Acute HIV and the North Carolina STAT Project Past, Present and Future Peter Leone, MD Associate Professor of Medicine University of North Carolina Medical Director North Carolina HIV/STD Prevention and Care

B D A C 9/10/05 Develops fever, ST, fatigue Local PMD gives Z-pack 8/15/05-8/30/05 A&B: Sex 3-4x D A 10/28/05 Develops fever, ST, oral ulcers, thrush Antibiotics given Requests HIV test 8/30/05 A,B,C: 3-way Partners B&C “steady” Sex 1-2x/wk 7/28/05 Develops HA, Fever Went to ER, LP, labs DX: RMSF, doxycycline given Symptoms worsen 2 Days later admitted HIV Ab neg Discharge Aseptic meningitis Possible RMSF C 10/15/05 B,C,D have 3-way 9/30/05 Develops fever, LAD,ST Local PMD gives Z-pack

B D 11/15/05 HIV+ (ELISA + WB: I) A C

12/1/05 HIV+ B D 11/15/05 HIV+ (ELISA + WB: I) A C 12/1/05 HIV+

B D A C 12/1/05 HIV+ 11/15/05 HIV+ (ELISA + WB: I) 12/20/05 HIV+

12/1/05 HIV+ B D 11/15/05 HIV+ (ELISA + WB: I) A 12/20/05 HIV+ C 12/1/05 HIV+ 5 infections could have been avoided if acute HIV infection considered at first presentation

Definition of Acute HIV Infection Time period following infection with HIV during which HIV virus can be detected in blood but antibodies to HIV are not OR Window period when routine HIV antibody tests (EIAs) are negative but HIV virus can be detected in blood

Primary HIV Infection Definition: Acute HIV infection + recent infection with HIV. Recent Infection: patients who are positive on HIV antibody testing (EIA), but have one of the following: A recent prior negative HIV test or Results of detuned antibody test suggesting recent infection.

Couthino et al., Bulletin of Mathematical Biology 2001

Detecting Acute HIV Infections Symptoms p24 Antigen HIV RNA HIV Ab Tests 0 1 2 3 4 5 6 7 8 9 10 Weeks Since Infection

PCR Testing of Pooled Sera to Identify Acute HIV Infection (seronegative, PCR positive) Source: ISSTDR, 2007 11

How do we pick-up Acute HIV infection if routine antibody tests are negative?

Acute Retroviral Syndrome 40-90% of new HIV infections are symptomatic Signs and symptoms typically begin 1-4 weeks following the exposure Symptoms can last from days to several weeks, but usually <14 days Pilcher C et al. N Engl J Med 2005;352:1873-1883 Kahn JO, Walker BD. N Engl J Med. 1998;339:33-39 Schacker T, et al. Ann Intern Med. 1996;125:257-264

Acute HIV Incubation Periods 31 Patients Average = 14 days Range: 5-30 days Sources: Pilcher, JAMA 2001; Borrow, Nat Med 1997; Schacker AIM 1996; Lindback, AIDS 2001 10 8 6 Frequency 4 2 7 14 21 28 Days from Sexual Exposure to Onset of Symptoms

Non-specific Mononucleosis-like Signs and Symptoms Fever Rash Oral ulcer Weight loss Loss of appetite Headache Fatigue Adenopathy Sore throat/ pharyngitis Muscle and/or joint pain Diarrhea GI upset/nausea/ vomiting

Common Signs & Symptoms Study of 160 patients with primary HIV infection in 3 countries % of patients Vanhems P et al. AIDS 2000; 14:0375-0381. 

Acute HIV and Symptoms Schacker Kinloch-de Loes NC STD Fever 93% 87% 48% Fatigue 93 26 37 Pharyngitis 70 48 30 Headache 55 39 26 Rash 15 GI Symptoms 37 Schacker TW, et al., AIM 1996 125:257-64

Common Mis-diagnoses Mononucleosis Rocky Mountain Spotted Fever Strep throat Influenza “Viral illness” Secondary syphilis

Primary HIV Infection: Pathogenesis Symptoms CD4 Cell Count (cells/mm³) Plasma HIV RNA CD4 Cell Count 4-8 Weeks Up to 12 Years 2-3 Years Primary HIV Progression AIDS

How do we pick-up Acute HIV infection if patients don’t have symptoms?

