Catherine Brennan, Abbott Diagnostics, Building AP20/Department 09NG, 100 Abbott Park Road, Abbott Park, IL 60068-6015; e-mail: email@example.com.
BACKGROUND: In this study, human immunodeficiency virus type 1 (HIV-1)-infected blood donors were evaluated for genetic subtype and drug resistance to determine the prevalence of divergent HIV strains in the US donor population.
STUDY DESIGN AND METHODS: Subtype was determined by phylogenetic analysis of viral sequences amplified by reverse transcription-polymerase chain reaction. The drug resistance profile of the protease and reverse transcriptase (RT) genes was determined using an HIV-1 genotyping system (ViroSeq).
RESULTS: From 1999 through 2005, 26 recently infected donors, defined as HIV-1 RNA–positive, antibody-negative (RNA+/Ab−), were identified (yield, 1:1.61 million). Over the same period, the frequency of anti-HIV–positive donors was 1:34,700. Twenty RNA+/Ab− specimens were evaluated; all were infected with HIV-1 subtype B. Drug resistance profiles obtained for 18 donors identified one strain with protease mutation L90M that confers resistance to nelfinavir and one with RT mutation Y188H that confers resistance to nevirapine. Genetic subtype was determined for 44 of 46 HIV antibody–reactive and confirmed-positive (Ab+) specimens. Three infections (6.8%) were due to circulating recombinant forms: 2 CRF01_AE and 1 CRF02_AG. In the Ab+ group, one strain was resistant to all nucleoside RT inhibitors and one had mutations that confer resistance to protease inhibitors.
CONCLUSION: The data show that antiretroviral drug–resistant HIV strains are being transmitted in the United States. Overall 6.5 percent (4 of 62) of HIV-1–infected donors harbored drug-resistant strains. HIV-1 non-B strains accounted for 4.7 percent (3 of 64) of the infections in donors. HIV-1 subtype B is still the predominant strain in the United States; however, non-B strains are increasing.
In this study, we evaluated human immunodeficiency virus type (HIV-1) strain diversity in two groups of US blood donors. The first group consisted of HIV-infected donors identified by HIV-1 nucleic acid amplification testing (NAT). These donors recently acquired their infection and thus provide representation of viruses that are currently being transmitted in the United States. The second group was HIV-infected donors that were identified based on the presence of anti-HIV antibodies; these represent established infections of unknown duration. Strain diversity was evaluated by determining genetic subtype and drug resistance profiles.
HIV-1 is composed of groups M (major), N (non-M non-O), and O (outlier) with group M accounting for the vast majority of HIV infections worldwide. Owing to a high level of genetic diversity, group M is further subclassified into subtypes (A-D, F-H, J, and K) and circulating recombinant forms (CRF01-43).1,2 The prevalence of HIV-1 subtype/CRFs varies geographically and is continuously changing driven by such factors as immigration, travel/ tourism, commerce, and military deployment.2 The first HIV-1 strain identified in the United States and France in the early 1980s was group M subtype B. Although subtype B is still the predominant strain in the US and Europe, strain diversity has increased. The prevalence of non-B strains in Europe has risen dramatically since the beginning of the epidemic. A study in France showed that 48 percent of newly diagnosed HIV-1 infections in 2003 were due to non-B strains.3 Non-B strains were found primarily in the heterosexual population and associated with individuals born in sub-Saharan Africa. However, 19 percent of French-born individuals were infected with non-B strains. Similar results are reported for other European countries and the United Kingdom.4,5
In the United States, most studies of HIV strain diversity have focused on selected populations: immigrant communities, military personnel, and blood donors.6-15 The prevalence of non-B strains varied greatly, depending on the selected population, from a low of 0.6 percent in HIV-infected patients in northern California to a high of 95 percent of HIV cases in an African-born community in Minnesota.8,13,14 In the only broad population-based study conducted in the United States, the Centers for Disease Control and Prevention (CDC) recently reported the prevalence of HIV-1 non-B strains was 5.1 percent in newly diagnosed HIV-1–infected individuals.16
HIV strain diversity driven by antiretroviral therapy has become a major concern. Mutations that confer resistance to drugs compromise the effectiveness of treatment and limit therapeutic options. As antiretroviral treatment has become more available, the transmission of drug-resistant HIV has increased.17-20 The appearance of drug-resistant strains in newly infected individuals occurred approximately 1 year after a drug was included in therapeutic regiments.17
MATERIALS AND METHODS
The study population consisted of individuals who donated blood between 1999 and 2005 at American Red Cross (ARC) centers in addition to one non-ARC center for which the ARC performs testing. All recently infected donors identified during this time period were included in the study. For the established infections, donors were randomly selected from stored specimens collected during this period based on the availability of sufficient volume. The ARC Institutional Review Board approved this retrospective study that utilized existing specimens obtained during blood donation that were unlinked to the donor.
