1553CHAPTER 202 Human Immunodeficiency Virus Disease: AIDS and Related Disorders

resistant, respectively, to T-cell activation–induced demethylation of

the CCR5 locus.

In worldwide populations, HHE and HHC are prevalent haplotypes,

whereas the ancestral HHA haplotype is more common in persons

of African ancestry. The associations of CCR5 haplotypes with HIV

acquisition and/or HIV disease course are largely consistent with their

effects on CCR5 gene expression. For example, homozygosity for the

CCR5-HHE haplotype is associated with an increased risk of acquiring

HIV, progressing rapidly to AIDS, and reduced immune recovery during ART. The HHA haplotype is associated with slower disease progression in African populations and has been speculated to be a basis for

why chimpanzees (who all carry the ancestral CCR5-HHA haplotype)

naturally infected with simian immunodeficiency virus resist disease

progression. The pairing of the HHC and CCR5 Δ32-bearing HHG*2

haplotypes (HHC/HHG*2 genotype) is associated with a lower risk of

acquiring HIV infection and slower rate of HIV disease progression,

whereas the pairing of the HHE haplotype with the HHG*2 haplotype

is associated with the opposite effects. The CCR2-64I–bearing HHF*2

haplotype is associated with a slower HIV disease course.

Consistent with these genetic associations, polymorphisms in genes

encoding ligands for CCR5 have also been associated with variable

HIV susceptibility and disease progression rates. Examples include

copy number variations of CCL3L1 and SNVs in CCL5. The sum of

these studies established a pivotal role of CCR5 and its ligands in HIVAIDS pathogenesis and, potentially, immune recovery.

The discovery that the CCR5 Δ32/Δ32 genotype is associated with

strong resistance to HIV infection, and that uninfected persons bearing this genotype did not appear to have impaired immunity, led to

the development of two kinds of novel therapies. First, it spurred the

development of a new class of therapies approved by the U.S. Food

and Drug Administration: entry inhibitors (e.g., maraviroc) that block

the interaction of CCR5 with the HIV envelope. Second, it led to the

evaluation of novel experimental cellular therapies. An HIV-infected

patient with acute myelogenous leukemia was given an allogeneic

stem cell transplantation from an HLA-compatible person whose cells

lacked expression of CCR5 due to the Δ32/Δ32 genotype. There was no

evidence of HIV-1 infection for 13 years in the patient who underwent

the transplant; the patient eventually died due to recurrence of leukemia. This observation provided a proof of concept for an HIV cure and

led to the development of additional novel cellular therapies involving

autologous transplantation of CD4+ T cells in which the CCR5 gene is

inactivated ex vivo using new gene editing procedures. Similar cellular

strategies have had mixed success, mainly due to the latent viral reservoir in various tissues.

DISCOVERY OF HLA CLASS I ALLELES THAT ASSOCIATE WITH VIROLOGIC CONTROL OF HIV INFECTION There is a strong association

between variations within the HLA-B gene with protective (e.g., HLAB*57 and HLA-B*27 alleles) or detrimental (e.g., HLA-B*35 allele)

outcomes during HIV infection. Carriage of the HLA-B*57 and/or

HLA-B*27 alleles is associated with slower disease progression. The

beneficial effects of these alleles may relate in part to their associations

with a lower virologic set point as well as to higher cell-mediated

immunity in HIV-infected persons. The protective effect of the HLAB*57 and HLA-B*27 alleles on the HIV disease course is underscored

by the finding that the prevalence of these alleles is higher among

persons with long-term nonprogression and persons who control HIV

replication spontaneously (elite controllers). In contrast, the HLA-B*35

allele has been associated with faster progression to AIDS and higher

viral load. The prevalence of the HLA-B alleles differs between populations. HLA-B*57:01 in Europeans and HLA-B*57:03 in persons of

African descent are the protective alleles. In some populations (e.g.,

Japanese) where the HLA-B*57/HLA-B*27 alleles are absent, HLA-B*51

is associated with a protective phenotype.

Possession of the protective HLA-B alleles is associated with

broader and stronger CD8+ T-cell responses to HIV epitopes. The

mechanisms underlying the differential effects of the HLA-B alleles

on the course of HIV disease may relate to differences in the ability of

antigen-presenting cells to present immunodominant HIV epitopes to

T helper or cytotoxic T lymphocytes in the context of MHC-encoded

molecules. This may result in differential immune responses that

influence viral replication. In this regard, the HLA-B alleles that impact

the course of HIV disease differ in their amino acid residues in the

HLA-B peptide-binding groove; this difference may play a critical role

in virologic control.

The HLA-B −21M allele does not influence HLA-B gene expression;

however, it is in linkage with HLA-B haplotypes that are associated with

higher HLA-A and HLA-E expression. Higher HLA-A levels associate

with poorer control of HIV as well as higher viral load, reduced CD4+

T-cell counts, and accelerated progression to AIDS. HLA-E is the ligand

80

20

8

60

0

Chr. 1 2 3 4 5 6 7 10

PSORS1C3 HLA-C HLA-B MICA HCP5 MICB

8 9 11 12 14

13

rs3131018

rs9264942

rs4418214

rs2395029

15

16

17

18

19

20

21

22

– Log10 (

P value)

Chr. 3p21

CCR5 locus

Chr. 6p21

MHC region

CCR3

HHD

HHE

HHF*1

HHF*2

HHG*1

HHG*2

HHC

HHB

HHA

Haplotype

V

V

V

V

V

V

I

V

V

A

A

A

A

A

A

A

G

G

G

T

T

T

G

G

G

G

G

G

G

G

G

A

A

A

A

A

T

T

T

T

C

C

C

C

C

A

A

A

G

A

A

A

A

A

C

C

C

C

C

T

T

C

C

C

C

T

C

C

C

C

C

C

wt

wt

wt

wt

wt

wt

wt

wt

∆32

CCR5 promoter

C

C

C

C

A

A

C

C

C C

C

C

C

C

C

T

T

T

HLA-C

M

M

M

V

M

M

V

V

V

Cw*08:02

Cw*07:02

B*57:01

B*52:01

B*27:05

B*14:02

B*07:02

B*35:01

G

R

R

R

R

R

R

R

R

E

E

E

E

N

N

E

N

N

M

S

S

C

C

S

S

Y

F

V

T

T

N

W

R

R

S

R

C

T

T

C

T

T

T

T

T

S

N

N

K

N

N

N

Q

N

G

G

G

G

G

G

G

G

T

HLA-B

allele 304 allele 63 67 70 97 62

CCR2 CCR5 CCRL2

rs1799864

rs2856758

rs2734648

rs1799987

rs1799988

rs41469351

rs1800023

rs1800024

rs333

rs1015164

A

A

A

A

A

G

A

G

A

FIGURE 202-26 Schema depicting haplotypes within two regions that contribute significantly to HIV-AIDS susceptibility. Top: Haplotypes (Left, CCR5; Right, HLA alleles).

Bottom: GWAS Manhattan plots schematized. Chr: chromosome. Horizontal dotted line: genome level significance threshold.

1554 PART 5 Infectious Diseases

for natural killer (NK) cell NKG2A, an inhibitory receptor. Engagement of NKG2A with HLA-E inhibits NK cells that would normally

be potent eliminators of virally infected cells. Thus, targeting NKG2A

might provide a therapeutic avenue for HIV treatment.

Investigators have also examined the influence of extended HLA

haplotypes (linked alleles) on the course of HIV disease. The extended

HLA ancestral haplotype (AH) 8.1 is defined by the presence of HLAA1, HLA-B8, and HLA-DR3 alleles. AH 8.1 is the most common ancestral haplotype in persons of European descent (present in 10%) and

is associated with multiple autoimmune diseases in HIV-seronegative

persons. These associations of AH 8.1 are thought to be due to a genetically determined hyperresponsiveness characterized by high TNF-α

production and lack of complement C4A. Strong epidemiologic data

indicate that carriage of AH 8.1 in HIV-seropositive persons is associated with a rapid decline in the number of CD4+ T cells and faster progression to AIDS development. Gene–gene interactions between HLA

alleles and other genes (e.g., killer cell immunoglobulin-like receptors)

also may influence HIV disease progression rates.