Our approach to Screening for AHI Specimen pooling Advantages Seamless (almost) incorporation into HIV testing Reduced cost No real change in specificity Universal application Disadvantages Requires large testing volume Small loss in sensitivity Logistics Time to Dx and locating patient

STAT Testing Protocol + - + - - + EIA/ Western Blot HIV RNA testing HIV Positive + - F/U Testing (Ab + HIV RNA) - + HIV Negative Acute HIV

Pooling and HIV RNA testing A B C D E F G H I 1 2 3 4 5 6 7 8 9 10 90 individual HIV antibody negative specimens 9 intermediate pools (10 specimens) 1 master pool (90 specimens)

Distribution of Viral Loads in Ab Negative VCT Specimens NC Testing Data 2002-2005 (n=58) 16 14 12 10 n 8 6 4 2 2 3 4 5 6 7 8 log HIV RNA cp/ml

Low viral load specimens 16 14 12 10 n 8 6 4 2 2 3 4 5 6 7 8 log HIV RNA cp/ml

STAT Index Case Protocol EIA/Ab (+) and WB (+) or EIA/Ab (-) RNA (+) STAT Case Possible acute HIV Infection Confirmed Acute HIV + STAT Notification Confirmatory Test HIV Antibody and RNA Testing EIA or Ab (+) EIA or Ab(-) False RNA Positive Repeat Testing Ab - Immediate contact Dr. Leone UNC ID – on call STAT Post-Exposure Protocol Contact < 72 hrs DIS Interview Referral to Care STAT Contact Protocol Contact < 8 weeks Contact > 8 weeks Routine Partner Notification Protocol 26

Notification of AHI in STAT 02-05 Time to notification improved to ~11 days from the time of testing (est. ~39D into 80D hyper-infectious period)

Disease Intervention Specialist Team UNC/Duke Collaborative The STAT System State Laboratory Laboratory Identification Disease Intervention Specialist Team Notification, Interviews, Confirmatory Testing, Transportation to Clinic UNC Weekly Case-Conference (Surveillance, Lab, DIS, UNC Evaluation Teams) Data collection UNC/Duke Collaborative Free Urgent clinical evaluation Recruitment to studies UNC Acute HIV Program Research Database UNC Specimen Repository -surveillance/research testing

Screening and Tracing Active Transmission (STAT) Program From 2003-2006, 79 cases identified 3 not located 1 refusal for PCRS 269 partners (from 75 AHI patients) identified within an 8-week exposure window 174 (65%) named 132 (76%) located 95 (35%) anonymous

STAT PCRS Outcomes (2003-2006) 46% (80) Counseled & Tested

Why focus on Acute HIV Infection?

HIV Epidemic in NC 7th leading cause of death for men and women ages 25-44 in 2004 Approximately 10,600 HIV-infected NC residents were unaware of their status in 2005 HIV incidence in the US and NC is stable or increasing NC ranked 2nd in the US for the number of AIDS cases from non-metropolitan areas

New Patients in the UNC ID Clinic The median CD4 count was 202 cells/mm3 for patients initiating HIV care. Majority (68%) initiated HIV care within 1 year of their first positive HIV test. 75% met guidelines for starting HIV treatment at their first visit. NC DHHS- HIV/STD Prevention & Care Branch

HIV viremia during early infection Peak viremia: 106-108 gEq/mL HIV RNA (plasma) Ramp-up viremia DT = 21.5 hrs HIV Antibody HIV p24 Ag p24 Ag EIA - Viral set-point: 102 -105 gEq/mL HIV MP-NAT - 1st gen HIV ID-NAT - 2nd gen 3rd gen “blip” viremia 11 16 22 10 20 30 40 50 60 70 80 90 100

Primary HIV-1 Infection 1000 800 600 400 200 Early Opportunistic Infections CD4 Cells + Late Opportunistic Infections 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Infection Time in Years

Earlier HIV Diagnosis Allows prompt entry into care Initiation of ART prior to CD4 decline <200 improves mortality and morbidity Management of STIs and other illness Short-term behavioral changes can have a large impact on HIV spread Improve natural history of disease with treatment during acute HIV infection?

