913© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/B978-0-08-097037-0.00072-5

In 1981, a report of five young, homosexual men with Pneumocystis carinii pneumonia and immunodeficiency was the first indication of the emergence of a new and lethal human pathogen (CDC, 1981; Gottlieb et al., 1981). This initial report was quickly followed by additional cases, in the United States and Europe, of previously healthy indi-viduals developing severe immunodeficiency resulting in lymphadenopathy, acquisition of opportunistic infections, such as P. carinii pneumonia, cytomegalovirus, mucosal candidiasis, and cryptococcal meningitis, and rare cancers such as Kaposi’s sarcoma. The main groups affected by this new disease were homosexual men, injection drug users, hemophiliacs, Haitians, and sexual partners and children of these risk groups. The risk groups indicated that the disease was caused by an infectious agent, trans-mitted sexually and through exposure to blood. The syndrome was designated acquired immune deficiency syndrome (AIDS). In 1983, the French research group headed by Luc Montagnier isolated a retrovirus from a lymph node of an AIDS patient that they designated lympho-adenopathy virus or LAV (Barré-Sinoussi et al., 1983). The following year a series of publications, from Robert Gallo’s laboratory in the United States, reported isolation and propagation in cell culture of a virus they designated human T-cell lymphotropic virus-III (HTLV-III), demonstrated that AIDS patients had anti-bodies to HTLV-III, and provided confirmation that the virus was the causative agent of AIDS (Gallo et al., 1984; Popovic et al., 1984; Sarangadharan et al., 1984). After much investigation, it was determined that LAV and HTLV-III were the same virus; in 1986, it was renamed human immunodeficiency virus type 1 (HIV-1). HIV-1 has caused a global pandemic. The World Health Organi-zation and UNAIDS estimate that the number of individuals living with HIV grew from 7 million in 1990 to 33 million in 2009 while the annual deaths due to AIDS increased from 0.3 million in 1990 to a peak of 2.2 million in 2005, followed by a decline to 1.8 million in 2009. There were 2.6 million new HIV infections, over 7000 infections a day, in 2009 (UNAIDS, 2010).

Etiologic AgentHIV is a member of the lentivirus genus of Retroviridae (Barré-Sinoussi et al., 1983; Freed et al., 2006). Lentivi-ruses are enveloped viruses with a distinct cylindrical or conical-shaped core. The hallmark of retroviruses is the use of the viral encoded enzyme, reverse transcriptase, which transcribes the viral RNA genome into DNA. Reverse transcription is followed by integration of the DNA into the host chromosomal DNA, thus establishing permanent infection of the host. The integrated proviral DNA serves as the template for transcription of genomic

RNA and spliced messenger RNA for expression of viral proteins. HIV virion contains a homodimer of linear, posi-tive sense, single-stranded RNA genome encoding gag, pro, pol, and env genes that are common to all lentivirus, and additional accessory genes. The HIV accessory genes are vif, vpr, vpu, tat, rev, and nef; vpu is unique to HIV-1 while HIV-2 has vpx which is a homolog for vpr. The accessory gene products are involved in viral replication, RNA transcription and processing, and virion assembly.

HIV is classified into type 1 and type 2. HIV-2 was discovered in 1986 in AIDS patients in West Africa (Clavel et al., 1986). HIV-1, the pandemic virus, arose from the cross-species transmission of simian viruses found in chimpanzees (Pan troglodyte troglodyte) and west-ern gorillas (Gorilla gorilla gorilla) into human (Keele et al., 2006; Plantier et al., 2009). HIV-2, which originated in sooty mangabeys found in West Africa, is found primarily in Western Africa and countries with colonial and com-mercial ties to that region (Gao et al., 1992; DeCock et al., 1993). HIV types are subclassified into groups with each group representing an independent transmission of virus into humans. For HIV-1, there are group M (main), N (nonM nonO), O (outlier), and P. Groups M and N are most closely related to simian immunodeficiency viruses (SIV) found in chimpanzees in Cameroon (Keele et al., 2006). Group P is most closely related to an SIV found in western gorillas in Cameroon (Plantier et al., 2009). Cur-rently, the SIV origin of group O is not identified. HIV-1 groups N, O, and P are rare strains while HIV-1 group M is circulating around the world. HIV-1 group M is further subclassified into subtypes (currently A–K) and circulating recombinant forms (CRF01–CRF54) (HIV Sequence Database). Subtypes are the results of viral evolution after a strain has been introduced and expanded exponentially within a limited geographical region or population (Peeters and Sharp, 2000). If more than one subtype is cir-culating within a population, recombination in dually infected individuals generates viral strains comprised of more than one subtype. Recombination between HIV-1 groups M and O has been documented. HIV rapid evolu-tion and recombination are driven by virus replication using reverse transcriptase.