POLYMORPHISMS IDENTIFIED BY GWAS THAT ASSOCIATE WITH VIROLOGIC CONTROL AND DISEASE PROGRESSION GWAS have not identified additional genetic variations that associate with the risk of HIV-1

acquisition, presumably due to a paucity of well-characterized risk

cohorts in which level of exposure has been quantified. By contrast,

large-scale GWAS have identified SNVs, especially in the MHC, that

influence HIV viral load, including in a large group of individuals

termed “HIV controllers (including elite controllers)” who spontaneously (without ART) control viral replication. GWAS in HIV-infected

persons of European ancestry identified four SNVs in genes in the

HLA class I loci that associated with virologic control. These SNVs are

within or in the vicinity of PSORS1C3, HLA-C, MICA, and HCP5 genes

(Fig. 202-26). As noted in this figure, the individual effects of these

alleles are difficult to discern because of linkage disequilibrium. The

protective effects of the SNVs in HCP5 and MICA may relate to their

linkage with known protective HLA-B alleles. The protective HCP5

allele is in linkage disequilibrium with the HLA-B*57:01 allele, and the

protective MICA allele tags with the HLA-B*57:01 and HLA-B*27:05

alleles. The protective HLA-C SNV is associated with higher HLA-C

expression, which has been associated with viral control and better

HIV outcomes. This protective SNV (rs9264942; T→C) resides 35 kb

upstream of the HLA-C gene and is in strong linkage disequilibrium

with a 3′-UTR indel263 SNV (rs67384697; G→deletion), generating

the T-G or C-deletion haplotypes (Fig. 202-27). miR-148a binds to

the 3′-UTR region encompassing the rs67384697 SNV and silences

HLA-C expression. Binding of miR-148a to the 3′-UTR is disrupted

on the mRNA transcribed from the C-deletion haplotype; this disruption associates with less silencing of the mRNA and therefore higher

HLA-C cell surface expression, which associates with better HIV

disease outcomes (Fig. 202-27). Conversely, binding of miR-148a to

the 3′-UTR is intact on the mRNA transcribed from the T-G haplotype; this binding associates with silencing of the mRNA and therefore

lower HLA-C cell surface expression associates with worse HIV disease

outcomes (Fig.  202-27). GWAS in persons of African descent have

identified an SNV (rs2523608) that tags the HLA-B*57:03 allele that

is known to associate with HIV-1 control and a slower disease course.

Together, these GWAS data underscore the importance of variations in

HLA class I loci in control of viral replication.

A recent GWAS suggested that an SNV (rs1015164G→A) approximately 34 kb downstream of the CCR5 loci associated with a higher

viral load set point (Fig. 202-26) and lower CD4+ T-cell counts in

therapy-naïve HIV-seropositive persons. rs1015164 maps to a lncRNA

gene in proximity to the CCRL2 gene (Fig. 202-26). The lncRNA is

transcribed from the antisense strand of CCR5 and was therefore

named CCR5AS. The rs1015164A allele associated with higher expression of CCR5AS in CD4+ T cells, which in turn was associated with

increased levels of CCR5 mRNA. Although the detrimental effect of the

rs1015164A allele was suggested to be independent of the detrimental

effects of the abovementioned CCR5-HHE haplotype, further investigation is warranted as the rs1015164A allele and CCR5-HHE haplotype

are in a high degree of linkage disequilibrium.

Most GWAS studies have been performed in European populations,

limiting generalizability to other populations. Additionally, GWAS are

generally not suitable for identifying rare variants (<1% prevalence).

Therefore, next-generation sequencing (NGS) approaches were suggested to identify these rare variants. However, a recent NGS study

suggests that exonic variants with large effect sizes are unlikely to have

a major contribution to host control of HIV infection. Mathematical

modeling revealed that variations in host genes may explain about

10% of the observed variability in HIV viral load, whereas viral genetic

diversity may explain 29% of the variability.

GENETIC ASSOCIATIONS WITH SPECIFIC AIDS AND NON-AIDS

CONDITIONS

Carotid artery disease Many of the non-AIDS events in HIV-seropositive

individuals resemble those attributable to immune senescence and

those found in the HIV-seronegative aging population. A functional

SNV in the ryanodine receptor 3 (RYR3) gene was found to be associated with an increased risk of common carotid intima–media thickness

(cIMT), which is a surrogate for subclinical atherosclerosis. Functional

studies on RYR3 and its isoforms demonstrate a major role of these

receptors in modulating endothelial function and atherogenesis via

calcium-signaling pathways, providing a biologically plausible mechanism by which the SNV in RYR3 may associate with increased cIMT

risk.

Kidney disease HIV-1–associated nephropathy (HIVAN) is a form

of focal sclerosing glomerulonephritis caused by direct infection of

kidney epithelial cells with HIV. HIVAN is more common in persons

of African descent. There is evidence that polymorphisms in the MYH9

gene and in the neighboring APOL1 gene are a strong determinant

of susceptibility to HIVAN in persons of African descent. The effect

of carrying two APOL1 risk alleles explains nearly 35% of HIVAN.

Overexpression of the APOL1 kidney risk variants may associate with

increased kidney cell death.

HIV-associated neurocognitive disorder HIV-associated neurocognitive disorder (HAND) comprises a spectrum of neurocognitive deficits due to

HIV infection. Variations in the apolipoprotein E (ApoE) gene have

strong associations with Alzheimer’s disease in the HIV-seronegative

population. In HIV-seropositive persons, possession of the E4/E4

genotype has been associated with dementia, peripheral neuropathy,

and impairment in cognition as well as immediate and delayed verbal

memory. Macrophage recruitment and activation play a central role

in the development of many of the HAND syndromes. Variations in

chemokines that play an influential role in macrophage activation

and recruitment, namely CCL2 (MCP-1) and CCL3 (MIP-1α), have

been shown to influence the risk of developing HAND. Variations in

mitochondrial genes also have been associated with a risk of AIDS and

mRNA

silencing

HLA-C expression

Viral control

HIV outcomes

Lower

Reduced

Worse

Higher

Increased

Better

miR-148a

Binding intact

T G

less

silencing

-35 kb

gDNA

T

Indel263

C

G

–

-35 kb

gDNA

T

Indel263

C

G

–

mRNA C –

HLA-C HLA-C

FIGURE 202-27 Linkage disequilibrium between two variants in the HLA-C locus and

their influence on binding of miR-148a to the 3-untranslated region (UTR). Altered

binding of miR-148a associates with HLA-C protein expression levels and, in turn,

viral control and HIV disease outcomes. Effects associated with T-G (left) and

C-del (right) haplotypes are depicted. C-del: C-deletion. The C-deletion haplotype

prevents binding of miR-148a to 3-UTR of HLA-C (less silencing). Kb, kilobase.

1555CHAPTER 202 Human Immunodeficiency Virus Disease: AIDS and Related Disorders

HAND. A GWAS identified a polymorphism in chromosome 14 in the

T-cell receptor α locus that may influence neurocognitive outcomes.

HIV-1-associated Pneumocystis pneumonia Human Apobec3 cytidine deaminases are intrinsic resistance factors to HIV-1. However, HIV-1

encodes a viral infectivity factor (Vif) that degrades APOBEC3 proteins. Association studies suggest a role of genetic variations in the

APOBEC3 family in HIV disease. A common haplotype derived from

6 SNVs in the APOBEC3F gene and tagged by a codon-changing variant is associated with a significantly lower viral load set point, slower

rate of progression to AIDS, and delayed development of Pneumocystis

jirovecii pneumonia (PCP). In addition, a coding SNV in the CCRL2

gene is associated with accelerated progression to AIDS and rapid

development of PCP.

HIV-related non-Hodgkin lymphoma (NHL) The relative risk of developing

NHL in HIV-seropositive persons is highly elevated compared with

the general population. NHL represents approximately 34% of all identified cancers in HIV-seropositive persons. A recent GWAS identified

a promoter SNV in the CXCL12 gene that was associated with higher

susceptibility to develop HIV-related NHL. The effect of this SNV is

likely causal as it creates new transcription factor binding sites, impacting CXCL12 expression.

ASSOCIATIONS WITH ART-RELATED ADVERSE EVENTS Abacavir, an

effective antiretroviral agent, is associated with significant risk of

hypersensitivity reactions (2–9% of cases). Interestingly, while the HLAB*57:01 allele is associated with a slower HIV disease course, possession of this allele is associated with a higher risk of abacavir-associated

hypersensitivity, possibly due to the abacavir-specific activation of

cytokine-producing CD8+ T cells only in HLA-B*57:01 carriers. Pharmacogenetic screening for the HLA-B*57:01 allele is recommended

before initiation of abacavir treatment.

The antiretroviral agent nevirapine is associated with hypersensitivity reactions in 6–10% of patients, including Stevens–Johnson

syndrome (SJS) and toxic epidermal necrolysis (TEN). rs5010528G, a

strong proxy for HLA-C*04:01 carriage, was associated with high risk

of SJS and TEN during nevirapine treatment. In addition, efavirenz

was among the first antiretroviral agents to be co-formulated into

single-pill regimens for mass rollout globally. Several genetic variants

in the drug-metabolizing enzyme CYP2B6 have been associated with

high efavirenz plasma concentrations and increased risk of adverse

neuropsychiatric effects. For example, homozygosity for one such variant, rs3745274 T/T, increases the risk of adverse reactions to efavirenz

up to five folds, and this risk genotype is much more common in Africans (13.7%) than Europeans (5.6%).

