HIV: Questions

Genital ulceration breaches the mucosa and draws in activated CD4 T cells bearing the CCR5 co-receptor, the very cells HIV infects, so HSV-2 raises HIV acquisition roughly threefold and contributes a substantial share of new HIV infections where both are common.

It does not reduce or abolish the risk, and the effect is seen in both sexes rather than men alone.

HIV crosses the blood-brain barrier chiefly within infected monocytes and macrophages, a “Trojan horse” mechanism, seeding perivascular macrophages and microglia as the productively infected brain cells.

Neurons lack CD4 and are not productively infected; their injury is indirect, via inflammatory mediators. The remaining routes (axonal transport, passive diffusion, endothelial infection) do not describe HIV neuroinvasion.

Interpreting M184V

M184V is the highest-yield NRTI (nucleoside reverse transcriptase inhibitor) resistance mutation: methionine at codon 184 of reverse transcriptase (RT) has been replaced by valine, through a single A-to-G change in the codon (ATG to GTG).

• Mechanism: discrimination at the YMDD motif; the mutated active site selects natural dCTP and excludes lamivudine (3TC) and emtricitabine (FTC).
• Susceptibility: very high-level (over 100-fold) resistance to 3TC and FTC.
• Re-sensitisation: re-sensitises the virus to zidovudine (AZT), with a modest effect on tenofovir; the mutated RT extends less efficiently past chain-terminating analogues.
• Fitness cost: the virus replicates measurably more slowly.

The implication is that 3TC should be retained. Even without direct activity against an M184V virus, the residual selective pressure and the fitness cost work against virological breakthrough.

Interpreting G118R

G118R is an integrase strand-transfer inhibitor (INSTI) resistance mutation, first described in dolutegravir monotherapy studies and later in cabotegravir pre-exposure prophylaxis (PrEP) breakthroughs and the rare programmatic dolutegravir-failure setting.

• Mechanism: active-site reshaping in integrase.
• Susceptibility: confers dolutegravir resistance, with bictegravir resistance also expected; cross-resistance to the first-generation INSTIs (raltegravir, elvitegravir) is variable.
• Fitness cost: measurable, as for R263K the mutant replicates less efficiently.
• Significance here: together with the detectable dolutegravir level it confirms true dolutegravir resistance rather than non-adherence, the unusual case the reflex drug-level workflow is designed to detect.

No protease mutations

The protease region is clean, so the protease-inhibitor class is fully open for the next regimen.

Management

This patient has failed dolutegravir-based second-line therapy with true resistance, so the regimen must be reconstructed.

• Switch dolutegravir to darunavir/ritonavir, the highest-barrier protease inhibitor and preferred given the absent PI mutations.
• Retain lamivudine (3TC) for the M184V fitness cost.
• Retain tenofovir if renal function permits: there is no TAM (excision) pattern to compromise it, and it also provides hepatitis B virus (HBV) cover.
• Apply to the ARV Drug Resistance Committee (ADReC) for third-line authorisation, since darunavir/ritonavir is accessed through that process.
• Provide intensive adherence support and repeat the viral load at 12 weeks.

This is extensive multi-class resistance from long-standing inadequate suppression on a protease-inhibitor regimen.

Principle. A third-line regimen needs at least two, ideally three, fully active drugs, anchored by a high-genetic-barrier agent that can tolerate one suboptimal partner.

Anchor. Pair two high-barrier agents from different classes:

• Darunavir/ritonavir, the highest-barrier protease inhibitor: despite the four major PI mutations, Stanford HIVdb usually still grades darunavir active unless three or more darunavir-specific mutations are present, so the sequence should be submitted for explicit interpretation.
• Dolutegravir, a fully active new class with a high genetic barrier.

Backbone. Retain lamivudine (3TC) for the M184V fitness cost. The M41L/L210W/T215Y TAM cluster substantially reduces both tenofovir and abacavir activity (Type-1 TAMs hit them hardest), so neither is reliably active; Stanford HIVdb interpretation guides whether tenofovir is worth retaining for residual activity or hepatitis B virus (HBV) cover.

Proposed regimen: tenofovir + lamivudine + darunavir/ritonavir + dolutegravir. The two fully active drugs are darunavir/ritonavir and dolutegravir; lamivudine is kept for the M184V effect and tenofovir for any residual activity and HBV cover.

Process. Apply to the ARV Drug Resistance Committee (ADReC) for third-line authorisation with the resistance report and clinical summary; provide intensive adherence support, without which any third-line regimen will fail in turn; repeat the viral load at 12 and 24 weeks; and avoid non-nucleoside reverse transcriptase inhibitors (NNRTIs) for life, since the archived K103N, V106M and Y181C re-emerge under any NNRTI pressure.

Interpretation. The NRTI (nucleoside reverse transcriptase inhibitor) mutations compromise both tenofovir and abacavir: M184V gives high-level lamivudine/emtricitabine resistance and a useful fitness cost, K65R reduces tenofovir and abacavir activity, and M41L is an early thymidine analogue mutation (TAM) of the excision pathway; zidovudine is relatively spared and is re-sensitised by K65R. K103N is archived from prior efavirenz use: no longer active pressure, but it persists in the reservoir and closes the NNRTI (non-nucleoside reverse transcriptase inhibitor) class for life. The protease region carries multiple major mutations, including the I50V darunavir/lopinavir signature, so lopinavir and most protease inhibitors are compromised; darunavir activity needs explicit Stanford HIVdb interpretation but is likely reduced.

Management. This is multi-class resistance requiring third-line salvage through the ARV Drug Resistance Committee (ADReC). Anchor with two high-barrier agents from different classes, dolutegravir (a fully active new class) and darunavir/ritonavir; recycle lamivudine (3TC) for the M184V fitness cost; and retain tenofovir only if Stanford HIVdb grades it active. Intensive adherence support is essential, since three years of inadequate suppression will otherwise defeat any new regimen.

The first thing to recognise is that the patient is not failing treatment. An “LDL” viral load means the assay cannot detect viral RNA above its limit (typically below 50 copies/mL): the patient is virologically suppressed. The CD4 of 203 is a separate question about immune reconstitution, not virological success.

Possible explanations for a discordant CD4 to VL picture in a suppressed patient:

Late start with a low CD4 nadir. Antiretroviral therapy (ART) was started at a low nadir CD4, perhaps below 200, and immune recovery has been slow. The lower the starting point, the slower and more limited the recovery: after two years of suppression a CD4 of 203 from a baseline of, say, 80 represents real progress, and recovery often takes 3 to 5 years to plateau.

Persistent immune activation. Even on suppressive ART, residual low-level inflammation (driven by microbial translocation from the gut, ongoing low-level viral replication, and other factors) limits peripheral CD4 reconstitution. Co-infections such as hepatitis C virus (HCV), hepatitis B and recurrent tuberculosis (TB) exacerbate this.

Spurious result. A single CD4 result is biologically variable: diurnal variation, recent vaccination, intercurrent infection (even minor viral illness), or laboratory variability can produce a result around the 200 threshold. A repeat sample after correcting any modifiable factor is sensible before drawing conclusions.

Management. Reassure the GP that virological suppression is the correct indicator of treatment success, and do not change the ART regimen on the basis of the CD4 count; the sensible steps are to screen for and treat co-morbidities (especially TB and HCV) and repeat the CD4 in 6 months.

Pair A: genetic distance 0.018

Possible reasons for a distance this low between two HIV sequences:

• Recent transmission between the two individuals, directly or through a short chain; the most parsimonious explanation well below the cut-off.
• A shared recent ancestor: both viruses descend from a recently established local cluster (a community founder effect).
• Convergent evolution: both have independently acquired the same resistance pattern under the same regimen; less likely below the cut-off, but possible for short, mutation-rich regions of pol.
• Sampling artefact: both individuals come from a network of limited viral diversity, so all sampled sequences are closely related.

A distance below 0.03 is not proof of transmission; it flags a pair for investigation, and epidemiological and clinical context must be added before any inference.

Pair B: woman and 2-year-old child, distance 0.012

This combination is highly suggestive of mother-to-child transmission: the distance is well below the cut-off and the age difference fits an infected child of the mother. The other explanations fit less well once a plausible mother-child link is on the table.

• Forensic value: genetic distance alone cannot confirm direct transmission, but a low value in this demographic combination strongly supports vertical transmission.
• Ethical considerations: linked-transmission investigation requires the consent and protection of both parties, with confidential use for surveillance only; phylogenetic linkage has been used controversially in legal settings, and the laboratory’s role is interpretive, not adjudicative.
• Practical action: confirm the programmatic linkage (was the mother known HIV-positive antenatally, was prevention of mother-to-child transmission, PMTCT, delivered), assess for PMTCT failure, ensure the mother is virally suppressed on ART and the child on an appropriate paediatric regimen, and intensify follow-up.

The same Sanger sequencing data that drives the resistance report can map molecular epidemiology and target prevention; the 0.03 cut-off is an operational screening threshold, not a diagnostic test.

An indeterminate qualitative HIV PCR is a low-level positive that does not meet the reporting threshold for a definitive positive (commonly defined by the laboratory as a cycle-threshold (CT) value above a set cut-off, e.g. CT above 36). It may represent: very early infection with low viral copy number; contamination during sample handling; technical issue with the assay; or, in an infant, passive transfer of maternal HIV nucleic acids in transplacentally acquired plasma.

The general approach

The first principle: do not ignore the result, and do not initiate antiretroviral therapy (ART) on a single indeterminate PCR. The next step depends on the infant’s testing history.

Step 1: review the infant’s prior HIV-PCR or viral-load results.

• Prior PCR positive, or prior VL detectable, or prior PCR indeterminate: the indeterminate result is corroborated by earlier evidence of infection. Treat as HIV-infected: initiate ART immediately and confirm on a fresh sample after starting therapy.
• Prior PCR negative, or no prior PCR: the indeterminate result stands alone. Repeat both the HIV PCR and a viral load urgently on a fresh sample.
  • Repeat PCR positive or indeterminate → treat as HIV-infected, initiate ART.
  • Repeat PCR negative and repeat VL undetectable → routine HIV-exposed follow-up; continue the standard EID schedule with the next PCR at 10 weeks.

Worked example: indeterminate PCR at birth, CT 38.1

A birth PCR with CT 38.1 is just above the threshold for a positive result. The clinical context matters:

• Maternal viral load at delivery. If the mother was virally unsuppressed (VL at or above 50 copies/mL on the most recent measurement), the indeterminate result is much more likely to represent true infection.
• Was prevention-of-mother-to-child-transmission (PMTCT) prophylaxis given? Dual nevirapine (NVP) plus zidovudine (AZT) prophylaxis at birth has been received by all HIV-exposed infants since the 2026 National Consolidated Guidelines (NCG) change. Continue the prophylaxis pending clarification.
• Was there any maternal antiretroviral exposure that might lower the infant’s HIV viral copy number at birth? Maternal ART (and infant prophylaxis itself) suppresses early infant viraemia and can produce a low-positive or indeterminate PCR pattern.

Immediate management:

• Repeat the HIV PCR and a viral load urgently, same week if possible.
• Continue the infant prophylaxis (NVP daily ± AZT twice daily) until the result is clarified.
• If maternal VL at delivery is at or above 50 copies/mL and the result is highly suspicious, consider starting infant ART pending confirmation rather than waiting.
• Counsel the mother: discuss the possible meanings honestly without making a premature diagnosis.

Subsequent action depends on the repeat:

• Repeat positive or indeterminate → start infant ART immediately; confirm with viral load; continue the EID follow-up schedule.
• Repeat negative, VL undetectable → return to routine HIV-exposed follow-up (PCR at 10 weeks, 6 months, etc.). Document the indeterminate result and the subsequent negative for clarity.

Indeterminate PCRs are not rare in early infant diagnosis: they represent a confluence of the test’s sensitivity at low copy numbers, the effect of PMTCT prophylaxis on early infant viraemia, and the inherent uncertainty of sub-threshold signals. A clear local algorithm prevents both over-treatment (initiating ART on a falsely positive result) and under-treatment (missing a true infection because the result was dismissed).

• (a) False. Inactivated influenza vaccine is recommended, not contraindicated, in pregnancy and in HIV at any CD4 count; it is non-live and safe, and both pregnancy and HIV raise the risk of severe influenza.
• (b) True. HPV vaccination is recommended for HIV-infected people regardless of CD4 count, ART use or viral load, including men who have sex with men up to about 40 years, because of their high HPV-associated cancer risk.
• (c) True. Hepatitis A vaccine is inactivated and safe in HIV; the standard two-dose schedule is appropriate, and chronic liver disease is itself an indication. Checking post-vaccination antibody is reasonable, as the response may be reduced.
• (d) False. The varicella vaccine is live and contraindicated at a CD4 count of 150 cells/uL. Give varicella-zoster immunoglobulin (VZIG) for post-exposure prophylaxis instead.

TRUE.

The standard inactivated HAV vaccine schedule is two doses, a priming dose at time zero and a booster at six to twelve months, and applies in HIV-positive patients, including those with chronic liver disease. The vaccine is inactivated whole virus and safe at any CD4 count.

Response is reduced in advanced immunosuppression (around 52% to 94% seroconversion versus over 99% in immunocompetent adults), so a third booster dose six to twelve months after the first is commonly recommended in this population to improve seroconversion.

Long-acting antiretroviral PrEP (pre-exposure prophylaxis) delivers inhibitory drug concentrations for weeks to months from a single administration, converting adherence from a daily-tablet problem into a clinic-visit problem.

Available agents:

• Long-acting injectable cabotegravir (CAB-LA): an integrase strand-transfer inhibitor formulated as a nanosuspension; given by intramuscular injection at month 0 and month 1 (the loading doses), then every 8 weeks. HPTN 083 (men who have sex with men, transgender women) and HPTN 084 (cisgender women in sub-Saharan Africa) showed superiority to daily oral TDF/FTC (tenofovir disoproxil fumarate/emtricitabine).
• Dapivirine vaginal ring: a monthly silicone ring delivering a non-nucleoside reverse transcriptase inhibitor (NNRTI) locally to the vaginal mucosa; modest (~30%) efficacy.
• Lenacapavir: first-in-class capsid inhibitor; two subcutaneous abdominal injections every 26 weeks, with brief oral loading. Purpose 1 and Purpose 2 showed near-complete prevention.

