Serologic testing for HIV

HIV antibody assays by ELISA and Western blot

Antibodies to HIV proteins may be detected by various methodologies; the current standards are enzyme-linked immunosorbent
assays (ELISAs) for screening and Western blot (WB) for confirmation. Other acceptable methodologies are particle cell agglutination
for screening, and immunofluorescence and radioimmunoblot assay for confirmation. ELISA is highly sensitive but has been fraught
with the major issues of false positives resulting from insufficient specificity and false negatives mainly from low levels of antibody in
very early infection, the so-called window period (Box 3.1). With each successive generation of assays, the rate of false positives has
fallen and the window period shrunk significantly. This improvement owes itself to the replacement of viral lysate by recombinant
antigens and the use of double-antigen sandwich to enhance capture of IgM and IgG antibodies. Its overall performance
characteristics compare very favourably with the best diagnostic tests in other areas of medicine.

HIV p24 antigen assay

The p24 protein is a product of the gag gene and resides in the viral core. Prior to the availability of viral load testing, p24 was used
for monitoring disease activity as well as diagnosis. It is specific as a diagnostic test but falls short on sensitivity, as a negative test is
not uncommon in genuine HIV infection. In general, the level of p24 rises early soon after infection but subsequently falls as specific
antibody develops, only to rise again in the advanced stage of AIDS. Nevertheless, its presence generally precedes that of antibody
and will shorten the window period if tested positive. It is now incorporated into the current, fourth-generation ELISA antibody test
that is used by the Public Health Laboratory Centre. This generation of ELISA combines testing for both p24 and antibodies to HIV-1
and HIV-2, detects IgM as well as IgG, and has an average window period of only 16 days. The sensitivity is >99.5% and specificity
>99% to both B and non-B subtypes. However, there is the theoretical possibility of a brief second window period, during which p24
falls and HIV-antibody remains at low, undetectable levels.
Virology tests for HIV

Both HIV RNA and proviral DNA are amenable to assay. The major application of RNA assay in HIV medicine is in the measurement of
viral load, w h i c h is essential to gauge the efficacy of antiretroviral therapy. Proviral DNA is incorporated in host cells, of which
the peripheral blood mononuclear cells (PBMC) are the most accessible for testing. Both proviral DNA and HIV RNA may be tested
for diagnosis. HIV isolation in lymphocyte culture is also diagnostic but has a long turnaround time and places extraordinary demands
on facilities and expertise. These virologic tests are mainly used for diagnosis in infants and patients in the window period. The
term, Nucleic acid amplification test (NAT), is also used to denote virologic testing algorithms for further reduction of the window
period in donated blood.

Use of NAT in blood donor screening

Blood borne pathogens in donated blood may escape detection if only serologic markers are used for screening. With the concurrent
use of NAT, this window period for HIV detection is reduced to about 11 days. They are implemented as standard in many developed
countries, including Hong Kong, to assure the safety of blood supply. In light of the high sensitivity of current serologic assays, the
cost-effectiveness of NAT in routine HIV testing is doubtful and will depend highly on the prevalence of HIV in the population.12 It
must be noted that NAT complements HIV antibody test, and is rarely used in isolation.

Indications for a diagnostic HIV test

For individual diagnosis, the standard testing algorithm is the two-step use of ELISA for screening and WB for confirmation. The
requesting physician should ensure that all tests are done in a quality laboratory. An HIV diagnostic test is indicated in people at
risk of the infection, including persons with:

(a) Behavioural risks of and potential exposure to HIV infection, such as injecting drug use, any form of cocaine use,
male-male sex, multiple sexual partners, and commercial sex. All sex partners of persons with these risk factors are also
at risk.
(b) Occupational exposure to a possibly infected source, normally referring to that in the health care setting such as
after a needlestick injury.

(c) Other sexually transmitted infections (STI) - It is known that either ulcerative or non-ulcerative STI facilitates HIV
transmission.

(d) Clinical conditions associated with HIV infection, varying from classical AIDS-defining conditions to tuberc ulosis,
herpes zoster in a young patient, oral thrush, unexplained fever, and lymphadenopathy.

(e) Receipt of blood or blood products between 1978 and 1985, the year when screening of donated blood and organs
began in Hong Kong.