The ARC screened donors for HIV antibody using HIVAB HIV-1/HIV-2 (rDNA) enzyme immunoassay (EIA; List 3A77, Abbott Laboratories, Abbott Park, IL). Specimens with repeat reactivity for HIV antibody were confirmed by HIV-1 Western blot (Calypte Biomedical, Rockville, MD). Donors were screened for HIV-1 RNA primarily in minipools of 16 specimens using an HIV-1/hepatitis C virus (HCV) assay (Procleix, Novartis/Chiron Corp., Emeryville, CA; and Gen-Probe, Inc., San Diego, CA); occasionally known HIV-seroreactive samples or fewer than 15 samples occurring at ends of shifts were tested individually. Reactive pools were resolved to the individual reactive donation; reactive individual donations were tested by discriminatory probe reagents to distinguish HIV-1 from HCV RNA reactivity. For HIV-1 discriminatory-reactive specimens, HIV-1 RNA confirmation was performed using qualitative polymerase chain reaction (PCR, Ultra Qual, National Genetics Institute [NGI], Los Angeles, CA). Viral loads were determined for PCR-positive samples using a product for the quantitation of HIV RNA (SuperQuant, NGI).21
The genetic subtype of the HIV-1 strain present in a specimen was determined as previously reported.22 Briefly, nucleic acid was extracted from specimen (plasma or serum; 0.2-2 mL) and subjected to reverse transcription (RT)-PCR amplification using HIV-1 group M primers in env gp41. For HIV-1 non-B subtypes, regions of gag p24 and pol integrase were also amplified. The PCR products were directly sequenced. Phylogenetic analysis was performed using a software package (PHYLIP, Version 3.5c, J. Felsenstein, University of Washington, Seattle, WA) with evolutionary distances estimated using Dnadist and phylogenetic relationships determined using Neighbor with an HIV-1 group N reference sequence as the outgroup. Trees were constructed using TreeView (R.D.M. Page, University of Glasgow, Glasgow, UK) and branch reproducibility was done using Seqboot (100 replicates) and Consense.
An HIV-1 genotyping system (ViroSeq, Software Version 2.6, Celera Diagnostics, Alameda, CA) was used to determine drug resistance profiles for the protease (PR) and reverse transcriptase (RT) genes. For some specimens with low viral loads, the extraction and amplification procedures were modified to obtain sufficient product for DNA sequencing. The PR-RT sequences were also utilized in phylogenetic analysis to determine genetic subtype.
During the time period of this study, 1999 through 2005, and screening of approximately 42 million donations by the ARC, 26 HIV-1 RNA–positive, antibody-negative (RNA+/Ab−) donors were identified for a yield of 1:1.61 million. The yield increased significantly over time with 16 of 26 identified in 2004 through 2005 (p = 0.003; Fig. 1). The increase reached a plateau in subsequent years, 2006 through 2007, having six and eight HIV RNA+/Ab− donors, respectively; the yield from 1999 through 2007 for HIV RNA+/Ab− was 1:1.38 million. During the 1999 through 2005 time period, 1205 HIV antibody–reactive and confirmed-positive (Ab+) donors were identified (1:34,700); extending the time period to include 2006 and 2007 has no impact on prevalence (1:34,900).
The RNA+/Ab− specimens represent donors who had been infected with HIV-1 shortly before donating blood, that is, recently acquired infections. Recent infection was substantiated by follow-up data obtained for 15 of the 26 donors (data not shown; partial data previously reported).21 Over a time period ranging from 1 to 10 weeks after the initial RNA+ result, 12 of the 15 donors seroconverted to HIV EIA–reactive with 8 confirmed by Western blot and the remaining 4 either Western blot–indeterminate or -negative. All 15 donors were repeatedly positive for the presence of HIV-1 RNA with 14 showing an increase in viral load during the follow-up time period. The HIV Ab+ donors in the study (n = 46) were defined as established HIV-1 infections; all had a full HIV-1 Western blot banding pattern indicating duration of infection was greater than 30 days at the time of donation (data not shown);23 all antibody-positive donors were also RNA-positive. The median viral load was 920 RNA copies per mL for the RNA+/Ab− group compared to 15,000 copies per mL for the Ab+ group (Table 1); the mean values for log viral load are not significantly different between the two groups. Figure 2 shows the viral load value distribution for the two donor groups. For the RNA+/Ab− group, viral load ranged from less than 100 to 4,400,000 RNA copies per mL whereas the range for the Ab+ group was less than 100 to 290,000 copies per mL. The low median viral load for the RNA+/Ab− group is another indication that the majority of the infections were detected during the early phase of infection before the peak of RNA levels.
