Objective: To investigate the association between an unexpected increase in the blood plasma HIV-1 viral load in chronically untreated HIV-infected patients and the occurrence of an HIV superinfection, we analyzed the HIV-1 quasispecies in plasma samples before and at peak level in 14 patients.
Results: Phylogenetic analysis of HIV-1 env-V3 fragments showed that in 2 patients a superinfection had occurred: their dominant V3 population at the peak level clustered separately from the V3 sequences in a sample predating the peak level. The rapid rise in viral load could be attributed to upper respiratory tract infections or a vaccination in 4 patients, suggesting that even minor health problems can result in significantly increased HIV-1 replication. In most other patients, no minor or major medical condition accompanied the rise in HIV-1 viral load, implying that in these patients the viral load increase was probably associated with disease progression.
Conclusion: This study suggests that an unexpected rapid rise in the plasma HIV-1 viral load of untreated patients can infrequently be ascribed to an HIV-1 superinfection.
From the *Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Amsterdam, The Netherlands; †Department of Internal Medicine, Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands; and the ‡International Antiviral Therapy Evaluation Centre (IATEC), Amsterdam, The Netherlands.
Received for publication February 19, 2007; accepted August 13, 2007.
Correspondence to: Antoinette C. van der Kuyl, PhD, Laboratory of Experimental Virology, Department of Medical Microbiology, CINIMA, Academic Medical Centre of the University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands (e-mail: email@example.com).
In treated HIV-1-infected individuals, a sudden rise in plasma viral load is mostly associated with noncompliance to highly active antiretroviral therapy (HAART) or with therapy withdrawal for other reasons. During therapy, intermittent episodes of low-level viremia, called “blips,” are common and can be associated with drug resistance.1-4 However, patients not treated with antiretroviral therapy can also experience unexpected rapid rises in the plasma viral load that sometimes resolve spontaneously later on. Opportunistic infections, sexually transmitted infections such as syphilis, and vaccinations can cause such an increase in the HIV-1 viral load.5-10 Alternatively, significant rises in viral load have been associated with a HIV-1 superinfection.11-17 To explore the association between sudden rises in plasma viral load and the occurrence of HIV-1 superinfection, we analyzed the HIV-1 quasispecies in 14 patients who were followed at our HIV outpatient clinic between 1996 and 2006 and who experienced an unexpected increase in plasma viral load that was not due to treatment interruption.
SUBJECTS AND METHODS
For this cross-sectional, retrospective study, patients were selected based on plasma viral load follow-up data collected during the years 1996 to 2006 from a group of 1596 HIV-1-seropositive individuals followed at the Academic Medical Center in Amsterdam, The Netherlands. Follow-up per patient varied from 0 to 28.1 years, with an average of 7.0 years. The occurrence of viral load increase was examined in 2005 and 2006. In this group the male to female ratio is approximately 4:1, and 77% to 79% of the patients were treated with HAART in 2005 to 2006. Patients selected either had never had any antiretroviral treatment or had stopped all antiviral treatment at least 1 year before the sudden increase in the viral load was detected, in order to exclude individuals with viral load increase due to treatment interruptions, changes, or failure.
During regular follow-up, plasma viral load measurements are on average performed at 3-month intervals. After some important event, such as a large increase in viral load, patients are monitored more often, usually every 4 weeks. Patients selected experienced a sudden, at least 5-fold rise in viral load from 1 follow-up moment to the next, eg, within 12 weeks of an earlier measurement, in the year 2005 or 2006. Individuals with a gradual rise of <5 times were excluded to circumvent patients with ongoing disease progression. Here we assumed that a sudden, substantial increase in the viral load represents an acute, new HIV infection. Whether or not the viral load decreased spontaneously after the peak level was not taken into account. In a primary HIV infection, the viral load normally decreases after the acute phase to a much lower level when specific immune responses are developed. Often, but not always, after HIV-1 superinfection the plasma viral load is persistently increased. Of the 14 patients selected, 10 had never been treated with any antiretroviral therapy, and the other 4 had stopped all therapy 1 to 3.5 years before the rapid rise in viral load. Patients A, D, F, G, I, K, L, and P initially presented at our clinic with a primary HIV-1 infection defined by the detection of antigen in an initial test and by an incomplete pattern on subsequent Western blot analysis.