HIV-2 is classified into groups A–H meaning that SIV strains from sooty mangabeys have crossed into humans at least eight times. However, only HIV-2 groups A and B appear to be transmitted from one human to another. Recombination between HIV-2 groups has been observed, but recombination between HIV-1 and HIV-2 has not been observed. While HIV-1 and HIV-2 share the same modes of transmission, HIV-2 infections are rare compared to HIV-1 infections. HIV-2-infected individuals have lower levels of viremia, are slower to progress to disease, and transmit at a lower rate than HIV-1-infected individu-als (DeCock et al., 1993).

HIV-1 and HIV-2: Causative Agents of AIDSCatherine A. Brennan (catherine.brennan@abbott.com)

Sushil G. Devare (sushil.devare@abbott.com)

C H A P T E R

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The high level of HIV strain genetic diversity and the ability of the virus to rapidly mutate create a complex challenge for diagnosis of HIV infections, optimal antivi-ral treatment, therapeutic monitoring, and vaccine development.

PathogenesisHIV is transmitted by sexual intercourse (via semen or vaginal secretions), by parenteral routes (e.g., blood trans-fusions, intravenous drug use, or infusion of blood prod-ucts) and by perinatal exposure (in utero or via colostrum). HIV infects and kills CD4+ T-lymphocytes leading to progressive loss of cell-mediated immunity, eventually resulting in the inability of the immune system to protect against opportunistic infections.

The course of disease can be divided into three phases: acute, chronic, and advanced. During the acute or early phase of infection, there is widespread infection of the gut-associated lymphoid tissue (Mehandru et al., 2004). Both resting and activated memory CD4 cells are infected and depleted resulting in loss of greater than half of the mem-ory CD4 cells in the gut while establishing a stable reservoir of HIV-infected memory cells for viral replication. Plasma viremia rises rapidly, reaching a peak of 106–107 RNA copies/mL about 2–4 weeks after infection (Fiebig et al., 2003). At the same time, infected individuals often experi-ence symptoms of viral infections that can last 3–4 weeks; symptoms include fever, rash, headache, pharyngitis, lymphadenopathy, and malaise. During acute infection, there is a transient decline in CD4+ T-lymphocytes and increase in CD8+ T-cells. Development of antibodies to HIV marks the end of the acute phase and beginning of the chronic or asymptomatic phase. The host immune response

to the HIV infection results in a decline in plasma viremia to a steady-state set point. The progression to disease is correlated with the viral set point. In the absence of anti-retroviral therapy, individuals with high-RNA set points (>104–105 RNA copies/mL) progress faster to AIDS than those with lower set points (<103–104 RNA copies/mL) (Lyles et al., 2000). The chronic phase can last as short as several months to more than 15 years during which there is ongoing viral replication and a slow, progressive decline in CD4+ cells from >500/mm3 to 200/mm3. Patients may experience fatigue, lymphadenopathy, and a variety of non-life-threatening illnesses such as oral candidiasis, her-pes zoster, night sweats, and weight loss. The onset of AIDS occurs when the CD4+ cell count drops below 200/mm3 marking collapse of the immune system. Plasma virus lev-els climb and opportunistic infections and malignancies become common. AIDS-defining illnesses include candi-diasis, recurring bacterial infections, invasive cervical cancer, crytococcosis, encephalopathy, histoplasmosis, Kaposi sarcoma, Pneumonia jirovecii, Mycobacterium, as well as others (Schneider et al., 2008).

