SUPPLEMENTARY DATA: METHODS

Study Patients and Disease Outcomes: In total, 64 patients were analyzed for dual HIV-1 infection. These included patients from the Multicenter AIDS Cohort Study (MACS) (N=32)¹, the Seattle Primary Infection Cohort (N=10)², and the South African female sex worker cohort (N=22)^3,4. Of these 64 patients, 6 were found to be dually infected, whereas 58 only had evidence of infection from a single source (i.e. singly infected). Thirty-four had AIDS disease progression outcome data. Five of these 34 patients with outcome data were dually HIV-1 infected: including the previously described patient B from the Seattle Primary Infection Cohort⁵, 3 patients (14⁶, 21, 31) from the MACS , and 1 patient (Du151) from the South African female sex worker cohort³.

The MACS patients included both rapid (N=21) and typical (N=11) progressors. Rapid disease progressors were defined as those that had an AIDS defining event⁷ within 5 years of seroconversion⁸ and typical progressors were defined as those that had an AIDS defining event or were still asymptomatic 5 or more years after seroconversion^9,10. Of the singly infected MACS patients, 7 received no antiretroviral therapy; 16 received intermittent monotherapy, 14 received intermittent dual nucleoside therapy, and 5 received highly active antiretroviral therapy (HAART) starting at 9.4, 11.7, 12.0, 12.8, and 14.9 years post seroconversion.

In the Seattle Primary Infection Cohort, 2 of the 10 patients were rapid progressors; 3 were asymptomatic when starting HAART (all within 5 years of seroconversion) and 3 were lost to follow up within 5 years of seroconversion; 1 was asymptomatic and started HAART at 7 years after seroconversion, and in one case we could not confirm the date of seroconversion (this patient was excluded from further analysis).

In the South African female sex worker cohort, patient Du151, was the only patient of a total of 22, to progress to AIDS prior to the cohort being disbanded at 2 years. An additional patient (Du179) from the South African cohort was found to be dually infected but did not have an AIDS defining event within the 2 years prior to the cohort being disbanded so could not be further evaluated (her CD4 count = 394/µl and viral load = 3.1 log copies/ml at 16.1 months post-seroconversion).

None of the dually infected patients received HAART prior to onset of AIDS; MACS patient 14 received intermittent monotherapy. MACS patient 21 received mono- and dual NRTI therapy at 1.8 and 3.3 years post seroconversion, respectively. Patient B received multiple antiretroviral agents after developing AIDS. None of the South African patients received ART. Opportunistic infections were treated with appropriate therapy.

Dates of seroconversion were estimated as the midpoint between the last HIV seronegative visit and the first seropositive visit. HIV-1 plasma viral loads were measured using standard RT-PCR or bDNA assays. As there is no consensus on whether a viral “set-point” occurs in patients with rapid disease progression, or the best method of its determination, we report the average of the first year values. In the MACS cohort as a whole, the ~1 year post-seroconversion plasma HIV viral load has been shown to be the best predictor of subsequent disease progression¹¹. In the 30 patients from the MACS cohort for whom viral load data was available, there was a positive correlation between “~1 year plasma viral load” and subsequent rate of disease progression (R²=0.30, p=0.0026), suggesting our group was representative of the cohort as a whole. Although there was a trend toward higher viral loads in the 5 dually infected patients compared to the singly infected patients, this was not statistically significant (mean log₁₀ copies/ml = 5.0 and 4.5, respectively, p=0.19, by Kruskal-Wallis test).

Statistical Analysis: Rates of disease progression were measured by time from seroconversion to a clinical AIDS defining event⁷ (n=27) or death (n=5), or persistent CD4+ T-cell count < 200/µl (n=27); a total of 34 patients reached at least one endpoint, 29 patients did not reach an AIDS endpoint and were censored at time of initiation of HAART or time of lost to follow up (Figure 2a and 2b). For Kaplan-Meier survival analysis, we censored patients using both a very conservative “intent to treat” analysis in which censored equaled AIDS (Figure 2a) or CD4+ T-cell count < 200/µl (Figure 2b) and a less conservative censored equaled excluded (data not show). Dual infection was significantly associated with rapid progression to both endpoints using both “intent to treat” and “censored equaled excluded” analyses (Wilcoxon test for difference in time to AIDS or CD4+ T-cell count < 200/µl between those with dual and those with single infection, p = 0.005 and p = 0.0025, respectively, for censored equals AIDS (“intent to treat”), Figure 2). There was an increased relative risk (adjusted for censoring) for dual versus single infection for time to AIDS (RR=2.9; 95% CI = 1.6-4.6; p=0.001) and time to CD4<200/µl (RR=3.6 ; 95% CI=1.9-6.4; p=0.002). In a sub-analysis of only the MACS patients (N=32; dual=3, single=29), who represented the majority of those patients who reached true endpoints, dual infection remained significantly associated with rapid progression to both endpoints (Wilcoxon test p = 0.002 and p = 0.0006, respectively). Statistical analysis was carried out using JMP software (SAS Institute, Cary, NC).

Patient Samples, Virus Isolation, HMA, PCR, Cloning and Sequencing: Patient samples were obtained approximately every 6 months throughout the course of infection and were screened for dual infection using a combination of heteroduplex mobility assay (HMA) and virus sequencing^12,13. Samples with slow migrating heteroduplexes on HMA, which are suggestive of dual infection, were confirmed to be dually infected by viral sequencing (see below). HIV-1 plasma viral RNA and PBMC viral DNA was isolated as previously described^5,13. Nested PCR of the HIV-1 C2-V3 region (South African FSW) or C2-V5 (Seattle PIC and MACS) envelope (env) region was performed using end-point dilution to avoid template resampling bias¹⁴ as previously described^5,13. PCR products were cloned and sequenced using standard methodology. Multiple HIV-1 env sequences were analyzed from each sample throughout the course of infection for each patient.