■ NEUROPATHOGENESIS IN HIV DISEASE

While there has been a remarkable decrease in the incidence of the

severe forms of HIV encephalopathy among those with access to

treatment in the era of effective ART, HIV-infected individuals can

still experience milder forms of neurocognitive impairment despite

adequate ART. Factors that contribute to the neurocognitive decline

include lack of complete control of HIV replication in the brain;

production of HIV proteins that may be neurotoxic; low CD4+ T-cell

nadir; chronic immune activation; comorbidities such as drug abuse,

microvascular disease, older age, and diabetes; and the potential for

neurotoxicity of certain antiretroviral drugs. HIV has been demonstrated in the brain and CSF of infected individuals with and without

neuropsychiatric abnormalities. As opposed to lymphoid tissues, there

are no resident lymphocytes in the brain. The main cell types that are

infected in the brain in vivo are the perivascular macrophages and

the microglial cells, which can sometimes form syncytia resulting in

multinucleated giant cells; low-level viral replication is also seen in

perivascular astrocytes. It has been proposed that monocytes that

have already been infected in the blood can migrate into the brain,

where they then reside as macrophages, or macrophages can be

directly infected while residing within the brain. The precise mechanisms whereby HIV enters the brain are unclear; however, they are

thought to relate, at least in part, to the ability of virus-infected and

immune-activated macrophages to induce adhesion molecules such

as E-selectin and vascular cell adhesion molecule 1 (VCAM-1) on

brain endothelium. Other studies have demonstrated that HIV gp120

enhances the expression of intercellular adhesion molecule 1 (ICAM1) in glial cells and HIV Tat protein can disrupt the tight junctions of

the brain endothelial cells to facilitate entry of HIV-infected cells into

the CNS. Virus isolates from the brain are preferentially R5 strains as

opposed to X4 strains; in this regard, HIV-infected individuals who are

heterozygous for CCR5-Δ32 appear to be relatively protected against

the development of HIV encephalopathy. Once HIV enters the brain

due to pressures of the local environment, it evolves to develop distinct

sequences in the env, tat, and LTR genes. These unique sequences have

been associated with neurocognitive dysfunction; however, it is unclear

if they are causal (see below).

HIV-infected individuals may manifest white matter lesions as well

as neuronal loss. The white matter lesions are due to axonal injury and

a disruption of the blood-brain barrier and not due to demyelination.

Given the absence of evidence of HIV infection of neurons, HIVmediated effects on neurons are thought to involve indirect pathways

whereby viral proteins, particularly gp120 and Tat, trigger the release of

endogenous neurotoxins from macrophages and to a lesser extent from

astrocytes. In addition, it has been demonstrated that both HIV-1 Nef

and Tat can induce chemotaxis of leukocytes, including monocytes,

into the CNS. Neurotoxins can be released from monocytes as a consequence of infection and/or immune activation. Monocyte-derived

neurotoxic factors have been reported to kill neurons via a variety

of mechanisms including activation of the N-methyl-d-aspartate

(NMDA) receptors and induction of oxidative stress. In addition, HIV

gp120 shed by virus-infected monocytes could cause neurotoxicity by

antagonizing the function of vasoactive intestinal peptide (VIP), by

elevating intracellular calcium levels, and by decreasing neurotrophic

factor levels in the cerebral cortex. A variety of monocyte-derived

cytokines can contribute directly or indirectly to the neurotoxic effects

in HIV infection; these include TNF-α, IL-1, IL-6, TGF-β, IFN-γ,

platelet-activating factor, and endothelin. Furthermore, among the

CC-chemokines, elevated levels of monocyte chemotactic protein-1

(MCP-1 or CCL-2) in the brain and CSF have been shown to correlate

best with the presence and degree of HIV encephalopathy in ART-naïve

patients. In addition, infection and/or activation of monocyte-lineage

cells can result in increased production of eicosanoids, quinolinic acid,

nitric oxide, excitatory amino acids such as l-cysteine and glutamate,

arachidonic acid, platelet-activating factor, free radicals, TNF-α, and

TGF-β, which may contribute to neurotoxicity. Astrocytes may play

diverse roles in HIV neuropathogenesis. Reactive gliosis or astrocytosis

has been demonstrated in the brains of HIV-infected individuals, and

TNF-α and IL-6 have been shown to induce astrocyte proliferation.

In addition, astrocyte-derived IL-6 can induce HIV expression in

infected cells in vitro. Furthermore, it has been suggested that astrocytes may downregulate macrophage-produced neurotoxins. Evidence

of neuronal injury can be demonstrated by measuring neurofilament

levels in CSF. Treatment with ART leads to improvement in neuropsychiatric manifestations and a decrease in these cytokine levels in

CSF, suggesting that they are driven by the virus or by its products.

However, even in patients on long-term ART, there may be evidence

of persistently activated lymphocytes in the CSF. It is unclear if these

lymphocytes may contribute to neuronal injury in the brain or are

critical for controlling the CNS viral reservoir. However, some individuals may develop a subacute encephalitis due to an IRIS reaction

(see below). This often occurs weeks or a few months after initiation

of ART in individuals with low CD4+ T-cell counts. It is thought that

the recovery of CD4+ T cells causes a lymphocyte response to the CNS

HIV reservoir. The contribution of host genetic factors to development

of neuropsychiatric manifestations of HIV infection has not been well

studied. However, evidence supports the role of several genetic factors

including the E4 allele for apoE in an increased risk of HIV-associated

neurocognitive disorders and peripheral neuropathy.

It has also been suggested that the CNS may serve as a relatively

sequestered site for a reservoir of latently infected cells that might be a

barrier for the eradication of virus by ART (see “The HIV Reservoir:

Obstacles to the Eradication of Virus,” above).

1556 PART 5 Infectious Diseases

■ PATHOGENESIS OF KAPOSI’S SARCOMA

There are at least four distinct epidemiologic forms of KS: (1) the classic form that occurs in older men of predominantly Mediterranean or

eastern European Jewish backgrounds with no recognized contributing

factors; (2) the equatorial African form that occurs in all ages, also

without any recognized precipitating factors; (3) the form associated

with organ transplantation and its attendant iatrogenic immunosuppressed state; and (4) the form associated with HIV-1 infection. In

the latter two forms, KS is an opportunistic disease; in HIV-infected

individuals, unlike typical opportunistic infections, its occurrence is

not strictly related to the level of depression of CD4+ T-cell counts.

The pathogenesis of KS is complex; fundamentally, it is an angioproliferative disease that is not a true neoplastic sarcoma, at least not in its

early stages. It is a manifestation of excessive proliferation of spindle

cells that are believed to be of vascular origin and have features in

common with endothelial and smooth-muscle cells. In HIV disease the

development of KS is dependent on the interplay of a variety of factors

including HIV-1 itself, human herpes virus 8 (HHV-8), immune activation, and cytokine secretion. Numerous epidemiologic and virologic

studies have clearly linked HHV-8, which is also referred to as Kaposi’s

sarcoma–associated herpesvirus (KSHV), to KS not only in HIV-infected individuals but also in individuals with the other forms of KS.

HHV-8 is a γ-herpesvirus related to EBV and herpesvirus saimiri. It

encodes a homologue to human IL-6 and, in addition to KS, has been

implicated in the pathogenesis of body cavity lymphoma, multiple

myeloma, and monoclonal gammopathy of undetermined significance.

Sequences of HHV-8 are found universally in the lesions of KS, and

patients with KS are virtually all seropositive for HHV-8. HHV-8 DNA

sequences can be found in the B cells of 30–50% of patients with KS

and 7% of patients with AIDS without clinically apparent KS.

Between 1 and 2% of eligible blood donors are positive for antibodies to HHV-8, while the prevalence of HHV-8 seropositivity in

HIV-infected men is 30–35%. The prevalence of HHV-8 seropositivity

in HIV-infected women is ~4%. This finding is reflective of the lower

incidence of KS in women. It has been debated whether HHV-8 is the

transforming agent in KS; the bulk of the cells in the tumor lesions of

KS are not neoplastic cells. However, it has been demonstrated that

endothelial cells can be transformed in vitro by HHV-8. In this regard,

HHV-8 possesses genes, including homologues of the IL-8 receptor,

Bcl-2, and cyclin D, that can potentially transform the host cell. Despite

the complexity of the pathogenic events associated with the development of KS in HIV-infected individuals, HHV-8 is the etiologic agent

of this disease. The initiation and/or propagation of KS requires an

activated state and is mediated, at least in part, by cytokines. A number of factors, including TNF-α, IL-1β, IL-6, granulocyte-macrophage

colony-stimulating factor (GM-CSF), basic fibroblast growth factor,

and oncostatin M, function in an autocrine and paracrine manner

to sustain the growth and chemotaxis of the KS spindle cells. In this

regard, KSHV-derived IL-6 has been demonstrated to induce proliferation of lymphoma cells and to inhibit the cytostatic effects of IFN-α

on KSHV-infected lymphoma cells.

