011

42. PREVENTION AND CONTROL CONT’D
• Needle and Syringe Programs: Providing clean needles and
syringes to injection drug users helps prevent HIV transmission
through sharing of contaminated needles
• Education and Awareness: Raising awareness about HIV/AIDS,
how it spreads, and how to prevent it is crucial in combating
the stigma and promoting safe behaviors
Pierce VM et al., 2011

43. PREVENTION AND CONTROL CONT’D
• Eliminating Mother-to-Child Transmission: Ensuring proper
medical care and treatment for pregnant women with HIV can
significantly reduce the risk of transmission to their babies.
• Promoting Health Equity: Addressing social determinants of
health and ensuring access to healthcare for vulnerable and
marginalized populations can contribute to HIV prevention
and control
Pierce VM et al., 2011

44. MANAGEMENT
• Antiretroviral therapy (ART) reduces the amount of HIV in the
blood (called viral load), reduces HIV related illness and
prevents transmission to others.
• People with HIV should take treatment as prescribed to avoid
transmission of the infection to others.
Kuhar DT et al., 2013

45. MANAGEMENT CONT’D
• Various antiretroviral drugs are used in different
combinations, and the treatment plan depends on factors such
as patients age , overall health, presence of coexisting
conditions, and the stage of the HIV infection.
• Regular monitoring of viral load and CD4 count help asses
treatment effectiveness and make necessary adjustment.
Kuhar DT et al., 2013

46. CONCLUSION
• HIV stands for Human Immunodeficiency, Virus is a type of
virus that attacks the immune system, which is the body’s
defense against infections and disease.
• HIV can be transmitted Vertically (from mother to child) and
Horizontally route (sexual intercourse, blood transfusion Etc)
• If not well managed HIV infection can leads to AIDS (Acquired
Immunodeficiency syndrome) which is an advanced stage of
the infection.
• sexually active individuals should be advised to use condoms,
and also know their HIV status and seek early treatment if
positive.
• The PCR is used in monitoring the progression of the virus and
the effectiveness of the Antiretroviral therapy.
Masciotra SQ et al., 2013

47. RECOMMENDATION
• Sexually active individual who cannot abstain from sex should be
advised to play safe ( by the use of condoms) and to maintain a
single sex partner.
• Health practitioners should be advised to always put on their
personal protective equipment (PPE) while working on infectious
samples.
• Cleaning, hygiene and proper waste management should be ensured
in the medical laboratory.
• Regular public awareness programs should be organized.
• Those infected with the virus should access their healthcare
providers for Regular monitoring of viral load and CD4 count help
asses treatment effectiveness and make necessary adjustment.
• FMC keffi should upgrade the fan VCT laboratory to Air condition,
to avoid spread of pathogens to those using the facility

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49. REFERENCE CONT’D
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50. REFERENCE CONT’D
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51. THANKSFORLISTENING