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Report Negative* Confirm Test 25

MACRONEUROLOGICAL EXAM 38

47b. Results of exam exclusive of neuropathy 46

Abridged Neuropsychological Assessment Test Battery 47

THE WORLD HEALTH ORGANIZATION 51

QUALITY OF LIFE (WHO QOL) -BREF 51

7. 
   To archive samples for future investigations including determination of viral evolution, host genetics and innate, cell-mediated and humoral immune responses in peripheral blood and mucosal compartments
   

4. Background
4.1 Introduction:
Increasing attention is being focused on very early events in HIV infection as these viruses and attendant, innate and adaptive immune responses may play a significant role in shaping disease burden and ultimately (untreated) disease outcome. Viruses from acute infection may be the most relevant for vaccine design as they will likely reveal higher rates of unique recombinant forms and rates of infection with two subtypes of viruses. In addition, recently transmitted viruses may contain variants that may be more susceptible to neutralization (“transmission bottleneck”) as suggested by studies of discordant transmission pairs. The analysis of the induced immune response and viral destruction of HIV-specific and other CD4+ T helper cells may define critical elements or interactions that may inform immunogen choice or configuration.
Early events in human HIV infection are speculative. Transmission typically occurs across a mucosal barrier [1] and is enhanced by agents or actions that compromise that barrier: trauma, infection, inflammation. Most commonly a CCR5 tropic virus presumably enters a target cell and is transported by that cell to regional lymph tissue (lymph node). Activation of the transporting cell results in integration and the exponential process of virus production, the infection of susceptible, activated targets and spread of cell free and cell associated virus through the body. The induction of HIV specific immune responses and CD4+ T helper cells is accompanied by preferential infection and destruction of this critical subset [2]. Studies from monkeys suggest early and rapid infection and destruction of gut-associated lymphoid tissue (GALT), a major reservoir of immune cells [3-7]. Similar disease is seen in humans studied to date, though the number of reported observations is not substantial, particularly for events early in disease [5, 8-10]. Roughly 3 weeks after initial infection, HIV specific, CD8+ cytotoxic T cells become detectable and viral load begins to decrease.
The information that may be obtained from acute HIV infection of humans is critical to several aspects of HIV pathogenesis, HIV vaccine development, and perhaps HIV treatment. Data from monkeys with acute SIV infection [6, 11-14] may not adequately model acute human disease for several reasons. First, animal models of both intravenous and mucosal transmission aim for a nearly 100% risk of infection. Hence very high multiplicities of infection are required (100 monkey infectious doses, 1000 tissue culture infectious doses, etc). Second, monkey strains are often chosen for pathogenicity, commonly used strains such as SIVmac239 or SIVmac251 cause very rapidly progressive disease, where a case of HIV disease may take 8-10 years to progress to AIDS, the SIV model systems progress much more rapidly. Third, the relationship of SIV and monkeys may not be equivalent to that of HIV and humans since the natural hosts for these viruses (old world monkey) maintain relatively high-levels of circulating virus without significant pathologic consequence. The advantages and disadvantages of the animal model must be balanced against the technical and logistical problems attendant with the detection of acute HIV infection in humans, logistical issues around enrollment, and questions about the "timing" of exposure and detection.
Persons with acute HIV-infection have an 8-20 times greater risk for transmitting HIV compared to those with chronic infection due to their high viral load [15-18]. They are often highly infectious during the window period for standard HIV testing. Identifying individuals with acute infection and intervening to change risk behavior can reduce HIV spread [19]. It has been estimated by preventing just one additional HIV transmission by an acutely-infected individual can save about $300,000 in lifetime treatment costs.
Neurological dysfunction in HIV-1 is manifest in various forms, ranging from early acute neurological disease to mild neurological dysfunction (MCMD) to frank dementia [20, 21]. An estimated 20-40% of infected individuals will develop HIV-associated dementia (HAD) or MCMD [20, 22]. Acute encephalitis has been reported in acutely infection persons with high viral loads [23]. There is limited information on how HIV affects the brain especially in early infection, knowledge of which can lead to development of appropriate interventions such as early HAART. HIV-1 is found in the brain associated with macrophages and microglia and likely enters the brain via infected monocytes/macrophages (M/M) early after infection [24]. Whether productive viral infection of the CNS is the result of this early viral entry or later seeding of the CNS by activated blood M/M, or a combination, is not fully understood [25]. There is a strong correlation between the number of activated macrophages in the brain, their products, and the neurological status of the patient [26], it is thought that progressive CNS damage may occur as a result of the accumulation of activated macrophages and the release of soluble factors [27, 28]. The role of the HIV-1 specific CD4+ and CD8+ response is likely important but currently poorly defined.
