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HIV drug resistance acquired through superinfection
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.....he had an incomplete response to therapy most likely because the superinfecting viral strain had a decreased susceptibility to two of the prescribed medications.....
AIDS: Volume 19(12) 12 August 2005 p 1251-1256
Smith, Davey Ma; Wong, Joseph Ka,b,d; Hightower, George Ka; Ignacio, Caroline Ca; Koelsch, Kersten Ka; Petropoulos, Christos Jc; Richman, Douglas Da,b; Little, Susan Ja
From the aUniversity of California San Diego, San Diego, California, USA
bVeterans Affairs San Diego Healthcare System, San Diego, California, USA
cViroLogic, Inc., South San Francisco
dVAMC, San Francisco, San Francisco, California, USA.
Abstract
Objective: HIV interclade B superinfection has previously been described in individuals initially infected with drug resistant virus who then become superinfected by a drug susceptible strain. We report an individual initially infected with a drug-sensitive clade B strain of HIV who was superinfected with another clade B strain resistant to two classes of antiretroviral drugs.
Methods and design: To differentiate superinfection from possible co-infection we applied three independent molecular techniques: dye-primer sequencing of a pol fragment, length polymorphism analysis of the V4-5 coding region of the env gene and clonal sequencing of the V3 coding region of the env gene. To assess viral fitness we performed replication capacity assays of the pol gene.
Results: These investigations supported the conclusion that this was a case of superinfection and not co-infection. Coincident with acquiring the new strain, the individual's viral load increased by about 10 000 copies/ml with a decrease of 150 x CD4 T cells/ml over the next 6 months. The greater in vivo fitness of the second virus was not supported by the replication capacity assay. Furthermore, superinfection negatively impacted this individual's treatment course. It was not known that he had acquired a drug resistant strain before entering a treatment study, and he had an incomplete response to therapy most likely because the superinfecting viral strain had a decreased susceptibility to two of the prescribed medications.
Conclusion: HIV drug resistance acquired through superinfection significantly lowers the likelihood of successful antiretroviral therapy and undermines the clinical value of a patient's prior drug resistance testing and lack of prior antiretroviral use.
Introduction
Superinfection with HIV is presumed to occur frequently because of the prevalence of interclade recombinants [1,2]. However, documented superinfection in humans has only recently been reported, first with different clades and subsequently, two instances of intraclade B superinfection [3-7]. Koelsch et al. reported a patient who was initially infected with drug-resistant HIV and then secondarily infected by drug-sensitive ('wild-type') virus. The superinfecting wild-type HIV replicated at high levels and masked from standard drug resistance tests the underlying presence of the initial drug-resistant virus [4]. We now report a patient initially infected with a drug-sensitive clade B strain of HIV who was superinfected with another clade B strain resistant to two important classes of antiretroviral drugs.
Author Discussion
Although unlikely, it is possible that the individual described in this report was coinfected with both a drug susceptible and drug resistant strain and that the drug resistant strain only emerged at a later time. However, four independent lines of molecular investigation: env cloning, sequencing, and phylogenetic analysis; dye-primer sequencing; length polymorphism detection; and pol sequencing and phylogenetic analysis provide evidence that the individual was initially infected with wild-type HIV and subsequently superinfected with a second strain resistant to two classes of antiretroviral drugs, nucleoside reverse transcriptase inhibitors and protease inhibitors. He maintained relatively good control of the first virus, with low HIV VL and high CD4 cell counts, without antiretroviral therapy. He subsequently experienced a rise in VL and a drop in CD4 cell count coincident with the appearance of the second virus, which is similar to previous reports of superinfection [4,16]. The greater fitness of the second virus in this individual, demonstrated by its predominance in vivo, was not supported by the replication capacity assay of the viral isolates. The replication capacity assay only interrogates (or assesses) replication fitness contributed by the pol coding region, while other genes such as env most likely play an important role in determining in vivo fitness of the virus [15].