Public Health Benefit Acute HIV is the most infectious period HIV RNA levels in the genital tract correspond to HIV RNA levels in the blood Diagnosis is often missed even when patients are symptomatic with acute HIV infection

Plasma Viral Load and HIV Transmission Risk 10 20 30 <400 400- 3500 3500- 10'000 10'000- 50'000 >50000 HIV-RNA load (cp/ml) % partners infected Rakai (Uganda) 453 HIV-disc. couples 11.6 % TR / year Quinn 2000, NEJM 342:921

Wawer, et al, JID 2005, 191:1403

Viral Loads at Initial Detection Pilcher C et al Viral Loads at Initial Detection Pilcher C et al. N Engl J Med 2005;352:1873-1883 10 Median Viral Loads 9 8 209,183 7 6 Log HIV RNA cp/ml 29,347 5 4 3 2 1 Established HIV+ (n=66) Acute HIV+ (n=21)

HIV transmission prob. per male-female act: fold-change relative to wk 16 (calculated after Chakraborty H, et al AIDS 2003) 16 14 12 10 Fold-change vs. wk 16 8 6 Based on our 4 2 1 2 4 8 12 16 Weeks from Testing Positive for AHI

Risk of Transmission 5 4 3 2 Acute Infection Asymptomatic Infection Reflects Genital Viral Burden (1/30- 1/200) HIV RNA in Semen (Log10 copies/ml) 4 (1/100- 1/1000) 3 (1/500 - 1/2000) (1/1000 - 1/10,000) 2 Acute Infection Asymptomatic Infection HIV Progression AIDS

Further Evidence That Primary HIV Infection Accounts for a Large Proportion of HIV Transmission Source: ISSTDR, 2007 43

Public Health Benefit Identify HIV transmission networks Allows real time prevention with index case and partners Awareness of HIV status has been associated with decreased sexual risk behaviors

Lessons for Public Health Acute HIV infection may be unexpectedly prevalent in common clinical scenarios Immediate rather than deferred testing is key HIV ELISA and HIV RNA Sexual partners of acutely HIV infected individuals are at a markedly increased per-act risk of acquiring HIV

Lessons for Public Health Linkage of acute HIV diagnosis with Emergent ID Consultation is paramount Interpretation and counseling on test results Extensive counseling of newly diagnosed patient Facilitate linkage to care and services Consideration of ART for interested patients Acutely infected individuals provide public health officials with a unique opportunity to understand complex sexual networks

Screening and Tracing Active Transmission (STAT) Program 2003 2004 2005 2006 Total Tests (publicly-funded clinics) 107,733 118,998 128,708 140,100 Antibody positive 581 552 571 592 Antibody negative, RNA+ (acute) 22 21 15

November 1, 2002 – May 28, 2008 Number of True RNA Positives 108 2 22 TOTAL 2002 2003 2004 2005 2006 2007 2008 Number of True RNA Positives 108 2 22 21 15 16 11 Number of Community Index Cases (acute and recent) 188 1 23 38 40 53 48

STAT Acutes by County (11/1/2002-2/1/2008) H H Case Count 1 2 (Burke, Franklin, Pitt, Henderson, Onslow, Martin) 3 (Buncombe, New Hanover) 4 (Robeson) 8 (Cumberland) 12 (Forsythe, Guilford) 15 (Wake) 15 (Mecklenburg) H Duke University Hospital H UNC Hospitals 49

Testing Site November 2002- May 2005 Tests Ab+ AHI (%) % of AHI HIV CTS 18,299 400 12 (2.9) 21 STD 117,804 526 27 (4.9) 48 FP 47,476 28 -- -- Prenatal/OB 47,598 39 2 (4.9) 3 Prison/Jail 7,158 57 4 (6.6) 7 Other 37,073 320 13(3.9) 22

The STD/HIV Connection Susceptibility: Genital ulcers provide portal of HIV entry Non-ulcerative STDs increase target cells STD treatment has been shown to slow the spread of HIV infection (individual & community) Infectiousness: Presence of another STD increases amount of HIV in genital secretions Treating STDs in PWHIV decreases the amount of HIV they shed how often they shed the virus