The only significant difference between the groups is the percent of first-time donors (p = 0.0357). The geometric mean values for log viral load were not significantly different.
Number of specimens
Donation time period (month/year)
December 1999-December 2005
March 2003-April 2005
First-time donors (%)
Median age (years)
Median viral load (RNA copies/mL)
Infections by HIV-1 non-B strain (%)
0 (0 of 20)
6.8 (3 of 44)
Infections by drug-resistant HIV-1 (%)
11.1 (2 of 18)
4.5 (2 of 44)
In the recently infected donor group, 81 percent were male with a median age of 28 years and 46 percent were first-time donors (Table 1). Donors with established infections were also predominantly male, 83 percent, with a similar median age, 32 years, but a higher percentage were first-time donors, 74 percent (Table 1). Risk factors for HIV infection were not revealed by the donors during the initial donor screening process nor during follow-up.
Stored specimens were available for 20 of the 26 RNA+/Ab− donors for subtype analysis. Phylogenetic analysis of env gp41 sequences obtained by RT-PCR amplification showed that all 20 infections were due to HIV-1 group M subtype B strains (ARC-N1-ARC-N20; Fig. 3A). Of the 46 Ab+ donors, env gp41 sequences were amplified from 44 specimens. For the two specimens that were RT-PCR–negative, one had a viral load of less than 100 RNA copies per mL, and for the other, insufficient specimen volume was available to determine the viral load. Phylogenetic analysis of the 44 sequences identified three infections (6.8%) that were caused by HIV-1 non–subtype B strains (ARC-A1-ARC-A46; Fig. 3B and Table 3). CRF01_AE strains were found in two donors; one a 21-year-old female living in Virginia (ID ARC-A25) and one a 45-year-old male living in California (ID ARC-A32). An infection with CRF02_AG was identified in an 18-year-old female in New Jersey (ID ARC-A3). Additional viral sequences from the gag p24, PR-RT, and pol integrase regions were amplified from the non-B infections, and the strain classification for each specimen was consistent across all four regions (data not shown). All three non-B infections were identified in first-time donors, and all had viral load values within 1 log of the median value for the established infection group (Table 2). Overall, 4.7 percent (3 of 64) of the HIV-infected donors were infected with an HIV-1 non-B strain.
Table 3. Donors infected with drug-resistant HIV-1
s/co = signal/cutoff: values of 1.0 or greater are reactive. All reactive specimens were confirmed by HIV-1 Western blot.
Drug resistance profile determined using ViroSeq. Mutations shown in bold either confer resistance or the possibility of resistance. Mutations not in bold must appear with at least one other mutation to confer possibility of resistance. All viruses are subtype B.
Last negative donation was on October 23, 1999.
Mutations confer high resistance to lamivudine, emtricitabine, zalcitabine, zidovudine, didanosine, stavudine, abacavir, and tenofovir.
Mutations confer high resistance to indinavir, saquinavir, ritonavir, nelfinavir, and atazanavir and possible resistance to amprenavir, fosamprenavir, and lopinavir plus ritonavir.
s/co = signal/cutoff: values of 1.0 or greater are reactive. All reactive specimens were confirmed by HIV-1 Western blot.
Subtype based on PR-RT and envelope (env) sequences.
Drug resistance profile determined using ViroSeq.
March 14, 2003
March 28, 2004
April 30, 2004
Drug resistance profiles for the protease and RT genes were determined using the ViroSeq genotyping system. Profiles were obtained for 18 of the 20 recently infected donors. Two donors (11.1%) harbored drug-resistant HIV-1 (Table 1 and 3). A 28-year-old male (ID ARC-N3) living in Pennsylvania was infected with a strain containing the RT mutation Y188H that confers resistance to the nonnucleoside RT inhibitor (NNRTI) nevirapine (Table 3). Donor ARC-N3 was a repeat donor whose previous HIV-nonreactive donation was 16 months earlier. Follow-up data for donor ARC-N3 showed that over a time period of 1 month, the donor seroconverted to EIA-reactive and Western blot–positive with increasing viral loads (data not shown). Donor ARC-N11, a 32-year-old male in California, was infected with an HIV-1 strain resistant to the protease inhibitor (PI) nelfinavir; mutation L90M alone confers resistance to nelfinavir and possible resistance to other PIs while L63P and V77I, mutations common in drug-naive individuals, must occur in the presence of other mutations to confer possible resistance to PIs (Table 3). Follow-up data 2 weeks after the initial RNA-positive donation showed donor ARC-N11 seroconverted to EIA-reactive with an indeterminate Western blot and increasing viral loads (data not shown). At the time of the initial RNA-positive donation, both donors had viral loads within 1 log of the median value for the recently infected group (Table 3). The two specimens that were negative in the ViroSeq assay had low viral loads, less than 100 and 720 RNA copies per mL. Phylogenetic analysis of the 18 PR-RT sequences classified all the recently acquired infections as HIV-1 subtype B strains consistent with the subtype classification based on env sequences (data not shown).