Diagnosis of HIV InfectionThe markers of HIV infection in blood evolve with the stage of infection (Fig. 1). During the acute phase, circu-lating virus first appears in blood at approximately 6–12 days after infection (Fiebig et al., 2003) and can be detected using assays that directly detect viral RNA or the viral cap-sid protein p24 antigen (Ag). The humoral immune response to HIV infection, leading to seroconversion, begins with the production of anti-HIV IgM antibodies (Ab) followed by an IgG response that is sustained throughout the chronic phase of infection. Following

0 10 20 30 40 50 60 70 80 90 100

HIV RNA

17

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3rd

Gen Ab

4th

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Anti-HIV IgM

FIGURE 1 Markers of HIV infection in blood. HIV-1 virion begins appearing in the blood 1–2 weeks after infection. The first detectable marker of infection is HIV RNA (dotted line) followed by p24 Ag (dashed line). p24 antigen appears before an antibody response is developed and can be detected by fourth-generation combination immunoassays. Over time, as antibody production occurs, p24 antigen becomes undetectable. The antibody response (solid line) to infection begins with the development of anti-HIV IgM at approximately 3 weeks; at this time, infection can be detected by third-generation HIV antibody assays. Anti-HIV IgG antibodies are detected at approximately 5 weeks and persist throughout chronic infection. Figure was adapted from Fiebig et al., 2003; Owen et al., 2008; Masciotra et al., 2011.

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seroconversion, p24 Ag is complexed with antibody and becomes undetectable; Ag can be detected during the chronic phase if the test specimen is pretreated to disrupt immune complexes. HIV-1 RNA remains detectable throughout the chronic phase except in elite suppressors who naturally control viral replication and those patients on effective antiretroviral therapy. HIV-2 infected individuals frequently have undetectable RNA levels (Damond et al., 2002). When patients have progressed to AIDS, seroreversion to antibody negative and the reap-pearance of p24 Ag can occur as the immune system fails and viral titers increase.

The primary method for diagnosis of HIV infection is the detection of anti-HIV antibodies. The first-generation HIV tests, developed in the mid-1980s, were indirect enzyme immunoassays (EIAs) that used viral lysate derived from cell-cultured HIV-1 to capture IgG Abs from a patient plasma or serum specimen and detected the captured antibody using an enzyme-labeled anti-human IgG conjugate. Second-generation HIV assays also used an indirect format, but assay sensitivity and specificity were improved by replacing the HIV-1 viral lysate with purified recombinant proteins and/or synthetic peptides for Ab capture. When HIV-2 was discovered, it was observed that not all HIV-2 infections were detected by the HIV-1 assays. The addition of HIV-2-specific recom-binant proteins or peptides to the assays allowed detection of IgG Abs to both HIV-1 and HIV-2. The development of direct immunoassays, so-called third-generation anti-gen sandwich assays, reduced the seroconversion window period (Owen et al., 2008). These assays detect both IgM and IgG Abs by using recombinant proteins and/or pep-tides for antibody capture as well as for the detection conjugates. The window period is further reduced when fourth-generation Ag/Ab combination assays are used for diagnostic testing (Masciotra et al., 2011). Fourth-generation assays combine detection of HIV p24 Ag (for detection of the infection during the acute phase prior to seroconver-sion) with third-generation Ab detection (for detection during the early and long-standing chronic phase). Since the late 1980s, stand alone HIV Ag assays have been com-mercially available but are not widely used for diagnostic testing because their ability to detect infection is limited to a period of a few weeks prior to and during seroconversion. HIV Ag assays have largely been replaced with RNA nucleic acid tests (NATs) for the detection of acute infec-tion and for blood donor screening. In 1999, blood banks in the United States began screening donations with both a third-generation antibody assay and HIV-1 RNA NAT. Blood donations are screened individually for Ab to HIV and then the Ab-negative donations are pooled (pool size of 16–24 donations) and screened for HIV-1 RNA to facilitate detection of window period infections (Stramer et al., 2004).

