STDS- recent diagnosis [email protected]

STDS- HIV : laboratory
Investigations
Dr. Kamal Jung Shahi
2nd year Resident
Dermatology (NGMC-TH)

Purpose of HIV testing
Information is useful for prophylaxis, medical
management and treatment of HIV and related illnesses.
To assure blood safety and donation safety.
To monitor trends of epidemic (sentinel surveillance etc.)
Identification of asymptomatic individuals .
To plan personal and family’s future if the result is
positive.
To motivate for behaviour modification through
counselling
 To induce behaviour change and prevent transmission by
counselling in those who test positive.
To diagnose clinically suspected cases.
To assess efficiency of intervention.

World Health Organization (WHO) estimated
that inadequate screening of blood products had
lead to over 1 million infections with HIV
worldwide
 HIV testing must always be performed after
pretest counseling and informed consent
Confidentiality should be maintained at all times

Indication
Clinical features suggestive of HIV infection.
Patients with tuberculosis, especially in young
patients.
Patients having sexually transmitted diseases.
Antenatal Care (ANC) patients.
Hepatitis B and C coinfection .
History of high-risk behaviour/transfusion.

Classification
 Antibody based tests (Indirect Demonstration)
 Viral detection( Direct Demonstration)

Antibody based tests
Screening tests
 ELISA (2-3 hours)
 Rapid tests (within minutes)
Supplemental/ confirmatory
 Immunofluorescent assay (IFA)
 Western blot
 Line immuno assay (LIA)
 Radio immuno precipitation assay (RIPA)

Viral detection
P24 ANTIGEN CAPTURE ASSAY
HIV RNA Viral Load Test
● RT – PCR
● Branched DNA ( b – DNA )
● Nucleic acid sequenced based assay
(NASBA).

STDS- recent diagnosis methods@1223.pptx

ELISA / EIA
Most commonly used screening test
Highly sensitive.
Results can be quantitative or qualitative
Can give rise to false positive results
Antibody detected
6-12 weeks-Earlier generation
3-4 weeks- Newer generation

PRINCIPLES OF ELISA
• ELISA use an enzyme to detect the binding of antigen (Ag) and antibody
(Ab).
• The enzyme convert a colorless substrate (chromogen) to a colored
product, indicating the presence of Ag:Ab binding.
 On the basis of the principle of the test ELISA can be divided into:
i. Indirect
ii. Competitive
iii. Sandwich
iv. Capture assays
 ELISA based on indirect method is used in most of the kits

TYPES SOURCE OF Ag Ag/Ab Detection
First generation ELISA Cultured viral lysate Antibody
(Poor sensitivity and
specificity)
Second generation Elisa Recombinant Ag Antibody
(Detection of HIV-1 and
HIV-2)
Third generation Elisa Synthetic peptides Antibody
(Detection of IgM and IgG,
improved sensitivity )
Fourth generation Elisa Synthetic peptides +
Recombinant
Glycopeptides
Both antigen (p24) and
antibody

FALSE POSITIVE RESULTS
Autoimmune disorders
 Hematological malignancies (Plasmacytoma)
Connective tissue disorders
 Acute rheumatic fever
Multiple pregnancies
 Multiple transfusions
Post-vaccination including HIV vaccine
 Positive rapid plasma reagin test
Technical errors, etc.

False negative results
Window period (up to 3 months)
 Immunosuppressive therapy
Replacement transfusion
 B-cell dysfunction
 Malignant disorder
 Late stage disease (immune collapse)
Technical

RAPID TESTS
Yield results in < 30 min
The results are read by naked eye.
Based on agglutination and Elisa Principle.
Sensitivity and specificity of these assays are
comparable with ELISA.
TYPES :
1.Dot blot assays/ tridot
2. Particle agglutination
3. HIV spot and comb tests
4. Fluorimetric microparticle technologies

Advantages:
 Rapid HIV assays have proven particularly useful for testing
pregnant women in labor who have not received prenatal
care.
 Easy to perform
 Rapid
 Do not require sophisticated equipments, technical expertise
 cost-effective.
Disadvantages:
 Subjective interpretation
 Difficult to read if the laboratorian is color
blind.