IMMUNE RESPONSE TO HIV

As detailed above and below, following the initial burst of viremia during primary infection, HIV-infected individuals mount robust immune

responses that in most cases substantially curtail the levels of plasma

viremia and likely contribute to delaying the ultimate development of clinically apparent disease for a median of 10 years in untreated individuals.

This immune response contains elements of both humoral and cell-mediated immunity involving both adaptive and innate immune responses

(Table 202-7; Fig. 202-28). It is directed against multiple antigenic determinants of the HIV virion as well as against viral proteins expressed on the

surface of infected cells. Ironically, those CD4+ T cells with T-cell receptors specific for HIV are theoretically those CD4+ T cells most likely to be

activated—and thus to serve as early targets for productive HIV infection

and the cell death or dysfunction associated with infection. Thus, an early

consequence of HIV infection is interference with and decrease of the

helper T-cell population needed to generate an effective immune response.

TABLE 202-7 Elements of the Immune Response to HIV

Humoral immunity

Binding antibodies

Neutralizing antibodies

 Type specific

 Group specific

 Broadly neutralizing

Antibodies participating in antibody-dependent cellular cytotoxicity (ADCC)

 Protective

 Pathogenic (bystander killing)

Enhancing antibodies

Complement

Cell-mediated immunity

Helper CD4+ T lymphocytes

Class I MHC–restricted cytotoxic CD8+ T lymphocytes

CD8+ T-cell–mediated inhibition (noncytolytic)

ADCC

Natural killer cells

Abbreviation: MHC, major histocompatibility complex.

Neutralizing antibody

Cytotoxic CD8+

T lymphocyte

Helper CD4+

T lymphocytes

Activation,

proliferation, cytokine

and chemokine release

Activation

Viral antigens

Lysis

Lysis

Class I MHC

Class II MHC

Free

CD4 gp120

Bystander

killing

Natural killer cells

Uninfected CD4+

T lymphocyte

HIV-infected CD4+

T lymphocyte

HIV-infected CD4+

T lymphocyte

Fc

receptor

ADCC

Cytokine

release

TCR

FIGURE 202-28 Schematic representation of the different immunologic effector

mechanisms thought to be active in the setting of HIV infection. Detailed

descriptions are given in the text. ADCC, antibody-dependent cellular cytotoxicity;

MHC, major histocompatibility complex; TCR, T-cell receptor.

1557CHAPTER 202 Human Immunodeficiency Virus Disease: AIDS and Related Disorders

Although a great deal of investigation has been directed toward

delineating and better understanding the components of this immune

response, it remains unclear which immunologic effector mechanisms

are most important in delaying progression of infection and which, if any,

play a role in the pathogenesis of HIV disease. This lack of knowledge has

also hampered the ability to develop an effective vaccine for HIV disease.

■ HUMORAL IMMUNE RESPONSE

Antibodies to HIV usually appear within 3–6 weeks and almost

invariably within 12 weeks of primary infection (Fig. 202-29); rare

exceptions are in individuals who have defects in the ability to produce

HIV-specific antibodies. Detection of these antibodies forms the basis

of many diagnostic screening tests for HIV infection. The appearance

of HIV-binding antibodies detected by ELISA and Western blot assays

occurs prior to the appearance of neutralizing antibodies; the latter

generally appear following the initial decreases in plasma viremia and

are more closely related to the appearance of HIV-specific CD8+ T

lymphocytes. The first antibodies detected are those directed against

the immunodominant region of the envelope gp41, followed by the

appearance of antibodies to the structural or gag protein p24 and the

gag precursor p55. Antibodies to p24 gag are followed by the appearance of antibodies to the outer envelope glycoprotein (gp120), the gag

protein p17, and the products of the pol gene (p31 and p66). In addition, one may see antibodies to the low-molecular-weight regulatory

proteins encoded by the HIV genes vpr, vpu, vif, rev, tat, and nef. On

rare occasions, levels of HIV-specific antibodies may decline during

treatment of acute HIV infection.

While antibodies to multiple antigens of HIV are produced, the precise functional significance of these different antibodies is unclear. The

only viral proteins that elicit neutralizing antibodies are the envelope

proteins gp120 and gp41. Antibodies directed toward the envelope

proteins of HIV have been characterized both as being protective and

as possibly contributing to the pathogenesis of HIV disease. Among the

protective antibodies are those that function to neutralize HIV directly

and prevent the spread of infection to additional cells, as well as those

that participate in ADCC. The first neutralizing antibodies are directed

against the autologous infecting virus and appear after approximately

12 to 24 weeks of infection. Due to its high rate of mutation the virus is

usually able to quickly escape these (and subsequent) neutralizing antibodies. One important mechanism of immune escape is the addition

of N-linked glycosylation sites, forming a glycan shield that interferes

with envelope recognition by these initial antibodies.

A number of broad and potent HIV-neutralizing envelope-specific

antibodies have been isolated from HIV-infected individuals in studies

designed to better understand the host response to HIV infection.

Approximately 20% of patients develop antibodies capable of neutralizing highly diverse strains. These usually appear 2 or more years

following infection in the face of continual viremia. These studies have

revealed at least five major sites within the HIV envelope trimer that

are able to elicit broadly neutralizing antibodies. These sites include

antibodies directed toward the CD4 binding site (CD4bs) of gp120,

those binding glycan-dependent epitopes in the V1/V2 region of

gp120, those near the base of the V3 region of gp120, those binding to

the gp120/gp41 bridge, and those binding to the membrane-proximal

region of gp41 (Fig. 202-30). Several of these antibodies contain

unique features including high levels of somatic hypermutation, selective germline gene usage (especially for CD4bs antibodies), and long

heavy chain complementary determining regions (especially CDRH3).

Of note, while these antibodies are broadly neutralizing in vitro, their

precise in vivo significance is unclear and the patients from whom they

were derived demonstrate evidence of ongoing viral replication unless

treated with ART.

The other major class of protective antibodies are those that participate in ADCC, a form of cell-mediated immunity (Chap. 342) in

which NK cells that bear Fc receptors are armed with specific anti-HIV

antibodies that bind to the NK cells via their Fc portion. These armed

NK cells then bind to and destroy cells expressing HIV antigens. The

levels of anti-envelope antibodies capable of mediating ADCC are

highest in the earlier stages of HIV infection. Antibodies to both gp120

and gp41 have been shown to participate in ADCC-mediated killing of

HIV-infected cells. In vitro, IL-2 can augment ADCC-mediated killing.

In addition to playing a role in host defense, HIV-specific antibodies

have also been implicated in disease pathogenesis. Antibodies directed

to gp41, when present in low titer, have been shown in vitro to be capable of facilitating infection of cells through an Fc receptor–mediated

mechanism known as antibody enhancement. Thus, the same regions

of the envelope protein of HIV that give rise to antibodies capable

of mediating ADCC can also elicit the production of antibodies that

can facilitate infection of cells in vitro. In addition, it has been postulated that anti-gp120 antibodies that participate in the ADCC killing

of HIV-infected cells might also kill uninfected CD4+ T cells if the

uninfected cells had bound free gp120, a phenomenon referred to as

bystander killing.

One of the most primitive components of the humoral immune

system is the complement system (Chap. 342). This element of innate

immunity consists of ~30 proteins that are found circulating in blood or

associated with cell membranes. While HIV alone is capable of directly

activating the complement cascade, the resulting lysis is weak due to

the presence of host cell regulatory proteins captured in the virion

envelope during budding. It is possible that complement-opsonized

HIV virions have increased infectivity in a manner analogous to

antibody-mediated enhancement.

Initial

viremia

Seroconversion,

ADCC, CTL

Broadly reactive

NAbs

Autologous

NAbs

0 1 2 3–10

Years infected

FIGURE 202-29 Relationship between initial HIV viremia and the development

of antibodies to HIV. Within 3 to 6 weeks of initial HIV infection, nonneutralizing

antibodies to HIV appear. These antibodies are capable of mediating antibodydependent cellular cytotoxicity (ADCC). The decline in plasma viremia generally

correlates with the appearance of cytotoxic T lymphocytes (CTL). After

approximately 3 months, autologous neutralizing antibodies (NAbs) capable of

neutralizing prior circulating strains of HIV appear. After 2 or more years, broadly

reactive NAbs appear. (Republished with permission of Annual Reviews, Inc. from

The Role of Antibodies in HIV Vaccines, JR Mascola and DC Monteori, 28:413, 2010;

permission conveyed through Copyright Clearance Center, Inc.)

V3 glycan

V1V2 glycan

Subunit

interface

Fusion

peptide

CD4

binding

site

Silent

face

center

Membrane

Proximal

External Region

FIGURE 202-30 Known targets of broadly neutralizing antibodies against HIV-1.

(Courtesy of J Stuckey, GY Chuang.)