Commercial platforms for HIV diagnostic testing range from laboratory-based, high-throughput, automated instrument systems to manual microtiter plate platforms to single-use rapid (or point-of-care) devices. Some rapid devices are designed for use in non-laboratory settings such as community outreach sites and physician’s offices, as well as, resource-limited settings. Assay technologies have expanded beyond EIAs that use enzymes to generate

a colorimetric, chemiluminescent, or fluorescent signal. Non-enzyme-based signals can be generated by conjugates directly labeled with a chemiluminescent compound or chromophore (e.g., colloidal gold, selenium).

Traditionally, specimens that gave a reactive result in the HIV diagnostic assay were retested using an HIV-1 western blot (WB) or immunofluorescence assay (IFA) to confirm the initial assay result. HIV-1 WB has been con-sidered the “gold standard” for HIV diagnosis because of its high specificity. The WB consists of viral lysate pro-teins separated by SDS polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane. The membrane is cut into strips, incubated with patient speci-men to capture Ab, followed by detection of Ab using an enzyme-labeled anti-human IgG conjugate. The criteria for HIV-1 WB positivity are based on the pattern of reac-tive viral-specific proteins; most criteria require reactivity to at least two viral proteins, one of which must be an env protein (gp41, gp120, or gp160). An alternative to WB is the line immunoassay. While using the same principle as a WB, the line immunoassay uses purified recombinant pro-teins and peptides derived from HIV-1 and HIV-2, which are applied as discrete bands on a membrane support. In an IFA, patient specimen is reacted to HIV-infected cells in wells on a microscope slide. Abs to HIV are captured on the surface of the infected cells and detected by a fl uorescent-labeled anti-human IgG conjugate using fluorescent microscopy. All these confirmatory assays rely on the detection of HIV-specific IgG antibodies and were appropriate methods when first- and second-generation HIV-1 EIAs were the only diagnostic tests available. How-ever, most current commercially available HIV immuno-assays have improved sensitivity (particularly during the early phase of infection), improved specificity, and can detect HIV-1 group M, group O, and HIV-2, making these confirmatory assays outmoded. As a result, many laboratories are retesting a reactive specimen with a sec-ond HIV immunoassay; typically an HIV Ab test that is different from the first test performed. If the second HIV assay is reactive, the patient is presumed HIV infected and followed up with additional clinical testing for disease staging; staging would include measuring CD4 cell count and determining baseline viral load using quantitative HIV RNA NAT. If the second HIV Ab test is nonreactive, the specimen is then tested with an HIV-1 RNA NAT to confirm or rule out acute/early infection. Alternatively RNA NAT could directly follow the first reactive Ab test.

When diagnosing a patient’s HIV status, the patient’s clinical profile and risk factors also need to be taken into consideration as diagnostic tests have limitations. All assays have a low frequency of false-positive results and the potential for false-negative results. HIV antibody assays differ in sensitivity during the early stage of infection (Owen et al., 2008; Masciotra et al., 2011). HIV-1 NAT does not detect HIV-2 RNA.

Therapeutic MonitoringIn recent years, highly active antiretroviral therapy and expanded access to treatment have had a significant impact by reducing HIV-related morbidity and mortality. Each of

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the current classes of antiretroviral drugs targets a differ-ent step in the HIV replication pathway; these include inhibitors of reverse transcriptase (blocking replication at the complementary DNA (cDNA) synthesis step), prote-ase (preventing processing of polyproteins needed for viral replication and assembly), integrase (blocking replication at the cDNA integration step), and virus entry (preventing fusion of viral and cellular membranes). Highly active antiretroviral therapy uses a combination of two to three different drug classes to suppress HIV replication and limit the development of drug-resistant virus. The crite-rion for initiation of treatment is a CD4 cell count of less than 350/mm3 (WHO websites and NIH websites). However, a recent study supports starting treatment ear-lier to reduce the rate of sexual transmission and clinical events (Cohen et al., 2011).