Potential impact of STI co-infection on detection of AHI HIV/STI Co-Infection Event HIV RNA + 4th gen. EIA 3rd gen. EIA week 1 week 2 week 3 week 4 GC Trichomoniasis Chlamydia Syphilis HSV ARS Symptoms McCoy 0-014

STI Co-infections 23 clients (30%) had a concurrent STI STD Type N (%) Men (n=13) Women (n=10) Gonorrhea 9 (39) 7 (54) 2 (20) Trichomoniasis 5 (22) (0) (50) Syphilis 4 (17) (31) Herpes 3 (13) (15) 1 (10) Chlamydia (8) Bacterial vaginosis - (30) GUD, unspecified (4) Other reported STD-related sx (7) (6) McCoy 0-014

STI Co-infections by Race, Gender, and Risk Category p = 0.03 STI Co-infection This chart further breaks down our findings down by race, gender, and risk category. Among MSM with acute HIV, all the co-infections occurred among non-Whites, a statistically significant difference. We didn’t have enough number to compare this for the other categories, but by frequencies along we can see that this trend held for heterosexual men. Among women, half of the non-white women were co-infected and about 2/3 of the white women. We would expect there to be more STI co-infections among heterosexuals in our population, as they belong to a larger and more diffuse network. In addition, the prevalence of STIs among heterosexuals is likely much greater. MSM comprise a smaller network, so STIs may not spread as rapidly. Similarly, by race, we know that in the South, non-whites are disproportionately impacted by sexually transmitted infections, and we see this in our data of new HIV infections. MSM Male Hetero Female McCoy 0-014

Missed Opportunities in STD Clinics HIV testing not offered to all Risk factors for HIV either not obtained or not recognized HIV testing not integrated into STD services Primary HIV Syndrome unrecognized by patients and clinicians Diagnostic test for Acute HIV Infections is not ordered

NC HIV Testing in STD Clinics HIV testing to be offered to all STD clients for each new visit regardless of when last HIV test performed DHHS policy to offer opt-out HIV testing 2005 estimate ~52% of NC STD clinic clients tested for HIV Wake County ( 2nd largest STD clinic in North Carolina) with ~80-85% with universal offering of HIV testing. Wake County HIV testing increased to ~90% with opt-out approach

Acute HIV and North Carolina STAT 57

Duke-UNC Acute HIV Infection Research Consortium Research opportunities for patients with Acute and Recent HIV Infection: “Treatment of Acute HIV Infection with Once Daily Atripla” (24 month treatment study which supplies Atripla) “Longitudinal Assessment of Acute/Recent HIV Infection” (Adds to limited scientific knowledge currently available regarding acute/recent infection) 58

Duke-UNC Acute HIV Infection Research Consortium 3) “CHAVI 001: Acute HIV-1 Infection Prospective Cohort Study” Acquire information to develop an HIV vaccine The most relevant information may come from people with acute HIV infection and their partners 59

2 7 CHAVI Index Cases by County of Residence, 6/2007-2/2008 n=18 2 1 2 Forsythe Guilford Durham Halifax Wake 1 2 2 1 U D 7 1 1 1 2 Randolph Martin Pitt Key D Duke University Hospital U Cumberland UNC Hospital 60

Other Partner Locations CHAVI Partners By County of Residence, 6/2007-2/2008 n=58 Durham Wake Hertford Bertie Forsythe Guilford Granville Northampton Pasquotank 1 1 1 1 1 3 2 1 D U 17 1 1 1 1 9 1 Mecklenburg 1 Lee Harnett Craven Martin Scotland Key D Duke University Hospital U Cumberland UNC Hospitals Other Partner Locations “NC”: 3 SC: 1 GA: 2 WA: 1 Abroad: 2 Unk: 6 61

Advatages to Dx and Care of AHI 1.An HIV diagnosis per se results in subsequent risk reduction 2. Initiation of HAART to reduce plasma and hence genital viral load thus reducing transmission potential 3. As we identify more undiagnosed HIV+ and more are successfully placed on HAART, transmission will shift even more to AHI 4. As frequency of HIV testing increases, we will idenitfy more AHI 5.Opportunity for short term behavior change (period of high infectivity of weeks)

