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The Probable Source of Both the Primary Multidrug-Resistant (MDR) HIV-1 Strain Found in a Patient with Rapid Progression to AIDS and a Second Recombinant MDR Strain Found in a Chronically HIV-1-Infected Patient
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The Journal of Infectious Diseases May 1, 2007;195:1250-1259
Gary Blick,1 Ron M. Kagan,2 Eoin Coakley,3 Christos Petropoulos,3 Laura Maroldo,3 Paola Greiger-Zanlungo,1,4 Scott Gretz,1 and Trish Garton1
1Circle Medical LLC, Norwalk, Connecticut; 2Quest Diagnostics, San Juan Capistrano, and 3Monogram Biosciences, San Francisco, California; 4New York Medical College/Mount Vernon Hospital, Mount Vernon
(See the editorial commentaries by; the editorial commentaries by Hecht et al., on pages 1239-41, and Allos and Schaffner, on pages 1245-7; and the brief report by Redd et al., on pages 1311-4.)
ABSTRACT
Background. Rapid progression to AIDS after acute infection with a multidrug-resistant (MDR), dual-tropic strain of human immunodeficiency virus type 1 (HIV-1) was reported in a New York City man (hereafter referred to as "NYC") who has sex with men. The probable source of this HIV-1 (hereafter referred to as "CT01") and the development of a recombinant MDR HIV-1 in the source's partner (hereafter referred to as "CT02") are described.
Methods. After identification of the epidemiological link of CT01 and CT02 to NYC, viral sequences and phenotypic analyses were compared. Confirmatory genotypic and phenotypic analyses, replicative capacity, and viral coreceptor use were assessed. Viral recombination was assessed using a sliding window technique and phylogenetic tree analysis.
Results. NYC and CT01 were linked historically and epidemiologically and were genetically confirmed from CT01's samples acquired 2 days before and subsequent to the transmission event. Genotypic, recombination, and phylogenetic analyses suggest CT02 became superinfected by CT01 with subsequent production of a recombinant panresistant HIV-1.
Conclusion. The probable source of a dual-tropic, MDR HIV-1 that was associated with rapid progression to AIDS is illustrated, suggesting progression was not explained by the HIV-1 variant alone. A probable second finding of a chronically infected host becoming superinfected with MDR HIV-1 with subsequent formation of a panresistant recombinant HIV-1 is described. This case illustrates the public health implications of unsafe sex between serodiscordant and seroconcordant partners.
Background
On 11 February 2005, the New York City Department of Health and Mental Hygiene issued a public health alert after a previously HIV-1-seronegative New York City man (hereafter referred to as "NYC") who has sex with men acquired a highly multidrug-resistant (MDR), dual-tropic HIV-1 infection and progressed to AIDS with absolute CD4 T cell counts <100 cells/L within months of his seroconversion. This case created a realistic public health concern as NYC continued to engage in high-risk sexual activities with several additional anonymous partners during the 2-month "window period" before testing HIV-1 seropositive, when HIV-1 viremia and potential infectivity may be greatest, thus raising the prospect of epidemic transmission of this unique MDR HIV-1 variant [1].
The advent of highly active antiretroviral therapy (HAART) has resulted in increasing numbers of individuals living with HIV/AIDS as rates of death and progression to AIDS have declined [2, 3]. This trend-combined with increasing use of the Internet to find sex partners; adverse trends in recreational substance abuse, particularly methamphetamine and erectile dysfunction (ED) medications; and an increased incidence of HIV-1-seropositive individuals engaging in unsafe sexual activities-has resulted in an increase in new HIV-1 infections and AIDS diagnoses in the United States [4-11]. This trend has been particularly evident with HIV-1-seropositive men who have sex with men (MSM), who are increasingly using HIV serostatus as a criterion for the selection of casual sex partners for unprotected anal receptive (UAR) and unprotected anal insertive (UAI) intercourse to reduce transmission of HIV-1 to seronegative individuals, a practice referred to as "serosorting." Additionally, with HIV-1 becoming progressively more resistant to HAART, MDR HIV-1 is being transmitted with increasing frequency to both HIV-1-seronegative and chronically HIV-1-infected individuals [11-23].