The need for therapeutic monitoring is driven by the high mutation rate caused by HIV replication via error-prone reverse transcriptase coupled with a high level of viral replication and turnover in an infected individual. Prior to the initiation of therapy, baseline viral load and drug resistance profiles are obtained. Baseline viral load helps with staging disease progression and is also needed to determine the effectiveness of subsequent treatment. A baseline resistance profile guides the choice of treatment regimen. In treatment-naive patients, the preexistence of drug-resistant virus can be due to naturally occurring ran-dom mutagenesis or to acquisition of virus from a partner that has developed drug resistance. In the United States, approximately 10% of newly diagnosed HIV infections are due to transmitted drug-resistant virus (Brennan et al., 2010; Wheeler et al., 2007). Following initiation of treatment, viral loads should drop as viral replication is blocked; with optimal response to treatment a patient viral load should become undetectable. Viral load is measured at regular intervals during treatment to monitor the con-tinued effectiveness of therapy. A rebound in viral load indicates treatment failure possibly due to the develop-ment of drug-resistant virus. At this point, the resistance profile is repeated to assess the need to change the treat-ment regimen.

Viral load is determined using quantitative HIV-1 RNA NAT, which typically quantifies RNA over a range from 20–40 copies/mL to 107 copies/mL. Currently, the most commonly used HIV-1 viral load assays are based on real-time RT-PCR amplification of viral RNA from patient sample; real-time assays detect amplified target at each amplification cycle using a fluorescent-labeled probe spe-cific for HIV. The alternative approach is a branched DNA method that uses signal amplification to detect captured viral RNA rather than amplifying the target; viral RNA is captured by hybridization and then detected by sequential hybridization to amplifier and enzyme-labeled probes.

Drug resistance monitoring can be done using either genotypic or phenotypic assays. Genotypic assays consist of RT-PCR amplification of the viral genome region of interest (typically protease-reverse transcriptase). This is most commonly followed by DNA sequencing of the amplicon and sequence evaluation for the presence of mutations associated with resistance to antiretroviral drugs. An alternative to DNA sequencing is a line assay that detects a specific set of resistance mutations using

hybridization of the amplicon (or amplified RNA tran-scripts) to oligonucleotides. The future may see a move to next-generation (or deep) sequencing methods that allow detection of low-frequency viral variants (Jabara et al., 2011). Phenotypic assays measure the ability of virus to replicate in the presence of drug. In one phenotypic assay, the viral gene of interest is amplified from patient sample by RT-PCR, cloned into a reference HIV strain, and viral replication is measured in the presence of drug. The assay determines change in susceptibility to drug relative to the wildtype reference strain.

SummaryHIV diagnostic testing is a key component of efforts to combat the HIV epidemic. Knowledge of HIV status allows individuals to take precautions to protect them-selves against the acquisition of HIV, to prevent transmission of HIV to their uninfected partners and chil-dren and to access life-prolonging antiretroviral therapy. Therapeutic monitoring allows optimal management of a patient’s treatment regimen.

References and Further ReadingBarré-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T., Charmaret, S.,

Gruest, J., Dauget, C., Axler-Blin, C., Vézinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Montagnier, L. Isolation of a T-lymphotropic retrovirus from a patient at risk for AIDS. Science 220, 868–870 (1983).

Brennan, C.A., Yamaguchi, J., Devare, S.G., Foster, G.A. and Stramer, S.L. Expanded evaluation of blood donors in the United States for human immuno-deficiency virus type 1 non-B subtypes and antiretroviral drug-resistant strains: 2005 through 2007. Transfusion 50, 2707–2712 (2010).

CDC. Pneumocystis pneumonia–Los Angeles. Morb. Mortal. Wkly. Rep. 30, 250–252 (1981).

