L12 Public Health HIV - Epidemiology, clinical care and microbiology overview

Overview

This lecture covers how HIV infection is diagnosed in the lab, how antiretroviral drugs target each stage of the HIV replication cycle, why HIV develops drug resistance so readily and why combination therapy is required, and the current approaches to preventing transmission and the barriers to a cure.

Diagnosis of HIV

After transmission, the three diagnostic markers appear in a fixed order: HIV RNA rises first and peaks during acute HIV syndrome, p24 antigen rises shortly after and peaks slightly later, and antibody appears last, during the window period, then stays high through most of infection before declining late in disease. The window period is the gap before a marker becomes detectable.

Four generations of HIV immunoassay exist:

• 1st generation (IgG-sensitive): anti-IgG detection reagent on a crude HIV lysate coating; detects IgG only.
• 2nd generation (IgG-sensitive): anti-IgG reagent on recombinant HIV proteins/peptides; detects IgG only.
• 3rd generation (IgM-sensitive): a “sandwich” EIA using an HIV protein reagent; detects both IgM and IgG.
• 4th generation (antigen-antibody combination): HIV protein and anti-p24 antibody reagents; detects IgG and IgM antibody plus p24 antigen.

Because it also detects p24 antigen, the 4th generation assay shortens the window period by about 5 days compared to a 3rd generation (antibody-only) assay.

Rapid point-of-care tests give an immediate result and, with a skilled operator, perform similarly to EIA. Problems: subjective reading, lack of automation, visual problems such as colour blindness, and potential technical error. The oral fluid rapid test (OraQuick) has an increased false positive rate.

Real-time PCR (RT-PCR) is used to quantify HIV RNA. Reverse transcriptase first converts the RNA template to cDNA (RNA-dependent DNA polymerase activity), its RNase H activity degrades the original RNA strand, then DNA-dependent DNA polymerase activity produces double-stranded cDNA. This cDNA is then amplified by standard PCR cycling (denaturation, annealing, extension), with each cycle roughly doubling copy number so that one starting copy becomes about 1.1x10^12 copies after 40 cycles. In real-time PCR, a fluorescent probe with a quencher is bound during annealing and cleaved during extension, releasing the fluorescent signal; the more starting virus copies present, the fewer cycles are needed to cross the fluorescence detection threshold (Ct). A sample starting with 10^8 copies crosses threshold around cycle 18, versus around cycle 34 for a sample starting with only 10^3 copies.

The CDC testing algorithm: an HIV-1/2 antigen/antibody combination immunoassay is run first. If negative, the result is reported as negative for HIV-1/2 antibodies and p24 antigen. If positive, a second immunoassay differentiates HIV-1 from HIV-2 antibodies. If this differentiation assay is negative or indeterminate for both, and acute infection is suspected, HIV-1 RT-PCR is used to confirm or exclude acute HIV-1 infection; otherwise serology is repeated in 2-3 weeks.

Testing in special circumstances:

• Blood donation screening needs high sensitivity, so both HIV RT-PCR and anti-HIV-1/2 immunoassay are used.
• Antenatal screening has a low pre-test probability, so an anti-HIV-1/2 immunoassay is used first, with repeat immunoassay and/or RT-PCR on a new sample if reactive.
• Infants cannot be tested by serology alone because transplacental maternal antibody is present, so HIV RT-PCR is used.

Summary: initial testing is a combined antigen/antibody (4th generation) immunoassay, confirmed with a second immunoassay that differentiates HIV-1 from HIV-2; rapid tests are available but need confirmation; quantitative HIV RNA RT-PCR is used for acute infection, blood bank screening, resolving positive antenatal serology (not essential), infants, and monitoring treatment.

HIV Replication Cycle and Antiretroviral Drug Classes

The HIV replication cycle proceeds through attachment, fusion, uncoating, reverse transcription, nuclear import, integration, transcription, nuclear export, translation (producing Gag/Pol, Gag, and the accessory proteins Vpu, Vif, Rev, Tat), assembly, budding, release, and maturation. Each major antiretroviral drug class blocks a different step:

• Entry inhibitors act at attachment/fusion. CCR5 inhibitors (e.g. maraviroc) prevent gp120 binding to the CCR5 co-receptor. Fusion inhibitors (e.g. enfuvirtide) are peptide analogues of the gp41 fusion domain that block membrane fusion.
• Reverse transcriptase inhibitors (RTIs) act at reverse transcription and are split into nucleoside analogues (NRTIs) and non-nucleoside inhibitors (NNRTIs). NRTIs such as zidovudine (AZT), a thymidine analogue, compete with natural dNTPs for HIV RT; because AZT lacks the 3’-OH group that thymidine has, its incorporation into the growing DNA strand causes chain termination. NNRTIs such as efavirenz and nevirapine instead bind a hydrophobic pocket near the RT catalytic site, blocking DNA polymerisation without being incorporated into the DNA.
• Integrase inhibitors (INSTIs, e.g. raltegravir) act at integration, preventing HIV cDNA from being inserted into the host genome.
• Protease inhibitors (e.g. nelfinavir) act at maturation. Gag and Pol require cleavage by HIV protease for virus assembly into an infectious virion; protease inhibitors are substrate analogues that bind the enzyme’s active site and block this cleavage.

Drug Resistance and the Need for Combination Therapy

HIV mutates at a high rate because both reverse transcriptase and host RNA polymerase II lack proofreading ability, giving roughly 0.25 mutations per cycle of replication. Combined with a high rate of viral turnover, about 10^10 new virions per day, this generates many variants each day and an accumulating population of viral variants known as a quasispecies. Multiple different mutations in HIV protease can each independently confer drug resistance.

Because resistant variants already exist at low frequency before treatment starts, monotherapy selects for them: as a single drug suppresses the wild-type virus, a pre-existing variant resistant to that one drug is not suppressed and comes to dominate the population, while variants resistant to other drugs remain unaffected. Starting three antiretroviral drugs together instead means each drug is active against the variants resistant to the other drugs, so no single pre-existing or emerging resistant variant can escape all three drugs simultaneously, allowing complete viral suppression.

Even with successful suppression, lifelong treatment is required: stopping treatment leads to rapid viral rebound from a latent reservoir, with plasma HIV RNA rising sharply and CD4 count falling within weeks of interruption. This latent reservoir, which current antiretroviral drugs do not purge, is the major barrier to a cure.

Prevention

• Behavioural measures, e.g. condom use.
• Circumcision reduces risk of infection by about 58%.
• Antenatal screening and treatment.
• Treatment as prevention (reducing infectivity in treated individuals).
• Post-exposure prophylaxis (PEP): started within 72 hours of exposure, continued for 4 weeks.
• Pre-exposure prophylaxis (PrEP): a daily oral pill gives about 75% (range 45-86%) protection depending on adherence; a long-acting injectable form (dosed monthly to twice yearly) is highly effective, giving 79-100% efficacy compared with oral PrEP.
• Microbicides: a dapivirine vaginal ring (monthly use, approved in South Africa in March 2022) reduces transmission by 27% to over 50%.
• Vaccines: experimental vaccines have been tested in 9 clinical efficacy trials to date with no significant protection shown.

Summary points

Antiretroviral drugs can inhibit HIV replication at multiple stages of the life-cycle, but the high mutation rate selects for resistant mutants, so three-drug regimens are required to prevent resistant clones emerging (mono- or dual-therapy fails rapidly, though a two-drug regimen containing an integrase inhibitor can be effective). Antiretroviral therapy (ART) has revolutionised patient outcomes and also contributes to prevention, through prevention of mother-to-child transmission, PEP and PrEP, and reduced infectivity in treated individuals. There is no effective vaccine on the horizon, and the latent reservoir remains the main barrier to cure.

Self-test

1. List the three HIV diagnostic markers in the order they become detectable after transmission, and define the window period.
2. Distinguish the four generations of HIV immunoassay by what they detect.
3. Why does a 4th generation immunoassay shorten the window period compared with a 3rd generation assay?
4. List two limitations of rapid point-of-care HIV tests.
5. Describe the steps by which reverse transcriptase converts HIV RNA into double-stranded cDNA.
6. Explain how a real-time PCR Ct value relates to the starting number of virus copies in a sample.
7. Outline the CDC testing algorithm from initial immunoassay to confirmation of acute HIV-1 infection.
8. For each of blood donation screening, antenatal screening, and infant testing, state which test(s) are used and why.
9. Name the antiretroviral drug class that acts at each of these HIV life-cycle steps: entry/fusion, reverse transcription, integration, maturation.
10. Explain the structural basis for chain termination by zidovudine (AZT), and contrast this mechanism with how NNRTIs work.
11. Explain why HIV develops drug resistance so readily, referring to mutation rate and viral turnover.
12. Describe what happens to wild-type and resistant viral populations under monotherapy versus triple-drug therapy, and explain why triple therapy prevents resistance emerging.
13. Why is lifelong antiretroviral treatment required even after viral suppression is achieved?
14. Compare the efficacy of oral PrEP with long-acting injectable PrEP, and state the effect of circumcision on infection risk.