Key practical considerations:

• Confirm HIV-negative status before each administration. A single sub-therapeutic antiretroviral can select resistant virus if given during unrecognised acute infection.
• Oral lead-in. A brief oral course of the active drug before the first injection confirms tolerability before committing to a long-acting agent.
• Injection-site reactions occur in around 60% with both CAB-LA and lenacapavir; usually self-limiting nodules, rarely causing discontinuation.
• Pharmacokinetic (PK) tail. Prolonged sub-therapeutic concentrations follow the last dose; exposure during this tail may fail to prevent infection yet still select resistance. Counsel on cessation, bridging with oral PrEP if risk continues.
• Breakthrough infection and resistance. Purpose 2 demonstrated capsid N74D in both breakthroughs; HPTN 083/084 demonstrated integrase resistance in CAB-LA-exposed seroconverters. Resistance genotyping must include the relevant region (capsid for lenacapavir; integrase for cabotegravir).
• LEVI syndrome: delayed and blunted seroconversion under long-acting PrEP that fails to prevent infection.
• Programmatic implementation. Cold-chain logistics, trained injectors, reliable patient recall systems and supply security determine whether long-acting PrEP succeeds at scale.
• Cost and access. Lenacapavir in particular is currently expensive; access is the live policy issue in South Africa and globally.

The dapivirine vaginal ring is a flexible silicone ring containing 25 mg of dapivirine, a non-nucleoside reverse transcriptase inhibitor (NNRTI). Worn continuously for one month then replaced, it gives slow, sustained local release at the cervicovaginal mucosa.

Evidence base. Two large randomised trials, ASPIRE (MTN-020) and The Ring Study (IPM-027), tested the ring in over 4,500 sub-Saharan African women and showed an approximately 27 to 31% reduction in HIV acquisition compared with placebo. Efficacy was clearly adherence-dependent: the most consistent users achieved a reduction of over 50%, while efficacy was lower in younger women whose adherence was typically poorer. The open-label extensions HOPE and DREAM showed greater real-world reductions, likely reflecting selection of more motivated users.

Regulatory status. The World Health Organization in 2021 recommended the dapivirine ring as a prevention option for women at substantial risk of HIV who are unable or unwilling to use oral pre-exposure prophylaxis (PrEP). Several countries, including South Africa, have approved it.

Strengths:

• Women-controlled: inserted and managed privately, valuable where partner dynamics constrain consistent pill-taking.
• Local delivery: minimal systemic exposure, no significant drug interactions, no renal monitoring.
• Long-acting: monthly replacement, no daily-pill burden.
• Continuous protection: no need to anticipate exposures.
• Acceptable to most trial participants.

Limitations:

• Modest efficacy compared with newer long-acting agents: around 30% in trials versus 96 to 100% with lenacapavir and superior efficacy with cabotegravir.
• HIV-1 only: HIV-2 is intrinsically resistant to NNRTIs.
• Only for women.
• Provides no contraceptive effect: dual prevention requires an additional method.
• Insertion technique and acceptability vary; some women find the device uncomfortable or culturally unacceptable.
• Public-sector uptake has been limited globally.

Place in current practice. The ring is a niche tool, for women who reject or cannot reliably take daily oral PrEP and cannot access long-acting injectables. Long-acting cabotegravir (CAB-LA) and lenacapavir have reduced its prominence, but its user-control and minimal systemic profile retain value in specific contexts.

The South African EID programme exists to identify HIV infection in infants exposed to HIV, as early as possible, so that antiretroviral therapy (ART) can be initiated before the rapid disease progression characteristic of paediatric HIV (untreated infant HIV mortality is up to 50% by two years of age).

Current EID schedule (National Consolidated Guidelines, NCG 2026)

• Birth PCR. Performed on all HIV-exposed infants within 48 hours of delivery.
• 10-week PCR. Aligned with the Expanded Programme on Immunisation (EPI) 10-week vaccination visit.
• 6-month PCR. Aligned with the maternal 6-month viral-load check.
• 18-month rapid antibody test. Universal for all children, regardless of HIV exposure status.
• Age-appropriate test 6 weeks after breastfeeding cessation.
• Any time the infant is unwell with features suggestive of HIV.

A positive HIV-PCR result in a child under 2 years must be confirmed on a fresh sample (or a baseline viral load) before reporting as definitive.

Evolution of the South African EID programme

• Pre-2008: a single PCR at 6 weeks for HIV-exposed infants. This identified most intrapartum infections but missed in-utero infections, and the 6-week wait delayed ART start.
• From 2008: birth PCR introduced initially for high-risk infants, progressively universalised. The motivation was to detect in-utero infection at the earliest possible point. Birth-positive infants, by definition infected before delivery, represent a small but high-risk group (intrauterine acquisition is associated with rapid disease progression).
• 2015: HIV PCR at birth made universal for all HIV-exposed infants.
• 2019 (vertical transmission prevention update): the 6-week PCR moved to 10 weeks to align with the EPI immunisation visit, reducing the number of clinic touch-points and improving programme efficiency. The 10-week timepoint also gives a wider detection window for intrapartum infections (which typically become PCR-positive after the first 4 to 6 weeks of life under nevirapine prophylaxis).
• 6-month PCR added alongside the maternal 6-month viral load (VL) check, to catch HIV infections acquired during breastfeeding while the maternal VL is being monitored anyway.
• 18-month rapid antibody test retained as the definitive end-of-EID test: the residual maternal immunoglobulin G (IgG) that confounds infant antibody testing has waned by 18 months, so a positive rapid test at this age is a true infant antibody, not a maternal one.
• Post-cessation test: any breastfeeding-acquired infection that emerged after the 6-month PCR is captured by a test 6 weeks after weaning.
• NCG 2026 retains this five-point schedule (birth, 10 wk, 6 mo, 18 mo, post-cessation), reinforces the 10-week/EPI alignment, and standardises indeterminate-PCR management as a national algorithm.

Rationale for each timepoint

• Birth PCR: detects in-utero infection (the highest-mortality group, infants infected before labour). Early diagnosis means ART by the first month.
• 10-week PCR: detects intrapartum infections (those acquired during labour), now visible above the suppressive effect of infant prophylaxis. EPI alignment reduces visits.
• 6-month PCR: detects breastfeeding-acquired infection during the period of exclusive breastfeeding; aligned with maternal VL monitoring.
• 18-month rapid antibody: the definitive diagnostic endpoint after maternal antibody wanes; also universal-population HIV screen.
• Post-cessation test: catches infections acquired in the late breastfeeding period, after the 6-month PCR.

Taken together, the schedule reflects three biological realities: maternal antibody persists until about 18 months (so antibody tests in infants are uninterpretable before then); prevention-of-mother-to-child-transmission (PMTCT) drug prophylaxis suppresses early infant viraemia (so the PCR window opens after about 4 to 6 weeks); and breastfeeding remains a transmission route until weaning (so a single early test cannot exclude infection in a breastfed infant). Stringing the timepoints together gives the programme a near-complete diagnostic safety net for HIV-exposed infants.

Broadly neutralising antibodies (bNAbs) neutralise a wide range of HIV-1 strains across subtypes. They develop in only a minority of infected people, and only after years, requiring extensive somatic hypermutation driven by prolonged exposure to a diversifying virus. They arise too late to control the infection in the person who makes them, but they define the conserved targets a vaccine must hit.

Targets. bNAbs recognise the small number of relatively conserved sites on the envelope trimer:

• the CD4-binding site (e.g. VRC01, 3BNC117);
• the V1/V2 apex (e.g. PG9, PG16);
• the V3-glycan supersite (e.g. PGT121, 2G12);
• the gp41 membrane-proximal external region (MPER) (e.g. 10E8, 4E10);
• the gp120–gp41 interface and the fusion peptide.

Why they are hard to elicit. The envelope defends these sites with a dense glycan shield of host sugars, conformational masking, hypervariable loops and sequence diversity, and the need for unusual, highly mutated antibody lineages. No immunogen has yet reliably induced bNAbs by vaccination.

Applications. bNAbs are being developed for prevention (the Antibody Mediated Prevention [AMP] trials showed VRC01 reduced acquisition of sensitive strains but not overall, showing that single antibodies are too narrow, so combinations or engineered multispecific antibodies are needed), for long-acting therapy, and as part of cure strategies, where they may both neutralise virus and recruit immune effectors to clear reservoir cells.

The gut-associated lymphoid tissue (GALT) holds most of the body’s CD4+ T cells, the majority of them activated, memory-phenotype, CCR5-expressing cells that are the ideal target for HIV. The GALT is therefore the site of the earliest and most massive CD4 depletion in infection: a large fraction of mucosal CD4+ T cells is destroyed within the first weeks, far exceeding the loss measurable in peripheral blood, and this depletion is only partially reversed by antiretroviral therapy.

The consequence is loss of mucosal immune integrity and breakdown of the epithelial barrier, letting microbial products (notably lipopolysaccharide) translocate from the gut lumen into the systemic circulation. This sustained antigenic stimulation is a major driver of the chronic immune activation that underlies disease progression and much of the non-AIDS morbidity (cardiovascular, neurocognitive) seen even in treated infection. The GALT is thus both an early casualty of HIV and an engine of its pathogenesis.

Pre-exposure prophylaxis (PrEP) is antiretroviral medication taken by HIV-negative people at substantial risk, to prevent acquisition. It is a cornerstone of biomedical HIV prevention.

Principle. Maintain inhibitory drug concentrations at exposure sites so encountered virus cannot establish productive infection. iPrEx, CAPRISA 004, Partners PrEP and TDF2 showed daily oral tenofovir/emtricitabine reduces acquisition in proportion to adherence (~70% with consistent use, over 90% with detectable drug levels).

Available agents:

• Daily oral TDF/FTC (tenofovir disoproxil fumarate/emtricitabine): the workhorse of public-sector PrEP globally; available through dedicated PrEP clinics in South Africa.
• Event-driven oral PrEP (2-1-1): TDF/FTC dosed before and after sexual contact, suitable for men who have sex with men with predictable, infrequent exposures.
• Dapivirine vaginal ring: a women-controlled monthly silicone ring; ~30% efficacy in trials.
• Long-acting cabotegravir (CAB-LA): intramuscular injection every two months after a loading dose; superior to daily oral PrEP in HPTN 083 and 084.
• Twice-yearly subcutaneous lenacapavir: the most efficacious PrEP agent yet studied; near-complete prevention in Purpose 1 (cisgender women, 100%) and Purpose 2 (men and gender-diverse people, 96%).

Target populations. Those at substantial risk of HIV acquisition: sex workers, men who have sex with men, transgender people, serodiscordant couples (until the partner is virally suppressed), adolescent girls and young women in high-prevalence settings, and people who inject drugs.

Considerations before initiation. Confirm HIV-negative status (and re-test at each visit); baseline renal function (for TDF-based PrEP); hepatitis B surface antigen (HBsAg, for ongoing tenofovir coverage); pregnancy is not a contraindication. Counsel on the requirement for adherence and on safer-sex practice during the start-up period.

Synergy with treatment as prevention. PrEP complements rather than replaces antiretroviral therapy (ART) for the HIV-positive partner. A virally suppressed partner does not transmit (undetectable equals untransmittable, U=U); PrEP protects against exposures from people not on suppressive ART.

Limitations and emerging issues. Adherence is the main challenge for oral PrEP, particularly in adolescent girls and young women. Long-acting agents address adherence but introduce specific issues: injection-site reactions, pharmacokinetic-tail resistance risk if discontinued, and the long-acting early viral inhibition (LEVI) phenomenon. Drug-resistance selection is rare on PrEP but possible if started during unrecognised acute infection. Access to the newest agents, lenacapavir in particular, is currently constrained by cost and supply.

Concern over dolutegravir (DTG) in pregnancy arose from a safety signal that attenuated as the data matured.

The 2018 Tsepamo signal. In May 2018 the Tsepamo birth-outcomes surveillance in Botswana reported an apparent excess of neural-tube defects (NTDs) in women on dolutegravir at conception: ~0.94% in DTG-exposed pregnancies versus ~0.12% in the unexposed (4 NTDs in 426 DTG-exposed births).

The initial response. The World Health Organization (WHO) issued interim guidance cautioning against DTG in women of child-bearing potential without effective contraception. National programmes, including South Africa’s, adjusted recommendations accordingly. Women of child-bearing potential who could not commit to reliable contraception were considered for non-DTG regimens during the period of uncertainty.

Subsequent evolution. As the cohort accrued more women and longer follow-up, the NTD signal attenuated. By the early 2020s the DTG-exposed NTD prevalence had fallen to ~0.19% versus ~0.11% in the unexposed, no longer statistically significant, and corroborated by independent data. It is now considered most likely a chance finding inflated by ascertainment bias in the small early dataset.

Current recommendations:

• DTG is the first-line antiretroviral throughout pregnancy and in women of child-bearing potential, without restriction.
• Effective contraception is no longer required specifically for NTD concerns (general prevention of mother-to-child transmission (PMTCT) and reproductive-health principles still apply).
• Conception on a DTG-containing regimen is supported as safe.
• Routine pre-conception folate intake (5 mg daily in resource-rich settings, or as locally available) remains recommended, in line with general pre-conception care.

Significance. The episode carried a real cost: women may have been switched to less effective or less tolerated regimens during the uncertain period, illustrating the limits of early signal detection in small cohorts and the value of prospective birth-outcomes surveillance.

Why DTG is preferred. Its high genetic barrier, rapid viral-load reduction and good tolerability make it the regimen of choice in women of child-bearing potential. Vertical transmission on maternal DTG-based regimens is at or below WHO elimination thresholds once maternal suppression is achieved.

The latent reservoir is the population of cells carrying integrated, transcriptionally silent but replication-competent provirus, from which infection rebounds if therapy stops. It is the central obstacle to curing HIV.

What and where. The best-characterised reservoir is the long-lived resting memory CD4+ T cell (central, transitional and effector memory subsets), but provirus also persists in tissue macrophages and in sanctuary sites such as lymphoid tissue and the central nervous system. It is established within days of infection, before therapy can realistically begin, which is why even very early ART does not prevent it.

Why it persists. Once integrated, the silent provirus is invisible to the immune system and unaffected by antiretrovirals (which block new infection, not established proviral DNA). The reservoir is maintained by the normal homeostatic proliferation and antigen-driven clonal expansion of the memory T cells that harbour it, so it is remarkably stable over decades.

Intact versus defective. The great majority of persisting proviruses are defective (hypermutated or deleted) and cannot rebound; only a small intact fraction is rebound-competent. Measuring the clinically relevant reservoir is therefore difficult: the quantitative viral outgrowth assay and the intact proviral DNA assay attempt to quantify the replication-competent pool, while total proviral DNA overestimates it.