Clavel, F., Guetard, D., Brun-Vézinet, F., Chamaret, S., Rey, M.A., Santos-Ferreira, M.O., Laurent, A.G., Dauguet, C., Katlama, C., Rouzioux, C., et al. Isolation of a new human retrovirus from West African patients with AIDS. Science 233, 343–346 (1986).

Cohen, M.S., Chen, Y.Q., McCauley, M., Gamble, T., Hosseinipour, M.C., Kumarasamy, N., Hakim, J.G., Kumwenda, J., Grinsztejn, B., Pilotto, J.H.S., Godbole, S.V., Mehendale, S., Chariyalertsak, S., Santos, B.R., Mayer, K.H., Hoffman, I.F., Eshleman, S.H., Piwowar-Manning, E., Wang, L., Makhema, J., Mills, L.A., de Bruyn, G., Sanne, I., Eron, J., Gallant, J., Havlir, D., Swindells, S., Ribaudo, H., Elharrar, V., Burns, D., Taha, T.E., Nielsen-Saines, K., Celentano, D., Essex, M. and Fleming, T.R. Prevention of HIV-1 infection with early antiretroviral therapy. N. Engl. J. Med. 365, 493–505 (2011).

Damond, F., Gueudin, M., Pueyo, S., Farfara, I., Robertson, D.L., Descamps, D., Chène, G., Matheron, S., Campa, P., Brun-Vézinet, F. and Simon, F. Plasma RNA viral load in human immunodeficiency virus type 2 subtype A and B infections. J. Clin. Microbiol. 40, 3654–3659 (2002).

DeCock, K.M., Adjorlolo, G., Ekpini, E., Sibailly, T., Kouadio, J., Maran, M., Brattegaard, K., Vetter, K.M., Dorrly, R. and Gayle, H.D. Epidemiology and transmission of HIV-2. Why there is no HIV-2 pandemic. JAMA 270, 2083–2086 (1993).

Fiebig, E.W., Wright, D.L., Rawal, B.D., Garrett, P.E., Schumacher, R.T., Peddada, L., Heldebrant, C., Smith, R., Conrad, A., Kleinman, S.H. and Busch, M.P. Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection. AIDS 17, 1871–1879 (2003).

Freed, E.O. and Martin, M.A. HIVs and their replication. In: Fields Virology 5th edn (eds Knipe, D.M. and Howley, P.M.), (Lippincott Williams & Wilkins, Philadelphia, PA, USA, 2006).

Gallo, R.C., Salahuddin, S.Z., Popovic, M., Shearer, G.M., Kaplan, M., Hayner, B.F., Palker, T.J., Redfield, R., Oleske, J., Safai, B., White, G., Foster, P. and Markham, P.D. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224, 500–502 (1984).

Gao, F., Yue, L., White, A.T., Pappas, P.G., Barchue, J., Hanson, A.P., Greene, B.M., Sharp, P.M., Shaw, G.M. and Hahn, B.H. Human infection by genetically diverse SIVSM-related HIV-2 in West Africa. Nature 358, 495–499 (1992).

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Gottlieb, M.S., Schroff, R., Schanker, H.M., Weisman, J.D., Fan, P.T., Wolf, R.A. and Saxon, A. Pneumocystis carinii pneumonia and mucosal candidiasis in previ-ously healthy homosexual men: evidence of a new acquired immunodeficiency. N. Engl. J. Med. 305, 1425–1431 (1981).

HIV Sequence Database. http://www.hiv.lanl.gov.Jabara, C.B., Jones, C.D., Roach, J., Anderson, J.A. and Swanstrom, R. Accurate

sampling and deep sequencing of the HIV-1 protease gene using a primer ID. Proc. Natl. Acad. Sci. USA 108, 20166–20171 (2011).

Keele, B.F., Van Heuverswyn, F., Li, Y., Bailes, E., Takehisa, J., Santiago, M.L., Bibollet-Ruche, F., Chen, Y., Wain, L.V., Lieogeos, F., Loul, S., Ngole, E.M., Bienvenue, Y., Delaporte, E., Brookfield, J.F.Y., Sharp, P.M., Shaw, G. m., Peeters, M. and Hahn, B.H. Chimpanzee reservoir of pandemic and nonpan-demic HIV-1. Science 313, 523–526 (2006).