1558 PART 5 Infectious Diseases

■ CELLULAR IMMUNE RESPONSE

T-cell–mediated immunity plays a major role in host defense against

most viral infections (Chap. 342) and is thought to be an important

component of the host immune response to HIV. T-cell immunity can

be divided into two major categories: that mediated by helper/inducer

CD4+ T cells and that mediated by cytotoxic/immunoregulatory CD8+

T cells.

HIV-specific CD4+ T cells can be detected in the majority of

HIV-infected patients through the use of flow cytometry to measure

intracellular cytokine production in response to MHC class II tetramers pulsed with HIV peptides or through lymphocyte proliferation

assays utilizing HIV antigens such as p24. These cells likely play a

critical role in the orchestration of the immune response to HIV by

providing help to HIV-specific B cells and CD8+ T cells. They may also

be capable of directly killing HIV-infected cells. HIV-specific CD4+

T cells may be preferential targets of HIV infection by HIV-infected

antigen-presenting cells during the generation of an immune response

to HIV (Fig. 202-28). However, they also are likely to undergo clonal

expansions in response to HIV antigens and thus survive as a population of cells despite the virus. No clear correlations exist between levels

of HIV-specific CD4+ T lymphocytes and plasma HIV RNA levels;

however, in the setting of high viral loads, CD4+ T-cell responses to

HIV antigens appear to shift from one of proliferation and IL-2 production to one of IFN-γ production. Thus, while a reverse correlation

exists between the level of p24-specific proliferation and levels of

plasma HIV viremia, the nature of the causal relationship between

these parameters is unclear.

MHC class I–restricted, HIV-specific CD8+ T cells have been

identified in the peripheral blood of patients with HIV-1 infection.

These cells include CTLs that produce perforins and granzyme, and

T cells that can be induced by HIV antigens to express an array of

cytokines such as IFN-γ, IL-2, MIP-1β, and TNF-α. Multiple HIV

antigens, including Gag, Env, Pol, Tat, Rev, and Nef, can elicit CD8+

T-cell responses. CTLs have been identified in the peripheral blood

of patients within weeks of HIV infection and prior to the appearance

of plasma virus. The selective pressure they exert on the evolution of

the population of circulating viruses reflects their potential role in

control of HIV infection. These CD8+ T lymphocytes, through their

HIV-specific antigen receptors, bind to and cause the lytic destruction

of target cells bearing autologous MHC class I molecules presenting

HIV antigens. Two types of CTL activity can be demonstrated in the

peripheral blood or lymph node mononuclear cells of HIV-infected

individuals. The first type directly lyses appropriate target cells in

culture without prior in vitro stimulation (spontaneous CTL activity).

The other type of CTL activity reflects the precursor frequency of CTLs

(CTLp); this type of CTL activity can be demonstrated by stimulation

of CD8+ T cells in vitro with a mitogen such as phytohemagglutinin

or anti-CD3 antibody.

In addition to CTLs, CD8+ T cells capable of being induced by HIV

antigens to express cytokines such as IFN-γ also appear in the setting

of HIV-1 infection. It is not clear whether these are the same or different effector pools compared with those cells mediating cytotoxicity; in

addition, the relative roles of each in host defense against HIV are not

fully understood. It does appear that these CD8+ T cells are driven to

in vivo expansion by HIV antigen. There is a direct correlation between

levels of CD8+ T cells capable of producing IFN-γ in response to HIV

antigens and plasma levels of HIV-1 RNA. Thus, while these cells are

clearly induced by HIV-1 infection, in most instances they are not

able to effectively control infection. One exception may be a subset of

patients who control viral replication in the absence of antiretroviral

drugs and are referred to as elite nonprogressors (see “Long-Term Survivors, Long-Term Nonprogressors, and Elite Controllers,” above). The

peripheral blood of these patients contains a population of CD8+ T

cells that undergo substantial in vitro proliferation in response to HIV

antigens and express perforin and granzyme.

At least three other forms of cell-mediated immunity to HIV have

been described: non-cytolytic CD8+ T-cell–mediated suppression of

HIV replication, ADCC, and NK cell activity. Non-cytolytic CD8+

T-cell–mediated suppression of HIV replication refers to the ability of

CD8+ T cells from an HIV-infected patient to inhibit the replication of

HIV in tissue culture without killing infected targets. There is no requirement for HLA compatibility between the CD8+ T cells and the HIV-infected cells. This effector mechanism is thus nonspecific and appears to

be mediated by soluble factor(s) including the CC-chemokines RANTES

(CCL5), MIP-1α (CCL3), and MIP-1β (CCL4). These CC-chemokines are potent suppressors of HIV replication and operate at least

in part via blockade of the HIV co-receptor (CCR5) for R5 (macrophage-tropic) strains of HIV-1 (see above). ADCC, as described above

in relation to humoral immunity, involves the killing of HIV-expressing cells by NK cells armed with specific antibodies directed against

HIV antigens. Finally, NK cells alone have been shown to be capable

of killing HIV-infected target cells in tissue culture. This primitive

cytotoxic mechanism of host defense is directed toward nonspecific

surveillance for neoplastic transformation and viral infection through

recognition of altered class I MHC molecules.

DIAGNOSIS AND LABORATORY

MONITORING OF HIV INFECTION

The establishment of HIV as the causative agent of AIDS and related

syndromes early in 1984 was followed by the rapid development of

sensitive screening tests for HIV infection. By March 1985, blood

donors in the United States were routinely screened for antibodies to

HIV. In 1996, blood banks in the United States added the p24 antigen

capture assay to the screening process to help identify the rare infected

individuals who were donating blood in the time (up to 3 months)

between infection and the development of antibodies. In 2002, the

ability to detect early infection with HIV was further enhanced by the

licensure of nucleic acid testing (NAT) as a routine part of blood donor

screening. These refinements decreased the interval between infection

and detection (window period) from 22 days for antibody testing to 16

days with p24 antigen testing and subsequently to 12 days with NAT.

The development of sensitive assays for monitoring levels of plasma

viremia ushered in a new era of being able to monitor the progression

of HIV disease more closely. Utilization of these tests, coupled with the

measurement of levels of CD4+ T lymphocytes in peripheral blood, is

essential in the management of patients with HIV infection.

■ DIAGNOSIS OF HIV INFECTION

The CDC has recommended that screening for HIV infection be

performed as a matter of routine health care. The diagnosis of HIV

infection depends on the demonstration of antibodies to HIV and/or

the direct detection of HIV or one of its components. As noted above,

antibodies to HIV generally appear in the circulation 3–12 weeks

following infection. In addition to laboratory-based screening tests,

several home tests are available.

The standard blood screening tests for HIV infection are based on

the detection of antibodies to HIV and/or the p24 antigen (see below)

of HIV. A common laboratory-based platform is the ELISA, also

referred to as an enzyme immunoassay (EIA). This solid-phase assay

is an extremely good screening test with a sensitivity of >99.5%. Most

diagnostic laboratories use commercial kits that contain antigens from

both HIV-1 and HIV-2 and thus can detect antibodies to either. These

kits use both natural and recombinant antigens and are continuously

updated to increase their sensitivity to newly discovered species, such

as group O viruses (Fig. 202-1). The fourth-generation EIA tests combine detection of antibodies to HIV-1 or HIV-2 with detection of the

p24 antigen of HIV. EIA tests are generally scored as positive (highly

reactive), negative (nonreactive), or indeterminate (partially reactive).

While the EIA is an extremely sensitive test, it is not optimal with

regard to specificity. This is particularly true in studies of low-risk

individuals, such as volunteer blood donors. In this latter population,

as few as 10% of EIA-positive individuals are subsequently confirmed

to have HIV infection. Among the factors associated with false-positive

EIA tests are antibodies to class II antigens (such as may be seen following pregnancy, blood transfusion, or transplantation), autoantibodies,

hepatic disease, recent influenza vaccination, acute viral infections,

and administration of an HIV vaccine. For these reasons, anyone

suspected of having HIV infection based on a positive or inconclusive

1559CHAPTER 202 Human Immunodeficiency Virus Disease: AIDS and Related Disorders

fourth-generation EIA result should have the result confirmed with

a more specific assay such as an HIV-1– or HIV-2–specific antibody

immunoassay, a Western blot, or a plasma HIV RNA level. One can

estimate whether an individual has a recent infection with HIV-1 by

comparing the results on a standard EIA test that will score positive

for all infected individuals with the results on an assay modified to be

less sensitive (“detuned assay”) that will score positive for individuals

with established HIV infection and negative for individuals with recent

infection. In rare instances, an HIV-infected individual treated early in

the course of infection may revert to a negative EIA. This does not indicate clearing of infection; rather, it signifies levels of ongoing exposure

to virus or viral proteins insufficient to maintain a measurable antibody

response. When these individuals have discontinued therapy, viruses

and antibodies have reappeared.

CDC recommendations indicate that a positive fourth-generation

assay confirmed by a second HIV-1– or HIV-2–specific immunoassay

or a plasma HIV RNA level is adequate for diagnosis. The Western blot,

which had previously been used for a confirmatory test, is no longer

used for this purpose.