Recent evidence has shown that differences between HIV-infected persons in HIV disease progression, immunological and virological status may be due to host factors (Neaton JD Nature Med 2008). Examples are CCL3L1-CCR5 genotype and gene duplication influencing HIV susceptibility, early immune damage from HIV and HIV disease progression. Ahuja et al. showed that patients with high CCL3L1 gene copies and non-detrimental CCR5 genotype had less CD4 loss following acute HIV infection. This favorable genotypic profile is also associated with improved CD4 recovery following HAART [29].
It is now possible to diagnose HIV infection within the first three weeks of onset using NAT [30]. Acute HIV infection was identified at a rate of 4.9 per 10,000 low risk blood donors in North Carolina using this strategy [15]. A recent study from China screened 11,461 high risk clients of sexual transmitted diseases (STD) clinics and found 5 with acute HIV infection by NAT giving an acute HIV infection prevalence of 1.2% [31]. In addition, use of an HIV testing algorithm employing an initial HIV antigen/antibody combination direct EIA using recombinant HIV antigens (4^th generation), followed by a less sensitive antibody-only EIA with viral lysate (1^st or 2^nd generation) on 4^th generation EIA-reactive samples, would also provide an indication of acute HIV infection if the 1^st or 2^nd generation test was non-reactive [32].
The Thai Red Cross AIDS Research Centre (TRCARC) in Bangkok established the Thai Red Cross Anonymous Clinic (TRCAC) on July 4, 1991. Patients are not asked for their names or addresses; instead, each patient is assigned an identification number. From 2000 – 2004, the TRCAC saw an average of 7,922 persons per year for VCT [33]. Seroprevalence averaged 15.9 ± 1.3% and was higher in women than in men. An anonymous questionnaire, linked to anonymous test results by the same numerical identifier, provides baseline demographic and risk factor data. In one month of 2006, about 550 clients used the Anonymous Clinic, and 13.4% (N = 74) were HIV antibody positive. A novel serological assay for measuring early HIV infection with subtypes B, E and D (BED test) was used to screen 83 seropositive samples, and 7 cases (8.4%) may be "recent converters" (it should be noted that the BED assay may overestimate incidence by misidentifying "late" AIDS cases as "early"). Therefore, each month at the Anonymous Clinic, there are approximately 74 men and women who test HIV positive and approximately 6 may be recently infected. An annualized acute HIV infection incidence of 2.7% was seen in a recent pilot study at the TRCAC (see preliminary result section).
4.2 Relevance to Military HIV Vaccine Program
The development of this cohort will enhance the overall missions of the vaccine program for the following reasons: first, understanding the pathogenesis and characterizing clinical events very early in non-subtype B HIV infection is critical to the evaluation of current T-cell based vaccines, hypothesized to delay disease progression. Longitudinal follow up of clinical (HIV disease progression and neurocognitive function), immunological (CD4, T cell subsets, T cell function, neutralizing antibody, cytokines) and virological (viral load, viral kinetics and sequence) in different compartments of acute HIV-infected subjects will provide an understanding of the optimal time (prior to significant immune dysfunction) for HIV vaccine response. Second, HIV sequencing close to the time of infection will provide an understanding of the epidemiology of transmitted HIV strains that may be important to the development of vaccines designed to prevent infection. Third, information on demographic and risk behaviors can allow tracking of contacts and aid in the planning of a vaccine program for high risk individuals and the development of retention tools.
Laboratory staging of acute HIV infection
Timing between acute HIV infection diagnosis and onset of infection can be categorized by utilizing different laboratory methods including RT PCR, p24 antigen detection assay, EIA (sensitive and less sensitive) and Western blot. In this protocol, laboratory staging for acute HIV infection will be performed according to Fiebig et al [34]. This protocol will be targeting enrollment of subjects with Fiebig stages I to III which will correspond with the reported average (95% CI) cumulative window period for HIV diagnosis of 5.0 (3.1-8.1), 10.3 (7.1-13.5) and 13.5 (10.0-17.0) days for Fiebig stages I, II and III respectively.