Drug resistance profiles were obtained for 44 of the 46 established infections and 2 (4.5%) were determined to be infected with drug-resistant HIV-1 strains (Table 1 and 3). Donor ARC-A24, a 43-year-old male in Pennsylvania, harbored a virus with multiple mutations in RT that together confer high resistance to all nucleoside RT inhibitors (NRTIs); four of six mutations associated with thymidine analogs were present (M41L, D67N, L210W, and T215Y), as well as additional mutations to other nucleoside analogues (Table 3). Donor ARC-A40, an 18-year-old male in Texas, had a virus with mutations in PR that confer resistance to several PIs and possible resistance to the remaining drugs in this class. Both donors were first-time donors, infected with subtype B strains, and had viral loads within 1 log of the median value for the established infections group (Table 3). The two Ab+ specimens that were negative in the ViroSeq assay were the same specimens that were RT-PCR–negative for env sequences. In the established infections group, subtype classification based on PR-RT sequences agreed with env subtype classification (data not shown).
The blood systems in the US implemented NAT in 1999 to screen blood donations for HIV-1 and HCV RNA.21 The goal was to improve the safety of the blood supply by identifying acute infections in the window phase before the presence of antibodies to the viral agents. The yield of HIV RNA+/Ab− donors has significantly increased during the period of 1999 through 2005 but then stabilized during 2006 through 2007; the reason for this increase is unknown. For HIV-1, RNA is detected in the blood approximately 8 to 12 days before an antibody response to the infection develops.21,23 It is estimated that the residual risk of HIV-1 transmission was reduced to 1 in 2 million for donations from repeat donors screened by HIV-1 NAT and third-generation HIV antibody assays compared to a risk of 1 in 1.5 million for donations when NAT was not used.24 Minipool HIV-1 NAT of blood donations is effective at detecting low-level viremia in the window period before seroconversion. For those RNA+/Ab− donors for whom follow-up data were available, most infections were detected before the peak of viremia. The RNA+/Ab− group had a median viral load (920 RNA copies/mL) consistent with Stage 1 infection, as defined by Fiebig and colleagues,23 which corresponds to the phase early in infection when virus levels are rapidly rising before peak viremia is achieved and before HIV p24 antigen and antibodies are detectable. The median viral load for the Ab+ group (15,000 copies/mL) is consistent with Stages V and VI when seroconversion is completed (Ab+, Western blot+) and viremia has decreased from peak to a lower steady-state level.23
The number of donors evaluated in this study is limited; a low number of recent infections were identified and the number of established infections was limited by the availability of stored volume. However, the demographics of the HIV-infected donors in this study are consistent with a previous study of US blood donors in 2002.25 HIV-infected donors tend to be young (20s and 30s) and are more likely to be male than female.25 For both of our donor groups (RNA+/Ab− and Ab+), first-time donors represented a disproportionate number of the HIV infections; only approximately 23 percent of donations are collected from first-time donors yet they accounted for the majority of HIV cases.21 The previous study of US donors showed that the prevalence of HIV was 15 times higher in first-time donors than in repeat donors.25
The prevalence of non-subtype B strains in HIV-infected US blood donors is increasing. Previous studies show that before 1986 only HIV-1 subtype B was present in the donor population, whereas from 1993 to 1996 and 1997 to 2000, non-B strains represented 0.8 and 2.2 percent of infections, respectively.6,10 This study of donations from HIV-infected donors collected between 1999 and 2005 shows the overall prevalence of non-B infections to be 4.7 percent (3 of 64) with all three non-B subtype donors identified in the established infection group. The increasing prevalence of non-B strains occurred despite donor deferral recommendations that exclude some donors from non-B endemic countries. The deferral of donors at risk for malaria restricts persons who recently lived in or traveled to regions of Central and South America, Africa, and Asia,26 whereas deferral of donors at risk for HIV-1 group O excludes persons born in or having a sexual partner from west central African countries.27 CRF01_AE, a strain found primarily in Thailand and southeast Asia, was identified in two donors.2 CRF02_AG, a strain endemic to west central Africa, was found in 1 donor.2 The fact that non-B strains were found only in the established infections group may indicate that donors did not meet a deferral criteria because of the passage of time or that donors were unaware of their risk status. Alternatively, non-B infections could have been acquired within the United States in a population that does not meet a current risk category, that is, US-born, heterosexual population. Although the small number of donations evaluated in this study limits the significance of findings, the fact that two of three non-B infections were identified in female donors is consistent with previous studies that associated non-B infections with heterosexual transmission.3,5
Our finding of a higher prevalence of non-B subtypes in established infections than in recently infected donors is in contrast to a French study. A study of HIV-infected blood donors identified in France from 1992 to 2006 reported that non-B subtypes were significantly associated with recent infections.28 This is probably due to the differences in HIV epidemiology between the two countries.