We report on subject "CT01," the probable source of this MDR HIV-1 strain transmitted after unprotected sex with NYC, and document the development of a probable unique recombinant MDR strain in the source's partner (hereafter referred to as "CT02") after years of serosorting.
SUBJECTS AND METHODS
The public health alert prompted confidential searches of reference laboratory databases for matches to the NYC sequence. Quest Diagnostics performed nucleotide (BLASTN) searches in its database of 142,000 clinical sequences from 1998 to 2005 to identify matches to NYC [24]. On 19 February 2005, Quest Diagnostics identified 1 epidemiologically linked male, CT01, with a 99% pol gene sequence match to NYC and confidentially alerted CT01's primary care physician (PCP) HIV specialist and the Connecticut Department of Public Health. CT01 and CT02 were contacted by their PCP and confidentially interviewed, and additional serum samples were obtained after written informed consent was obtained. CT01 and CT02 also consented to allow archived samples to be used and clinical summaries to be confidentially obtained from their medical records for the purpose of this investigation. On 19 March 2005, Laboratory Corporation of America (LabCorp) identified 1 sequence in its database with a 98.5% pol gene match to NYC, that of CT02, and confidentially notified CT02's PCP.
Genotypic sequence data for 1 time point from NYC (2005), 8 time points from CT01 (1999, 2000, 2001, 2002, and 2003), and 2 time points for CT02 (2001 and 2004) were obtained from the Quest Diagnostics database. These 11 nucleotide sequences have been deposited in GenBank, the National Institutes of Health genetic sequence database, under accession numbers EF042794-EF042804 (available at: http://www.ncbi.nlm.nih.gov/Genbank/). A 2002 genotype report (GenoSure) of protease (PR)- and reverse transcriptase (RT)-associated resistance mutations for CT02 was obtained from LabCorp, and 2004 PhenoSenseGT mutational profiles (Monogram Biosciences) from CT01 (October 2004) and CT02 (September 2004) were obtained [25-27].
The susceptibility of CT01's HIV-1 to antiretroviral (ARV) drugs was determined by resequencing the viral pol gene, sequencing the gp41 env gene, and performing confirmatory phenotypic analyses (PhenoSenseGT). Genotypic and phenotypic studies of NYC's HIV-1 susceptibility to ARV drugs were performed and reported elsewhere [1].
Coreceptor usage was assessed using the Monogram Biosciences Trofile, in which patient-derived env genes are transfected with a luciferase-containing env-defective HIV vector into producer cells. The resultant recombinant virus is incubated with cells expressing CD4 and CCR5 or CXCR4. The magnitude of a single round of viral replication is measured as luciferase activity (relative light units [RLUs], in log10 units), and each virus is assigned an R5RLU and X4RLU value. Replication capacity (RC) was measured using a modified PhenoSense assay (Monogram Biosciences) [28-30].
Recombination analysis was performed with the Recombination Identification Program (RIP; version 2.0; available at: http://www.hiv.lanl.gov/content/hiv-db/RIPPER/RIP.html), which compared the last nucleotide sequence from CT02 (January 2004) to the background sequences from CT02 (September 2001), partner CT01 (May 1999, July 1999, February 2000, January 2001, December 2001, May 2002, and May 2003), and NYC (January 2005). The comparison utilized a sliding window of 200 nt and a 95% significance threshold. Recombination analysis was initially performed unmasked and then repeated using 500 bootstrap replicates and masking 21 PR- and 28 RT-associated resistance codons (International AIDS Society-USA; March-April 2005) before the reanalysis [31, 32]. Neighbor-joining (NJ) phylogenetic trees of the resistance-codon masked and aligned nucleotide sequences upstream and downstream of the proposed recombination site were constructed with Clustal X (version 1.8.3) [33].
Phylogenetic trees were constructed following BLASTN searches with NYC, and the 8 related sequences from CT01 were utilized to identify any additional matches in the Quest Diagnostics database [24]. Multiple alignments and NJ trees were constructed with Clustal X comparing nucleotide and translated sequences from the viral pol gene of NYC to sequences obtained from CT01 from 1999 to 2004 and from CT02 from 2001 to 2004 [33]. The highest scoring nucleotide matches were corroborated by protein database searches using Protein Blast (BLASTP; version 2.2.2) and Hidden Markov Model (HMM; version 2.3.2) searches [24, 34].
