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Intermittent HIV-1 Viremia (Blips) and Drug Resistance in 10 Patients Receiving HAART
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10 patients were studied, a rather small study. It appears that most of the blips were low in magnitude, <100 c/ml. So it's important to bear in mind that this study did not look at patients with viral load increases to above 100 or 200 c/ml.
Richard E. Nettles, MD; Tara L. Kieffer, PhD; Patty Kwon, BA; Daphne Monie, PhD; Yefei Han, BS; Teresa Parsons, PhD; Joseph Cofrancesco, Jr, MD; Joel E. Gallant, MD, MPH; Thomas C. Quinn, MD; Brooks Jackson, MD; Charles Flexner, MD; Kathryn Carson, ScM; Stuart Ray, MD; Deborah Persaud, MD; Robert F. Siliciano, MD, PhD
JAMA. Feb 16, 2005 2005;293:817-829.
ABSTRACT
Context: Many patients infected with human immunodeficiency virus type 1 (HIV-1) and receiving highly active antiretroviral therapy experience intermittent episodes of detectable viremia ("blips"), which may raise concerns about drug resistance, lead to costly repeat measurements of viral RNA, and sometimes trigger alterations in therapy.
Objective: To test the hypothesis that blips represent random biological and statistical variation around mean steady-state HIV-1 RNA levels slightly below 50 copies/mL rather than biologically significant elevations in viremia.
Design, Setting, and Patients: Between June 19, 2003, and February 9, 2004, patients receiving therapy underwent intensive sampling (every 2-3 days) over 3 to 4 months to define the frequency, magnitude, and duration of blips and their association with drug levels and other clinical variables. Blips were defined as HIV-1 RNA measurements greater than or equal to 50 copies/mL preceded and followed by measurements less than 50 copies/mL without a change in treatment. To determine whether blips result from or lead to drug resistance, an ultrasensitive genotyping assay was used to detect drug resistance mutations before, during, and after blips. Patients were 10 HIV-1-infected asymptomatic adults recruited by clinicians and followed up in the Moore Clinic at the Johns Hopkins Hospital. Patients had suppression of viremia to below 50 copies/mL while receiving a stable antiretroviral regimen for 6 months or longer.
Main Outcome Measures: At each time point, plasma HIV-1 RNA levels were measured in 2 independent laboratories and drug resistance mutations were analyzed by clonal sequencing.
Results: With the intensive sampling, blips were detected in 9 of 10 patients. Statistical analysis was consistent with random assay variation around a mean viral load below 50 copies/mL. Blips were not concordant on independent testing and had a short duration (median, <3 days) and low magnitude (median, 79 copies/mL). Blip frequency was not associated with demographic, clinical, or treatment variables. Blips did not occur in relation to illness, vaccination, or directly measured antiretroviral drug concentrations. Blips were marginally associated (P = .08) with reported episodes of nonadherence. Most importantly, in approximately 1000 independent clones sequenced for both protease and reverse transcriptase, no new resistance mutations were seen before, during, or shortly after blips.
Blips were low in magnitude (median, 79 copies/mL; range, 51-201 copies/mL), with a clustering of values toward the 50 copies/mL limit and only 1 value above 200 copies/mL. Brief blips of low magnitude are also consistent with the random-variation hypothesis. (ed: so it sounds like blips were <200 c/ml so there was no comparison with blips >200 c/ml, an important point).
Conclusion: Most blips in this population appear to represent random biological and statistical variation around mean HIV-1 levels below 50 copies/mL rather than clinically significant elevations in viremia.
AUTHOR DISCUSSION
Given that in adherent patients HAART decreases viremia to a new steady-state level slightly below 50 copies/mL (eg, 15-20 copies/mL),23 we hypothesized that normal biological and statistical variation would result in occasional values above 50 copies/mL without a clinically significant elevation in plasma HIV-1 RNA levels. Our analysis of the frequency, magnitude, reproducibility, duration, clinical associations, and genotypic consequences of blips are all consistent with this hypothesis.
With respect to frequency, blips were detected in a greater percentage of the patients than in previous studies,8-13 probably because of the intensive sampling used here. In previous studies, blips were detected in 11% to 46% of patients.8-11,13 Since the sampling interval we used (every 2-3 days) is on the same time scale as the decay rate of virus-producing cells (t1/2, approximately 1 day),54 it is unlikely that significant elevations in viremia were missed. Based on the statistical arguments presented above, the number of blips detected is likely to be directly related to the frequency of sampling. However, the fraction of positive viral RNA measurements will be related to the patient's steady-state level of viremia regardless of sampling frequency. Blips occurred with a frequency (3.6% of measurements) that was similar to that observed prior to entry (4.8%) and that was consistent with random variation around a steady-state viral RNA level of 10 to 20 copies/mL (Table 2). Of course, this steady-state level will vary among patients, accounting for the variation in blip frequency observed herein (0 blips in patient 140 and 5 blips in patient 154) and elsewhere.14, 55
The intensive sampling used herein allowed a more precise definition of blip duration. Most of the blips consisted of a single measurement preceded and followed by measurements below 50 copies/mL. The short duration of most blips documented herein is in contradistinction to longer estimates made with mathematical models that infer the shape of blips from clinical data with sparse sampling.14 The magnitude of observed blips was low (median, 79 copies/mL), with clustering of values close to the detection limit. This is consistent with the tail of a normal log10 distribution centered around 20 copies/mL. Blip magnitude correlated poorly between laboratories. The poor reproducibility of blips below 200 copies/mL has been noted previously.56 At a true viral RNA level of 20 copies/mL, any pair of duplicate or consecutive measurements would rarely be concordantly positive (probability = 2.2% [derived from data in Table 2]), consistent with the poor reproducibility ({kappa} = 4.4%) and short duration (median, 2.5 days) observed herein.
