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Incidence of Zidovudine-Resistant Human Immunodeficiency Virus Isolated from Patients before, during, and after Therapy Sally Land, Catherine McGavin, Ron Lucas, and Chris Birch
Virology Department and Medical Division, Fairfield Hospital. Australia
Zidovudine (azidothymidine) has been used extensively for the treatment of human immunodeficiency virus (HIV) infection [I], and many groups have demonstrated that HIV isolated from individuals receiving long-term therapy is resistant to the drug in vitro [2-5]. Although no common clinical manifestations have been linked with the emergence of resistant virus, the clinical deterioration frequently observed after many months of good health after commencement of zidovudine therapy often coincides with the isolation of resistant virus, To examine the pattern of development of resistance in more detail, we followed 237 patients receiving the drug for up to 3 years.
Materials and Methods Patients. HIV isolates were obtained from 237 individuals attending Fairfield Hospital as in- or outpatients. Most (95%) were male homosexuals, but isolates from female sex partners, intravenous drug users, and hemophiliacs were also included. The group comprised both asymptomatic (30%) and symptomatic (70%) individuals. Treatment regimen. Dosage and continuity of zidovudine therapy varied from patient to patient depending on the year the drug was first administered and the degree of hematologic toxicity subsequently experienced. The drug became available in Australia in 1987, and patients initially were given 1200 mg! day. The advent of adverse side effects necessitated dose reduction to 600 mg/day after 9 months of treatment for --80% ofthe patients. Pauses in therapy (=::;3 weeks) were necessary for some patients, usually during treatment of opportunistic infections. For the purpose of analysis such breaks were disregarded. After
Received 17 October 1991; revised II May 1992. Reprints or correspondence: Dr. Sally Land. Virology Department. Fairfield Hospital. Yarra Bend Rd .. Fairfield 3078, Victoria. Australia.
The Journal of Infectious Diseases
1992;166:1139-42
© 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6605-0025$01.00
August 1989, when evidence from other studies [6, 7] indicated that lower doses ofthe drug were effective in slowing the progression of disease, most patients were treated with 500 mg/day. Virus isolates. HIV was isolated from peripheral blood mononuclear leukocytes (PMNL) [8]. Briefly, infected PMNL were cocultured with an equal number of phytohemagglutininstimulated donor PMNL in RPMI 1640 containing 10% heat-inactivated fetal calf serum and 10% recombinant human interleukin-2 (IL-2) medium. Virus replication was detected using an HIV antigen EIA (Vironostika; Organon Teknika, Turnhout, Belgium). Specific blocking antibody was used to confirm all positive test results. Aliquots of clarified culture supernatant were stored at -70°C and used in subsequent drug sensitivity assays. The infectivity titer of each isolate was established using the assay described below, in the absence ofzidovudine. Reference isolates of known zidovudine sensitivity (AO18 H112-2 and AOl2 G762-3 [pretherapy] and AOl8 G910-6 and AOl2 G691-6 [posttherapy] provided by D. Richman [2]) were obtained through the AIDS Research and Reference Reagent Program, AIDS Program, National Institutes of Health (NIH; Bethesda, MD). Zidovudine sensitivity assay. Zidovudine (Wellcome Foundation, London) was dissolved in dimethyl sulfoxide to 10 mg/ mL, and working dilutions were prepared in IL-2 medium at the start of each assay. Isolates were tested for sensitivity to the drug by the method of Land et al. [3]. To facilitate testing of large numbers of isolates, the cell culture and reverse transcriptase (R T) assays were done in 96-well microtiter plates. Donor PMNL (l0 5 cells/well) were infected with 100 TCID so of virus for 2 h before addition of 5.0, 0.5, or 0.05 J.Lg/mL zidovudine. All assays were done in quadruplicate, and each isolate was simultaneously titrated to check input dose. Known sensitive and resistant isolates were included in each assay. Drug concentrations were maintained through half-volume medium changes and through subsequent addition of donor PMNL (lOS/well) 7 days after infection. Virion-associated RT activity was assayed at day 10 [9]. Virus was precipitated from 150 J-LL ofculture supernatant by the addition of 50 J-LL of 30%polyethylene glycol 6000 at 4°C overnight in a microtiter plate (96-U Immunoplate; Nunc, Kamstrup, Denmark). Sealed plates, housed in airtight boxes. were centri-
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The zidovudine sensitivity of 372 isolates of human immunodeficiency virus (HIV) obtained from 237 patients before, during, and after treatment with zidovudine was examined. Virus resistant to >0.5 J.Lg/mL (1.87 p,M) zidovudine was isolated from most patients (93%) after 36 months of therapy. Zidovudine-sensitive virus was isolated from 5 of 15 patients who had ended antiretroviral therapy but had previously shed resistant virus. The emergence of sensitive virus after end of therapy appeared to be influenced by both the duration of treatment and the time off drug. Patients with resistant virus tended to have low CD4 cell counts and HIV antigenemia at the commencement of therapy, suggesting that these two factors are important in the development of drug resistance.