RESULTS
Historical confirmation: CT01 and CT02 patient profiles. CT01 and CT02 have been involved in a long-term relationship and have engaged in unprotected oral, UAI, and UAR intercourse with each other and others. CT01 tested HIV-1 positive in 1993 and began sequential ARV monotherapy in 1995 followed by dual ARV therapy and has ultimately been treated with 14 ARV medications. In October 2003, resistance testing demonstrated sensitivity to only efavirenz (EFV) and delavirdine (DLV). His CD4 T cell count was 271 cells/uL, his CD4 T cell percentage was 14%, and his plasma HIV-1 RNA was 48,843 copies/mL at that time (figure 1). In February 2004, he began treatment with enfurvitide (ENF) and EFV, along with stavudine (d4T), didanosine (ddI), abacavir (ABC), and ritonavir-boosted saquinavir. CT01's viral load suppressed to <400 copies/mL from April through September 2004 when he developed detectable HIV-1 viremia (5077 copies/mL) during a period of nonadherence. A PhenoSenseGT profile was obtained on 20 October 2004 when CT01's viral load was 1452 copies/mL. Throughout 2005, CT01 had stable CD4 T cell counts and HIV-1 RNA consistently <400 copies/mL.
CT02, the partner of CT01, was diagnosed as HIV-1 positive in 1994, began ARV therapy in 1996, and has been treated with 11 prior ARV drugs. On 27 August 2002, before beginning his first protease inhibitor (PI)-based HAART, a virtual phenotype assay (GenoSure; LabCorp) was performed. This nucleotide sequence was later identified in the LabCorp database as being genotypically related to that of NYC. CT02 has never achieved sustained viral suppression, and his CD4 T cell counts have been slowly declining since 2002, with HIV-1 viremia remaining < log10 5.0 copies/mL (figure 1).
On 22 October 2004, 2 days after CT01 had virologic and immunologic analyses performed, CT01 and CT02 met NYC at a New York City sex club, informed NYC that they were both HIV-1 positive, and participated in unsafe sexual activities while using methamphetamine. CT01 engaged in UAI intercourse with ejaculation on 2 occasions with NYC, whereas CT02 engaged in UAI intercourse without ejaculation with NYC. In November 2004, NYC developed symptoms consistent with primary HIV-1 infection (PHI) and was diagnosed as HIV-1 seropositive by mid-December. In mid-January 2005, NYC stated he "believed he was infected ... in the third week of October 2004" and recalled "the Connecticut couple" and other demographic, anatomical, and dermatological specifics that linked NYC with CT01 and CT02 historically [1].
Genotypic and phenotypic analyses: CT01 and NYC. Genotyping studies of NYC's HIV-1 revealed high-level resistance to all 3 ARV classes with phenotypic susceptibility to EFV, DLV, and ENF [1]. CT01's PhenoSenseGT, performed October 2004, revealed broad-level genotypic resistance to all nucleoside reverse transcriptase inhibitors (NRTIs) and nucleotide reverse transcriptase inhibitors (NtRTIs), all PIs, DLV, and nevirapine (NVP), and susceptibility to EFV. The protease mutational patterns of viral sequences from NYC and CT01 are compared in figure 2. The mutations found at 18 different codons are identical. The mutations for NRTIs/NtRTIs and non-NRTIs are compared in figure 3. The mutations found at 14 different codons are also comparable, although mixtures of viral species detected at aa 184 and 215 and different substitutions at aa 40 and 210 are noted for NYC. A mutation unique to NYC was found at aa 219, whereas mutations unique to CT01 were found at aa 69 and 70. Genotypic resistance analysis performed on CT01's HIV-1 in May 2003 revealed nearly identical PR and RT sequences to those found in October 2004.