In 8 patients, the viral load decreased again spontaneously, 3 others initiated HAART immediately, and in 1 patient the load did not decrease significantly but HAART was not initiated because his CD4 cell count remained stable. For 2 patients, no data were available. Two of the patients in whom the viral load decreased spontaneously nonetheless initiated HAART 3 to 4 months after the viral load peak. The CD4 cell count at the HIV-1 load peak did not change significantly in 10 patients and decreased in 2 patients (Table 1). For 2 patients, no CD4 counts could be retrieved. Because acute syphilis infection has been related to an increase in the HIV-1 plasma viral load,8-10 syphilis serology was also tested (Table 1). Additionally, medical records were searched to find any events coinciding with the defining peak level.
HIV-1 Plasma Viral Load Determination
Blood plasma HIV-1 RNA was measured using the Versant HIV-1 RNA 3.0 assay (bDNA) (Bayer Diagnostics Division, Tarrytown, NY), which has a detection level of 40 copies/mL. Samples with a viral load above 5 × 105 copies/mL were diluted 1:10 and tested again because the assay is not validated above this value. The other samples were only tested once, inasmuch as this assay has excellent reproducibility and is approved for clinical use.18
Amplification and Sequencing
Samples taken before and at the peak level were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) amplification and population sequencing of a 1302-nucleotide pol fragment using the ViroSeq HIV-1 genotyping kit version 2.0 (Celera Diagnostics, Alameda, CA). Furthermore, a 264-nucleotide V3 fragment of the HIV-1 env gene was amplified and sequenced.19 Amplification products of V3 were cloned with the Topo TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced with the BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). Electrophoresis and data collection were performed on an ABI Prism 3100 genetic analyzer (Applied Biosystems). Sixteen clones were analyzed per sample. The lower limit of detection of this method is 8% of a minority viral population. A 720-nucleotide fragment of the HIV-1 gag gene, encompassing most of p17 and the first part of p24, was amplified, cloned, and sequenced from 2 patients to collect additional evidence for an HIV-1 superinfection.20
Sequences were aligned with reference HIV-1 pol, env-V3, and gag sequences (from the Los Alamos National Laboratory, available at: http://hiv-web.lanl.gov) using ClustalW, available in BioEdit Sequence Alignment Editor Version 7.0.1 (Ibis Biosciences, Carlsbad, CA; available at: http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Nucleotide distances were estimated with the Tamura-Nei21 distance, using the gamma model to correct for multiple hits and to account for different rates of substitution between nucleotides and the inequality of nucleotide frequencies. The gamma shape parameter α for HIV-1 env-V3 (0.38) and HIV-1 gag (0.25) were taken from Leitner et al.22 Neighbor-joining (NJ) trees based on Tamura-Nei distances were constructed with the MEGA 3.1 software package (available at: http://www.megasoftware.net), and 1000 bootstrap replicates were analyzed. Additional phylogenetic analyses were done with the parallel version of MrBayes 3.1 (available at: http://mrbayes.net), modified so that the program uses the Scalable Parallel Random Number Generators Library (available at: http://sprng.cs.fsu.edu/) to generate independent streams of random numbers. MrBayes 3.1 was run at the SARA High Performance Computing facilities (available at: http://www.sara.nl).
The HIV-1 quasispecies was analyzed to detect the occurrence of an HIV-1 superinfection in 14 cross-sectionally selected patients who experienced an unexpected increase in the plasma viral load in 2005 to 2006 that was not due to treatment interruption (Table 1). All patients were followed at our HIV outpatient clinic between 1996 and 2006, and serial samples were available.
None of the patients showed a switch from nonsyncytial-forming (CCR5-using) to syncytial-forming (CXCR4-using) viruses at the peak level as determined from the deduced env-V3 amino acid sequences (not shown). Phylogenetic analysis of pol and env-V3 fragments showed that the sequences of the 2 time points clustered together with high bootstrap values in 12 patients, suggesting that these patients had not acquired an HIV superinfection at the second time point (not shown). However, in 2 patients, L and P, the dominant population of both pol and env fragments at the peak level was completely different from the earlier time and formed a separate, highly supported cluster in the phylogenetic trees (not shown). All viruses were subtype B strains. At the second time from patient L, only 2 of 16 V3 clones represented the first virus, whereas 13 of 16 clones were from a novel subtype B strain. One fragment was found to be a recombinant between the 2 strains. The patient was HIV-1 infected at this maximum measured level for half a year. Further analysis showed that in another sample predating the peak, only the V3 sequence of the first strain was demonstrable, whereas in a fourth sample postdating the peak, the second strain was exclusively detectable. All clones obtained at the peak level from patient P were from a completely different subtype B strain compared to the measurement before the viral load peak (not shown). Patient P was also known to be HIV positive for approximately half a year, identical to patient L. To confirm the HIV-1 superinfection in these patients, a 720-nucleotide fragment of the HIV-1 gag gene was amplified and sequenced.20 Phylogenetic analysis of these gag fragments confirmed the acquisition of novel, distinct subtype B strains in both patients at the defining peak level (not shown).