Conclusion Make HIV testing routine Opt-out HIV Testing for all STD clients Screen all STD clients for AHI Include AHI in the Differential Dx of Acute Viral Syndrome in all Sexually Active Adults

Conclusion AHI is a true Public Health Emergency! AHI detection and case investigation puts identification of HIV at leading edge of transmission Opportunity for both early diagnosis and prevention Report all AHI cases within 24 hrs

laboratory

Given this VL distribution: Analytical vs. Clinical Sensitivity LL, cp/ml Ab- HIV N=58 Se (Ab-) All HIV N=1437 Se (all) 1000 56 96.5 1435 99.9 3000 54 93.1 1433 99.7 5000 52 89.6 1431 99.6 10000 49 84.5 1428 99.4 Ab only 1379 95.9

Requirement for Analytical Sensitivity is Less Stringent than for VL Monitoring To be recommended as part of (all) general HIV testing, a NAAT would likely need ~95% detection at viral loads the equivalent of 5,000 to 10,000 HIV RNA copies per mL Better sensitivity required for effective analysis of pooled specimens

Detection of Acute HIV Acute HIV infections (first 2-3 months) are estimated to account for as much as half of all HIV transmission (Wawer at al JID 2005) They represent 0-10% of detectable infections presenting for HIV testing Real-time recognition of acute infections creates opportunities for highly targeted treatment, prevention and surveillance activities

Detection of Acute HIV “Detuned” assays can identify recent seroconversion, but with a 1-2 month delay from infection. These also do not identify additional cases over routine antibody tests. Real-time diagnosis of acute HIV depends on the identification of HIV antigens (e.g., p24) or nucleic acids (NAAT) in the absence of HIV antibodies.

Pilcher, CD et al. JAMA 2002;288:216-221 The Gold Standard for Acute Screening is RNA Group Testing of Ab - Specimens Ab screen - + - Ab confirm + NAAT screen - + Established HIV Positive Possible Acute HIV HIV Negative Pilcher, CD et al. JAMA 2002;288:216-221

Testing to Identify Acute HIV NAAT is highly sensitive and with pooling, may be made specific. However, even pooled NAAT may be inefficient in high prevalence areas (>5%) and is technically demanding. ‘Fourth generation’ HIV ELISAs detect both antigen and antibody simultaneously Easy to perform Equipment available in most HIV laboratories

Window Periods for HIV Tests Stekler J. et al CID 2007

Commercial Assays Comparative Timing of Detection of Acute HIV Infection Source HPA -UK = combined antigen-antibody = immunometric = Class specific antibody capture = antiglobulin / indirect

Reducing time to case identification

Summary: Pooling vs. Individual NAAT Pooled screening (even with ‘minipools’) makes testing possible by reducing costs and improving predictive value More complex but more efficient for through put and cost Single specimen NAAT screening should be reserved for situations where the pre-test likelihood of acute HIV infection is >/= 1% (e.g., suspected AHI, ?ED/urgent care screening)

Opportunities for New Technologies and Approaches Need to reduce time to identification of AHI NC median time to identification is ~9D Fast Track can reduce time to 2-4 days Current POC HIV tests only test for Ab 4th generation EIA can reduce time to Dx and reduce cost Strategy may need to combine individual NAAT or discrepant POC 3rd generation EIA to identify AHI

Rapid Antibody Testing The Good Makes testing feasible in non-traditional settings Highly effective for outreach situations (needle exchange, bathhouse testing, “street-corner” outreach) Increases receipt of positive HIV test results Where HIV results notification (PCRS) not in place May increase requests for HIV testing The Not So Good Confidentiality Cost 2-3x ELISA Ab tests May defer resource allocation/personal to HIV negatives May miss AHI Requires Confirmation

Alternative Approaches North Carolina AHI referral network Educate community providers about AHI Educate high risk community about AHI Linkage of ED testing to ID clinic and local health departments…… strongly encourage partnerships . EDs will test if burden for referral to care is met. Raise awareness of 3rd generation EIA +/ WB I as possible AHI