There was no association between blips and demographic, treatment, or HIV-associated clinical factors. Furthermore, blips were unrelated to intercurrent illnesses, vaccination, or decreases in antiretroviral drug concentrations. Blips were marginally associated with self-reported nonadherence (P = .08). Again, these findings are consistent with the hypothesis presented above. Recent work by Miller et al57 also found no link between blips and nonadherence. Extensive analysis of drug concentrations over time revealed wide intrapatient variation but no correlation between drug concentrations and blips, raising concerns about the usefulness of therapeutic drug monitoring in the management of patients experiencing blips.
Despite previous reports that blips represent resistant virus,18-19,22 our analysis of a total of 951 and 1079 independent clones for the protease and RT regions, respectively, failed to identify any new genotypic resistance before, during, or immediately after blips. One potential explanation for the discrepancy is that the extensive sampling used here allowed a more precise definition of preexisting resistance so that the appearance of new mutations could be more accurately assessed.
These findings provide an explanation for the work of Havlir et al8 and others9-11 demonstrating that blips do not predict virological failure. We suggest that isolated low-level positive viral RNA measurements may not be cause for clinical concern. Of course, consistently detectable viremia can be associated with resistance,6, 58-59 and further studies will be needed to define when detectable viremia should trigger a change in therapy. Given a steady-state level of 20 copies/mL, 96.4% of blips due to random variation will fall below 200 copies/mL. Therefore, blips with a magnitude of greater than 200 copies/mL or blips that are detected in at least 2 independent or consecutive measurements may be more of a cause for concern.
In conclusion, among patients with suppression of viremia to below 50 copies/mL, most blips appear to represent normal biological and statistical variation around mean levels that are below 50 copies/mL rather than clinically significant elevations in the level of viral replication. These conclusions are based on an intensive study of a small group of patients and may not be representative of all patients. Thepatients studied had started therapy with low CD4 cell counts and high viral load levels, and it will be important to confirm these results in patients who start therapy earlier in the course of infection.
RESULTS
Patient Demographics and Treatment Histories
To analyze blips, we studied 10 patients receiving HAART who had stable suppression of viremia to below 50 copies/mL (Table 1). The patients ranged in age from 39 to 59 years. Seven were men and 3 were women; 7 were black and 3 were white. The study was carried out between June 19, 2003, and February 9, 2004. Most patients had started HAART after reaching low CD4 nadirs with high viral RNA levels. Some had received prior nonsuppressive antiretroviral therapy, but all were eventually started on a HAART regimen that produced prolonged suppression of viremia to below 50 copies/mL (median, 34 months; range, 11-79 months). With their current regimens, 4 patients had blips detected during routine clinical care prior to entry, and there were 6 total blips out of 125 prior viral RNA measurements (4.8%). These blips were detected a mean of 15.7 months (range, 2-30 months) before study entry, and in all cases viral loads returned to below 50 copies/mL without a change in therapy. The median prior blip magnitude was 94 copies/mL (range, 61-108). In these patients, viral load measurements were generally performed every 3 months as part of routine clinical care and were not influenced at all by participation in the study because these viral load measurements predated study participation.
Blip Frequency and Dynamics
To capture as many blips as possible, we obtained plasma samples every 2 to 3 days for 3 to 4 months for duplicate HIV-1 RNA measurements in 2 independent laboratories. The higher value was used because any value above 50 copies/mL may be considered a blip by clinicians and we wanted to capture as many blips as possible. All patients completed 36 study visits. Patients were permitted to take breaks from the study protocol when the General Clinical Research Center was closed for holidays and when patients requested time for out-of-town travel. In general, we attempted to gather data in a "3-visit" or "weekly" clustered fashion to minimize isolated study visits. For study conclusions, it was considered more important to have a large number of study visits clustered within a relatively short time vs having strictly consecutive study visits. The mean and median times to completion of the 36 study visits were 99.4 days and 97.5 days, respectively (range, 88-127 days). The minimum possible time to complete the study was 82 days. Mean and median numbers of gaps in consecutive study visits were 3.3 and 3, respectively.