Lyles, R.H., Muñoz, A., Yamashita, T.E., Bazmi, H., Detels, R., Rinaldo, C.R., Margolick, J.B., Phair, J.P. and Mellors, J.W. Natural history of human immunodeficiency virus type 1 viremia after seroconversion and proximal to AIDS in a large cohort of homosexual men. J. Infect. Dis. 181, 872–880 (2000).

Masciotra, S., McDougal, J.S., Feldman, J., Sprinkle, P., Wesolowski, L. and Owen, S.M. Evaluation of an alternative HIV diagnostic algorithm using specimens from seroconversion panels and persons with established HIV infec-tions. J. Clin. Virol. 52, S17–S22 (2011).

Mehandru, S., Poles, M.A., Tenner-Raczk, K., Horowitz, A., Hurley, A., Hogan, C., Boden, D., Racz, P. and Markowitz, M. Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract. J. Exp. Med. 200, 761–770 (2004).

NIH website. www.aidsinfo.nih.gov/guidelines.Owen, S.M., Yang, C., Spira, T., Ou, C.Y., Pau, C.P., Parekh, B.S., Candal, D.,

Kuehl, D., Kennedy, M.S., Rudolph, D., Luo, W., Delatorre, N., Masciotra, S., Kalish, M.L., Cowart, F., Barnett, T., Lal, R. and McDougal, J.S. Alternative algorithms for human immunodeficiency virus infection diagnosis using tests that are licensed in the United States. J. Clin. Microbiol. 46, 1588–1595 (2008).

Peeters, M. and Sharp, P.M. Genetic diversity of HIV-1: the moving target. AIDS 14, s129–s140 (2000).

Plantier, J.-C., Leoz, M., Dickerson, J.E., DeOliveira, F., Cordonnier, F., Lemée, V., Damond, F., Robertson, D.L. and Simon, F. A new human immunodeficiency virus derived from gorillas. Nature Med. 15, 871–872 (2009).

Popovic, M., Sarangadharan, M.G., Read, E. and Gallo, R.C. Detection, isolation and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224, 497–500 (1984).

Sarangadharan, M.G., Popovic, Bruch, L., Schüpbach, J. and Gallo, R.C. Antibodies reactive with human T-lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 224, 506–508 (1984).

Schneider, E., Whitmore, S., Glynn, K.M., Dominguez, K., Mitsch, A. and McKenna, M.T. Revised surveillance case definitions for HIV infection among adults, adolescents, and children aged <18 months and for HIV infection and AIDS among children aged 18 months to <13 years—United States, 2008. MMWR Recomm. Rep. 57(RR-10), 1–12 (2008).

Stramer, S.L., Glynn, S.A., Kleinman, S.H., Strong, M., Caglioti, S., Wright, D.J., Dodd, R.Y. and Busch, M.P. Detection of HIV-1 and HCV infections among antibody-negative blood donors by nucleic acid-amplification testing. N. Engl. J. Med. 351, 760–768 (2004).

UNAIDS Global Report (2010). http://www.unaids.org/globalreport/Global_report.htm.

Wheeler, W., Mahle, K., Bodnar, U., Kline, R., Hall, I., McKenna, M. The US Variant, Atypical and Resistant HIV Surveillance (VARHS) Group. Antiretroviral drug-resistance mutations and subtypes in drug-naive persons newly diagnosed with HIV-1 infection, US March 2003 to October 2006. In: Program and abstracts of the 14th Conference on Retroviruses and Opportunistic Infections, February 25–28 2007, Los Angeles, CA. Poster 648.

WHO website. Antiretroviral therapy for HIV infection in adults and adolescents: recommendations for a public health approach 2010 revision. http://www.who.int/hiv/pub/arv/adult2010/en/