Phenotypic analysis of NYC's virus (January 2005) revealed resistance to ABC, ddI, tenofovir (TDF), and d4T; high-level resistance to all PIs, emtricitabine (FTC), lamivudine (3TC), zidovudine (AZT), and NVP; and susceptibility to EFV (0.6-fold change), ENF (0.3-fold change), and DLV [1]. CT01's HIV-1 phenotypic assay demonstrated similar resistance to ABC, ddI, TDF, and d4T; high-level resistance to all PIs (18-fold to above maximum measurable drug resistance [MAX]), FTC, 3TC, NVP (all >MAX), and AZT; susceptibility to DLV; and hypersusceptibility to EFV (0.2-fold change). Phenotypic susceptibility analysis to ENF was not performed for CT01 in October 2004, but subsequent analysis in November 2005 revealed susceptibility to ENF.
BLASTN searches with the first 1116 nt of the NYC sequence and 8 sequences from CT01 ("patient B") epidemiologically linked to NYC showed nucleotide similarities of 96%-98% for these and 3 other patients' sequences: "patient C" (2 sequences) and "patient D" (1 sequence) [35]. The 100 highest scoring BLASTN hits for these 12 sequences yielded a nonredundant data set of 291 sequences. The distribution of scores within the NYC-related cluster was well separated from scores between cluster and background sequences. In an NJ phylogenetic tree, these 12 sequences were clustered together in 200/200 bootstrap replicates (figure 4). Sequence similarities (±SD) within the NYC-related cluster were 97.8% ± 0.7% (range, 96.3%-99.1%). BLASTN searches failed to identify additional sequences with a similar high degree of similarity to members of the cluster in the Nichols Institute Chantilly reference laboratory database (8010 sequences; October 2001 to March 2005). We performed BLASTP searches using the translated cluster sequences (PR 1-99 and RT 1-273) against the translated sequence database. A compilation of the top 100 protein hits from each of the 12 queries yielded a nonredundant protein data set of 364 sequences. Phylogenetic analysis again showed that the 12 NYC-related sequences did not cluster with any other sequences (129/200 bootstrap replicates; data not shown). An HMM profile search using an HMM profile built from the translated cluster sequences against the translated database did not identify additional sequences matching this cluster (data not shown).
Genotypic, phenotypic, and recombination analyses: CT02. Two intriguing findings were observed in CT02's genotypic resistance tests performed between September 2001 and January 2004. First, the PR sequence from CT02 obtained in September 2001 demonstrated 3 polymorphisms without any primary or secondary PR mutations (figure 2), whereas the RT mutational pattern contained the Q151M MDR mutational complex along with a K103N mutation and mutations at 20 additional amino acid positions (figure 3). However, genotypic analysis performed in August 2002 on CT02's virus revealed it to be virtually indistinguishable from that of CT01's virus analyzed genotypically from May 1999 through October 2004 with the same mutations found at 34 RT and PR codons, except for the absence of RT mutations at aa 40 and 208 (figures 2 and 3). CT02's genotypic pattern in August 2002 is virtually unique and divergent from that identified in September 2001, with the exception of RT mutations present at positions 67 and 118 and PR mutations at 62, 63, and 64 found at both time points.
NJ trees of the nucleotide sequences upstream (nt positions 1-366, encompassing all the PR and the first 23 RT codons) and downstream (nt positions 367-1116 encompassing RT codons 24-273) of the proposed recombination site are shown in figure 6. The upstream sequence from NYC clusters with the sequences from CT01 and the most recent sequence from CT02 (CT02-0104), whereas the earlier upstream sequence from CT02 (CT02-0901) is divergent (figure 6A). In contrast, the 2 sequences for CT02 downstream from the proposed recombination site are closely related and diverge from CT01 and NYC (figure 6B). These findings strongly suggest that a recombination event occurred by January 2004 after superinfection of CT02 before 2002.
Second, the viral sequences from CT02 in January 2004 and September 2004 contained RT sequences indistinguishable from CT02's virus in September 2001, whereas the PR mutations were identical to those found in CT02 in August 2002. These findings suggest that the predominant viral sequence present in CT02 at January 2004 may be the product of a recombination event that occurred in CT02 before January 2004, possibly after superinfection of CT02 with the virus from CT01 at some time before August 2002. The remote possibility also exists that both CT01 and CT02 became superinfected simultaneously by another individual infected with MDR HIV before the first detection of this viral strain in CT01 in 1999. Both CT01 and CT02 confirmed serosorting with each other and other partners during the entire period in question. Additionally, CT02 was a participant in HIV-1 clinical trials from 2002 to 2005 with documented >95% adherence to HAART.