Blips were detected in 9 of 10 patients. Of 713 viral RNA measurements, 26 (3.6%) were above 50 copies/mL. Together these constituted 18 total blips, with consecutive positive measurements counted as a single blip. Patients experienced a median of 2 blips (range, 0-5). The observed proportion of positive assay results was consistent with random variation around a mean level of 10 to 20 copies/mL. Nine blips were detected by one laboratory, 8 by the other laboratory, and 1 by both. Thus, although there was no difference in the sensitivity of assays used by the 2 laboratories, concordance was poor ({kappa}=4.4%). This result is expected if blips result from random variation around a mean substantially below 50 copies/mL.
Frequent sampling also allowed us to better estimate the true duration and magnitude of blips. Fifteen of 18 blips represented isolated measurements above 50 copies/mL, with the subsequent measurement negative. Thus, the typical blip was brief (median duration, 2.5 days; range, 2-11.5 days). Only 1 patient (patient 154) experienced blips that persisted for more than 1 consecutive study visit. Blips were low in magnitude (median, 79 copies/mL; range, 51-201 copies/mL), with a clustering of val ues toward the 50 copies/mL limit and only 1 value above 200 copies/mL. Brief blips of low magnitude are also consistent with the random-variation hypothesis.
Association of Blips With Immune Activation, Adherence, and Drug Concentrations
Blip frequency was not associated with demographic parameters such as sex, race, and age. There was no association with clinical parameters such as CD4 cell nadir, CD4 cell count at entry, pretreatment viral load, duration of infection, duration of virological suppression, and number of prior blips. Blips were not associated with therapeutic variables such as the number of drugs in the current regimen (Table 3). Blips were not observed with intercurrent illnesses (pharyngitis/sinusitis, cold/upper respiratory tract infection, gout flare, oral herpes outbreak, or gastrointestinal tract upset), or influenza vaccination (given during the study to 9 of 10 patients). Blips were marginally (P = .08) associated with patient-reported nonadherence.
To determine whether blips were temporally associated with decreased drug concentrations, the protease inhibitor and nonnucleoside RT inhibitor concentrations in plasma were measured at each time point in each patient. Large intrapatient fluctuations in drug concentrations were noted in some patients (patients 99, 136, and 148). Importantly, there was no association between low drug concentrations and blips (P = .22 by {chi}2 test). Most blips (78%) occurred when drug levels were above the suggested trough concentrations (Figure 1).
Genotypic Analysis of Blips
Although plasma virus levels were below 50 copies/mL at most time points, the protease and RT regions of plasma viral RNA were successfully amplified and sequenced before, during, and after blips in 9 of the 10 patients. An average of 4 to 5 clones were obtained per time point, for a total of 951 independent protease clones (830 in nonblip samples, 121 in blip samples) and 1079 independent RT clones (916 in nonblip samples, 163 in blip samples). As is shown in Figure 2, virus detected during blips did not have new drug resistance mutations. The virus detected during blips was either wild type or had mutations that were present in the baseline sampling of plasma and the cellular reservoir or in plasma samples obtained at time points prior to the blip. These results are compatible with the idea that there may be no accumulation of new drug resistance mutations associated with blips.
Phylogenetic analysis and genotypic data for a representative patient (136) are shown in Figure 3. Clones obtained from the resting CD4 cell reservoir at baseline and from the 36 plasma samples clustered together away from sequences from other patients. Virus present during a blip was not phylogenetically distinct from nonblip samples, indicating a lack of viral evolution during blips. Some blip sequences were identical to sequences in the cellular reservoir. Phylogenetic analysis using network trees44 also failed to show increased divergence of blip sequences. Analysis of genetic diversity (theta) at each time point did not show increased diversity during blips. The median theta for all patients was 0.0051 (interquartile range, 0.002-0.010) for nonblip visits and 0.0061 (interquartile range, 0.003-0.012) for blip visits (P = .37). Most importantly, no drug resistance mutations were detected in any of the sequences from this patient, who had received no prior nonsuppressive therapy before starting HAART.
In contrast to patient 136, the remaining 8 patients had received prior nonsuppressive therapy, and resistance mutations attributable to the nonsuppressive therapy could be detected (Figure 2). However, no new drug resistance mutations were seen in the 121 protease sequences and the 163 RT sequences obtained during blips in this study. All blip sequences were either wild type or contained mutations that were seen prior to the blip (Figure 2). Blip samples did not have a higher proportion of resistant clones. The degree of resistance was not associated with blip magnitude or frequency. The patient with the blip of greatest magnitude (patient 136) had only wild-type clones, and the patient with the most frequent blips (patient 154) had only 1 major protease mutation (I84V) and no RT mutations that would confer resistance to the current regimen. The K103N mutation detected during 1 blip was selected by prior therapy with efavirenz. During the study period, the patient was not receiving any drug that would select for this mutation.
The genotypic analysis also suggested that blips do not lead to resistance. New resistance mutations were not found immediately after blips. In 455 independent protease sequences and 575 independent RT sequences obtained in the 30 days following a blip, no new resistance mutations were detected. In 1 patient (patient 99), the protease mutation M46I appeared 8 weeks after a blip and then disappeared. Given the patient's history of poor adherence and prior exposure to multiple protease inhibitors, this is likely to be an archival mutation not detected in baseline sampling. Taken together, these results refute the notion that resistance arises during or immediately after blips.
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