According to Table 3 below these will be subjects who have

Fiebig stage I: positive nucleic acid testing or NAT, negative EIA by both sensitive and less sensitive methods, negative p24 antigen

Fiebig stage II: positive NAT, negative EIA by both sensitive and less sensitive methods, positive p24 antigen

Fiebig stage III: positive NAT, positive EIA by sensitive method, negative EIA by less sensitive method, positive p24 antigen
Table 3: Fiebig staging of acute HIV infection[34]



Preliminary Study progress Report
We conducted the SEARCH 004 (WRAIR #1307) study to evaluate the prevalence and incidence of acute HIV infection at the TRCARC. Between March 2006 and September 2007, we screened 6426 VCT stored samples from the TRCAC for acute HIV infection by two methods. First, 4^th generation EIA (Abbott AxSYM HIV Ag/AB Combo, Wiesbaden, Germany) non-reactive samples (n=5402) were pooled (40 subjects per pool), and NAT was performed using Roche Amplicor v1.5 ultrasensitive assay (Roche Diagnostics, Branchburg, New Jersey), according to methods previously described (1), with a modification in which pools were tested in a qualitative format. Samples from reactive pools were then tested individually according to the manufacturer’s instructions. Acute HIV infection samples were 4^th generation-negative, NAT positive. Secondly, 4^th generation-positive samples (n=1024) were tested with US FDA approved 1st generation HIV-1 EIA (Vironostika HIV-1 Microelisa System, Organon Teknika, Durham, NC), which is not IgM sensitive and measures HIV antibody only. Acute HIV-infection samples were defined as 4th generation EIA reactive, 1^st generation EIA non-reactive and NAT positive. Laboratory staging for acute HIV infection was performed according to Fiebig et al (4). Multiregion hybridization assays (MHAbce) [35] and TRUGENE® HIV-1 genotyping (Bayer, Leverkusen, Germany) were performed to determine HIV subtype and resistance respectively. The study was approved by the Chulalongkorn University, the University of Hawaii and the Walter Reed Army Institute for Research institutional review boards.
Eleven of 6426 subjects had acute HIV-1 infection. Seven of 5402 4^th generation-negative samples were NAT positive. Four of 1024 4^th generation-positive samples were non-reactive using 1^st generation EIA. The acute HIV infection prevalence was 20.3 per 10,000 persons at risk (95% CI, 10.1-36.4), and the estimated HIV incidence was 2.7 per 100 person-years (95% CI, 2.2-4.3). Nine of the 11 acutely infected subjects were men. The median age of all subjects was 28 years (range 17-45). Reported risk factors included heterosexual sex (3), MSM (6), bisexual sex (1) and unknown (1). Only two subjects reported condom use at all times. Median HIV RNA (range) was 99,601 (130 to >100,000) copies/ml. Three samples were untypable using MHAbce genotyping, and of the 8 typable samples, 6/8 were CRF01_AE, 1/8 was B, and 1/8 was a CRF01_AE and B recombinant. TRUGENE HIV-1 genotyping showed 2/10 without resistance and 8/10 with mutations in the protease gene which are probably natural polymorphisms of CRF_01AE.
The SEARCH 004 study showed that the prevalence of acute HIV infection in our population is higher than that reported in low-risk blood donors in the United States and in clients of STD clinics in China [15, 31]. The acute HIV-infection subjects in our study were mostly young MSM. This supports the current epidemiologic trends in Thailand where the observed prevalence of HIV among MSM in Bangkok rose from 17% in 2003 to 28% in 2005 [36]. Our study was in agreement with two recent studies which indicated that the occurrence of non-CRF01_AE strains in Thailand might be increasing among high-risk groups [37, 38].