Answers

Reveal answers

1. RNA rises and peaks first (during acute HIV syndrome), p24 antigen rises and peaks shortly after, and antibody appears last (during the window period) and stays elevated through most of infection. The window period is the time after transmission before a given marker becomes detectable.
2. 1st generation detects IgG only, using a crude HIV lysate coating; 2nd generation detects IgG only, using recombinant HIV proteins; 3rd generation detects both IgM and IgG, using a sandwich EIA format; 4th generation detects IgG and IgM antibody plus p24 antigen.
3. Because it also detects p24 antigen, which appears in blood before antibody does, a 4th generation assay can pick up infection earlier than an antibody-only 3rd generation assay, shortening the window period by about 5 days.
4. Any two of: subjective reading, lack of automation, visual problems such as colour blindness, potential for technical error, or increased false positives with oral fluid testing (OraQuick).
5. Reverse transcriptase’s RNA-dependent DNA polymerase activity uses the RNA as a template to synthesise a complementary DNA strand, forming an RNA/cDNA duplex; its RNase H activity then degrades the original RNA strand; finally its DNA-dependent DNA polymerase activity synthesises the second DNA strand, producing double-stranded cDNA.
6. A higher starting number of virus copies means the fluorescence signal reaches the detection threshold (Ct) after fewer amplification cycles; a sample with 10^8 copies crosses threshold around cycle 18, while one with only 10^3 copies takes until around cycle 34. Ct is therefore inversely related to starting viral load.
7. An HIV-1/2 antigen/antibody combination immunoassay is run first. If negative, the result is HIV-1/2 antibody and p24 antigen negative. If positive, a differentiation immunoassay distinguishes HIV-1 from HIV-2 antibodies; if both are negative or indeterminate and acute infection is suspected, HIV-1 RT-PCR is used to confirm or exclude acute HIV-1 infection.
8. Blood donations: both HIV RT-PCR and anti-HIV-1/2 immunoassay, because high sensitivity is essential. Antenatal screening: anti-HIV-1/2 immunoassay first (low pre-test probability), with repeat immunoassay and/or RT-PCR if reactive. Infants: HIV RT-PCR, because maternal antibody crossing the placenta makes serology unreliable.
9. Entry/fusion: entry inhibitors (CCR5 inhibitors, fusion inhibitors). Reverse transcription: reverse transcriptase inhibitors (NRTIs, NNRTIs). Integration: integrase inhibitors (INSTIs). Maturation: protease inhibitors.
10. Thymidine has a 3’-OH group needed to extend the DNA chain; AZT substitutes an azide group at that position, so once incorporated it cannot form the next phosphodiester bond, terminating the chain. NNRTIs do not get incorporated into DNA at all; instead they bind a hydrophobic pocket near the RT active site and block its polymerase activity directly.
11. Both HIV reverse transcriptase and host RNA polymerase II lack proofreading, producing about 0.25 mutations per replication cycle, and about 10^10 new virions are generated per day; together these produce many distinct variants daily, accumulating into a diverse quasispecies.
12. Under monotherapy, the single drug eliminates wild-type virus but a pre-existing variant already resistant to that drug is unaffected and comes to dominate the population. Under triple-drug therapy, each drug remains active against the variants resistant to the other two drugs, so no single existing or emerging variant is resistant to all three simultaneously, and the whole population, wild-type and resistant variants, can be eliminated.
13. Even after suppression, HIV persists in a latent reservoir; stopping treatment causes rapid viral rebound from this reservoir (rising plasma RNA and falling CD4 count within weeks), so treatment must continue indefinitely to keep the virus suppressed.
14. Oral PrEP gives about 75% (45-86%) protection depending on adherence, while long-acting injectable PrEP is more effective, giving 79-100% efficacy compared with oral PrEP. Circumcision reduces risk of infection by about 58%.