We performed recombination analysis with the RIP 2.0 program to further define the nature of this proposed recombination event. The analysis (figure 5) showed that the January 2004 sequence from CT02 matched the CT01 and NYC background sequences for the first 366 nt but diverged from these background sequences for the remaining nucleotide positions, which bore a close resemblance to the earlier September 2001 sequence from CT02.
RC and viral tropism. The RC of NYC's HIV-1 was previously reported to be 136% (range, 86%-215%), comparable to wild-type drug-susceptible virus [1]. The RC of CT01's HIV-1 was analyzed from the 20 October 2004 sample and was determined to be 41% (range, 21%-65%). Differences in RT mutations at 4 major codon positions (D67D/N, M184V/I, L210L/G/M/R/V/W, and K219E) were found in NYC's virus, compared with that of CT01. The RC of CT02's suspected recombinant HIV-1 analyzed in September 2004 was 6.8% (range, 4.3%-11%).
With regard to HIV-1 coreceptor use, the HIV-1 quasispecies found in NYC were collectively dual tropic; however, the viral population consisted of equal portions of both CCR5-tropic and dual-tropic viruses. Initial analysis of CT01's October 2004 sample revealed a CCR5-tropic virus. The specific RLU measurements were R5 = 471,643 and X4 = 76 on the CD4/CCR5 and CD4/CXCR4 cells, respectively. The level of X4 activity was at background. Subsequent analysis of CT01's viral tropism, performed from samples collected in October and November 2005, revealed an R5/X4-tropic virus.
DISCUSSION
Markowitz et al. described an infection with a unique MDR HIV-1 variant followed by rapid clinical progression to AIDS in an MSM residing in New York City after unsafe sex and methamphetamine use [1]. As a direct result of a public health alert issued 11 February 2005 and the subsequent reporting of details surrounding NYC presented in a timely manner at a medical conference and in the medical literature, we were able to identify and describe the probable source (CT01) and details surrounding the transmission of this unique triple-ARV-class MDR, dual-tropic HIV-1 that was transmitted on 22 October 2004 [1, 36].
The public health alert was issued after the rapid clinical progression to AIDS in NYC, raising the possibility that his clinical course could be explained by the unique properties of this HIV-1 variant. Phylogenetic analysis illustrates that this variant probably evolved within CT01 before May 1999. CT01 and CT02 have experienced a relatively indolent clinical course while receiving ARV therapy for >10 years, raising the possibility that other factors, such as NYC's acknowledged methamphetamine abuse and unsafe sexual activities and/or genetic or immunological host factors, possibly in combination with the unique HIV-1 properties, may have contributed to NYC's rapid clinical decline. These possibilities could have been elucidated if any one of NYC's sexual contacts during the 2 months after acute infection had been identified, diagnosed with PHI, and followed clinically.
NYC and CT01 have been linked historically, epidemiologically, and genetically. However, the Centers for Disease Control and Prevention recently reported on 2 additional chronically infected individuals who have nucleotide sequences with >95% homology to NYC's and CT01's pol sequence and who reported engaging in sexual activities at the same New York City venue, raising the possibilities that either CT01 superinfected these individuals at some undetermined time, NYC superinfected these individuals during the about 2 months of continued unsafe sexual activities after PHI, or either or both of these individuals or another as yet unidentified individual could have dual infected or superinfected NYC [37].
NYC was infected with a mixture of CCR5-tropic and dual-tropic HIV-1 populations, whereas CT01's HIV-1 population in October 2004 was predominantly CCR5 tropic [1]. This difference might be explained by differences in compartmental tropism, because plasma, not seminal fluid, HIV-1 was analyzed for coreceptor tropism. Subsequent sampling of CT01's virus in 2005 revealed a dual/mixed-tropic population in plasma. Biologic shift in coreceptor tropism over time has been described [38, 39]. Additionally, previous data highlighted the potential impact of treatment interruption as a driver of tropism switches, suggesting that these differences might be explained by CT01's purported treatment nonadherence [40].