This superinfection negatively affected this subject's disease and course of treatment. It was not known that he had acquired a drug-resistant virus before he entered the trial for treatment-naive patients. During the ACTG 5095 trial, he received the combination therapy of zidovudine, lamivudine, and abacavir and his VL decreased from 65 954 copies/ml to 263 copies/ml, but never became undetectable (Fig. 1). This incomplete response may have resulted from the patient's intermittent compliance with his medications secondary to nausea during the trial or it may have been that the prescribed treatment regimen was suboptimal since there was unrecognized reduced susceptibility to two of the medications used (M184V mutation in reverse transcriptase).
This first observation of superinfection with multidrug-resistant virus was initially recognized as a failure of antiretroviral therapy, which raises the possibility that some patients who fail antiretroviral therapy might do so because of superinfection rather than the de novo evolution of drug resistance. At present, it appears unlikely that superinfection contributes substantially to a significant number of cases of drug resistance in clinical practice since a recent retrospective study by Gonzalez et al. comparing viral sequences from patients undergoing serial resistance genotyping found no evidence for such cases. However, it is unclear whether any baseline viral sequences were compared in that study [12].
HIV drug resistance acquired through superinfection significantly lowers the likelihood of successful antiretroviral therapy. It undermines the clinical value of a patient's prior drug resistance testing and lack of prior antiretroviral use. Vigilant personal protection, through safe sex practices or clean needle use for injection drugs, must be counseled to patients already infected with HIV even if their risk exposures are with other HIV infected people.
Case report
In September 2001, a 31-year-old man was enrolled into the San Diego Acute HIV Infection and Early Disease Research Program (AIEDRP) after recently testing positive for HIV at a community clinic. He previously had a negative HIV test in October 2000, but reported having had symptoms consistent with acute HIV infection in April 2001. A repeat HIV ELISA in September 2001 was positive, but the detuned assay was negative, consistent with infection within 6 months. His reported risk factor was unprotected receptive anal intercourse with partners of unknown HIV serostatus. At presentation, his HIV viral load (VL) was 1203 copies/ml (AMPLICOR HIV-1 MONITOR Test, v.1.5, Roche Diagnostics, Basel, Switzerland) and his CD4 cell count was 571 x /l (34% of total T cells). Genotypic and phenotypic assays (Viroseq HIV-1 Genotyping System, Celera Diagnostics, Alameda, California, USA and PhenoSense HIV, Virologic, Inc., South San Francisco, California, USA) detected no drug resistance and the patient elected to defer antiretroviral therapy. Blood samples were collected weekly for 4 weeks, monthly for 5 months and every 8 weeks thereafter.
Initially, the patient's HIV VL remained low (140-2927 copies/ml) until 260 days after presentation when it increased to 11 611 copies/ml. It continued to increase to 142 566 copies/ml 4 months later, which corresponded to a drop in CD4 cell count to 331 x /l. A month later, he entered a double-blind randomized treatment trial, AIDS Clinical Trials Group (ACTG) 5095 [8]. After the trial was closed and unblinded, his treatment regimen was identified as lamivudine, zidovudine, and abacavir (Trizivir, GlaxoSmithKline, Research Triangle Park, North Carolina, USA). He reported that because of nausea, he was only intermittently compliant to the antiretroviral therapy. His VL remained detectable throughout the period of follow-up. After voluntary withdrawal from the trial at 4 months, testing revealed high-level drug resistance, including resistance to protease inhibitors (L90M) and other new polymorphisms in protease (M36I, L63P), although protease inhibitors were not used in the trial. When questioned after resistance was detected, he denied taking any other antiretroviral medications, but he did report frequent unprotected anal receptive intercourse since his initial HIV diagnosis, with partners of unknown HIV serostatus.
Results
HIV superinfection was suspected when retrospective drug resistance testing (260 days after enrollment into the AIEDRP cohort) on previously collected samples detected acquisition of resistance to protease inhibitors, to which the patient was never exposed. In retrospect, this switch in drug susceptibility occurred 4 months before the subject entered the treatment trial and in conjunction with a rise in plasma HIV RNA and drop in CD4 cell count. Both viral isolates were clade B. To discriminate superinfection or coinfection from the selection of drug resistance, cloning of the V3 region of HIV envelope was performed on samples collected before and after the emergence of drug resistance. Phylogenetic reconstruction revealed no intermingling of sequences between the two time-points, and genetic distances between the two clusters of sequences were greater then 10%, indicating two epidemiologically distinct strains [10-12].