In this study, we wanted to address the question “How many people with a sudden rise in the plasma viral load have an HIV-1 superinfection?” by examining a small, representative subset of HIV-1 infected individuals visiting our hospital. Of course, this small study size limits the conclusions drawn from it, so the estimate of HIV-1 superinfection occurrence in the total cohort is very imprecise. But it does show that there are many reasons for a sudden, substantial increase in the HIV-1 plasma viral load, and that an HIV-1 superinfection is at best only a minor explanation. From 14 patients experiencing such a sudden rise in the HIV-1 blood plasma viral load, 2 increases were found to be due to an HIV superinfection. Both superinfected patients reported continuing unsafe sexual practices after their primary HIV-1 infection. The main risk factor for HIV-1 superinfection is probably risk exposure, which is dependent upon 2 aspects: unsafe sexual behavior and HIV prevalence. If HIV prevalence is low or safe sex is practiced, an HIV superinfection becomes less likely. Other protective factors presumably are a broad-neutralizing antibody response15,16,23 and the use of ART,24 although superinfection with a drug-resistant virus under therapy has been described.25 Cytotoxic T-cell lymphocyte responses probably play a lesser role in superinfection prevention, as a broad and effective CD8 T-cell response did not protect against HIV-1 superinfection.12,16
Although the lower limit of detection of our cloning method is only 8% of a minority viral population, it is questionable that we missed any superinfections because of this. It is unlikely that an incoming virus comprising <8% of the total population will give rise to a massive increase in the plasma viral load of the virus already in residence.
The medical records of the other 12 patients were searched retrospectively to find any explanation for the peaking of their HIV-1 plasma viral load. In 3 patients (B, H, and N), the HIV-1 viral load peak coincided with an upper respiratory tract infection, whereas 1 patient (patient I) had had vaccinations for a holiday in the tropics at that time. For 6 other patients, their medical records revealed no condition that could explain the rapid increase in HIV-1 copy numbers. This suggests that the rise in viral load was associated with disease progression. Because this study was performed retrospectively, it was impractical to query the patients for any events at the time of the peak level load that had not been filed. For 2 other patients, medical records were lacking. Five patients had positive syphilis serology compatible with an old infection during the viral load increase (Table 1), but this has never been associated with HIV-1 viral load rises. The only patient (L) with an active syphilis infection that could have been associated with an increase in HIV-1 copy numbers experienced a simultaneous HIV-1 superinfection. It is possible that this active syphilis infection facilitated the second HIV-1 infection.
This study implies that a sudden rise in plasma viral load is not a very reliable predictor of an HIV-1 superinfection, as it only found such a relationship in 14% of the patients with a sudden rise in plasma viral load. Furthermore, neither the increase factor nor the absolute number of HIV-1 copies was indicative of an HIV-1 superinfection (Table 1). HIV-1 coinfections (second infection before seroconversion) and superinfections (second infection after seroconversion) are notoriously difficult to detect, both clinically and in the laboratory. However, because many dual infections have been associated with either a higher viral set point or with disease progression,26-28 there is a clinical demand for an easy detection technique. Recently, we described a novel method that uses ambiguity in the ViroSeq genotyping sequence of the HIV-1 pol gene as an indicator of HIV-1 dual infections.29 This method was found to predict HIV-1 double infections in approximately half of the suspected cases and could thus prove more reliable than relying on unexpected viral load peaks to forecast double infections. Additionally, this method29 can be used to detect dual infections at any time point, whereas unexpected increases in the plasma viral load can at best only suggest superinfections, not HIV coinfections.
In conclusion, unexpected plasma viral load peaks occurring in chronically HIV-1-infected patients are infrequently associated with an HIV-1 superinfection. Minor medical conditions such as upper respiratory tract infections or vaccinations seem more likely to increase HIV-1 replication. Our 2 superinfection cases occurred within a half year of the original HIV-1 infection, consistent with prior observations.17 Thus, suddenly increased HIV-1 levels in the first 1 to 2 years after primary infection may be more suggestive for HIV-1 superinfection than those occurring later.
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