Clinical consequence. Stopping suppressive ART is followed by viral rebound within about two to three weeks, confirming lifelong infection. Cure research targets the reservoir directly: latency reversal (“shock and kill”), latency-promoting “block and lock”, broadly neutralising antibodies, and gene therapy.

Pre-exposure prophylaxis (PrEP) can drive HIV drug resistance in three distinct ways, and as long-acting PrEP scales up this is shifting from a theoretical concern to an active surveillance issue.

1. PrEP started during unrecognised acute infection. The principal mechanism. Single-agent or dual-NRTI (nucleoside reverse transcriptase inhibitor) PrEP given to a person already harbouring HIV becomes functional monotherapy or dual therapy and rapidly selects resistance: M184V to lamivudine/emtricitabine, K65R to tenofovir. Pre-PrEP fourth-generation antigen/antibody testing reduces but does not eliminate this risk, because the diagnostic window precedes detectability; nucleic-acid testing where available shortens the window.

2. Long-acting injectables and the pharmacokinetic (PK) tail. Long-acting cabotegravir and lenacapavir produce months of sub-therapeutic drug exposure after the final injection. A person who acquires HIV during this tail experiences single-class drug pressure on early viral replication, which readily selects resistance to that class:

• HPTN 083 and 084 documented integrase-strand-transfer-inhibitor resistance in cabotegravir-PrEP breakthroughs.
• Purpose 2 documented capsid N74D in both of its lenacapavir-PrEP breakthroughs.
• In treatment trials of lenacapavir (CAPELLA, CALIBRATE), M66I is the dominant emergent capsid mutation.

3. LEVI syndrome. Long-acting early viral inhibition, an atypical primary infection under long-acting PrEP, produces blunted viraemia, delayed seroconversion and prolonged single-class drug pressure; it both delays diagnosis and provides ideal conditions for resistance emergence.

Laboratory and surveillance implications:

• Pre-PrEP testing should use the most sensitive available assay, with nucleic-acid testing in high-risk contexts.
• Anyone seroconverting on long-acting PrEP needs resistance genotyping of the relevant gene region: the integrase region for cabotegravir-exposed seroconverters, and the capsid region for lenacapavir-exposed seroconverters. Capsid genotyping is not part of the standard South African pol genotype, and the capability is being developed.
• The subsequent treatment regimen must avoid the implicated class.

Population level. Transmitted-drug-resistance surveillance may eventually show an upward signal (M184V from oral PrEP, integrase mutations from cabotegravir, capsid mutations from lenacapavir). Oral-PrEP resistance has been uncommon in clinical trials, but every long-acting breakthrough forecloses one treatment class. The defences are PrEP adherence, rigorous HIV testing at every visit, and a low threshold for nucleic-acid testing in suspected breakthrough.

Lenacapavir is the first approved member of a new antiretroviral class, the capsid inhibitors.

Mechanism. Lenacapavir binds multiple sites on the HIV-1 capsid hexamer interface, disrupting capsid function at three stages of replication:

• Early post-entry: interferes with uncoating, reverse transcription and nuclear import of the pre-integration complex.
• Capsid stability: alters uncoating kinetics, exposing the genome prematurely or in the wrong cellular location.
• Late assembly: disrupts capsid lattice assembly during virion morphogenesis, producing immature, non-infectious particles.

This multi-step mechanism underlies its very high potency (picomolar half-maximal inhibitory concentration, IC₅₀) and high genetic barrier, since multiple, often costly, mutations are required to escape.

HIV-associated nephropathy (HIVAN) is the classic direct renal disease of HIV, histologically a collapsing focal segmental glomerulosclerosis (FSGS) with microcystic tubular dilatation. Its pathogenesis is a two-hit process: direct viral injury to the renal epithelium acting on a genetically susceptible kidney, neither hit sufficient alone.

The viral hit. The kidney acts as an HIV reservoir: podocytes and tubular epithelial cells harbour virus and express viral genes, even though they are not productively infected in the usual CD4/CCR5-dependent way. Local expression of nef and vpr drives the injury:

• nef activates Src-family kinase and MAPK (mitogen-activated protein kinase) signalling, driving the normally growth-arrested podocyte to dedifferentiate and proliferate; this dysregulated proliferation collapses the glomerular tuft, the defining lesion.
• vpr causes apoptosis and G2 cell-cycle arrest, injuring podocytes and tubular epithelial cells; the tubular injury produces the microcysts.

The host hit. HIVAN occurs overwhelmingly in people of African ancestry, because of high-risk APOL1 variants (G1 and G2). The risk is recessive: two risk alleles are needed, and they sensitise the podocyte to injury. This genetic architecture is why HIVAN is a major cause of chronic kidney disease in the South African and broader sub-Saharan epidemic.

The interaction. Most people carrying two APOL1 risk alleles never develop HIVAN, and the virus rarely causes it without that susceptibility. Uncontrolled viraemia is the trigger, so HIVAN clusters in advanced, untreated infection (low CD4, high viral load), and antiretroviral suppression can reverse early disease, confirming the causal role of active viral gene expression.

The drugs used to prevent vertical HIV transmission are highly effective, and they make early infant diagnosis harder. The same antiretroviral exposure that prevents infection also suppresses the viral signal in an exposed-but-infected infant, blunting and delaying the diagnostic markers EID relies on. The problems sit on three axes: maternal antiretroviral therapy (ART), infant prophylaxis, and the timing of each test.

Maternal ART during pregnancy and breastfeeding

A mother on suppressive tenofovir/lamivudine/dolutegravir (TLD) throughout pregnancy and breastfeeding:

• Reduces but does not abolish vertical transmission. Residual transmission risk remains around 1%.
• Lowers infant viraemia in any infected infant, slowing the emergence of detectable HIV RNA/DNA. PCR results may take longer to become reliably positive.
• Selects (rarely) for resistance in the infant, complicating any future paediatric ART decisions.

Infant prophylaxis

The current South African regimen (dual nevirapine (NVP) plus zidovudine (AZT) for all HIV-exposed infants at birth, then stratified by maternal delivery viral load (VL)) is:

• Highly effective at preventing infection.
• A confounder for EID. NVP and AZT directly suppress whichever infant viraemia exists, blunting the PCR signal at and around the next testing timepoints.
• A possible cause of indeterminate results. A low-positive PCR in a prophylaxed infant may be the diagnostic signature of an infection that has been partially but not fully suppressed by the prophylaxis.

Timing problems

• Birth PCR. Detects in-utero infection, but in an infant on prophylaxis the viral signal may be borderline, producing indeterminate results that need confirmation.
• 10-week PCR. Aligned with the Expanded Programme on Immunisation (EPI), designed to detect intrapartum infection, but in an infant on extended NVP (higher-risk infant prophylaxis is continued through and beyond this timepoint), even an intrapartum infection may show below-threshold viraemia at 10 weeks.
• 6-month PCR. Detects breastfeeding-acquired infection during the exclusive breastfeeding period, but again, NVP prophylaxis continuing through breastfeeding suppresses any emerging infant viraemia.
• Post-cessation test. Captures infection that emerged in late breastfeeding: once prophylaxis is stopped, the diagnostic window for any remaining infection re-opens.
• 18-month antibody. Now reliable as the definitive endpoint because maternal antibody has waned.

Specific diagnostic pitfalls

• Increased indeterminate PCR rate. Particularly at birth, and in higher-risk infants on extended dual prophylaxis. Algorithmic management is essential.
• Delayed seroconversion in an infected infant. Maternal antibody persists for up to 18 months, masking infant antibody until then; infant prophylaxis may further blunt the infant’s own antibody development.
• False reassurance from interim negatives. A negative PCR at 10 weeks does not exclude intrapartum infection if prophylaxis is still actively suppressing viraemia. Hence the layered schedule.
• Transmission late in breastfeeding may not be detected until the post-cessation test (potentially many months after acquisition).

Programme-level implications

The South African EID schedule is constructed precisely to see around these prophylaxis-induced diagnostic gaps. Multiple testing timepoints (birth, 10 weeks, 6 months, post-cessation, 18 months) cover the periods during which prophylaxis is active and the periods during which it is not. The post-breastfeeding-cessation test is critical: it is the window after which prophylaxis no longer affects detection. Indeterminate-PCR algorithms standardise management of borderline results, and the 18-month rapid antibody as the universal endpoint catches anything missed.

Mothers need to understand that prophylaxis works, but the diagnostic timeline is longer than it would be without prophylaxis, and the EID schedule is not over until the 18-month test or 6 weeks after weaning is complete. Premature reassurance (“the baby is HIV-negative”) at an interim timepoint is the commonest counselling error; defer the definitive statement until the end of the schedule.

Dolutegravir’s high genetic barrier makes clinically meaningful resistance uncommon, so the core task on virological failure is to distinguish true resistance from the much commoner non-adherence. South African practice has an algorithmic workflow for exactly this.

The clinical question. Two scenarios must be separated:

• Non-adherence: the drug is not being taken, no resistance is selected, and wild-type virus rebounds under inadequate pressure; genotyping returns wild-type or only minor mutations.
• True failure with resistance: the patient is adherent but the drug is overcome, genuinely rare with dolutegravir but rising in southern African cohorts, particularly viraemic switchers and paediatric patients.

Management differs entirely, adherence support versus regimen change, and genotyping is expensive and uninformative in the non-adherent.

The reflex dolutegravir drug-level workflow:

1. Two consecutive viral loads at or above 1,000 copies/mL after at least nine months on a dolutegravir-based regimen with documented adherence support.
2. The next sample, drawn in EDTA (ethylenediaminetetraacetic acid), goes for viral load and resistance testing.
3. A reflex dolutegravir drug level (DLT) is run first.
4. If dolutegravir is undetectable: reported as non-adherence, no genotype, patient returns for enhanced adherence counselling.
5. If detectable: full drug-resistance test across reverse transcriptase, protease and integrase regions.
6. The clinician refers to the ARV Drug Resistance Committee (ADReC) for third-line authorisation if indicated.

First-line (TLD 1). Emergent dolutegravir resistance on first-line TLD (tenofovir, lamivudine and dolutegravir) is uncommon but rising. The principal mutations are R263K (rare, with a fitness cost, the main dolutegravir-monotherapy escape) and the Q148H/R/K pathway with two or more secondaries from G140S/A, E138K, L74I/M, E92Q, T97A and N155H; Q148K plus two or more secondaries compromises dolutegravir even at double dose.

Second-line (TLD 2). A patient on TLD 2 is more likely to have already accumulated NRTI (nucleoside reverse transcriptase inhibitor) resistance, typically M184V, sometimes K65R or thymidine analogue mutations (TAMs), from previous regimens; these must be interpreted alongside any integrase findings.

Practical points:

• Sequencing needs a viral load above 1,000 copies/mL; lower loads can fail amplification.
• Sample on the failing regimen or within four weeks of stopping, since mutations fade once selective pressure is removed (M184V within about a year; protease-inhibitor and NNRTI mutations over about three years).
• Archived NNRTI (non-nucleoside reverse transcriptase inhibitor) mutations from prior efavirenz may not appear on current sequencing but persist in the reservoir.

Programmatic implications. Genotyping is focused on the small subset with genuine dolutegravir failure while the larger non-adherent group is channelled into adherence support, a cost-effective use of resistance testing at scale.

A dried blood spot (DBS) is a small volume of whole blood (typically obtained by capillary fingerprick or heel-prick) collected onto filter paper, dried, and shipped at ambient temperature. DBS extends molecular diagnostic capacity to settings where plasma logistics (refrigerated transport, rapid processing, large sample volumes) are impractical.

Applications

• HIV viral-load monitoring in remote or hard-to-reach sites.
• Drug-resistance surveillance (pre-treatment and acquired drug resistance studies, particularly the WHO HIVResNet programme).
• Early infant diagnosis in some settings (although South Africa primarily uses plasma EDTA for EID, DBS is acceptable).
• National seroprevalence and HIV-incidence surveys.
• Birth cohort and longitudinal study sampling where repeated venous draws are impractical.

Advantages

• Simple collection: no venepuncture skills required at the collection site.
• Stable at ambient temperature: can be transported by ordinary post or courier.
• Small volume: suitable for paediatric and capillary sampling.
• Cost-effective at scale: much cheaper logistically than plasma.

Challenges for viral-load testing

• Lower limit of detection. DBS VL assays typically require a viral load above 1,000 copies/mL to reliably amplify; the South African plasma assay threshold for suppression (below 50 copies/mL) cannot be reliably read off DBS. DBS cannot distinguish between truly suppressed and persistent low-level viraemia.
• Proviral DNA contamination. Whole-blood DBS contains both cell-free RNA and cell-associated proviral DNA. Most DBS VL assays detect both, leading to overestimation of viral load in suppressed patients, a critical limitation. Specialised RNA-only extraction protocols can mitigate this.
• Sample quality issues: under-spotting, blood mixed with sweat or alcohol, prolonged ambient exposure, humidity damage during transport.
• Inter-assay variability is higher than plasma.
• Subtype-specific sensitivity varies across DBS platforms.

Challenges for drug-resistance testing

• Higher VL threshold for sequencing success: typically VL above 1,000 copies/mL is needed for reliable Sanger amplification from DBS, similar to plasma.
• Sub-optimal amplification rates: DBS DRT amplification rates are typically lower than plasma (around 70 to 80% vs 90 to 95% for plasma).
• Proviral DNA contamination can confound RT/protease sequencing, picking up archived resistance mutations that are not present in the current plasma virus, relevant for surveillance interpretation.
• Subtype-specific primers may need optimisation.
• DBS is acceptable for surveillance (where archived mutations are arguably informative) but is not preferred for clinical case management.

South African programmatic context

The South African programme has used DBS primarily for surveillance and operational research rather than for routine clinical viral-load monitoring or resistance testing. Plasma EDTA remains the standard for clinical care. DBS is useful in:

• Hard-to-reach populations: mobile clinics, prison health services, remote rural sites.
• WHO HIVResNet surveillance: South Africa contributes pre-treatment and acquired drug-resistance data via DBS-based methodologies.
• Cohort studies: birth cohorts, key-population research.

DBS is a powerful tool for extending diagnostic and surveillance reach, but it is not a substitute for plasma in clinical case management. The South African virology laboratory should maintain DBS capability as a parallel competency for the surveillance and operational-research roles it serves, while continuing to drive clinical viral-load and resistance testing on plasma, particularly given the suppression threshold (below 50 copies/mL) that current South African practice demands.