Chemiluminescense
 This test is based on
Enzyme ImmunoAssay (EIA)
 Principle : emission of light
with limited emission of
heat as a result of a
chemical reaction.
 The final product formed in
this test produce
luminescence, which is
measured
 It is more sensitive method
than ELISA

WESTERN BLOT (WB)
It is a more specific assay.
Most commonly used confirmatory test.
Based on the fact that the antibodies against
HIV have different molecular weights.
The known antigen is separated using
polyacrylamide gel electrophoresis.

Creating Western Blot Strips
HIV lysate
proteins are
separated by
size using gel
electrophoresis
Proteins are
transferred
(blotted) onto the
surface of a
membrane
Strips are
incubated with
patient serum
and antihuman
IgG conjugated
with an enzyme
(and
chromagen)
The
membrane is
cut into strips

Interpretation
Negative - no bands
Positive - various criteria
Intermediate - bands present but doesnot satisfy
criteria
• When Western blot is indeterminate
• Repeat Western Blot in 4 – 6 weeks or HIV RNA
should be performed.

Criteria for a positive Western Blot
Organization Criteria
WHO 2 env with/without gag/pol
CDC Any two . p24, gp41, gp120/160

Advantages
 Specific interaction of antibody and antigen
can be directly visualized.
Disadvantages
 Technically demanding
 Expensive
 Subject to interpretation
• Presence or absence of bands
• Intensity of those bands

Lineimmuno assay (LIA)
 Recombinant or synthetic peptide antigens
are applied on a nitrocellulose strip
 This use of "artificial" antigens decreases the
presence of contaminating substances derived from
cell culture that can cause interference and
sometimes false reactions.
 Accuracy is equivalent to the Western blot.

Strategies of HIV testing in India
 Strategy I:
ELISA negative- HIV free
ELISA Positive- The unit of blood testing reactive (positive) is
discarded.
 Strategy II:
ELISA negative- HIV free
ELISA Positive- it is subjected to a second ELISA which utilizes a
system different from the first one
Used for surveillance and for diagnosis only if some AIDS indicator
disease is present.
 Strategy III:
It is similar to strategy II
Third reactive ELISA test
Asymptomatic individuals indulging in high risk behavior.

VIRAL DETECTION
 p24 antigen capture test
HIV RNA VIRAL LOAD TESTING
 HIV culture

Indication
 Acute HIV infection
 Indeterminate serology
 Neonatal infection

P24 ANTIGEN
The antigen test detects HIV free antigen
(p24) in the serum.
P24 is a core antigen which appears early
during the course of HIV
 Early as two weeks after infection
The same concept as in ELISA
Low sensitivity
Detects down to 15 pg/mL of p24 Antigen

This test is useful:
 During window period.
To detect HIV infection in newborn because
diagnosis is difficult due to presence of
maternal antibodies.
During late disease when patient is
symptomatic

HIV RNA VIRAL LOAD TESTING
a) RT – PCR
b) Branched DNA ( b – DNA )
c) Nucleic acid sequenced based assay (NASBA)
Sensitivity and specificity is above 96%
These kits shorten the window period
between infection and detectability to about
12 days.

INDICATIONS FOR VIRAL LOAD
TESTING
Early diagnosis of HIV infection
Diagnosis of HIV in neonates
Assess need for therapy
Monitor effectiveness of therapy
Determine prognosis

RT – PCR
Principle : PCR amplification of C dna
generated from viral RNA.
Detect up to 50 – 75 copies of HIV RNA / ml of
plasma.
Useful for amplifying defined areas of the HIV
genome for sequence analysis- sequence
diversity and microbial resistance to anti-
retroviral agents.

RT-PCR Process:
• Preparation of RNA.
• Conversion of RNA to cDNA using nucleotides,
reverse transcriptase and magnesium ions.
• Amplification of DNA using specific primers.
• Binding of amplified products to biotinylated probes
in microwell plate.
• Subsequent reaction with avidin-labelled
peroxidase.
• Use of standard and inbuilt control ensures
calculation of copies of virus per ml of plasma.
using Roche amplification assays viral load of 200
copies/ml can be detected. The sensitivity can be
further improved to 50 copies/ml.