Although the results obtained from the US blood donor population cannot be directly extrapolated to the general population because donor selection excludes high-risk groups, reducing the prevalence of HIV by 80 percent, data generated from blood donors contribute to a better understanding of the diversity of HIV in the United States.24 The prevalence of HIV-1 non-B strains in US blood donors is similar to that in the US diagnostic population consisting of individuals diagnosed primarily at sexually transmitted disease clinics and counseling and testing sites. A recent report from the CDC on more than 3000 HIV infections, diagnosed between 2003 and 2006 in 11 states, showed the prevalence of HIV-1 non-B strains was 5.1 percent with the most common being subtype C and CRF02_AG.16 In France, non-B subtypes represented 48 percent of newly diagnosed HIV-1 infections in the general population in 2003 compared to 26 percent of recently infected blood donors identified between 1992 and 2006.3,28
Drug-resistant HIV-1 was identified in 6.5 percent (4 of 62) of the HIV-infected donors: 11 percent (2 of 18) in recently infected donors and 4.5 percent (2 of 44) in established infections. The recently acquired infections showed drug resistance to a single drug within a drug class. In contrast, the resistant viruses present in the established infections showed resistance to multiple drugs in a single drug class. No multiclass resistance was identified in either group. The recently infected donors with drug-resistant virus are clearly cases of transmitted drug resistance; both donors seroconverted after their initial RNA-positive test result. The established infections could be either transmitted or acquired resistance. It is assumed that the donors were unaware of their HIV status and antiretroviral drug–naive, which would indicate that drug-resistant virus was transmitted. However, this assumption cannot be confirmed.
The prevalence of drug-resistant virus in HIV-infected US blood donors is similar to rates reported in several studies that evaluated transmitted drug-resistant HIV-1 in recently infected individuals in the United States.17,18,20,29-31 Although each study used a different algorithm to define drug resistance, the overall conclusions of the studies are similar. The studies conducted between 1995 and 2004 were composed predominantly of males with a risk factor of men having sex with men. Resistance, based on genotyping mutations, varied from 6 to 24 percent of infections. Transmission of drug-resistant HIV increased over time with the highest increase due to strains with mutations that are associated with resistance to NNRTIs. However, the most prevalent drug-resistant strains contained mutations that confer resistance to NRTIs. Drug resistance primarily affected only one drug class, and resistance to multiple drug classes was rare. These studies identified the most common mutations associated with each drug class; mutations at T215 (T215/Y/F, T215D/N/S) and M41L in RT are the most common mutations for NRTI resistance, K103M and Y181C for NNRTI resistance, and L90M for PI resistance. The broad-based national surveillance study conducted by the CDC identified drug resistance mutations in 10.4 percent of HIV infections diagnosed between 2003 and 2006.16 In this survey, NRTI resistance mutations were present in 3.6 percent of infections, NNRTI in 6.9 percent, and PI in 2.4 percent.
In the past there were concerns about the detection of divergent HIV strains by serologic and nucleic acid–based assays.32-34 Screening of blood donations by two different and sensitive methods appears effective and minimizes any impact of genetic variation on HIV-1 NAT and/or HIV antibody assays. The non-B infections were detected by both the NAT and antibody assay as were the drug-resistant HIV-1 strains. Monitoring the occurrence of drug-resistant HIV and genetically divergent non-B strains in blood donors provides a means to assess changes in the US HIV epidemic.
Genebank accession numbers for the sequences reported here are EU711431-EU711562. The authors thank Sara Allmond and Shimian Zou for assistance and John Hackett for critical review of the manuscript.