A guideline for the use of these serologic tests in attempting to make

a diagnosis of HIV infection is depicted in Fig. 202-31. In patients

in whom HIV infection is suspected, the appropriate initial test is

a fourth-generation HIV-1/2 antigen antibody immunoassay. If the

result is negative, unless there is strong reason to suspect early HIV

infection (as in a patient exposed within the previous 3 months), the

diagnosis is ruled out and retesting should be performed only as clinically indicated. If the immunoassay is indeterminate or positive, the

test should be repeated. If the repeat is negative on two occasions, one

can assume that the initial positive reading was due to a technical error

in the performance of the assay and that the patient is negative. If the

repeat is indeterminate or positive, one should proceed to an HIV-1/

HIV-2 antibody differentiation immunoassay such as the Bio-Rad

Genius. If testing is positive for HIV-1 and/or HIV-2 one may make

a diagnosis of HIV-1 and/or HIV-2 infection. If the HIV-1/HIV-2

antibody testing is negative or indeterminate one should proceed to

HIV-1 RNA testing with a nucleic acid test (NAT; see below). If the

NAT is positive, in the presence of a negative antibody test, one can

make a diagnosis of acute HIV-1 infection. If the NAT test is negative

for HIV-1 one should consider additional testing for HIV-2 RNA. One

can conclude a false-positive fourth-generation test in the setting of

repeated negative or indeterminate HIV-2/HIV-2 antibody tests in the

setting of negative NAT tests.

In addition to these standard laboratorybased assays for detecting antibodies to

HIV, a series of point-of-care tests can provide results in 1 to 60 minutes. While the

sensitivity and specificity of these tests are

generally quite high, it is generally recommended that any positive results be confirmed with standard laboratory testing.

Currently one rapid test kit is available for

use at home (OraQuick) as well as several

tests for which the sample is obtained at

home and mailed to the lab. A positive

result with any of these tests should be followed with confirmatory laboratory testing

by a healthcare professional.

A variety of laboratory tests are available for the direct detection of HIV or its

components (Table 202-8). These tests may

be of considerable help in making a diagnosis of HIV infection when the antibody

determination assays are indeterminate. In

addition, the tests detecting levels of HIV

RNA can be used to determine prognosis

and to assess the response to antiretroviral

therapies. The simplest, least expensive, and

most rarely used of the direct detection

tests is the p24 antigen capture assay. This

is an EIA-type assay in which the solid

phase consists of antibodies to the p24

antigen of HIV. It detects the viral protein

p24 in the blood of HIV-infected individuals where it exists either as free antigen

or complexed to anti-p24 antibodies. It

is currently part of the fourth-generation

HIV-1/2 antigen antibody immunoassay

test recommend for initial screening. Overall, ~30% of individuals with untreated

HIV infection have detectable levels of free

p24 antigen. This increases to ~50% when

samples are treated with a weak acid to

dissociate antigen-antibody complexes.

Throughout the course of HIV infection,

an equilibrium exists between p24 antigen

and anti-p24 antibodies. During the first

few weeks of infection, before an immune

response develops, there is a brisk rise in

p24 antigen levels. After the development

of anti-p24 antibodies, these levels decline.

+

–

indicates reactive test result

indicates nonreactive test result

NAT: nucleic acid test

Repeat

HIV-1/HIV-2

EIA

Retest in

3–6 months

if clinically

indicated

HIV-1

Western

blot

Diagnosis

of HIV-1

infection

Negative for HIV-1 and HIV-2

antibodies and p24Ag HIV-1/HIV-2 antibody

differentiation immunoassay

HIV-2

Western

blot

Screening

HIV–1/HIV-2

EIA

HIV-2

EIA Repeat in 4–6 weeks*

Diagnosis

of HIV-2

infection

+

+

+

+

+

+

+

–

–

–

–

–

–

Indeterminate

Indeterminate

A SEROLOGIC TESTS IN THE DIAGNOSIS OF HIV-1 OR HIV-2 INFECTION

B HIV-1/2 ANTIGEN/ANTIBODY COMBINATION IMMUNOASSAY

HIV-1 antibodies

detected

+

–

HIV-1

HIV-2

HIV-2 antibodies

detected

–

+

HIV-1

HIV-2

HIV antibodies

detected

+

+

HIV-1

HIV-2

HIV-1 NAT

HIV-1 NAT

Acute HIV-1

infection

HIV-1 NAT –

Negative for

HIV-1

–

HIV-1 or indeterminate –

HIV-2

FIGURE 202-31 Serologic tests for the diagnosis of HIV-1 or HIV-2 infection. A. Algorithm including the use of a

Western blot. *

Stable indeterminate Western blot 4–6 weeks later makes HIV infection unlikely. However, it should

be repeated twice at 3-month intervals to rule out HIV infection. Alternatively, one may test for HIV-1 p24 antigen

or HIV RNA. EIA, enzyme immunoassay. B. CDC algorithm not including the use of a Western blot. (Adapted from

stacks.cdc.gov/view/cdc/23446.)

1560 PART 5 Infectious Diseases

Late in the course of infection, when circulating levels of virus are

high, p24 antigen levels also increase, particularly when detected by

techniques involving dissociation of antigen-antibody complexes. The

p24 antigen capture assay has its greatest use as a screening test for

HIV infection in patients suspected of having the acute HIV syndrome

(see below), as high levels of p24 antigen are present prior to the development of antibodies. Its use as a stand-alone test for routine blood

donor screening for HIV infection has been replaced by use of NAT or

“fourth-generation” assays that combine antigen and antibody testing.

The ability to measure and monitor levels of HIV RNA in the plasma

of patients with HIV infection has been of extraordinary value in

furthering our understanding of the pathogenesis of HIV infection, in

monitoring the response to ART, and in providing a diagnostic tool in

settings where measurements of anti-HIV antibodies may be misleading, such as in acute infection and neonatal infection. In addition to the

commercially available tests for measuring HIV RNA, DNA PCR assays

also are employed by research laboratories for making a diagnosis of

HIV infection by amplifying HIV proviral DNA from peripheral blood

mononuclear cells. The commercially available RNA detection tests

have a sensitivity of 40–80 copies of HIV RNA per milliliter of plasma.

Research laboratory–based RNA assays can detect as few as one HIV

RNA copy per milliliter, while the DNA PCR tests can detect proviral

DNA at a frequency of one copy per 10,000–100,000 cells. Thus, these

tests are extremely sensitive. One frequent consequence of a high

degree of sensitivity is some loss of specificity, and false-positive results

have been reported with each of these techniques. For this reason, a

positive EIA with a positive HIV RNA assay can be considered the

“gold standard” for a diagnosis of HIV infection, and the interpretation

of other test results must be done with this in mind.

In the RT-PCR technique, following DNAse treatment, a cDNA

copy is made of all RNA species present in plasma. Because HIV is an

RNA virus, this will result in the production of DNA copies of the HIV

genome in amounts proportional to the amount of HIV RNA present

in plasma. This cDNA is then amplified and characterized using standard PCR techniques, employing primer pairs that can distinguish

genomic cDNA from messenger cDNA.

In addition to being diagnostic and prognostic tools, RT-PCR

and DNA-PCR also are useful for amplifying defined areas of the

HIV genome for sequence analysis and have become an important

technique for studies of sequence diversity and microbial resistance

to antiretroviral agents. In patients with a positive or indeterminate

EIA test and an indeterminate Western blot, and in patients in whom

serologic testing may be unreliable (such as patients with hypogammaglobulinemia or advanced HIV disease), these tests for quantitating

HIV RNA in plasma or detecting proviral DNA in peripheral blood

mononuclear cells are valuable tools for making a diagnosis of HIV

infection; however, they should be used for diagnosis only when standard serologic testing has failed to provide a definitive result.

■ LABORATORY MONITORING OF PATIENTS WITH

HIV INFECTION

The integration of clinical and laboratory data is essential to optimal

management of patients with HIV infection. The close relationship

between clinical manifestations of HIV infection and CD4+ T-cell

count has made measurement of CD4+ T-cell numbers a routine part

of the evaluation of HIV-infected individuals. The discovery of HIV

as the cause of AIDS led to the development of sensitive tests that

allow one to monitor the levels of HIV in the blood. Determinations of

peripheral blood CD4+ T-cell counts and measurements of the plasma

levels of HIV RNA provide a powerful set of tools for determining

prognosis and monitoring response to therapy.