A functional cure (better termed sustained virological remission) means durable, ART-free control of HIV: an undetectable viral load with no disease progression, without eradicating the virus. The latent reservoir persists but replication stays controlled. It contrasts with a sterilising cure, in which replication-competent virus is eliminated entirely, as in the Berlin and London patients after CCR5-Δ32 allogeneic stem-cell transplantation. Those cases are proof of concept that lifelong ART-free control is achievable, but the procedure is far too toxic to be a scalable cure.

Two approaches to functional cure have experimental support:

• Very early ART producing post-treatment control. In the VISCONTI cohort, a subset of people treated during acute infection maintained viral control for years after stopping therapy, attributed to a smaller, less diverse reservoir seeded before wide dissemination.
• Broadly neutralising antibodies (bNAbs). Combination bNAb infusion (for example 3BNC117 with 10-1074) has maintained ART-free suppression through analytical treatment interruption in a subset of people, and may also help clear reservoir cells by recruiting immune effectors.

Other reservoir-directed strategies under investigation include latency reversal (“shock and kill”), latency-promoting “block and lock”, and gene editing of CCR5.

Long-acting early viral inhibition (LEVI) syndrome describes the atypical clinical-laboratory picture that occurs when HIV is acquired despite long-acting antiretroviral PrEP, when the PrEP agent partially suppresses but does not abolish very early viral replication. The PrEP fails to prevent infection but profoundly distorts the early course of infection.

Mechanism. Inhibitory concentrations of a long-acting agent at the moment of acquisition suppress (without eradicating) the first rounds of viral replication. The infection establishes, but the viraemia is blunted and intermittent and the host humoral response is delayed and weakened in step with reduced antigenic stimulus.

Diagnostic implications:

• Antibody assays (including fourth-generation Ag/Ab combos) may be negative or only weakly reactive for weeks to months, well beyond the usual 2 to 4 week diagnostic window.
• HIV RNA is often detectable, but at very low levels, sometimes intermittently positive and negative on serial testing.
• Fiebig staging is distorted; patients may sit in early Fiebig stages for prolonged periods.
• Risk of missed or delayed diagnosis: a person continuing PrEP with intermittent low-level viraemia may be mistaken for an adherent PrEP user with assay noise. Routine 4th-gen testing for breakthrough monitoring is inadequate.
• Risk of drug-resistance selection: sub-therapeutic exposure to a single antiretroviral (ARV) class for weeks drives selection of resistance mutations (capsid N74D for lenacapavir; integrase mutations for cabotegravir).

Management implications:

• High suspicion in any long-acting PrEP user with seroconversion-like symptoms or any positive HIV nucleic-acid test, however low.
• Confirm by HIV RNA / nucleic-acid amplification test (NAAT) rather than relying on serology.
• Switch to a treatment regimen that avoids the implicated PrEP-agent class.
• Resistance genotyping including the relevant region (capsid for lenacapavir; integrase for cabotegravir).
• Specialist consultation: LEVI is an emerging clinical pattern requiring centralised expertise.

Progression implications. The natural history under LEVI is not fully characterised. Reservoir seeding and chronic infection still occur, but at a different rhythm. Long-term virological and immunological outcomes are an active area of study.

HIV-exposed but uninfected (HEU) infants consistently show higher rates of infectious-disease morbidity, hospitalisation and mortality than HIV-unexposed infants in cohort studies, even in the post antiretroviral therapy (ART) era. The phenomenon is well described in southern African populations and has multiple causes.

Underlying reasons

1. Reduced placental transfer of maternal antibody. HIV-positive mothers transfer lower titres of protective immunoglobulin G (IgG) to their infants, for measles, pertussis, Streptococcus pneumoniae, and other pathogens, because of disrupted placental Fc-receptor function. HEU infants are functionally less protected during the first 6 months of life.

2. Antenatal co-morbidity and immune activation. HEU infants are more likely to have been born to mothers carrying HIV-driven immune activation and chronic inflammation during pregnancy, which affects placental function and transmits a pro-inflammatory milieu to the fetus. Co-morbidities cluster with maternal HIV: tuberculosis (TB), syphilis, anaemia and malnutrition.

3. Higher prematurity and low-birthweight rates. HIV-positive mothers have higher rates of preterm delivery and low birth weight, both established drivers of infant morbidity. Tenofovir use in pregnancy has been associated with marginally lower birth weight in some cohorts.

4. Socioeconomic exposure. HEU infants face greater infectious exposure through poverty, food insecurity, household HIV burden (siblings and parents on ART, with intermittent illness and high TB exposure), maternal hospitalisation disrupting infant care, and reduced access to support (stigma, female-headed households without paternal involvement).

Measures to improve outcomes

• Maternal viral suppression throughout pregnancy and breastfeeding. The single most important determinant of both transmission risk and HEU outcomes; keeping the mother well is the single most effective intervention for the infant.
• EPI immunisations strictly on schedule to compensate for reduced placental antibody transfer, with pneumococcal and Haemophilus influenzae vaccines particularly important given reduced maternal antibody.
• Exclusive breastfeeding for the first 6 months, then continued breastfeeding with appropriate complementary feeding. Exclusive breastfeeding is now strongly recommended over formula feeding for HEU infants: the immune and microbiological protection outweighs the small residual transmission risk on suppressive maternal ART.
• Cotrimoxazole preventive therapy where indicated. The 2026 National Consolidated Guidelines (NCG) withdrew routine cotrimoxazole from HEU infants, driven by reduced transmission rates and concerns about antimicrobial pressure; it remains indicated for higher-risk infants and where the infant’s HIV status is not yet excluded, and the withdrawal will require monitoring of post-implementation HEU morbidity to confirm safety.

Next-generation sequencing (NGS) for HIV drug resistance promises minority-variant detection down to about 1%, well below the 20% threshold of Sanger sequencing, but its clinical implementation requires careful validation across several domains.

Technical and pre-analytical challenges:

• PCR amplification bias. Plasma HIV RNA is low-input, and early PCR (polymerase chain reaction) cycles can disproportionately amplify whichever templates cycle first. Primer ID tagging, unique molecular identifiers ligated to each input template, corrects for this bias and allows true variant frequencies to be calculated.
• Sequencing-platform errors. Each NGS platform (Illumina, Ion Torrent, Nanopore) has characteristic error patterns that must be characterised against known-truth controls; APOBEC-mediated G-to-A hypermutation can be misread as resistance mutations.
• Library-preparation reproducibility. Inter-batch and inter-operator variability must be quantified and controlled.

Bioinformatics challenges:

• Read alignment and consensus calling. HIV’s high genetic diversity, particularly in subtype C and circulating recombinant forms, complicates alignment to reference sequences such as HXB2.
• Variant-calling thresholds. Defining the minimum reportable variant frequency is methodologically contentious: too low reports noise, too high loses the value of minority-variant detection.
• Pipeline standardisation. Validated tools exist (HyDRA, MiCall and others) but produce subtly different outputs from the same raw data.

Clinical interpretation challenges:

• The significance of minority variants is not fully established: variants at 5 to 15% are increasingly recognised as meaningful for low-barrier classes (the non-nucleoside reverse transcriptase inhibitors, NNRTIs), while variants below 1% have uncertain impact.
• Algorithm compatibility. Stanford HIVdb and the IAS-USA mutation list were designed for consensus-level interpretation; adapting them to minority-variant data is ongoing.
• Subtype-specific variation. Subtype-specific polymorphisms can be misread as resistance, particularly in subtype C.

Quality assurance and regulatory challenges:

• Reference standards. No universal NGS reference panels exist for HIV resistance; international quality programmes (QCMD, the Quality Control for Molecular Diagnostics scheme) are developing material but coverage is limited.
• Accreditation. Most frameworks (SANAS, the South African National Accreditation System) need extension to cover NGS-specific requirements.
• Validation studies must demonstrate accuracy, precision, reproducibility, limit of detection, linearity and clinical correlation against an established reference method, typically Sanger.

Practical and economic challenges:

• Cost per sample remains higher than Sanger, though the gap is narrowing.
• Turnaround time depends on batching, so clinical urgency may not align with sequencing economics.
• Bioinformatics expertise is a substantial resource requirement for in-house NGS.

Sanger remains the clinical standard. Routine clinical NGS requires validation across all these domains alongside bioinformatics and quality-management capacity.

Both qualitative and quantitative nucleic-acid tests amplify HIV nucleic acid by reverse-transcription PCR (or transcription-mediated amplification, TMA), but they answer different clinical questions.

Comparison

Similarities

Both assays:

• Use reverse-transcription PCR or TMA to detect HIV nucleic acid.
• Are subject to PCR inhibition (heparin, haemoglobin, urine, faecal contamination, etc.); internal controls flag failed runs.
• Are vulnerable to subtype-specific primer mismatches if not properly validated for the local subtype (subtype C in South Africa).
• Detect virus earlier than serology, closing the diagnostic window.
• Require careful specimen handling to preserve RNA integrity (timely separation of plasma; appropriate shipping).

Key differences

• Purpose. Qualitative PCR answers “infected or not”; quantitative viral load (VL) answers “how well is therapy working”.
• Quantification. Qualitative outputs no number; quantitative outputs a precise viral concentration that drives clinical action (repeat-and-persistent-check eligibility at below 50 copies/mL; low-level viraemia between 50 and 999 copies/mL; failure assessment at 1,000 copies/mL or above).
• Indeterminate handling. Qualitative PCR has a defined indeterminate category and algorithm; quantitative VL produces a clean numerical result.
• Confirmation requirement. A positive qualitative result in a child under 2 always requires confirmation; a quantitative VL is a clinical trajectory, not a single-result diagnosis.

Clinical implications

• Early infant diagnosis uses qualitative PCR because the question is binary (infected or not) and the test must operate near the diagnostic threshold.
• ART monitoring uses quantitative VL because the clinically meaningful information is the numerical trajectory (rising, falling, stable around suppression).
• DBS is acceptable for both purposes in surveillance contexts but has greater quantitative inaccuracy.
• A qualitative result is not a substitute for a viral load in an established patient on ART, and vice versa for an infant diagnosis.

A fourth-generation assay detects both p24 antigen and HIV antibody, shortening the window to about two weeks after infection, earlier than antibody-only tests because p24 antigenaemia precedes seroconversion. (Markers appear in order: HIV RNA at roughly 10 to 11 days, then p24 antigen, then IgM at about three weeks, then mature IgG over two to six weeks.)

A negative fourth-generation result during the window does not exclude HIV infection. During the preceding eclipse period no marker is detectable, and in the days around it a person may be infected, and highly infectious, yet test negative.

Clinically, therefore:

• If there has been a recent high-risk exposure, repeat testing after the window (commonly at four to six weeks, with a final test per the local algorithm), or test HIV RNA (nucleic acid) to detect infection earlier.
• Counsel that a negative test within the window is not reassuring, and advise precautions against onward transmission meanwhile.
• If the exposure was within 72 hours, consider post-exposure prophylaxis (PEP) rather than waiting to retest.
• Long-acting injectable pre-exposure prophylaxis (PrEP), such as cabotegravir, can blunt and delay both antigenaemia and the antibody response, prolonging the effective window and risking a missed or late diagnosis.

A small minority of untreated people deviate from the usual course of progressive CD4 decline. Long-term non-progressors stay clinically well with preserved CD4 counts for many years (often more than 7 to 10 years) despite detectable viraemia. Elite controllers, rarer still (well under 1%), spontaneously suppress viraemia below the limit of detection (under 50 copies/mL) off all therapy; viraemic controllers hold a low but detectable load (under about 2,000 copies/mL). The atypical course is multifactorial, with viral, host-genetic and immunological contributions.

Viral factors. A few controllers carry replication-defective or attenuated virus. In the Sydney Blood Bank Cohort a nef-deleted strain produced slow progression in the donor and recipients, establishing nef as a virulence factor. Escape mutations in conserved Gag epitopes that carry a fitness cost also slow replication. In most controllers, though, the virus is fully pathogenic and the explanation lies with the host.

Host genetic factors. Protective HLA class I alleles (B*57:03, B*81:01 and B*27) present conserved Gag epitopes and drive highly effective CD8 cytotoxic T-cell (CTL) responses, the strongest known genetic correlate of control. Heterozygosity for CCR5-Δ32 lowers co-receptor density and slows progression, while homozygosity gives near-complete resistance to CCR5-tropic virus. Certain killer-cell immunoglobulin-like receptor (KIR) genotypes, such as KIR3DS1/3DL1 with their HLA-Bw4 ligands, strengthen natural-killer-cell control.

Immunological factors. Control tracks with polyfunctional, high-avidity CD8 CTL responses against conserved Gag, preserved HIV-specific CD4 helper responses, and lower generalised immune activation with better-preserved lymph-node architecture, the opposite of the chronic activation that drives typical progression.

Control is not cure. Even elite controllers keep a latent reservoir, show low-level replication and residual inflammation, carry excess cardiovascular and other non-AIDS morbidity, and a proportion eventually lose control. The phenomenon still guides vaccine and cure research, since it shows that durable immune control of HIV is biologically possible.

Dolutegravir (DTG) is a second-generation integrase strand-transfer inhibitor that has become the anchor of first-line antiretroviral therapy (ART) worldwide and the keystone of the South African ART programme.

Class and mechanism. DTG is an integrase strand-transfer inhibitor (INSTI). It binds the integrase active site and blocks the strand-transfer step that inserts proviral DNA into host chromatin; without integration, productive infection cannot proceed.

Pharmacology:

• Standard adult dose: 50 mg once daily, orally.
• Bioavailable with or without food (food slightly delays absorption without impairing efficacy).
• Metabolism via UGT1A1 (major) and CYP3A4 (minor); minimal renal elimination.
• Plasma half-life around 14 hours, supporting once-daily dosing.
• High genetic barrier: the R263K resistance mutation arises rarely and carries a measurable fitness cost.

Key interactions:

• Rifampicin (a strong UGT1A1/CYP3A4 inducer): add a second 50 mg tablet twelve hours after the TLD dose, until two weeks past rifampicin.
• Polyvalent cations (Ca²⁺, Fe²⁺, Mg²⁺, Al³⁺) chelate DTG in the gut; time antacids at least six hours before or two hours after DTG, though calcium and iron with food can be co-administered.
• Enzyme-inducing anticonvulsants (carbamazepine, phenytoin, phenobarbital) also need dose doubling; valproate, lamotrigine or levetiracetam are preferred.
• Metformin: DTG raises its levels; cap metformin (typically 2 g/day).