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Concise Communications
Results Development of zidovudine resistance. Table I shows the zidovudine sensitivity profiles of 362 isolates from 235 patients. Usually I or 2 isolates were available from each patient, although up to 6 isolates were obtained from some. All of the 77 pretherapy isolates tested were completely inhibited (ID IOO) by 0.5 Jlg/mL (1.87 J,LM) zidovudine. To assist us in discriminating between sensitive and resistant isolates, we tested 25 of the pretherapy isolates against an expanded range of zidovudine (0.0027-0.5 Jlg/mL). None was inhibited by 0.0027 J,Lg/mL, and only 3 were inhibited by 0.0 I J,Lg/mL. A further 17 were inhibited by 0.05 J,Lg/mL while 5 of25 required 0.5 J,Lg/mL for complete inhibition. Therefore, we considered isolates that replicated in the
Table 1. Concentration ofzidovudine required to completely inhibit HIV replication (ID lOo) by duration of therapy. Duration of therapy (months)
No. (%) of isolates with 10 100 in
0 1-5 6-9
10-12 13-18 19-36 NOTE.
~g/mL
0.05
0.5
5.0
>5.0
%of resistant" isolates
65 (84) 26 (54) 16 (27) 6 (15) 3 (5) 3 (4)
12 (16) 16 (33) 15 (25) 6 (15) 8 (12) 2 (3)
5 (10) 21 (35) 22 (55) 26 (41) 45 (61)
I (2) 8 (13) 6 (15) 27(42) 23 (32)
0 12 48 70 83 93
362 isolates were tested from 235 patients.
* Resistance is defined as replication in presence of>0.5 ~g/mL zidovudine (see Results).
40
...c .! ...as (I)
...a.o o
z
•
Zidovudine-Resistant
•
Zidovudine-5ensitive
30
20
10
o 300
CD4/ul Figure 1. Relationship between CD4 cell counts at start oftherapy and development ofzidovudine-resistant HIV.
presence of 0.5 J,Lg/mL to be zidovudine-resistant. In addition, we assayed 4 isolates of known zidovudine sensitivity obtained from the NIH repository. ID so of the drug-sensitive (pretherapy) isolates was 0.027 Jlg/mL (0.1 J,LM) and IDIOO was 0.5 Jlg/mL. The two posttherapy isolates tested were resistant to the drug, with IDsos of 2.0 and 4.2 J,Lg/mL (7.6 and 15.8 JlM) and IDIOOs of >5.0 Jlg/mL. Therefore, the assay we used detected a 76- to > IOO-fold difference in sensitivity between the reference sensitive and resistant isolates at both the ID so and ID IOOlevels. There was a clear progression to resistance with increasing duration of therapy (see table I). Whereas only 12% of the isolates tested were resistant after 5 months of therapy, 93% were resistant by 36 months. Even though most patients yielded resistant virus by 12 months after start ofzidovudine treatment, we found 16 isolates that were sensitive to the drug after this time (see table I) and 7 were still sensitive after 36 months. CD4 cell counts from the patients from whom the isolates were obtained were 20-960/JlL. Correlation of surrogate markers with development of resistance. CD4 cell levels at the start of therapy were available for I 17 patients. There was a significant trend for individuals with low CD4 cell counts «200/J,LL) to develop resistant virus within the initial 18 months of treatment, unlike patients with counts >200/JlL (P < .001, X Z test, see figure I). Of 49 patients with CD4 cell counts < 100/JlL, resistant virus could be isolated from 40 (82%). Twenty-five (71 %) of 35 patients with counts of 100-200/J,LL also yielded resistant virus. These isolation rates were reduced in patients with higher counts (60% and 29% in those with 200-299 and >300 cells/rd. respectively). Analysis ofCD4 cell counts at any time point after the beginning of therapy did not alter this pattern, nor did analysis by percentage of CD4 lymphocytes in the total white cell population rather than absolute CD4 cell numbers (data not shown).