Cost-effectiveness of the STAT Program: Decision Tree Analysis The expected savings from averting new HIV cases offset 22% of the testing costs Overall cost per QALY of $4,345 Conclusion: the program appears to be well below the cost effectiveness threshold of $50,000 which is often used as an indicator of good public health investment opportunities in the US. Still, cost a barrier for new programs

Targeting NAAT Screening by Site Over 2 years, at 135 public testing sites in NC, 325 acute and recent infections were identified among 224,124 testing clients (66% females, 4% MSM) Only 1/3 acute clients had HIV symptoms at testing There were no cases in 48 of 100 counties Targeted Screening: If NAAT used only in HIV C&T, STD, prison, and field visit sites in counties with  1 case, 95.4% of acute cases identified testing only 54.0% of the population with NAAT Testing only in STD clinics identified 40.1% of cases while testing 41.4% of the population.

Targeting is necessary; but be wary of preconceptions It is possible to construct a targeting algorithm for NAAT testing based on knowledge of local incidence, prevalence and individual risk factors associated with having recent infection “Detuned” test results can be used to develop NAAT targeting criteria A priori assumptions about who to test with NAAT are likely to be incorrect (i.e., limiting testing to only “high risk” clinics, or to symptomatic clients would be counterproductive)

Opportunities for New Approaches Need to reduce time to identification of AHI NC median time to identification is ~9D Fast Track can reduce time to 2-4 days We are implementing Fast track to all STD clinics based on symptoms and requiring STAT clinician approval

Fast Track Targeted AHI Testing : Screen all clients for HIV Ab Target Problem: Which symptoms (fever?) What time period (2-4 wks)? What duration ( >2 days)? Symptoms at best will detect 40% - Targeted testing Risk based ( i.e. MSM, anal/vaginal sex in past 2 weeks,etc ) Symptoms based (Fever + for >2 days within past 4 weeks) Site based ( prevalence 0.5% or type STD,CTS, etc.) 3. Need for further research to define symptom screen and develop predictive models for targeted AHI testing

Opportunities for New Technology Current POC HIV tests only test for Ab 4th generation EIA can reduce time to Dx and reduce cost Plan to do real time side by side comparison of NAAT pooling with 4th generation assay May need to combine individual NAAT or discrepant POC 3rd generation EIA to identify AHI

Biology

Jeffrey A. Anderson, MD-PhD University of North Carolina Determining the Genetic Linkage of HIV-1 Subtype B Transmission Pairs: Analyses of Viral env Sequences From Donor and Recipient Jeffrey A. Anderson, MD-PhD University of North Carolina

Background DONOR RECIPIENT A genetic bottleneck occurs during mucosal transmission, resulting in a subset of viruses responsible for transmission of HIV. DONOR RECIPIENT

Background Determining the genetic composition of the transmitted virus is critical to developing insight into disease progression, HIV pathogenesis, and candidate vaccines. Key questions: From the donor quasispecies, what are the properties of the specific variant(s) being transmitted? Are genital tract secretions a separate compartment from blood plasma?

3 MSM Transmission pairs from CHAVI 001: Donor vs. Recipient # of env amplicons blood # of env amplicons semen Sampling Time Weeks Post-infection ELISA WB Stage 9 11 + Chronic 20 22 32 2 5 - Fiebig 1/2 Fiebig 5/6 29 25 1 174 D1 150 R1 148 D2 40 R2 22 + Chronic 36 2 4 - NA Fiebig 1/2 Fiebig 5/6 43 29 9 11 + Chronic 14 22 36 2 5 6-7 - Fiebig 1/2 Fiebig 5/6 1 28 17 135 D3 81 R3 269 86

Experimental design Identify patients with acute HIV-1 infection, and sexual partners through contact tracing After informed consent, obtain blood plasma and semen/cervicovaginal lavage Isolate HIV-1 viral RNA from blood/semen/CVL fluid Generate a copy of the viral DNA and amplify by PCR Direct DNA sequence analysis to determine characteristics of HIV