Adverse effects:

• Common but mild: insomnia, headache, mild gastrointestinal (GI) upset.
• Creatinine artefact: DTG inhibits tubular creatinine secretion (OCT2/MATE1) without affecting true glomerular filtration rate (GFR). A rise of up to about 30 µmol/L in the first weeks is benign; investigate only larger or later rises.
• Weight gain, well-recognised, but now understood as unmasking of weight previously suppressed by HIV and efavirenz (EFV); DTG should not be switched off for weight gain alone.
• Pregnancy and women of child-bearing potential: the 2018 Tsepamo neural-tube-defect signal has not been confirmed in mature data; DTG is used without restriction in pregnancy, breastfeeding and safe conception.
• Rare hepatitis and hypersensitivity.

Role in the South African ART programme. Dolutegravir was rolled out as the anchor of the TLD (tenofovir / lamivudine / dolutegravir) fixed-dose combination from 2019 to 2020, replacing the efavirenz-based TEE regimen. TLD is now the first-line regimen for adults and adolescents from 30 kg and 10 years, including in pregnancy. The paediatric equivalent ALD (abacavir / lamivudine / dolutegravir) and the dispersible pALD formulation extend the regimen down to neonates of 2 kg and 37 weeks’ gestational age. DTG sits on the public-sector Essential Medicines List and is delivered through routine facility pharmacy without specialist authorisation.

Resistance. DTG monotherapy or low-adherence dolutegravir-based therapy occasionally selects the R263K mutation (low-level resistance, fitness cost) or, with prior INSTI exposure, the Q148H pathway (higher-level resistance). Recent South African practice introduces reflex DTG drug-level testing as a gatekeeper before resistance genotyping in patients meeting virological failure criteria: undetectable DTG implies adherence failure rather than true resistance.

South African post-exposure prophylaxis (PEP) is a 28-day course of TLD, started as soon as possible after exposure with an upper limit of 72 hours.

Begin empirically (the source’s HIV result is not required before starting) and stop only if the source proves HIV-negative on confirmed testing. A single tenofovir dose, cabotegravir injections, a seven-day triple-dose course, and withholding PEP on the basis of hepatitis B vaccination are all wrong. Baseline HIV, hepatitis B surface antigen (HBsAg) and anti-hepatitis C virus (anti-HCV) testing of the exposed person is part of the workup; consider separately the need for hepatitis B virus (HBV) PEP (hepatitis B immunoglobulin plus accelerated vaccination) if the source is HBsAg-positive and the exposed person is not immune.

K103N disrupts the hydrophobic binding pocket of the non-nucleoside reverse transcriptase inhibitors (NNRTIs), so efavirenz and nevirapine are both lost while other classes remain active: cross-resistance is the rule within a class and the exception between classes.

Drugs targeting different viral proteins, the nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (PIs), integrase strand transfer inhibitors (INSTIs), capsid inhibitors and entry inhibitors, keep working. K103N does not reach every class, does not touch tenofovir or lamivudine, does not spare nevirapine, and does not affect dolutegravir.

M184V confers high-level resistance to lamivudine and emtricitabine while reducing replicative fitness and re-sensitising the virus to zidovudine; it also modestly increases tenofovir susceptibility.

It does not affect dolutegravir, spares neither lamivudine nor emtricitabine, and does not abolish the whole nucleoside reverse transcriptase inhibitor (NRTI) class. This is why lamivudine is recycled into second-line regimens: the residual selective pressure on M184V helps suppress the virus and preserves NRTI activity in the backbone.

Retaining lamivudine (3TC) after M184V keeps selecting for a virus that replicates measurably less efficiently than wild-type, and the discrimination at the tyrosine-methionine-aspartate-aspartate (YMDD) motif re-sensitises the virus to zidovudine (AZT), with a modest effect on tenofovir.

Although M184V abolishes the antiviral activity of lamivudine, holding it in the regimen maintains the selective pressure that keeps M184V in the dominant population, to the patient’s benefit. Higher doses do not restore lamivudine activity; M184V is common and durable under drug pressure, not transient; anti-hepatitis-B activity is a genuine benefit in co-infection but not the primary rationale; and cost is not the reason.

In drug-sensitive extra-neurological tuberculosis (TB) with CD4 below 50 cells/µL, ART starts within 2 weeks of TB treatment, reflecting the high short-term mortality of untreated HIV at low CD4.

At CD4 at or above 50 the start is deferred to 8 weeks, which does not apply here. Same-day initiation is not the rule with TB co-treatment; deferring until TB treatment ends abandons a severely immunocompromised patient; and immune reconstitution inflammatory syndrome (IRIS) is a risk of early ART, not a trigger for it. The earlier start here is offset by prophylactic prednisone (40 mg for 2 weeks then 20 mg for 2 weeks) when CD4 is at or below 100 and ART begins within 30 days of TB treatment.

Vivid dreams, depression, dizziness and mood changes are typical central nervous system (CNS) effects of efavirenz, most marked in the first weeks and usually improving thereafter.

They are not encephalopathy demanding immediate cessation, virological failure, nucleoside-analogue mitochondrial toxicity or a rifampicin interaction. Take at night to mitigate; severe psychosis or persistent severe mood disturbance warrants switching.

Simvastatin (and lovastatin) are heavily CYP3A4-metabolised, so boosted protease inhibitors (PIs) raise their concentrations to a level that risks rhabdomyolysis, making them contraindicated.

Atorvastatin may be used at low dose, and pravastatin, fluvastatin and rosuvastatin are the safer choices.

Atazanavir inhibits UGT1A1, producing a benign indirect (unconjugated) hyperbilirubinaemia occasionally visible as scleral icterus or jaundice.

Normal aminotransferases exclude drug-induced hepatitis; the picture is not obstructive cholestasis, haemolysis or sepsis-related cholestasis. It is a cosmetic effect, not liver injury, does not require discontinuation, and is sometimes used as a marker of adherence.

Polyvalent cations (calcium, aluminium, magnesium, iron) chelate dolutegravir in the gut, so the antacid must be separated by at least six hours before or two hours after the dose.

Stopping, dose-doubling, co-administering or switching to a proton-pump inhibitor are all wrong: proton-pump inhibitors do not address the chelation, and calcium or iron taken with food may be co-administered.

Seroconversion on lenacapavir pre-exposure prophylaxis (PrEP) mandates resistance genotyping that includes the capsid region, since breakthrough virus selects the capsid N74D signature (M66I and Q67H are also reported).

Standard reverse-transcriptase, protease or integrase genotyping alone would miss the relevant resistance, and deferring or omitting testing is inappropriate; the capsid assay is not yet routine in South African virology laboratories, and capsid inhibitors should be avoided in the subsequent regimen.

Rifampicin induces UGT1A1 and CYP3A4 and substantially reduces dolutegravir exposure, so dolutegravir is doubled with a supplementary 50 mg tablet twelve hours after the TLD dose.

The supplementary dose continues until two weeks past the rifampicin so that enzyme induction has waned. Leaving TLD unchanged underdoses dolutegravir, while switching to a protease inhibitor, halving the dose or stopping antiretrovirals are all inappropriate.

Dolutegravir inhibits tubular secretion of creatinine via OCT2 and MATE1 without altering true glomerular filtration rate (GFR), so a rise of up to about 30 µmol/L in the first weeks is expected and benign.

This asymptomatic early rise is neither acute tubular necrosis, pre-renal injury, tenofovir-induced Fanconi syndrome nor HIV-associated nephropathy. Investigate only larger rises, those occurring later, or those accompanied by tubular wasting, proteinuria or symptoms.

Stanford HIVdb returns one of five levels per drug: susceptible, potential low-level, low-level, intermediate, and high-level resistance.

The algorithm uses an additive mutation penalty score: each recognised drug-resistance mutation contributes a penalty for every drug it affects, and the totals map to the five levels. The penalty scores are grounded in the International Antiviral Society-USA (IAS-USA) Drug Resistance Mutations List, updated annually. The two-, three-, four- and seven-level options do not match the Stanford scheme.

The standard HIV mutation notation is [wild-type amino acid][codon number][mutant amino acid]: M184V reads as methionine (M) at codon 184 of reverse transcriptase (RT) replaced by valine (V).

At RT codon 184 the underlying triplet changes from ATG (methionine) to GTG (valine), a single A-to-G nucleotide substitution. The other options misread the notation: it encodes an amino acid change, not a methylation change, a protein-abundance factor, a single base substitution, or a transmitted polymorphism status.

Boosted protease inhibitors reduce the efficacy of hormonal contraception, so dual contraception is advised, with an intrauterine device a favoured option.

The interaction lowers contraceptive hormone levels rather than raising protease-inhibitor levels or causing hyperkalaemia, and separating the dosing times does not overcome it.

Reaching 95% diagnosed, 95% on antiretroviral therapy (ART) and 95% virally suppressed multiplies out to population-level viral suppression of ~86%, the threshold above which onward transmission collapses.

The 95% and 90% values are individual cascade pillars, ~73% is South African TLD (tenofovir, lamivudine and dolutegravir) coverage, and ~77% is the current South African all-PLHIV suppression figure.

AHD is any one of CD4 at or below 200 cells/µL, World Health Organization (WHO) clinical stage 3 or 4, or age under 5 years (automatic unless stable on antiretroviral therapy for at least 12 months).

The threshold is 200, not 50, and stage 3 counts as well as stage 4, so the single-criterion options are too narrow; viral load and hospitalisation alone do not define AHD. The classification triggers the nine-step package: reflex cryptococcal antigen (CrAg), urine tuberculosis lipoarabinomannan (TB-LAM), cotrimoxazole prophylaxis, tuberculosis preventive therapy, pre-emptive fluconazole, rapid ART and intensified follow-up. About one in five people living with HIV present with AHD in South Africa.

Entry requires CD4 plus a chemokine co-receptor: CCR5 (R5 viruses) or CXCR4 (X4 viruses).

R5 viruses dominate transmission and early disease while X4 viruses tend to emerge later, and dual-tropic strains use either; this is the step blocked by the CCR5 antagonist maraviroc. DC-SIGN (dendritic-cell-specific ICAM-3-grabbing non-integrin) captures virus but is not the entry co-receptor, and CD8 and CD3 are unrelated T-cell markers.

AIDS is defined by a CD4 count below 200 cells/µL or by an AIDS-defining condition.

Specific opportunistic infections cluster at lower counts: Pneumocystis pneumonia around 200, and cryptococcal disease, cytomegalovirus (CMV) retinitis and disseminated Mycobacterium avium complex (MAC) below about 100.

Any positive HIV PCR in a child under 2 years must be confirmed on a fresh sample before a definitive report.

A single positive neither confirms nor excludes infection, so reporting on it, discharging, or deferring to a 6-month or 18-month test are all wrong. South African practice substitutes a viral load (VL) for the confirmatory PCR, giving confirmation and a baseline VL at once. Antiretroviral therapy (ART) starts immediately without waiting for the confirmatory result, but the report rests on the confirmed sample.

A maternal delivery VL below 50 copies/mL is low risk, so the infant receives nevirapine (NVP) daily for 6 weeks.

Prophylaxis is not omitted, 12 weeks of NVP or the zidovudine (AZT)-plus-NVP regimen is the higher-risk option (maternal VL at or above 50 while breastfeeding), and full combination therapy is treatment, not prophylaxis. Every exposed infant first gets dual NVP plus AZT at birth until the delivery VL is known; here the below-50 result stops the AZT and leaves 6 weeks of NVP.

The immunity threshold is HBsAb at or above 10 mIU/mL, so a value of 4 is non-immune and needs a full three-dose HBV vaccination at 0, 1 and 6 months.

The HBsAb is not protective, a single booster is insufficient in a never-immune non-responder, the vaccine is safe rather than contraindicated in HIV, and immunoglobulin is not the action here. Recheck HBsAb 2 months after the last dose; if still below 10, repeat the three-dose course, then refer if it remains below 10.

In South African practice, immunosuppressed patients (including HIV-positive patients with reduced CD4 counts or chronic liver disease) receive the standard two-dose HAV vaccine schedule plus a third booster dose at 6 to 12 months after the first dose, to improve seroconversion.

Seroconversion in HIV-positive patients falls to between 52% and 94% compared with over 99% in immunocompetent adults, which is the rationale for the extra dose. The inactivated vaccine is safe at any CD4 count.

A patient with HIV who is on effective ART, clinically well and virologically suppressed is not considered immunocompromised for the purposes of rabies vaccination. The standard 2-dose IM PrEP schedule on days 0 and 7 applies, with normal expected seroconversion.

The extended 3-dose schedule (days 0, 7, 21–28) is reserved for genuinely immunocompromised patients: symptomatic HIV with low CD4 counts, active cancer chemotherapy, high-dose corticosteroids (≥20 mg/day prednisone for ≥2 weeks), or transplant immunosuppression. Inactivated rabies vaccine is safe at any CD4 count.

Around 40 million people are living with HIV globally, the headline figure that frames the modern response.

The ~21 million figure is the eastern and southern African subtotal, ~7.8 million is South Africa’s national burden, ~1.3 million is annual new infections, and ~232,000 is South Africa’s yearly incidence.

V106M arises from a single nucleotide change in subtype C virus, whereas subtype B needs two changes, so it is selected much more readily in subtype-C populations under efavirenz pressure, directly relevant to South Africa given subtype C dominance.

V106M confers high-level resistance to nevirapine and efavirenz. It is a non-nucleoside reverse transcriptase inhibitor (NNRTI) mutation, so it does not touch dolutegravir or re-sensitise the virus to lamivudine; it is not the commonest transmitted mutation worldwide (that is K103N), and it is not confined to paediatric infection.

Proton-pump inhibitors suppress gastric acid enough to drop atazanavir levels below therapeutic, so they are contraindicated.

Atazanavir is absorbed only in an acidic stomach and must be taken with food; H2-receptor blockers and antacids need careful temporal separation. Loperamide, metformin, paracetamol and amoxicillin carry no comparable interaction.

Gag is cleaved into the structural proteins: matrix (p17), capsid (p24), nucleocapsid (p7) and p6.

The pol gene encodes the enzymes (protease, reverse transcriptase, integrase) and env encodes gp120 and gp41; Tat, Rev, Nef, Vif, Vpr and Vpu are regulatory and accessory proteins from other reading frames.