CT01 had a pol RC of 41% (range, 21%-65%) 2 days before the transmission event, and CT02 had an RC of 6.8% (range, 4.3%-11%) about 5 weeks before, whereas NYC had a pol RC of 136% (range, 86%-215%) 3 months later. These RC differences may be explained by the differences in RT mutations between the 2 isolates. The loss of a homogenous resistant population in NYC's virus through the emergence of mixtures at major RT codons may have occurred without selective ARV drug pressure on NYC's virus before HIV-1 diagnosis.
Dual HIV infection, the presence of 2 distinct HIV-1 strains in a host, occurs by either coinfection, in which a host is infected by 2 heterologous HIV-1 strains simultaneously or around the same period of time before infection with the first strain has been established, or superinfection, in which the host is sequentially infected by a second different viral strain after initial infection and an immune response has occurred [13-23, 41-45]. The phenomenon of superinfection has been controversial and an area of intense research, with several cohorts failing to demonstrate evidence of superinfection, whereas others argue that superinfection is an uncommon event that has been observed and described rarely [46, 47]. Intersubtype superinfections have been more commonly described, because distinct clades differ significantly from each other by as much as 30% in the env gene and up to 15% in the core proteins and thus can be more frequently detected [13, 14, 17-19, 41].
Through 2005, only 23 cases of HIV-1 superinfection had been described [13-23, 41-45]. HIV-1 recombination, whereby cells infected with 2 different strains produce heterozygous virions that provide an opportunity for recombination to occur during reverse transcription, has previously been described between different HIV-1 subtypes in individuals infected by recognized circulating recombinant forms and, less commonly, in the setting of primary infection [15-17, 20, 23, 41]. Recombination of distinctly different HIV-1 strains after superinfection has been postulated and described recently for the first time in an untreated chronically infected woman [18]. We describe the first case whereby a chronically HIV-1-infected, heavily ARV therapy-experienced individual (CT02) with subtype B MDR HIV-1 may have become superinfected by a second distinctly different subtype B MDR HIV-1 after years of serosorting with other chronically HIV-1-infected partners, including CT01, followed by formation of a recombinant HIV-1 strain in CT02 that was resistant to all Food and Drug Administration-approved ARV drugs. It is unlikely that CT02 infected CT01 before 1998, because CT01 began receiving PI-based HAART in 1998, only after which time his PI-resistant strain could have developed. These data are limited by the lack of additional genotypic analyses for CT02 between 1998 and 2001 and by sequencing data being restricted to the pol gene alone.
There are several noteworthy implications associated with this case study. First, this report describes transmission of MDR HIV-1 among HIV-1-serodiscordant individuals through unsafe sex, reflecting recent adverse trends in MSM in new HIV/AIDS diagnoses; recreational substance abuse, particularly with methamphetamine and ED medication; and unsafe sexual activities [5-8]. NYC reportedly engaged in UAR intercourse with both CT01 and CT02 despite knowing they were both HIV-1 positive, and all 3 individuals reported using methamphetamine and ED medication during the night in question.
Second, this report depicts the potential risk of transmission of MDR HIV-1 among HIV-1-seroconcordant individuals while serosorting, the practice of using HIV serostatus as a criterion for the selection of casual sex partners. Serosorting can represent an important strategy to reduce HIV-1 transmission to uninfected individuals when it is used to correctly identify HIV-seroconcordant sex partners [9]. However, studies indicate that perceived HIV seroconcordance is a strong predictor of unprotected sex, and individuals often misperceive the serostatus of their sex partners [4, 5].
Last, the public health impact of transmission of highly MDR HIV-1 into the general population could be catastrophic, because these strains are frequently untreatable with currently approved HAART.
What remains uncertain is how commonly superinfection occurs, and this ambiguity has been shown to affect an individual's perceived risk of unprotected sex. As the HIV-1-positive community engages in serosorting with increasing frequency, it is the responsibility of health care providers and public health workers to counsel HIV-1-positive individuals about the need to practice safer sex techniques with other HIV-1-positive individuals because of the risks of becoming HIV-1 superinfected, producing viral recombinant strains, and acquiring sexually transmitted diseases.
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