To distinguish further between coinfection and superinfection, length polymorphism detection (GeneScan) of the V4-5 region of HIV envelope from HIV RNA extracted from blood samples collected at the two time-points (September 2001 and April 2002) was also performed. The assay can discern minority variants that make up 1-5% of the viral population [4,13]. The viral populations differed by a two-codon (six-base pair) insertion in the region at the second time-point with homogenous populations at both time-points (data not shown). To further confirm the superinfection, dye-primer sequencing of a 500-base pair fragment of the HIV pol, which spanned the 184 region of reverse transcriptase, was also performed. This method is reported to detect a minor variant that makes up as little as 5-10% of the total viral population [4,9]. In the two samples obtained before the genotypic switch (11 September 20, 4 February 2002) only 184M (wild-type) virus was detected. In the sample collected from the time-point when drug resistance was first observed (29 April 2002), only 184V (lamivudine resistant) virus was detected. In the sample collected 104 days later (13 August 2002) the 184M (wild-type) had re-emerged but as a reversion in the context of the superinfecting virus. There were no mixtures of drug-sensitive and drug-resistant viruses at any time-point (data not shown). Phylogenetic analysis of pol sequences, obtained through population based sequencing (Viroseq), revealed that all sequences collected before 29 April 2002 clustered separately from all sequences obtained on and after 29 April 2002.
Replication capacity (RC) of the viral isolates using a pol sequence in a recombinant assay also changed between time-points [14]. The first virus had an RC of 57%, while the second virus had an RC of 18%, which is consistent with the presence of the M184V mutation and protease mutations [14,15]. At the next time-point (13 August 2002) when the 184V mutation had reverted to 184M, the RC was 63% even though the protease mutations remained. This change in RC did not explain the rise in VL seen after the second HIV infection, suggesting viral fitness based on HIV genes outside of the pol coding region.
Methods
Clonal env sequencing
HIV RNA was isolated from the subject's blood plasma from two time-points 228 days apart (11 September 2001, 29 April 2002) using the QIAamp Viral RNA extraction kit (QIAGEN Inc., Valencia, California, USA) according to manufacturer's instructions. Nested PCR of the V3 coding region was performed using previously described primers and methods [4]. PCR products were cloned using TOPO Cloning vectors (Invitrogen Corporation, Carlsbad, California, USA) per the manufacturer's instructions. Fifteen clones were sequenced using dye terminator sequencing and analyzed using the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, California, USA).
Phylogenetic analysis of pol and env sequences
Sequences were initially compiled, aligned, and edited in BioEdit using CLUSTAL W alignment tool. The alignment was then manually edited to preserve frame insertions and deletions. Phylogenetic analyses were performed using Phylogeny Inference Package software (PHYLIP) and FastDNAml (J. Felsenstein, 1993. PHYLIP version 3.5c. University of Washington, Seattle; G. Olsen, University of Illinois) starting from a neighbor-joining tree using the previously measured parameters from the maximum likelihood tree. To evaluate for possible sample contamination sequences from standard lab strains were included in the phylogenetic analysis.
Length polymorphism analysis
The V4-5 coding region of the env gene from HIV RNA was amplified by a nested PCR using previously described primers and protocols [4]. These fluorescently labeled PCR products were separated by capillary electrophoresis using an automated sequencer (ABI PRISM 3100) and analyzed by GeneScan Analysis Software (Applied Biosystems, Foster City, California, USA). These experiments were performed in triplicate.
Dye-primer sequencing
From HIV RNA isolated from blood plasma, a 500-base region of the pol gene was sequenced using ABI PRISM BigDye Primer v.3.0 kits with -20 M13 and M13 primers (Applied Biosystems, Foster City, California, USA) according to the manufacturer's protocols. Base mixtures were determined from ABI trace files using Vector NTI v.5.0 (InforMax, Frederick, Maryland, USA). Values from forward and reverse strand sequencing were averaged and correlated with those obtained from control samples [9].