5. Study Design
5.1 Overview
This study is a prospective cohort study designed to follow acute HIV infected individuals. Study participants will be identified and followed during an 6-year period. They will be monitored for clinical, CD4, HIV RNA, and safety laboratory tests. They will receive counseling on HIV-infection and will be asked to complete a questionnaire on HIV risk behavior. Archival of PBMCs and plasma will be performed. Optional procedures that require separate consenting include collection of genital secretion, cerebrospinal fluid, and colon biopsy tissue, brain MRI/MRS, host genetic testing and tracking of and offering VCT to subject’s sexual contacts. The procedures of recruitment and follow up are shown in Tables 1 and 2 respectively. Participants will be referred to their physicians or the physicians at the HIV clinic at the TRCARC for HIV care. This protocol allows co-enrollment in other treatment protocols.
Acute HIV infection is defined as subjects who have the following laboratory profile:

1. Negative 4^th generation EIA/positive NAT

2. Positive 4^th generation EIA/negative EIA (which is not IgM sensitive)/positive NAT
5.2 Endpoints
Primary endpoints or variables:

• 
  Number of HIV and non-HIV related clinical events
  

Secondary endpoints:

• 
  HIV-1 plasma viral RNA measurements and CD4 counts during follow-up
  
• 
  HIV-specific CD4+ and CD8+ responses, viral kinetics and genotyping
  
• 
  Number and distribution of T cell subsets in blood and colon mucosa
  
• 
  Incidence and pattern of neurocognitive impairment, MRI/MRS evidence of inflammation (myoinositol, choline) finding, and CSF evidence of neuronal injury (tau) and inflammation
  
• 
  HIV RNA, viral kinetics and genotyping in genital and neurologic compartment
  
• 
  HIV risk behavior
  
• 
  Willingness of acute HIV-infected subjects to allow the tracking of their sexual contacts for VCT
  

5.3 Sample Size
This site will enroll up to 75 acute HIV-infected subjects inclusive of withdrawal and/or participants lost to follow-up from approximately 40,000 that will be screened over about 4 years. This is based on the number of acute HIV-infected subjects in the SEARCH 004 study in which there were 11 acute HIV-infected subjects from approximately 6000 subjects screened. Should recruitment of 75 participants not be reached within the study period, a study extension will be requested only if funding is available. Approval of ethical review bodies will be sought prior to study extension.
5.4 Study Group Descriptions and Determinants of Study Group Design
Acutely HIV-infected male and female clients aged 18 years or above will be enrolled. This study has no control group.
5.5 Population to be studied
The population seeking VCT at the TRCAC will be screened. This consists of both men and women of different ages, economic stratus and educational level: a large portion of whom are at high risk for HIV infection through commercial sex work and as MSM. Normally clients attending the TRCAC are not asked for their names, addresses, a national identification number or any other identification documentation. Each client is assigned a unique identification number to encode and link samples. The specimens cannot be linked to the tested individual unless they agree to provide contact information. For this study, we will ask the VCT clients to provide contact information in order to notify them should they be confirmed to have acute HIV infection.
Routinely, clients at the TRCAC are informed of their HIV EIA results on the same day. Another blood sample is taken immediately for confirmatory testing. They are then asked to come back in 1 week for confirmatory results. Clients are not asked for their names or contact information. This study differs to the TRCAC routine practice in that the study team must be able to contact clients shortly (within 2-3 days) after their first HIV EIA test; hence, the necessity for the procedures specified above.
Approximately 75 male and female clients ages 18 years and above who have laboratory findings consistent with acute HIV infection will be enrolled. Male subjects will be offered free condoms. Female subjects of child bearing age will be offered free condoms and hormonal contraception. Pregnant clients will not be excluded and the investigators will coordinate with their doctors to ensure that they receive appropriate obstetric and HIV prevention of mother to child transmission care. Pregnant clients will not be offered the following optional procedures: collection of genital secretions, CSF and colonic tissue and brain MRI/MRS.