CD4+ T-Cell Counts The CD4+ T-cell count is the laboratory test

generally accepted as the best indicator of the immediate state of immunologic competence of the patient with HIV infection. This measurement has been shown to correlate very well with the level of immunologic

competence. Patients with CD4+ T-cell counts <200/μL are at high risk

of disease from P. jirovecii, while patients with CD4+ T-cell counts <50/

μL are also at high risk of disease from CMV, mycobacteria of the M.

avium complex (MAC), and/or T. gondii (Fig. 202-32). Once the CD4+

T-cell count is <200/μL, patients should be placed on a regimen for P.

jirovecii prophylaxis. Once the count is <50/μL, primary prophylaxis for

MAC infection is indicated unless the patient is immediately started on

ART. As with any laboratory measurement, one may wish to obtain two

determinations prior to any significant changes in patient management

based on CD4+ T-cell count alone. Patients with HIV infection should

have CD4+ T-cell measurements performed at the time of diagnosis

and every 3–6 months thereafter. More frequent measurements should

be made if a declining trend is noted. For patients who have been on

ART for at least 2 years with HIV RNA levels persistently <50 copies/

mL and CD4 counts 300-500/μL, monitoring may be decreased to every

year. For those with CD4 counts >500/μL, the monitoring of the CD4

count is felt by many to be optional. There are a handful of clinical situations in which the CD4+ T-cell count may be misleading. Patients with

HTLV-1/HIV co-infection may have elevated CD4+ T-cell counts that

do not accurately reflect their degree of immune competence. In patients

with hypersplenism or those who have undergone splenectomy, and in

patients receiving medications that suppress the bone marrow such as

IFN-α, the CD4+ T-cell percentage may be a more reliable indication of

immune function than the CD4+ T-cell count. A CD4+ T-cell percentage of 15 is comparable to a CD4+ T-cell count of 200/μL.

HIV RNA Determinations Facilitated by highly sensitive techniques for the precise quantitation of small amounts of nucleic acids,

the measurement of serum or plasma levels of HIV RNA has become

an essential component in the monitoring of patients with HIV

infection. As discussed in “Diagnosis of HIV Infection,” above, the

most used technique is the RT-PCR assay. This assay generates data

in the form of number of copies of HIV RNA per milliliter of serum

or plasma and can reliably detect as few as 40 copies of HIV RNA per

milliliter of plasma. Research-based assays can detect down to one copy

per milliliter. While it is common practice to describe levels of HIV

RNA below these cut-offs as “undetectable,” this is a term that should

be avoided as it is imprecise and leaves the false impression that the

level of virus is 0. By utilizing more sensitive, nested PCR techniques

and by studying tissue levels of virus as well as plasma levels, HIV

RNA can be detected in virtually every patient with HIV infection.

TABLE 202-8 Characteristics of Tests for Direct Detection of HIV

TEST TECHNIQUE SENSITIVITYa COST/TESTb

Immune complex–dissociated p24

antigen capture assay

Measurement of levels of HIV-1 core protein in an EIA-based format

following dissociation of antigen-antibody complexes by weak acid

treatment

Positive in 50% of patients; detects

down to 15 pg/mL of p24 protein

$1–2

HIV RNA by PCR Target amplification of HIV-1 RNA via reverse transcription followed by PCR Reliable to 40 copies/mL of HIV RNA $75–150

HIV RNA by bDNA Measurement of levels of particle-associated HIV RNA in a nucleic acid

capture assay employing signal amplification

Reliable to 50 copies/mL of HIV RNA $75–150

HIV RNA by TMA Target amplification of HIV-1 RNA via reverse transcription followed by T7

RNA polymerase

Reliable to 100 copies/mL of HIV RNA $225

HIV RNA by NASBA Isothermal nucleic acid amplification with internal controls Reliable to 80 copies/mL of HIV RNA $75–150

a

Sensitivity figures refer to those approved by the U.S. Food and Drug Administration. b

Prices may be lower in large-volume settings.

Abbreviations: bDNA, branched DNA; cDNA, complementary DNA; EIA, enzyme immunoassay; NASBA, nucleic acid sequence–based amplification; PCR, polymerase chain

reaction; TMA, transcription-mediated amplification.

1561CHAPTER 202 Human Immunodeficiency Virus Disease: AIDS and Related Disorders

the short-term ability to decrease viral

load by ~0.5 log compared with changing drugs merely based on drug history.

In addition to the use of resistance testing to help in the selection of new drugs

in patients with virologic failure, it may

also be of value in selecting an initial

regimen for treatment of therapy-naïve

individuals. This is particularly true

in geographic areas with a high level

of background resistance. The patient

needs to have an HIV-1 RNA level

above 500–1000 copies/mL for an accurate resistance determination. Resistance assays lose their consistency at

lower levels of plasma viremia.

Co-Receptor Tropism Assays

Following the licensure of maraviroc

as the first CCR5 antagonist for the

treatment of HIV infection (see below),

it became necessary to be able to determine whether a patient’s virus was likely

to respond to this treatment. Patients

tend to have CCR5-tropic virus early

in the course of infection, with a trend

toward CXCR4 viruses later in disease.

The antiretroviral agent maraviroc is effective only against CCR5-tropic

viruses. Because the genotypic determinants of cellular tropism are

poorly defined, a phenotypic assay is necessary to determine this property of HIV. The Trofile assay (Monogram Biosciences) is available to

make this determination. This assay clones the envelope regions of the

patient’s virus into an indicator virus that is then used to infect target

cells expressing either CCR5 or CXCR4 as their co-receptor. The assay

takes weeks to perform and is expensive. Another, less costly option is

to obtain a genotypic assay of the V3 region of HIV-1 and then employ

a computer algorithm to predict viral tropism from the sequence.

While this approach is less expensive than the classic phenotypic assay,

there are fewer data to validate its predictive value.

Other Tests A variety of other laboratory tests have been studied as

potential markers of HIV disease activity. Among these are quantitative

culture of replication-competent HIV from plasma, peripheral blood

mononuclear cells, or resting memory CD4+ T cells; circulating levels

of β2

-microglobulin, soluble IL-2 receptor, IgA, acid-labile endogenous

IFN, or TNF-α; and the presence or absence of activation markers such

as CD38, HLA-DR, and PD-1 on CD4+ or CD8+ T cells. Nonspecific

serologic markers of inflammation and/or coagulation such as IL-6,

d-dimer, and sCD14 have been shown to have a high correlation with

all-cause mortality (Table 202-9). While these measurements have

value as markers of disease activity and help to increase our understanding of the pathogenesis of HIV disease, they do not currently play

a major role in the monitoring of patients with HIV infection.

CLINICAL MANIFESTATIONS

The clinical consequences of HIV infection encompass a spectrum

ranging from an acute syndrome associated with primary infection

to a prolonged asymptomatic state to advanced disease. It is best to

There  are a few notable exceptions to this that involve patients who

underwent cytoreductive therapy followed by bone marrow transplant

from CCR5Δ32 homozygous donors.

Measurements of changes in HIV RNA levels over time have been of

great value in delineating the relationship between levels of virus and

rates of disease progression (Fig. 202-22), the rates of viral turnover, the

relationship between immune system activation and viral replication,

and the time to development of drug resistance. HIV RNA measurements are greatly influenced by the state of activation of the immune

system and may fluctuate greatly in the setting of secondary infections

or immunization. For these reasons, decisions based on HIV RNA levels should never be made on a single determination. Measurements of

plasma HIV RNA levels should be made at the time of HIV diagnosis

and every 3–6 months thereafter in the untreated patient. Following

the initiation of therapy or any change in therapy, plasma HIV RNA

levels should be monitored approximately every 4 weeks until the effectiveness of the therapeutic regimen is determined by the development

of a new steady-state level of HIV RNA. In most instances of effective

antiretroviral therapy, the plasma level of HIV RNA will drop to

<50 copies/mL within 6 months of the initiation of treatment. During

therapy, levels of HIV RNA should be monitored every 3–6 months to

evaluate the continuing effectiveness of therapy.