Dolutegravir binds the active site of integrase and blocks the strand-transfer step that splices proviral DNA into host chromatin, so without integration the provirus cannot be transcribed.

It does not act on reverse transcription, protease-mediated maturation, capsid uncoating or co-receptor-mediated fusion. Dolutegravir (DTG) has a high genetic barrier: R263K arises rarely and carries a fitness cost.

Dolutegravir is an integrase strand-transfer inhibitor (INSTI), blocking insertion of proviral DNA into host chromatin.

Its high potency, good tolerability and high genetic barrier to resistance make it the preferred first-line anchor drug. The distractors name steps handled by other drug classes: reverse transcriptase, protease, fusion and capsid inhibitors.

Drug-resistant variants typically carry a fitness cost, so once selective drug pressure is removed, fit wild-type virus outcompetes them and the resistant variants fade from the dominant sequence, the “memory effect”.

M184V fades within about a year; protease inhibitor (PI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations over about three years; some thymidine analogue mutations (TAMs) are more persistent. Resistance archives persist in the integrated reservoir, but a consensus-sequence test of plasma ribonucleic acid (RNA) will miss them, so testing is done on the failing regimen or within four weeks of stopping. The distractors misstate sample stability, algorithm requirements, integrase sequenceability and polymerase chain reaction (PCR) amplification, none of which explains the timing rule.

Protease inhibitors block cleavage of the Gag and Gag-Pol polyproteins, so the structural proteins stay uncleaved and the resulting virions are non-infectious.

They act at maturation, not at reverse transcription, integration, uncoating or fusion. All protease inhibitors (PIs) in clinical use require pharmacoenhancement with low-dose ritonavir (the “/r” suffix) or cobicistat.

Error-prone reverse transcription, combined with an enormous replicative output (on the order of ten billion virions a day), generates a quasispecies, a swarm of related variants within each host.

This standing diversity is the raw material for immune escape and for the rapid selection of drug resistance, so the distractors (uniformity, reduced mutation, or loss of core functions) are the opposite of what high error rates produce.

HIV-2 derives from the sooty mangabey virus SIVsm (simian immunodeficiency virus of the sooty mangabey).

It shares only about 50 to 60% of its sequence with HIV-1, which arose separately from chimpanzee and gorilla lineages, and it is less transmissible and slower to progress.

Without a functional CCR5 co-receptor, R5 virus cannot enter, conferring near-complete resistance to infection.

This is the basis of the CCR5-Δ32 allogeneic stem-cell-transplant cures; CXCR4-using (X4) virus could in principle still enter, and heterozygotes are partially protected and progress more slowly. The distractors invert the phenotype or attach it to the wrong target.

AGYW incidence fell approximately three-fold between 2017 and 2022, from 1.17% to 0.39%, one of the most substantial population-level prevention successes in the global response.

Option E reverses the direction, and the other options misstate the magnitude of the decline.

The cascade began as 90-90-90 in 2014 and was later upgraded to 95-95-95 when the original 2020 milestone came into view and the 2030 horizon needed a more ambitious anchor.

The other options confuse the cascade with the 2030 mortality goal, a national survey, vertical-transmission elimination and long-acting pre-exposure prophylaxis (PrEP), none of which is its origin.

Global new infections have plateaued at ~1.3 million per year, well above the UNAIDS interim target of 370,000 by 2025, so the world is not yet on track.

The 370,000 figure is the target itself rather than the actual count, ~232,000 is South Africa’s national incidence, and the remaining values are implausible.

South Africa carries the largest single national HIV burden in the world, an estimated ~7.8 million people living with HIV, and runs one of the largest medication programmes on earth.

The ~21 million figure is the eastern and southern African regional total, ~40 million is the global figure, and the smaller values are annual incidence counts.

The operational endpoint is a 90% reduction in new infections and AIDS-related deaths against the 2010 baseline, with continued decline thereafter to sustain the gain.

The other options misstate the reduction, the reference year or the metric; the goal is a threat-elimination target, not zero infections or universal suppression.

Tenofovir suppresses both HIV and HBV, so abrupt cessation of tenofovir disoproxil fumarate (TDF), lamivudine (3TC) or emtricitabine (FTC) can precipitate a severe, sometimes fatal hepatitis B flare in a co-infected patient.

HIV rebound, resistance from the drug tail, renal injury and prodrug withdrawal are not the driving concern. Check hepatitis B surface antigen (HBsAg) before stopping any of these agents; if TDF must be replaced (for example eGFR 30 to 50), the substitute is tenofovir alafenamide (TAF) plus FTC plus dolutegravir, which preserves HBV cover.

Cryptococcal meningitis defers ART 4 to 6 weeks after antifungal therapy starts, reducing the risk of paradoxical cryptococcal immune reconstitution inflammatory syndrome (IRIS) in the central nervous system.

Immediate or 2-week starts are too early for meningitis, waiting for antifungal completion is too long, and ART is not contraindicated. Tuberculous meningitis has a comparable 4-to-8-week window, whereas asymptomatic antigenaemia with a negative lumbar puncture starts ART immediately alongside pre-emptive fluconazole (1,200 mg daily for 14 days).

K103N is the commonest transmitted non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance mutation in South African surveillance and globally, conferring high-level resistance to efavirenz and nevirapine.

Pre-treatment NNRTI resistance rose above 10% in several South African surveys, a major reason first-line therapy moved from an efavirenz-based to a dolutegravir-based regimen. K103N alone has little effect on etravirine or rilpivirine (L100I plus K103N/S together markedly reduces rilpivirine). The distractors M184V, K65R, R263K and Q148H are selected under drug pressure and are not typical transmitted mutations at this frequency.

In the CAPELLA (multidrug-resistant treatment) and CALIBRATE (treatment-naive) trials of lenacapavir, M66I was the dominant emergent capsid mutation in patients with virological failure.

Other capsid mutations described under lenacapavir pressure include Q67H, L56I, K70N/H, N74D/S, A105T and T107N; combinations such as Q67H with N74S can produce fold-losses of more than a thousand. Detection requires capsid-region sequencing, which is not part of the standard South African pol genotype. N74D is the pre-exposure prophylaxis (PrEP) breakthrough signature, R263K an integrase mutation, and M184V a reverse transcriptase mutation, none of which is the dominant lenacapavir treatment-failure mutation.

Both Purpose 2 breakthrough infections under twice-yearly subcutaneous lenacapavir pre-exposure prophylaxis (PrEP) developed the capsid N74D mutation.

The dominant emergent mutation in lenacapavir treatment trials (CAPELLA, CALIBRATE) is M66I, not N74D. The PrEP-breakthrough signature flags the need for capsid-region resistance testing in any lenacapavir-PrEP seroconverter, and the next regimen should avoid the capsid-inhibitor class. M184V and K103N are reverse transcriptase mutations and R263K an integrase mutation, none relevant to capsid resistance.

The ABCDE differential runs Adherence, Bugs, Correct dose for weight, Drug interactions, then Resistance, so resistance testing is the last step, reached only once the others are excluded.

The distractors substitute lifestyle or generic factors (body mass index, diet, exercise, counselling, disclosure) that do not belong in the viraemia work-up. In detail: adherence is the dominant cause of detectable viraemia on tenofovir/lamivudine/dolutegravir (TLD); bugs means concurrent infection (tuberculosis, sexually transmitted infections, recent vaccination); correct dose matters especially in paediatrics; drug interactions include rifampicin, anticonvulsants and polyvalent cations. This complements the reflex dolutegravir (DTG) drug-level workflow.

Incidence is rising in the Middle East and North Africa, in Latin America and in eastern Europe and central Asia, a marker of the epidemic’s regional heterogeneity.

The other options name regions where incidence is falling or countries that have already reached the 95-95-95 target, not regions of rising incidence.

In subtype C the codon context upstream of K65 carries a poly-A run that facilitates the AAA-to-AGA nucleotide change required for K65R, so the mutation emerges more readily in subtype C than in subtype B under tenofovir disoproxil fumarate (TDF) pressure, relevant because subtype C predominates in South Africa.

The fold-loss of TDF susceptibility (about two-fold) is similar across subtypes; the difference is in mutation accessibility, not resistance magnitude, so it neither confers higher-level resistance nor loses clinical significance. K65R does not stay rare in subtype C, and it increases zidovudine (AZT) susceptibility rather than causing greater fitness loss than in subtype B.

KICS is a severe systemic inflammatory illness with high interleukin-6 and interleukin-10 and a high KSHV blood viral load, often with Kaposi sarcoma but without the lymph-node pathology of multicentric Castleman disease.

It is neither a discrete tumour nor a congenital, inherited or vaccine-related condition.

Lenacapavir is the first-in-class capsid inhibitor, disrupting capsid function at uncoating, nuclear import and assembly.

Its very long half-life allows six-monthly subcutaneous dosing for both treatment and prevention. The distractors name the established, older drug-target classes.

M184V sits in the tyrosine-methionine-aspartate-aspartate (YMDD) catalytic loop of reverse transcriptase (RT), where the valine substitution tightens the active site so natural deoxycytidine triphosphate (dCTP) is preferentially incorporated and the chain-terminating analogues are excluded: discrimination.

The mutation reduces lamivudine (3TC) and emtricitabine (FTC) susceptibility by more than a hundred-fold, reduces viral fitness, and re-sensitises the virus to zidovudine (AZT), with a modest effect on tenofovir. Excision is the mechanism of the thymidine analogue mutations, not M184V; allosteric disruption describes the non-nucleoside reverse transcriptase inhibitors (NNRTIs); protease cleavage and host apolipoprotein-B mRNA-editing enzyme (APOBEC) binding are not resistance mechanisms of this mutation.

Next-generation sequencing (NGS) reads thousands of individual template molecules per position in parallel, each read derived from a separate input template, so minority drug-resistance mutations down to about 1% (and sometimes lower) can be reliably called.

This is roughly twenty-fold more sensitive than Sanger sequencing, which resolves variants only at about 20% or more, and is changing the clinical relevance of low-frequency variants, particularly for low-barrier drug classes such as the non-nucleoside reverse transcriptase inhibitors (NNRTIs). The higher thresholds of about 10%, 20%, 50% and 90% understate NGS sensitivity.

Prophylactic prednisone is reserved for the highest-risk patients: CD4 at or below 100 cells/µL with ART started within 30 days of TB treatment, given as 40 mg daily for 2 weeks then 20 mg daily for 2 weeks.

The other options are too broad: prophylaxis is not for every HIV-TB patient, nor defined by drug-resistant or extrapulmonary TB alone, and CD4 above 200 with delayed ART is low-risk. Established non-neurological IRIS is instead treated reactively (prednisone 1.5 mg/kg for 2 weeks then 0.75 mg/kg for 2 weeks); IRIS is a diagnosis of exclusion, ruling out multidrug-resistant or extensively drug-resistant TB and non-adherence.

Purpose 2 showed a large reduction in HIV acquisition, about 96% versus background incidence and about 89% versus daily F/TDF (emtricitabine/tenofovir disoproxil fumarate).

Across 3,265 participants in the US, Brazil, Thailand, South Africa, Peru, Argentina and Mexico, there were 2 infections in the 2,179-participant lenacapavir arm, both developing the capsid N74D resistance mutation. The trial was neither futile nor placebo-equivalent, and incidence was lower, not higher, on lenacapavir.

R263K is the main escape mutation observed under dolutegravir monotherapy, and the mutant carries a measurable fitness cost.

It is rare in clinical failure of a tenofovir-lamivudine-dolutegravir (TLD) regimen, because dolutegravir’s high genetic barrier means clinically meaningful resistance usually requires the Q148-plus-secondaries pathway. Detection requires sequencing the integrase region, part of the South African drug-resistance test when dolutegravir is the failing anchor, so it is not seen on reverse transcriptase sequencing. R263K does not touch the protease inhibitors, is not common in non-adherent patients, and does not re-sensitise the virus to first-generation integrase inhibitors.

Sanger sequencing reads the consensus of the polymerase chain reaction (PCR) amplified template population, so a minority variant appears on the chromatogram as a secondary peak only when it reaches about 20% or more of the population.

Below that threshold it is lost in the wild-type signal. This is why next-generation (deep) sequencing, which can detect variants at about 1% or lower, is increasingly used in research and reference settings. The lower thresholds of about 1% and 5% overstate Sanger sensitivity, while 50% and 95% understate it.

Both target reverse transcriptase (RT), by different mechanisms.

Tenofovir is a nucleotide RT inhibitor, a chain-terminating analogue, whereas efavirenz is a non-nucleoside RT inhibitor acting allosterically. The distractors name other drug-target enzymes and structures.

In South African practice, tenofovir disoproxil fumarate (TDF) is avoided once the estimated glomerular filtration rate (eGFR) falls below 50 mL/min/1.73 m².

TDF is renally cleared and causes proximal tubular injury and reduced bone mineral density. The usual substitute is abacavir; TAF/FTC (tenofovir alafenamide/emtricitabine) is reserved for hepatitis B virus (HBV) co-infection with reduced renal function.

Abacavir hypersensitivity is strongly linked to the HLA-B*5701 allele, presenting within the first six weeks with fever, rash, gastrointestinal, respiratory and constitutional symptoms.

Re-challenge after a recognised reaction is contraindicated and can be fatal. It is not Stevens-Johnson syndrome (linked instead to HLA-B*15:02), drug-induced lupus, first-dose anaphylaxis or interstitial nephritis. The allele is rare in Black African populations (under 1%), so routine HLA-B*5701 screening is not part of South African public-sector practice.

The EID schedule places HIV PCR at birth, 10 weeks and 6 months, so the two-timepoint and 10-week-only options miss tests and monthly testing is not the schedule.

Alongside the three PCRs, a universal rapid antibody test follows at 18 months, with an age-appropriate test 6 weeks after breastfeeding stops and testing whenever the infant is unwell. The 10-week PCR was moved from the earlier 6-week timing to align with the Expanded Programme on Immunisation (EPI) vaccination visit; the 6-month PCR aligns with the maternal 6-month viral load.

The original 90-90-90 framework has been upgraded to 95-95-95 by 2030: 95% of people living with HIV diagnosed, 95% of those diagnosed on antiretroviral therapy (ART), and 95% of those on ART virally suppressed.

The 90-90-90 targets (2020) are superseded; 100-100-100 and 70-70-70 are not real targets; and the framework does set explicit numeric goals. Achieved in cascade, these targets deliver population-level suppression sufficient to end HIV as a public-health threat, and South Africa has committed to them in the 2026 National Consolidated Guidelines.