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fuged at 3000 g for 30 min, and supernatant was removed by inversion. Virus pellets were solubilized by addition of 15 J,LL of 0.5% Triton X-lOO, and 90 J,LL was added ofa standard reaction mix cocktail, which included poly r(A)· d(T)1O as the template primer, MgClz, and [3H]thymidine triphosphate. After 2 h at 37°C, the reaction mixtures were transferred to a microtiter plate containing three 5-mm DE-81 disks/well and incubated at room temperature for 30 min. After the disks were washed extensively (5 times in 5% wt/vol Na zHP0 4 , once in distilled water, and once in 70% vol/vol ethanol) using an EIA plate washer on low suction, incorporated label was quantitated using a ,B-radiation counter. The sensitivity of this micro RT assay was equivalent to the standard method [8] (results not shown). Levels of incorporation five times greater than background were considered positive. Isolates that were not inhibited by 0.5 J,Lg/mL zidovudine were considered resistant (see Results). CD4 cell and HIV antigen evaluation. CD4 lymphocytes in erythrocyte-lysed whole blood were stained (IMK Plus Kit; Becton Dickinson Immunocytometry Systems, Mountain View, CA) and counted in a FACScan (Becton Dickinson) flow cytometer. Serum samples were tested for HIV antigen using a solidphase, sandwich-type EIA (Abbott Laboratories, Abbott Park,IL).
JID 1992; 166 (November)
lID 1992: I66 (November)
Concise Communications
Discussion This study followed the development of zidovudine resistance in a representative population ofHIV-infected individuals in Melbourne. It examined the zidovudine susceptibility of 372 HIV isolates obtained before, during, and in several cases after treatment of 237 individuals with the drug. The treatment regimen varied, depending on the recommended dosage at the time each patient first received the drug, compliance after initiation of therapy, and tolerance of side effects. Chronically poor compliers were excluded from the study; however, data from patients experiencing breaks in therapy of 1-3 weeks were included in the analysis. We have developed a laboratory procedure that can be used for routinely testing the drug sensitivity of HIV isolates, and the assay has been validated by detection of the RT sequence changes commonly associated with zidovudine resistance [11] (results not shown). The use of PMNL is optimal for performance of these assays because only about onethird of the HIV isolates we obtain replicate in MT-2 cells or the other continuous T lymphoblastoid cell lines available to us (results not shown). Other groups have used plaque assays in CD4-transfected HeLa cells to do similar sensitivity evaluations [2], but the failure of many isolates to replicate in these cells [12] limits them for use in routine drug sensitivity testing. By defining the end point of our assay as complete inhibition of RT activity (ID IOO) , a cutoff for sensitivity of 0.5
JLg/mL (1.87 JLM) was established. All pretherapy isolates, including 2 zidovudine-sensitive reference isolates, were inhibited by this concentration of the drug. This cutoff is higher than that reported by other groups, who used 50% inhibition end points in either plaque-reduction or RT assays [2, 4]. Our results confirm that HIV isolated from patients on long-term zidovudine therapy is usually resistant to the drug in vitro. While in a minority of cases resistant virus can be isolated during the first few months of treatment, the typical time for the development of resistance is 6-9 months, after which nearly half of the patients yield resistant virus. While development of resistance is not an absolute consequence of therapy during the period covered by this study, most individuals (93%) shed resistant virus after 3 years. Our results, like those of others, also showed a strong correlation between ability to isolate resistant virus and CD4 cell counts