HIV Resistance Testing The availability of multiple antiretroviral

drugs as treatment options has generated a great deal of interest in

the potential for measuring the sensitivity of an individual’s HIV viral

quasispecies to different antiretroviral agents. HIV resistance testing

can be done through either genotypic or phenotypic measurements. In

the genotypic assays, sequence analyses of the HIV genomes obtained

from patients are compared with sequences of viruses with known

antiretroviral resistance profiles. In the phenotypic assays, the in vivo

growth of patient-derived viral isolates or genetically constructed pseudoviruses is compared with the growth of reference strains of the virus

in the presence or absence of different antiretroviral drugs. These tests

are quite good at identifying those antiretroviral agents that have been

utilized in the past and suggesting agents that may be of future value in

a given patient. Resistance testing is recommended at the time of initial diagnosis and, if therapy is not initiated at that time, at the time of

initiation of ART. Drug resistance testing is also indicated in the setting

of virologic failure and should be performed while the patient is still on

the failing regimen because of the propensity for the pool of HIV quasispecies to rapidly revert to wild-type in the absence of the selective

pressures of ART. In the hands of experts, resistance testing enhances

Opportunistic illness

CD4 (cells/µL3)

HSV HZos Crp KS Cry Can PCP NHL DEM PML WS Tox CMV PCP2 MAC

*

*

* *

* *

* *

*

* * *

*

*

* 100

200

300

0

FIGURE 202-32 Relationship between CD4+ T-cell counts and the development of opportunistic diseases. Boxplot of

the median (line inside the box), first quartile (bottom of the box), third quartile (top of the box), and mean (asterisk)

CD4+ lymphocyte count at the time of the development of opportunistic disease. Can, candidal esophagitis; CMV,

cytomegalovirus infection; Crp, cryptosporidiosis; Cry, cryptococcal meningitis; DEM, AIDS dementia complex; HSV,

herpes simplex virus infection; HZos, herpes zoster; KS, Kaposi’s sarcoma; MAC, Mycobacterium avium complex

bacteremia; NHL, non-Hodgkin’s lymphoma; PCP, primary Pneumocystis jirovecii pneumonia; PCP2, secondary P. jirovecii

pneumonia; PML, progressive multifocal leukoencephalopathy; Tox, Toxoplasma gondii encephalitis; WS, wasting

syndrome. (From Annals of Internal Medicine, RD Moore, RE Chaisson: Natural History of Opportunistic Disease in an

HIV-Infected Urban Clinical Cohort. 124(7):633-642, 1996. Copyright © 1996 American College of Physicians. All Rights

Reserved. Reprinted with the permission of American College of Physicians, Inc.)

TABLE 202-9 Association between High-Sensitivity CRP, Il-6, and

d-Dimer with All-Cause Mortality in Patients with HIV Infection

UNADJUSTED ADJUSTED

MARKER

ODDS RATIO

(FOURTH/FIRST) P

ODDS RATIO

(FOURTH/FIRST) P

Hs-CRP 2.0 .05 2.8 .03

IL-6 8.3 <.0001 11.8 <.0001

d-dimer 12.4 <.0001 26.5 <.0001

Abbreviations: Hs-CRP, high-sensitivity C-reactive protein; IL-6, interleukin 6.

Source: From LH Kuller et al: PLoS Med 5:e203, 2008.

1562 PART 5 Infectious Diseases

regard HIV disease as beginning at the time of primary infection and

progressing through various stages. As mentioned above, active virus

replication and progressive immunologic impairment occur throughout the course of HIV infection in most patients. Except for the rare,

true, “elite” virus controllers or long-term nonprogressors (see “LongTerm Survivors, Long-Term Nonprogressors, and Elite Controllers,”

above), HIV disease in untreated patients inexorably progresses even

during the clinically latent stage. Since the mid-1990s, ART has had a

major impact on preventing and reversing the progression of disease

over extended periods of time in a substantial proportion of adequately

treated patients. Today, a person diagnosed with HIV infection and

treated with ART has a close to normal life expectancy.

■ ACUTE HIV INFECTION

It is estimated that 50–70% of individuals with HIV infection experience an acute clinical syndrome ~3–6 weeks after primary infection

(Fig. 202-33). Varying degrees of clinical severity have been reported,

and although it has been suggested that symptomatic seroconversion

leading to the seeking of medical attention indicates an increased

risk for an accelerated course of disease, there does not appear to be

a correlation between the level of the initial burst of viremia in acute

HIV infection and the subsequent course of disease. The typical clinical findings in the acute HIV syndrome are listed in Table 202-10;

they occur along with a burst of plasma viremia. It has been reported

that several symptoms of the acute HIV syndrome (fever, skin rash,

pharyngitis, and myalgia) occur less frequently in those infected by

injection drug use compared with those infected by sexual contact. The

syndrome is typical of an acute viral syndrome and has been likened

to acute infectious mononucleosis. Symptoms usually persist for one

to several weeks and gradually subside as an immune response to HIV

develops and the levels of plasma viremia decrease. Opportunistic

infections have been reported during this stage of infection, reflecting

the immunodeficiency that results from reduced numbers of CD4+ T

cells and likely also from the dysfunction of CD4+ T cells owing to viral

protein and endogenous cytokine-induced perturbations of cells (Table

202-5) associated with the extremely high levels of plasma viremia. The

Fiebig staging system has been used to describe the different stages of

acute HIV infection, ranging from stage 1 (HIV RNA positive alone)

to stage VI (HIV RNA and full Western blot positive). A number of

immunologic abnormalities accompany the acute HIV syndrome,

including multiphasic perturbations of the numbers of circulating

lymphocyte subsets. The numbers of total lymphocytes and T-cell

subsets (CD4+ and CD8+) are initially reduced. An inversion of the

CD4+/CD8+ T-cell ratio occurs later because of a rise in the number of

CD8+ T cells. In fact, there may be a selective and transient expansion

of CD8+ T-cell subsets, as determined by T-cell receptor analysis (see

above). The total circulating CD8+ T-cell count may remain elevated

or return to normal; however, CD4+ T-cell levels usually remain

somewhat depressed, although there may be a slight rebound toward

normal. Lymphadenopathy occurs in ~70% of individuals with primary HIV infection. Most patients recover spontaneously from this

syndrome and many are left with only a mildly depressed CD4+ T-cell

count that remains stable for a variable period before beginning its progressive decline; in some individuals, the CD4+ T-cell count returns to

the normal range. Approximately 10% of patients manifest a fulminant

course of immunologic and clinical deterioration after primary infection, even after the disappearance of initial symptoms. In most patients,

primary infection with or without the acute syndrome is followed by a

prolonged period of clinical latency or smoldering low disease activity.

■ THE ASYMPTOMATIC STAGE—CLINICAL LATENCY

Although the length of time from initial infection to the development of clinical disease varies greatly, the median time for untreated

patients is ~10 years. As emphasized above, HIV disease with active

virus replication is ongoing and progressive during this asymptomatic

period. The rate of disease progression is directly correlated with HIV

RNA levels. Patients with high levels of HIV RNA in plasma progress

to symptomatic disease faster than do patients with low levels of HIV

RNA (Fig. 202-22). Some patients referred to as long-term nonprogressors show little if any decline in CD4+ T-cell counts over extended periods of time. These patients generally have extremely low levels of HIV

RNA; a subset, referred to as elite nonprogressors, exhibits HIV RNA

levels <50 copies/mL. Certain other patients remain entirely asymptomatic even though their CD4+ T-cell counts show a steady progressive decline to extremely low levels. In these patients, the appearance of

an opportunistic disease may be the first manifestation of HIV infection. During the asymptomatic period of HIV infection, the average

rate of CD4+ T-cell decline is ~50/μL per year in an untreated patient.

When the CD4+ T-cell count falls to <200/μL, the resulting state of

immunodeficiency is severe enough to place the patient at high risk

for opportunistic infections and neoplasms and, hence, for clinically

apparent disease.

■ SYMPTOMATIC DISEASE

Symptoms of HIV disease can appear at any time during the course of

HIV infection. Generally, the spectrum of illnesses that one observes

changes as the CD4+ T-cell count declines. The more severe and

life-threatening complications of HIV infection occur in patients

with CD4+ T-cell counts <200/μL. A diagnosis of AIDS is made in

any individual age 6 years and older with HIV infection and a CD4+

T-cell count <200/μL (stage 3, Table 202-2) and in anyone with HIV

infection who develops one of the HIV-associated diseases considered to be indicative of a severe defect in cell-mediated immunity

(Table 202-1). While the causative agents of the secondary infections

are characteristically opportunistic organisms such as P. jirovecii, atypical mycobacteria, CMV, and other organisms that do not ordinarily

cause disease in the absence of a compromised immune system, they

also include several common bacterial and mycobacterial pathogens.

Following the widespread use of ART and implementation of guidelines for the prevention of opportunistic infections (Table 202-11),

Plasma viremia

 (wide dissemination

 of virus)

Acute

 syndrome

Retrafficking of

 lymphocytes

1 week–3 months

1–2 weeks

3–6 weeks

Immune response to HIV

Curtailment

 of plasma

 viremia

Primary Infection

Establishment of

 chronic, persistent

 infection in

 lymphoid tissue

Clinical latency

FIGURE 202-33 The acute HIV syndrome. See text for detailed description.

(From G Pantaleo, C Graziosi, AS Fauci: The Immunopathogenesis of Human

Immunodeficiency Virus Infection. N Engl J Med 328:327, 1993. Copyright © 1993

Massachusetts Medical Society. Reprinted with permission from Massachusetts

Medical Society.)

TABLE 202-10 Clinical Findings in the Acute HIV Syndrome

General Neurologic

Fever Meningitis

Pharyngitis Encephalitis

Lymphadenopathy Peripheral neuropathy

Headache/retroorbital pain Myelopathy

Arthralgias/myalgias Dermatologic

Lethargy/malaise Erythematous maculopapular rash

Anorexia/weight loss Mucocutaneous ulceration

Nausea/vomiting/diarrhea

Source: Reproduced with permission from B Tindall, DA Cooper: Primary HIV

infection: Host responses and intervention strategies. AIDS 5:1, 1991.