HIV RNA by bDNA
 Measurement of levels of particle associated
 HIV RNA in a nucleic acid capture assay
employing signal amplification
 Reliable to 75 copies/mL of HIV RNA
 Research laboratories amplifying HIV proviral
DNA from peripheral blood mononuclear cells.

NUCLEIC ACID AMPLIFICATION TESTING (NAAT)
It amplifies the HIV viral RNA and detects viral
genes instead of viral antibodies or antigens
New HIV screening method
Reliable to 176 copies/mL of HIV RNA

ORASURE – (SALIVA) HIV TESTS
Noninvasively collected specimens of oral
fluids are used.
Fluid - oral mucosal transudate.
This system obtains antibodies that are
comparable to or exceed those from serum
samples.
This test first employs ELISA, and then WB.

ORAQUICK ADVANCE RAPID HIV TEST
It provides results in 20 min.
The blood, plasma, or oral fluid is mixed in a
vial with developing solution
 The results are read from a stick-like testing
device.

URINE TESTS
Intact IgG antibodies
The collection of urine is simple, noninvasive,
and inexpensive.
The USA FDA has approved an ELISA and WB
for use to test urine for antibodies to HIV-1.

HIV DIAGNOSIS IN NEWBORNS
 If DNA PCR is positive within the first 48 hours of life, it
indicates in-utero infection.
 In intrapartum infection, PCR is negative at 48 hours and
positive within one month.
 Following methods can be done:
I. Demonstration of IgA and IgM antibodies: Presence of
IgA at 3 months of age using western blot test sensitivity
is 97.6%
II. Estimation of p24 antigen: p24 is core antigen that
appears early in course of infection. So use for diagnosis
during window period.
III. In vitro antibody production: Involves culture of B- cells
and the supernatant is tested for production of
antibodies against HIV viral proteins.

HIV DIAGNOSIS IN NEWBORNS
In case of neonatal diagnosis, PCR should be
done at 48 hours, 1 week, 3 months, and 6
months.
It should always be confirmed by serology at
18 months by ELISA.

LABORATORY DIAGNOSIS DURING WINDOW
PERIOD
 PCR
 p24 antigen assay
 Viral culture
Nucleic acid amplification test

Need of laboratory diagnosis in window phase
Following untested blood transfusion.
Risky heterosexual/homosexual exposure.
 Needle stick injury (contaminated

Markers for assessment and
monitoring of therapeutic response
CD4 T cell count
HIV RNA levels

CD4 T cell count
Measured as a product of the percentage CD4
cells and the total lymphocyte count
Best indicator of immunological function of
the patient.
CD4 T cells < 200/µl AIDS defining condition
< 350/µl indication to start ART
< 50/µl Prophylaxis for MAC
(mycobacterium avium complex)

CD4 counts should be performed at time of
diagnosis and 3 – 6 months there after.
Adequate viral suppression is defined as an
increase in CD4 Cell counts 100 – 150 cells/mm3
per year.
A decline in CD4 Cells by 25% is an indication to
change ART

HIV RNA levels
Should be measured
Before initiation of therapy
2 – 8 weeks after initiation or any change in
therapy
Once HIV RNA levels has stabilized then monitor
every 3 – 4 months
Effective therapy: decrease in viral load below
50 copies/ml – in 16 – 24 months

Drug resistance testing
 Measures the sensitivity of HIV virions to different anti-
retrovirals
Genotypic assays:
 Detects resistance mutations.
 Involves sequencing of the entire virus.
 Genome of the patient is then compared with sequences of
viruses with known antiretroviral resistance .
Phenotypic assays:
 Measures the ability of the virus to grow at different drug
concentrations
 The rate is compared with growth rate of reference strains

CDC recommendations for drug
resistance testing
In acute / chronic HIV infection when patient
enters into care.
Virologic failure / suboptimal viral
suppression.
HIV infected pregnant women.

Refrence :
• Laboratory diagnosis of HIV. Sharma A,
Marfatia YS. Article 2005.
• Bolognia text book
• Rooks text book of dermatology 9th edition
2016.
THANK YOU

STDS- recent diagnosis [email protected]

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