DIAGNOSIS OF HIV

• All infants born to HIV positive mothers test antibody positive at birth because of passive transfer of HIV antibody across the placenta

• Seroreverters : Most uninfected infants without ongoing exposure lose maternal antibody by 6-12 months of age

• Less than 18 months : Diagnosis can also be made by the presence of anti HIV Ig A or Ig M in the infants circulation

• However anti HIV Ig A or Ig M both are nonspecific and insensitive

• There are 3 tests by which HIV in children less than 18 months can be diagnosed

1 HIV DNA PCR 2 HIV RNA PCR 3 HIV CULTURE

• HIV DNA PCR is the preferred technique 40% have positive results within 1st two

days of life >90% have positive results within 14 days

of life

• Highly sensitive and specific

• HIV CULTURE: - as sensitive as HIV DNA PCR - expensive and not easily available - requires upto 4 wks to do the test

• HIV RNA PCR: - less sensitive than HIV DNA PCR - negative results cannot be used to exclude

HIV infection

Less than 6 months old

Send DBS of babies b/w 6 weeks – 6 months

HIV – 1 DNA detected

Send Whole Blood HIV DNA PCR

HIV DNA detected Not Detected

Infant is HIV 1 Infected

Follow advisory 2

Send another sample for whole blood DNA PCR

Not detected

Follow advisory 3

Detected

Not detected : follow next slide

HIV DNA not detected on DBS

• If child develops symptoms of HIV < 6 months, repeat DBS• If asymptomatic, then repeat DBS at 6 months of age

HIV detected Not detected

Send whole blood for HIV DNA PCR

HIV detected

Infant is HIV infected

Continue Advisory 2

Not detected

BF in 6 weeks before test

Follow advisory 3

Not BF in last 6 weeks

Child is HIV non infected

Child of 6-18 months born to HIV positive mother

Do Rapid HIV test and prepare DBS for HIV-1 DNA PCR

Rapid test positive

Send DBS of child for HIV-1 DNA PCR

HIV – 1 DNA detected

Follow advisory 1Send Whole Blood Specimen for HIV-1 DNA PCR

HIV -1 DNA detected

Not Detected

Infant is HIV-1 Infected

Advisory 2

Not Detected

Repeat Whole Blood HIV-1 DNA PCR

HIV -1 DNA detected

Infant is HIV-1 Infected

Not Detected

Rapid test negative

Breastfeed in the 6 weeks before test

• Infant is probably not infected, but at high risk• Repeat Rapid HIV test 6 weeks after last breast milk feeding or If the child develops symptoms of HIV infection• If Rapid test positive, follow previous chart.• If negative, repeat Rapid test at 12 months• Continue Cotrimoxazole until definitely negative

Not Breast feed

Infant is HIV-1 uninfected

• Stop Cotrimoxazole• Discontinue ART if started • Avoid putting baby to Breast

• ADVISORY 1

• Start Cotrimoxazole if not already started

• Assess and encourage Breast Feeding if replacement feeding not started

• ADVISORY 2

• Continue Cotrimoxazole

• Manage OI

• Start ARV Therapy

• If Breast Feed continue BF and avoid mixed feeding

• ADVISORY 3

• Infant is probably not infected but is at risk

• Repeat HIV DNA PCR by DBS at 6 months, 6 weeks after last BF and if child develops symptoms of HIV

•Continue Cotrimoxazole until definitively negative

• After 18 months diagnosis can be made by 1 ELISA 2 western blot

ELISA Testing

• First serological test developed to detect HIV infection.– Easy to perform.– Easily adapted to batch testing.– Highly sensitive and specific.

• Antibodies detected in ELISA include those directed against: p24, gp120, gp160 and gp41, detected first in infection and appear in most individuals

ELISA Testing

• ELISA tests useful for:– Screening blood products.– Diagnosing and monitoring patients.– Determining prevalence of infection.– Research investigations.

Western Blot• Most popular confirmatory test.– Utilizes a lysate prepared from HIV virus.– The lysate is electrophoresed to separate out the HIV

proteins (antigens).– The paper is cut into strips and reacted with test sera.– After incubation and washing anti-antibody tagged with

radioisotope or enzyme is added.– Specific bands form where antibody has reacted with

different antigens.– Most critical reagent of test is purest quality HIV antigen.– The following antigens must be present: p17, p24, p31,

gp41, p51, p55, p66, gp120 and gp160.

Western Blot

• Antibodies to p24 and p55 appear earliest but decrease or become undetectable.

• Antibodies to gp31, gp41, gp 120, and gp160 appear later but are present throughout all stages of the disease.

Western Blot

• Interpretation of results.– No bands, negative.– In order to be interpreted as positive a minimum

of 3 bands directed against the following antigens must be present: p24, p31, gp41 or gp120/160.

• CDC criteria require 2 bands of the following: p24, gp41 or gp120/160.

Indirect Western immunoblot for HIV diagnosis

Two or more bands are required to be considered positive.