Chromatograms from a single DNA sequence

Donor env blood plasma populations are heterogeneous

Recipient env blood plasma populations are homogeneous 22 identical sequences 33 identical sequences 3 2 3 10 2 3 R1 R2 R3

Phylogenetic analysis of D1/R1 Blood and semen populations are well-mixed

Phylogenetic analysis of D1/R1 Blood and semen populations are well-mixed However, a subset of duplicated semen amplicons suggests selective outgrowth * * * * * *

Phylogenetic analysis of D1/R1 Blood and semen populations are well-mixed However, a subset of duplicated semen amplicons suggests selective outgrowth No blood amplicons were duplicated * * * Unique Duplicate * 42 21 11 Blood Semen * P < 0.0001 *

Phylogenetic analysis of D1/R1 Blood and semen populations are well-mixed However, a subset of duplicated semen amplicons suggests selective outgrowth No blood amplicons were duplicated * * * Unique Duplicate * 42 21 11 Blood Semen * P < 0.0001 4. R1 is clearly genetically linked to D1 semen (99% nt identity), and did not arise from a duplicated semen sequence *

Summary Genetic linkage of 3 subtype B transmission pairs was confirmed by SGA and DNA sequence analysis. All donor (D1-D3) populations had heterogeneous env populations, although D1 had low heterogeneity. A single variant was transmitted to each recipient (R1-R3). Semen populations were well-dispersed among blood populations. Clusters of duplicated sequences in semen of D1 and D3 suggest outgrowth of specific variants. These data suggest that semen sequences, in general, represent sequences present in blood; however, semen populations can be disrupted by selective outgrowth. Analyses of additional transmission pairs are ongoing and will lead to a greater knowledge of: compartmentalization of viral sequences within semen vs. blood the specific viral variant(s) transmitted from donor to recipient viral sequences important for HIV-1 vaccine design

Acknowledgments Ron Swanstrom and lab members Beatrice Hahn Brandon Keele Jesus Salazar Susan Fiscus and lab Julie Nelson Myron Cohen Lihua Ping Kristen Dang and Christina Burch CHAVI 001 Clinical Core NC Dept. of Health and Human Services DIS Training Program and Officers

North Carolina may have lower attribution of AHI on Transmission 27 individuals (12%) were in closely related (<1% divergence) clusters Still, a 4-6 week period accounts for 10-15% of Transmission Frost s et al. CROI 2007

Network Analysis: Project SNAP Acutely/Recently infected MSM and high risk HIV-negative men recruited for in-depth ACASI interview and qualitative interview Respondent driven sampling to derive sexual and social network (2 generations) Better understanding of network formation, HIV/STD transmission, sex partner selection and Internet use among NC MSM

SPREAD THE WORD - NOT HIV ACUTE HIV SYNDROME If you have an STD, Get Tested for HIV. Early Detection is Best! Learn to Recognize IT. Tell a Friend. Acute HIV is Easily Misdiagnosed. IT CAN BE MISTAKEN FOR COMMON ILLNESSES Common Symptoms of Acute HIV: High Fever Rash Fatigue Swollen Glands Sore Throat Nausea/Vomiting Night Sweats Symptoms usually appear about 2 weeks after exposure What Puts You At Risk? Unprotected Sex Sharing Needles The Acute HIV Program 919-966-8533 If you suspect you may have Acute HIV, get tested at your Local Health Department or at your doctor’s office. FREE Screening for acute HIV is done on all HIV tests done through the NC Health Departments Screening for acute HIV can be done at your doctor’s office – ask for an HIV RNA test in addition to the standard HIV antibody test. SPREAD THE WORD - NOT HIV

Conclusions HIV antibody screening is a necessary first step in targeting prevention activities Assays able to detect antibody-negative infections should be incorporated into current HIV screening/testing NAAT may not be reasonable for low-risk ‘routine’ screening in well patients and low prevalence populations Models for establishing criteria for targeting NAAT are need 4th generation EIAs may present an alternative for diagnosis of acute HIV infection and merit urgent large-scale clinical evaluations

Window Periods for HIV Tests Stekler J. et al CID 2007

Tests to DX HIV Antibody ELISA $47 Western Blot $212 p24 Antigen $38 Individual HIV RNA PCR $218