Phylogenetic Analysis: All sequences were assessed for potential sample mix-up and contamination as previously described¹⁵. HIV-1 sequences were aligned with reference sequences from the Los Alamos National Laboratory HIV Database (http://hiv-web.lanl.gov/) with CLUSTALW¹⁶ followed by manual adjustment using MacClade (version 4)¹⁷. Pairwise evolutionary nucleotide distances (excluding gaps in the pairwise alignment) were estimated using both distance based and maximum likelihood methods. Neighbor-joining and maximum likelihood methods were used to estimate evolutionary models and phylogenetic trees (PAUP* v4.0)¹⁸. The criteria used for definition of dual HIV-1 infection were as follows: 1) HIV sequences from an individual patient were required to cluster into two distinct monophyletic clades (excluding recombinants) without known epidemiologic linkage (i.e. no direct or known transmission history from one patient to another); and 2) These clades were required to be no closer to each other, in a phylogenetic tree, than multiple random unlinked sequences from the HIV-1 database; and 3) The mean pairwise nucleotide distance between the two distinct clades was required to be within the range of pairwise nucleotide distances generated from randomly selected unlinked sequences in the HIV-1 database. All HIV-1 sequences from the MACS and Seattle PIC were subtype B, all HIV-1 sequences from the S. Africa FSW cohort were subtype C.

Determination of HIV-1 Superinfection: HIV-1 superinfection^19-23 in MACS patient 21 was determined using a combination of HMA and viral sequence analysis as described above. HIV-1 plasma viral RNA and viral DNA from peripheral blood mononuclear cells (PBMC) from 0.27, 0.76, 1.3 and 3.3 years post seroconversion were analyzed (see figure 1). HIV-1 C2-V5 env region HMA from the 0.27 and 0.76 year time points demonstrated a viral variant with a unique length polymorphism that was distinct from that of viral variants from 1.3 and 3.3 years post-seroconversion suggesting superinfection. Phylogenetic analysis of 131 HIV-1 C2-V5 env region sequences from plasma and PBMC confirmed superinfection in that there was no evidence of the superinfecting strain prior to 1.3 years post-seroconversion (see figure 1). Furthermore, PCR, using primers specific for the superinfecting strain showed no evidence of it in plasma or PBMC prior to 1.3 years post-SC at a limit of detection of 1 copy per µg of PBMC DNA. PCR using primers specific for the initial strain showed no evidence of it in plasma after 0.8 years post-SC, however in PBMC the initial infecting strain was detectable at low levels (11 copies per 10⁶ PBMC) at 1.3 years post-SC. Sample mix-up was excluded by molecular HLA class II typing of the pre- and post- superinfection samples²⁴.

FIGURE LEGENDS:

Figure 1. Phylogenetic tree demonstrating dual HIV-1 infection in patients B, 14, 31, Du151 and 21. C2-V3 envelope (env) sequences (265bp) were used to construct a Neighbor-Joining tree using the HKY85 model of evolution (implemented in PAUP* v4.0). Sequences from dually infected patients are shown in symbols (Note: for clarity only a small subset of the total of 650 HIV sequences obtained from these 5 patients are shown). Superinfection of patient 21 was detected at 1.3 years post-seroconversion; filled stars represent sequences found pre-superinfection (pre-SI) (0.3 and 0.8 years post-seroconversion) and open stars represent sequences found post-superinfection (post-SI) (1.3 and 3.3 years post-seroconversion). Outgroup sequences from the Los Alamos HIV Database are shown as black lines. Patients B, 14, 31, and 21 cluster with HIV-1 subtype B reference sequences, patient Du151 clusters with HIV-1 subtype C reference sequences. Nucleotide distances are shown with scale bar.

Figure 2- panel a. Kaplan-Meier curves comparing time from seroconversion to AIDS/death (n=32) in the dually infected (N=6; 1 dually infected subject was lost to follow-up at 2 years) and the singly infected patients (n=57). 63 of 64 total patients were included (in 1 patient we could not confirm date of seroconversion). Patients who started HAART (n=8) or were lost to follow-up (n=23) prior to reaching an endpoint (AIDS) were censored to those dates, where censor equals AIDS event. (Note: The unusually steep drop in the single infection survival curves (in contrast to previously published reports) is due to censoring of many of the patients at 2 years and that the MACS patients studied were mostly rapid progressors). P-value shown for Wilcoxon test of dual versus singly infected patients for time to AIDS/death.

Figure 2- panel b. Kaplan-Meier curves comparing time from seroconversion to persistent CD4+ T-cells count <200/µl (n=27) in the dually infected (N=6; 1 dually infected subject was lost to follow-up at 2 years) and the singly infected patients (n=57). Sixty-three of 64 total patients were included (in 1 patient we could not confirm date of seroconversion). Patients who started HAART (n=7) or were lost to follow-up (n=29) prior to reaching an endpoint (CD4+ T-cells count <200/µl) were censored to those dates, where censor equals CD4+ T-cells count <200/µl. [Note: The unusually steep drop in the single infection survival curves (in contrast to previously published reports) is due to censoring of many of the patients at 2 years and that the MACS patients studied were mostly rapid progressors.] P-value shown for Wilcoxon test of dual versus singly infected patients for time to CD4+ T-cells count <200/µl.

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