Vpu (unique to HIV-1) antagonises tetherin (BST-2), which would otherwise hold budding virions at the cell surface; it also degrades CD4.

Vif counters APOBEC3G, and the HIV-2 and SIV protein Vpx counters SAMHD1; TRIM5α and MxA are restriction factors Vpu does not target.

The trimeric envelope spike is made of gp120 (surface, binds CD4) and gp41 (transmembrane, drives fusion).

Both are cleaved from the gp160 precursor, which is not itself the mature spike. The other options name internal structural proteins, enzymes, or influenza glycoproteins.

HIV is diploid, carrying two identical copies of positive-sense single-stranded RNA (about 9.7 kb each), flanked by long terminal repeats.

After entry, reverse transcriptase copies the RNA into double-stranded DNA. The distractors describe other virus classes: a DNA genome, a single negative-sense strand, a segmented orthomyxovirus genome, or the partially double-stranded circular DNA of hepatitis B.

The pol gene encodes protease, reverse transcriptase and integrase.

These are targeted respectively by the protease inhibitors, the nucleoside and non-nucleoside reverse transcriptase (RT) inhibitors, and the integrase strand-transfer inhibitors. The distractors mix in enzymes HIV does not encode, such as neuraminidase, helicase and thymidine kinase.

HIV-1 group M, the pandemic lineage, arose from chimpanzee SIVcpz (simian immunodeficiency virus of the chimpanzee).

Group N also came from SIVcpz, groups O and P from gorilla SIVgor, and HIV-2 (a separate virus) from sooty mangabey SIVsm.

Purpose 1 recorded 0 infections among 2,134 women in the lenacapavir arm, a 100% reduction versus background incidence and the highest pre-exposure prophylaxis (PrEP) efficacy yet recorded.

Among 5,338 women across South African and Ugandan sites, participants were randomised 2:2:1 to twice-yearly subcutaneous lenacapavir, daily F/TAF (emtricitabine/tenofovir alafenamide) or daily F/TDF (emtricitabine/tenofovir disoproxil fumarate). Efficacy was not modest, inferior or absent, and the trial was not halted for safety; it also illustrated the adherence problem with daily oral PrEP in this population.

Q148H, R or K alone, or with one secondary mutation, usually leaves dolutegravir clinically active because of its high genetic barrier; the picture changes when two or more secondaries accumulate (from G140S/A, E138K, L74I/M, E92Q, T97A and N155H), particularly with Q148K.

At that point dolutegravir efficacy is compromised even with double dosing, and the regimen must be reconstructed. Raltegravir and elvitegravir are compromised by Q148 with even a single secondary. A single Q148 mutation is not sufficient for dolutegravir, Q148 does affect dolutegravir, M184V is a reverse transcriptase mutation irrelevant to this integrase pathway, and both oral dolutegravir and cabotegravir are affected.

TLD is the once-daily fixed-dose tablet of tenofovir disoproxil 300 mg, lamivudine 300 mg and dolutegravir 50 mg.

The paediatric equivalent ALD substitutes abacavir for tenofovir, and the older efavirenz-based regimen was TEE (tenofovir, emtricitabine, efavirenz); the lopinavir/ritonavir and darunavir/ritonavir options name protease inhibitors, not the current first-line third agent.

The three-test serial strategy exists to hold the positive predictive value (PPV) at 99% or higher, minimising false-positive misdiagnosis. A highly sensitive screening test first identifies all reactive individuals; two confirmatory tests of different manufacture then rule out false-reactive results. Only a sample reactive on the screening test and both confirmatory tests is reported HIV positive.

Raising sensitivity is the role of the screening test, not the third; a surveillance case count and HIV-2-specific testing are unrelated to the algorithm’s purpose. A sample reactive on the screening test and confirmatory test 1 but non-reactive on confirmatory test 2 is reported as discrepant and referred for enzyme-linked immunosorbent assay (ELISA) reflex testing. The third test’s job is therefore to secure the PPV, not merely to break a tie between the first two.

Genotypic resistance testing requires enough viral ribonucleic acid (RNA) to amplify the pol gene by polymerase chain reaction (PCR), so the conventional threshold is above 1,000 copies/mL, below which amplification frequently fails.

South African virological-failure criteria use the same 1,000 copies/mL threshold (two consecutive measurements after at least nine months on a dolutegravir-based regimen with adherence support). Newer methods can sometimes succeed down to about 200 copies/mL, but 1,000 remains the operational standard. The 50 and 200 copies/mL options are suppression and low-level-viraemia markers, not amplification thresholds; 10,000 and 100,000 copies/mL are unnecessarily high.

TAMs were selected under zidovudine (AZT) and stavudine pressure and act by excision: once an analogue is incorporated into the nascent chain and stops extension, the TAM-mutated reverse transcriptase (RT) uses a phosphorolytic reaction to remove it and resume synthesis.

TAMs accumulate stepwise, and full-strength resistance usually needs three or four. Type-1 TAMs (41/210/215) affect tenofovir and abacavir more than Type-2 (67/70/215F/219). TAMs and the discrimination mutations (for example K65R) are often antagonistic, the virus tending to use one pathway or the other. Discrimination is the M184V mechanism, not excision; allosteric disruption describes the NNRTIs; host degradation and drug export are not TAM mechanisms.

Nevirapine’s defining safety signals are hepatotoxicity and severe rash, the latter including Stevens-Johnson syndrome, both most likely in the first weeks of initiation.

Anaemia and neutropenia belong to zidovudine, renal tubular injury and Fanconi syndrome to tenofovir, mitochondrial neuropathy and lactic acidosis to the older nucleoside analogues, and central nervous system (CNS) effects to efavirenz. These signals moved South African practice away from nevirapine in adult initiation; residual uses are the preterm neonatal regimen and infant prophylaxis for HIV-exposed infants.

HIV-1 is a retrovirus (family Retroviridae, genus Lentivirus), Baltimore Group VI: single-stranded RNA viruses that reverse-transcribe into DNA.

Group VII holds the DNA viruses that reverse-transcribe (hepatitis B), the common distractor, so option E pairs the right family with the wrong group. The remaining families and groups belong to unrelated viruses.

The Advanced HIV Disease (AHD) package triggers reflex serum CrAg at CD4 at or below 200 cells/µL, the same threshold that defines AHD.

The other cut-offs (50, 100, 350, 500 cells/µL) are not the trigger. Any positive serum CrAg requires a lumbar puncture (LP): a positive cerebrospinal fluid (CSF) CrAg is cryptococcal meningitis (treat and defer antiretroviral therapy 4 to 6 weeks); a negative CSF CrAg is asymptomatic antigenaemia (pre-emptive fluconazole 1,200 mg daily for 14 days and immediate ART).

TLD 1 covers the ART-naïve patient started on a dolutegravir (DTG)-based regimen and the patient switched from a non-DTG first-line (typically TEE) with a viral load (VL) below 50 copies/mL in the last 12 months, which is exactly this patient.

TLD 2 is for a switch from a non-DTG first-line with VL at or above 50, or from a protease inhibitor (PI)-based second-line (VL below 50, or VL at or above 1,000 without a genotype). TLD 3 follows a genotypic resistance test showing PI resistance, authorised by the ARV Drug Resistance Committee (ADReC). ALD 2 is the paediatric abacavir-based series, and “recycled TLD” is not a category.

The 2026 change moved the first routine VL from 6 months to 3 dispensing cycles (DCs), about 3 months on antiretroviral therapy (ART).

The 1-, 6- and 12-month options are the earlier or wrong timings, and testing is routine rather than symptom-triggered. Subsequent VLs follow at 10 DCs (about 10 to 12 months), 22 DCs (about 24 months), then every 12 DCs annually; breastfeeding women test every 6 DCs from delivery. The eGFR and creatinine schedule was pulled earlier to align with the new VL timing.

A central 2026 change dropped the higher-risk threshold from a maternal viral load (VL) at or above 1,000 copies/mL to at or above 50 copies/mL, extending dual prophylaxis to a far larger fraction of exposed infants.

The higher thresholds (200, 500, 1,000 and 10,000 copies/mL) are the old or invented cut-offs. Higher-risk infants receive nevirapine (NVP) for at least 12 weeks plus zidovudine (AZT) for 6 weeks if breastfed (NVP and AZT each for 6 weeks if formula-fed); every exposed infant gets dual NVP plus AZT at birth until the delivery VL is known.

Confirmed virological failure needs two consecutive viral loads (VLs) at or above 1,000 copies/mL, after at least 9 months on a DTG-based regimen, with at least two documented adherence interventions.

A single high VL, a threshold of 50 or 200 copies/mL, and a doubling VL all fall short of the definition. The criteria reflect that DTG resistance is rare and most detectable viraemia is non-adherence, which the reflex DTG drug-level workflow confirms or refutes before any genotype.

A 2026 change withdrew CPT from HIV-exposed uninfected infants and reserved it for confirmed HIV infection meeting the age, CD4 or stage criteria.

CPT is not for every HIV-positive person regardless of CD4 or stage, nor limited to active Pneumocystis pneumonia, nor given to exposed uninfected or breastfeeding infants. In detail: all HIV-positive infants under 1 year whatever the CD4 or stage; children 1 to 5 years with CD4 at or below 25% or World Health Organization (WHO) stage 3 or 4; those over 5 and adults with CD4 at or below 200 or WHO stage 3 or 4. Stop once CD4 rises above 200 (or above 25% in under-5s).

The default adult TPT regimen is 12H: isoniazid (INH) 300 mg daily with pyridoxine 25 mg daily, for 12 months.

3HP (weekly INH plus rifapentine, 12 doses) is an alternative only for clients 25 kg or more already on dolutegravir (DTG) with a recent suppressed viral load, so not for new antiretroviral therapy (ART) starters. Rifampicin monotherapy and bedaquiline are not TPT regimens, and TPT does not require confirmed active-TB exposure. TPT in pregnancy is now indicated only with Advanced HIV Disease (CD4 at or below 200 or World Health Organization stage 3 or 4).

The Children’s Act permits any child aged 12 or older to self-consent to HIV testing, and a child under 12 with sufficient maturity to understand the test (the mature-minor doctrine) may also self-consent.

A child under 12 without that capacity needs consent from a parent, caregiver or designated adult, so option E is too absolute. The 16-, 18- and 7-year thresholds are not the ones set in the framework.

Approximately 73% of treated adults are on a dolutegravir-containing regimen, and drug-resistance mutations in viraemic samples rose from 27.4% (2017) to 36.2% (2022), predominantly NNRTI (non-nucleoside reverse transcriptase inhibitor)-related.

The resistance is the legacy of the long efavirenz-based first-line era, not integrase or protease mutations, and the other options misstate the coverage, the direction of the trend or the resistance class.

U=U means a person with a sustained viral load below the limit of detection does not transmit HIV sexually, the mechanism behind treatment as prevention at scale.

The other options describe unrelated ideas; undetectable status suppresses transmission but is not a cure, and does not equate to programme-wide testing or coverage.

Lenacapavir showed 100% efficacy in cisgender women in Purpose 1 and 96% efficacy in cisgender men and gender-diverse populations in Purpose 2, the most dramatic single advance in HIV prevention in a decade.

The other options understate these results; the trial figures were near-total protection, not the mid-range or cascade-derived percentages listed.

APOBEC3G is a host cytidine deaminase causing lethal G-to-A hypermutation of nascent viral DNA; HIV-1 Vif targets it for proteasomal degradation.

Option D describes tetherin countered by Vpu, and option E describes SAMHD1 countered by Vpx; the others pair invented functions with the wrong antagonist.

The Strategy targets 20 million people accessing antiretroviral-based prevention, pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) at scale by 2030.

The 40 million figure is the parallel treatment-and-suppression target, while the other values are regional or national burden figures rather than prevention targets.

Eastern and southern Africa carry just over half of the global total, approximately 21 million people living with HIV (PLHIV), and remain the regional epicentre.

The other proportions understate or overstate the regional share; western and central Africa, Asia, the Americas and Europe together make up the remaining half.

Translated to all people living with HIV, around 77% are virally suppressed, approaching but not yet at the 86% population threshold.

The ~93% and ~90% figures are individual cascade pillars, ~86% is the target threshold itself, and ~73% is TLD (tenofovir, lamivudine and dolutegravir) coverage.

Estimated annual incidence was ~0.44%, around 232,000 new infections per year, down from ~250,000 in 2017.

The ~0.39% and ~1.17% figures are adolescent-girl-and-young-women incidence at each survey, while 12.7% is national prevalence, not incidence.

SABSSM VI put national HIV prevalence at 12.7%, down from 14.0% in 2017.

The 16.4% and 8.8% figures are the female and male prevalence, and 17.4% and 7.4% are the highest and lowest provincial values, not the national rate.

Prevalence was markedly higher in women, at females 16.4% versus males 8.8%.

Option A reverses the sexes, 12.7% and 14.0% are national figures, and 34.2% and 27.1% are the age-specific peak-prevalence values.

The persistent gap is the first 95, undiagnosed people living with HIV, concentrated in men (85% diagnosed versus 92% in women) and youth aged 15 to 24 (73%).

The second and third targets are effectively achieved, so the gap is at diagnosis, not treatment coverage or suppression, and it centres on men and youth rather than women or older adults.

The non-nucleoside reverse transcriptase inhibitors have a low genetic barrier: a single mutation such as K103N confers high-level, often class-wide resistance, which is why they were displaced from first line.

Boosted protease inhibitors and second-generation integrase inhibitors (dolutegravir) have high barriers needing several stepwise mutations, and tenofovir resistance also requires accumulation.

HIV-2 is naturally resistant to the non-nucleoside reverse transcriptase inhibitors (NNRTIs) and to enfuvirtide.

Some protease inhibitors are also less active against it, but it responds to nucleoside analogues and integrase inhibitors, so it is neither pan-susceptible nor restricted to a single class. The regimen and the viral-load assay both need choosing with HIV-2 in mind, since many HIV-1 assays miss it.

The epidemic, AIDS-associated form became the commonest cancer of the HIV epidemic and is frequently aggressive and visceral.

The classic, endemic and iatrogenic forms arise in other settings, and primary effusion lymphoma is a separate KSHV-associated malignancy, not a form of Kaposi sarcoma.

Subtype C predominates in southern Africa and India and accounts for roughly half of all infections globally.