References
1. Blackard JT, Cohen DE, Mayer KH. Human immunodeficiency virus superinfection and recombination: current state of knowledge and potential clinical consequences. Clin Infect Dis 2002; 34:1108-1114.
2. Goulder PJ, Rowland-Jones SL, McMichael AJ, Walker BD. Anti-HIV cellular immunity: recent advances towards vaccine design. AIDS 1999; 13(Suppl A):S121-S136.
3. Altfeld M, Allen TM, Yu XG, Johnston MN, Agrawal D, Korber BT, et al. HIV-1 superinfection despite broad CD8+ T-cell responses containing replication of the primary virus. Nature 2002; 420:434-439.
4. Koelsch KK, Smith DM, Little SJ, Ignacio CC, Macaranas TR, Brown AJ, et al. Clade B HIV-1 superinfection with wild-type virus after primary infection with drug-resistant clade B virus. AIDS 2003; 17:F11-F16.
5. Jost S, Bernard MC, Kaiser L, Yerly S, Hirschel B, Samri A, et al. A patient with HIV-1 superinfection. N Engl J Med 2002; 347:731-736.
6. Ramos A, Hu DJ, Nguyen L, Phan KO, Vanichseni S, Promadej N, et al. Intersubtype human immunodeficiency virus type 1 superinfection following seroconversion to primary infection in two injection drug users. J Virol 2002; 76:7444-7452.
7. Yang O, Daar E, Jamieson B, Balamurugan A, Smith D, Pitt J, Petropoulos C, Richman D, Little S, Leigh-Brown A. HIV-1 Clade B superinfection: evidence for differential immune containment of distinct clade b strains. J Virol 2005; 79:860-868.
8. Gulick RM, Ribaudo HJ, Shikuma CM, Lustgarten S, Squires KE, Meyer WA 3rd, et al, AIDS Clinical Trials Group Study A5095 Team. Triple-nucleoside regimens versus efavirenz-containing regimens for the initial treatment of HIV-1 infection. N Engl J Med 2004; 350:1850-1861.
9. Strain MC, Gunthard HF, Havlir DV, Ignacio CC, Smith DM, Leigh-Brown AJ, et al. Heterogeneous clearance rates of long-lived lymphocytes infected with HIV: intrinsic stability predicts lifelong persistence. Proc Natl Acad Sci USA 2003; 100:4819-4824.
10. Albert J, Wahlberg J, Leitner T, Escanilla D, Uhlen M. Analysis of a rape case by direct sequencing of the human immunodeficiency virus type 1 pol and gag genes. J Virol 1994; 68:5918-5924.
11. Yirrell DL, Robertson P, Goldberg DJ, McMenamin J, Cameron S, Leigh Brown AJ. Molecular investigation into outbreak of HIV in a Scottish prison. BMJ 1997; 314:1446-1450.
12. Gonzales MJ, Delwart E, Rhee SY, Tsui R, Zolopa AR, Taylor J, et al. Lack of detectable human immunodeficiency virus type 1 superinfection during 1072 person-years of observation. J Infect Dis 2003; 188:397-405.
13. Zhang L, Chung C, Hu BS, He T, Guo Y, Kim AJ, et al. Genetic characterization of rebounding HIV-1 after cessation of highly active antiretroviral therapy. J Clin Invest 2000; 106:839-845.
14. Deeks SG, Wrin T, Liegler T, Hoh R, Hayden M, Barbour JD, et al. Virologic and immunologic consequences of discontinuing combination antiretroviral-drug therapy in HIV-infected patients with detectable viremia. N Engl J Med 2001; 344:472-480.
15. Simon V, Padte N, Murray D, Vanderhoeven J, Wrin T, Parkin N, et al. Infectivity and replication capacity of drug-resistant human immunodeficiency virus type 1 variants isolated during primary infection. J Virol 2003; 77:7736-7745.
16. Gottlieb GS, Nickle DC, Jensen MA, Wong KG, Grobler J, Li F, Liu SL, et al. Dual HIV-1 infection associated with rapid disease progression. Lancet 2004; 363:619-622.
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