Subtype B dominates Europe and the Americas, and the recombinant CRF01_AE dominates South-East Asia.

Rev binds the Rev-response element (RRE) and exports unspliced and partially spliced transcripts, switching the infection to its late phase.

Tat is the transcriptional transactivator; Nef, Vif and Vpr are accessory proteins with unrelated roles.

HIV RNA is detectable first, from about 10 to 11 days, so nucleic-acid testing closes the window earliest.

The markers then appear in order: p24 antigen, IgM at about three weeks, and mature IgG at two to six weeks; fourth-generation assays (p24 plus antibody) detect at about two weeks. A falling CD4 count is a downstream consequence, not an early diagnostic marker.

Provincial prevalence ranged from a high of Mpumalanga 17.4% to a low of Western Cape 7.4%.

KwaZulu-Natal at 16.0% and the Northern Cape at 8.9% sit inside that range, and option C reverses the two extreme provinces.

Phenotypic testing measures susceptibility directly: the patient’s pol gene is cloned into a laboratory backbone, the recombinant virus is grown in cell culture against each drug, and the fifty-percent inhibitory concentration (IC50) is compared to wild-type to give a fold-change.

It is slow and expensive and is reserved for research, drug development and complex cases. Genotypic testing sequences pol from plasma ribonucleic acid (RNA) and predicts susceptibility from the mutations found: fast, cheap, and the workhorse of practice worldwide, including the South African public sector. The distractors invert the two methods, reduce the difference to cost alone, or wrongly make phenotyping the routine South African method.

Triple elimination covers HIV, syphilis and hepatitis B, integrated into a single antenatal and postnatal framework rather than three parallel ones.

The other options swap in tuberculosis, hepatitis C or congenital pathogens that are not part of the triple-elimination package.

Transplacental maternal IgG persists in the infant for up to about 18 months, so a reactive antibody test may simply reflect maternal antibody rather than infant infection.

Early infant diagnosis therefore relies on HIV nucleic-acid testing by polymerase chain reaction (PCR); serology becomes interpretable from around 18 months. The distractors describe phenomena that do not occur.

The viral reverse transcriptase (RT) has no proofreading and introduces roughly one mutation per genome per replication cycle; combined with very high replicative output, this produces a quasispecies, a population of closely related but non-identical variants within a single host.

Drug-resistance mutations exist within this swarm before any drug is given, and drug pressure under inadequate adherence then selects them. The distractors name related but distinct concepts: the reservoir is the latent integrated provirus, the archive is what persists in the reservoir, the provirus is the integrated DNA form, and the consensus is the single dominant sequence reported.

Zidovudine is retained for the preterm neonatal regimen (AZT/3TC/NVP) and for renal failure with concurrent abacavir hypersensitivity (AZT/3TC/DTG).

It is not indicated by HIV-2 status, patient preference, routine needlestick prophylaxis or hepatitis B co-infection alone. Anaemia and neutropenia are the watchpoints (monitor full blood count during the first months); macrocytosis is expected and benign.

A negative HIV serology at 6 months after exposure in a healthcare worker who received timely and complete post-exposure prophylaxis (PEP) is the expected and reassuring result. It effectively rules out HIV transmission from the exposure.

Interpretation of the 6-month negative

• The PEP worked, or transmission never occurred. Either is reassuring. The exposure (mucocutaneous splash to the eye) carries a transmission risk of approximately 0.09% per single contact, low even without PEP, and effectively zero with timely 28-day tenofovir/lamivudine/dolutegravir (TLD).
• Window-period testing has been completed. The South African PEP follow-up testing schedule for occupational exposure is HIV testing at 6 weeks, 3 months and 6 months (the 6-month test is the standard endpoint, with 12-month testing reserved for hepatitis C virus (HCV)-coinfected sources).
• Antibody response in PEP-treated patients. PEP itself does not produce HIV antibodies. The negative serology at 6 months means the worker did not seroconvert, confirming no transmitted infection.
• Long-acting PrEP caveat. This worker received TLD for PEP (not long-acting). The atypical-seroconversion (“LEVI syndrome”) pattern that complicates testing after long-acting pre-exposure prophylaxis (PrEP) failure does not apply here; the negative test can be taken at face value.

Management going forward

The worker is HIV-negative. Specific actions:

• Document the negative result in the occupational health record and close the exposure file.
• Reassure the worker: the exposure was managed appropriately and there is no HIV.
• Brief counselling on prevention going forward: adherence to standard precautions, prompt occupational-health reporting of future exposures.
• Mental-health debriefing if not already done: a 6-month period of uncertainty following an occupational exposure is significant. Offer counselling and employee assistance programme (EAP) referral if appropriate.
• Vaccination and immunisation review: confirm the worker’s hepatitis B virus (HBV) vaccination is current; if the source was HBV-positive and the worker was non-immune, that has been managed separately at the time of exposure.

Reflection for the programme

The case is an exemplar of how the South African occupational PEP pathway should work:

• Rapid initiation within 72 hours (in this case within 4 hours).
• Complete 28-day course of TLD.
• Structured serial follow-up at 6 weeks, 3 months and 6 months.
• Documentation and closure at the 6-month timepoint.

The detail of the SA PEP regimen and follow-up schedule sits in the standalone PEP 2019 guideline to which the National Consolidated Guidelines (NCG) defer; this case illustrates that pathway in action.

What if the 6-month test had been positive?

A positive HIV result at 6 months in a PEP-treated worker would prompt:

• Repeat HIV testing on a fresh sample to confirm.
• HIV viral load and CD4 as baseline for antiretroviral therapy (ART) initiation.
• Genotypic resistance testing: possibility of transmitted resistance from the source (especially if the source was on ART) or PEP-induced resistance during sub-therapeutic exposure.
• Initiation of ART: TLD as first-line in current practice, but with regimen choice informed by the resistance result.
• Investigation of the exposure: was the PEP regimen appropriate? Was there a missed exposure? Documentation of programme-level lessons.
• Counselling and occupational support: disclosure considerations, work restrictions (none required by NCG; the Health Professions Council of South Africa (HPCSA) position should be consulted), psychological support.

A negative 6-month HIV serology after a timely 28-day course of TLD PEP for an eye-splash exposure from a known HIV-positive source is the expected and reassuring outcome. Routine management closes the file; no further HIV testing is required.

Interpretation. A single detectable viral load of 50 to 199 copies/mL after sustained suppression is a viral blip: a transient result that returns to undetectable on repeat. Blips occur in ~10 to 30% of long-term suppressed patients and reflect stochastic release from the latent reservoir, intercurrent infection or vaccination, or assay variability near the detection limit. A single blip is not evidence of virological failure or drug resistance.

Action.

• Repeat the viral load (at the next routine visit, or in 4 to 6 weeks if pregnant or breastfeeding) and continue the current regimen unchanged.
• Do not request resistance testing on a single result below 1,000 copies/mL: genotyping needs a viral load above 1,000 copies/mL, and this does not meet the South African resistance-testing criteria.
• Reinforce adherence and counsel without alarm: the regimen is working and the repeat is almost always undetectable.

If the repeat is also detectable (50 to 999 copies/mL), the pattern is persistent low-level viraemia, which warrants intensified adherence support and a lower threshold for resistance testing. Sustained results at or above 1,000 copies/mL, after at least 9 months on a dolutegravir-based regimen with documented adherence support, define virological failure.

Clinical scenario. An HIV-exposed infant has the following PCR series:

• November 2017 (birth): HIV PCR indeterminate
• February 2018 (~10 weeks): HIV PCR negative
• September 2018 (~6 months): HIV PCR negative

Result-by-result interpretation

Result 1: birth PCR, indeterminate. An indeterminate result at birth is a low-level positive (commonly defined by a cycle-threshold (CT) value above the laboratory’s positive cut-off). The result requires immediate follow-up: review of the infant’s prior testing (none, in this case, as this is the birth test), urgent repeat HIV PCR and a viral load (VL) to clarify, continuation of nevirapine (NVP) plus zidovudine (AZT) prophylaxis, and clinical assessment of risk factors (maternal VL at delivery; prevention-of-mother-to-child-transmission (PMTCT) prophylaxis status; possible PMTCT failure indicators).

The possible explanations for an indeterminate birth PCR are: very early in-utero infection at low viral copy number; PMTCT prophylaxis (NVP given in late pregnancy and at birth) producing partial suppression; sample contamination; transfer of maternal nucleic acids in transplacental plasma; technical/threshold artefact.

The clinical management of the indeterminate result is what comes next.

Result 2: 10-week PCR (February 2018), negative. The 10-week test is the Expanded Programme on Immunisation (EPI)-aligned PCR that screens primarily for intrapartum-acquired infection. A negative result at this timepoint:

• Argues strongly against an established intrapartum infection.
• Effectively resolves the November 2017 indeterminate result: the indeterminate at birth was almost certainly not true infection (or was, but at a copy number that infant prophylaxis suppressed below subsequent detection). With prior negative testing, the standard National Consolidated Guidelines (NCG) algorithm would have closed the issue here.
• Returns the infant to routine HIV-exposed follow-up.
• Continues breastfeeding prophylaxis if still indicated by maternal VL.

A useful caveat: a negative 10-week PCR does not exclude HIV acquired during the intrapartum or early postnatal period if PMTCT prophylaxis is still actively suppressing infant viraemia. This is one reason the EID schedule continues with a 6-month PCR.

Result 3: 6-month PCR (September 2018), negative. The 6-month PCR is timed to detect breastfeeding-acquired infection that emerges during the period of exclusive or mixed breastfeeding. A negative result:

• Indicates no detectable HIV at 6 months, covering breastfeeding-acquired infection up to that point.
• Aligns with the maternal 6-month VL check, allowing simultaneous reassessment of vertical transmission risk.
• Does not exclude infection acquired after 6 months, particularly if breastfeeding continues, hence the post-cessation testing requirement (and the 18-month antibody test) that complete the early infant diagnosis (EID) schedule.

Overall interpretation

The infant has had an indeterminate birth result that was not confirmed on follow-up testing. The two subsequent negative PCRs at 10 weeks and 6 months effectively rule out vertically acquired infection up to 6 months. The infant is presumed HIV-negative for the purposes of clinical decision-making, while remaining on routine HIV-exposed follow-up (including the post-breastfeeding-cessation test and the 18-month rapid antibody).

Counselling and ongoing care

• Reassurance to the mother: the most likely interpretation is no vertical transmission, with the indeterminate birth result being a sub-threshold artefact or transient maternal nucleic-acid carryover.
• Continued breastfeeding management: the infant needs ongoing prophylaxis if the mother is unsuppressed and breastfeeding; the prophylaxis is reviewed at each maternal VL check.
• Complete the schedule: testing at 18 months and 6 weeks post-cessation of breastfeeding is mandatory for definitive clearance from the EID programme.
• Maternal care: confirm the mother’s ongoing antiretroviral therapy (ART) suppression, address any adherence issues.

This case illustrates how the layered EID schedule delivers progressively stronger confidence about an infant’s HIV status: no single test result alone is conclusive, but the series builds toward a defensible conclusion.

HIV is transmitted by three principal routes:

• Sexual: the dominant route worldwide, and overwhelmingly heterosexual in the sub-Saharan African epidemic. Receptive anal intercourse carries the highest per-act risk; vaginal, insertive and oral exposures follow.
• Parenteral (blood-borne): sharing of injecting equipment, occupational needlestick injury (~0.3% per percutaneous exposure to infected blood), and transfusion of infected blood or products (now rare where donations are screened).
• Vertical (mother-to-child): in utero, intrapartum, and through breastfeeding, the route targeted by prevention-of-mother-to-child-transmission programmes.

Per-exposure risk is raised by a high plasma viral load (hence the danger of acute infection), genital ulcers or other sexually transmitted infections, and the absence of male circumcision. Conversely, a person with a sustained viral load below 200 copies/mL does not transmit HIV sexually, the basis of treatment as prevention.

The 95-95-95 targets (95% of people living with HIV diagnosed, 95% of those diagnosed on antiretroviral therapy (ART), and 95% of those on ART virally suppressed) define the operational programme goal for ending HIV as a public-health threat by 2030. The earlier UNAIDS 90-90-90 framework was upgraded to reflect what is now achievable with current tools. The virology laboratory underpins all three pillars.

Pillar 1: 95% diagnosed

The laboratory provides the testing infrastructure for diagnosis at scale:

• The three-test serial rapid algorithm in primary care, validated and quality-assured, backed by external quality assurance to maintain the 99% positive predictive value the algorithm depends on.
• Fourth-generation antigen/antibody assays for laboratory confirmation, and HIV PCR for early infant diagnosis (birth, 10-week, 6-month).
• Surveillance and seroprevalence studies (including dried blood spot surveys) to track progress against the first 95.

Pillar 2: 95% of diagnosed on ART

The laboratory enables rapid same-day ART initiation through:

• Baseline workup at the point of diagnosis: hepatitis B surface antigen (HBsAg), syphilis, creatinine/estimated glomerular filtration rate (eGFR), CD4, tuberculosis nucleic acid amplification test (TB-NAAT), pregnancy test.
• Reflex cryptococcal antigen (CrAg) below CD4 200 and TB lipoarabinomannan (TB-LAM) for advanced HIV disease (AHD) identification, enabling rapid ART for those eligible and appropriate deferral for those who are not.
• Pre-treatment drug-resistance surveillance (transmitted drug-resistance studies) to inform regimen choice.

Pillar 3: 95% of those on ART virally suppressed

This is the pillar where the laboratory does the largest volume of work:

• Routine viral-load (VL) monitoring at scale: South Africa runs millions of HIV VL tests per year. The National Consolidated Guidelines (NCG) 2026 schedule (VL at 3, 10 and 22 dispensing cycles, then annually; breastfeeding 6-monthly) is feasible only because of centralised high-throughput laboratory capacity.
• The 2026 reflex dolutegravir (DTG) drug-level testing workflow: the laboratory measures DTG levels on every drug-resistance sample, gate-keeping unnecessary resistance testing in non-adherent patients.
• Genotypic drug-resistance testing for confirmed virological failure on a DTG-based regimen (integrase, reverse transcriptase and protease regions), plus dried blood spot VL where plasma logistics fail.

Cross-cutting laboratory roles

Underpinning all three pillars are surveillance reporting to the National Department of Health and UNAIDS for the 95-95-95 estimates, operational research informing programme evolution, and quality management systems (SANAS accreditation, proficiency testing, external quality assurance).