| |
Improved Single Copy Assays for Quantification of Persistent HIV-1 Viremia in Patients on Suppressive Antiretroviral Therapy
|
| |
| |
Download the PDF here
J. Clin. Microbiol
published online ahead of print on 3 September 2014
Anthony R. Cillo,*1 David Vagratian,1 Margaret Bedison,1 Elizabeth Anderson,2 Mary F. Kearney,2 Elizabeth Fyne,1 Dianna Koontz,1 John M. Coffin,3 Michael Piatak,2 John W. Mellors,#1
1University of Pittsburgh, Pittsburgh, PA, USA; 2HIV Drug Resistance Program, NCI9 Frederick, MD, USA; 3Tufts University, Boston, MA; 4SAIC, Frederick, MD, USA
"In summary, we have developed and validated an improved single-copy assay (iSCA) with an improved ability to detect and quantify low levels of plasma viremia. iSCA will undoubtedly be useful for future studies, such as an endpoint of clinical trials with interventions aimed at reducing the size of the HIV-1 reservoir. As the field of HIV-1 cure research continues to evolve, the ability to accurately and precisely quantify residual plasma viremia will be important to evaluate whether novel treatments have caused a change in viremia and whether HIV-1 RNA in plasma remains detectable by the most sensitive means possible after potentially curative interventions."
"The correlation between gSCA and levels of infectious virus suggests that HIV-1 RNA in the plasma may originate from stochastic reactivation of latently infected cells, and that changes in levels of HIV-1 RNA in plasma following an intervention may be reflective of changes in the size of the latent reservoir. Future studies should seek to further elucidate the relationship between persistent plasma viremia and the infectious virus reservoir."
Abstract
A quantitative real-time PCR (qRT-PCR) assay with single-copy sensitivity targeting HIV-1 gag RNA (gSCA) has been used widely to quantify plasma viremia below the limit of detection of clinical assays in patients on effective antiretroviral therapy (ART), but viral RNA in 15-30% of samples amplifies inefficiently because of primer/probe mismatches. We sought to develop improved single-copy assays with increased sensitivity by improving nucleic acid recovery, designing qRT-PCR primers and probe for a highly conserved region of integrase in the HIV-1 pol gene (iSCA), and increasing the plasma volume tested (Mega-iSCA). We evaluated gSCA versus iSCA in paired plasma samples from 10 consecutive patients with viremia >1000 copies/mL and 25 consecutive patients on suppressive ART. Three of 10 viremic samples amplified inefficiently with gSCA compared to the Roche Cobas Ampliprep/TaqMan 2.0, whereas all 10 samples amplified efficiently with iSCA. Among 25 samples from patients on suppressive ART, 8 of 12 samples that were negative for HIV-1 RNA by gSCA had detectable HIV-1 RNA by iSCA, and iSCA detected 3-fold or higher HIV-1 RNA levels compared to gSCA in 10 of 25 samples. Large volume plasma samples (>20 mL) from 7 patients were assayed using Mega-iSCA, and HIV-1 RNA was quantifiable in 6, including 4 of 5 that were negative by standard volume iSCA. These improved assays with superior sensitivity will be useful for evaluating whether in vivo interventions can reduce plasma viremia and for assessing relationships between residual viremia and other virologic parameters including the inducible proviral reservoir.
Introduction
Infection with human immunodeficiency virus type 1 (HIV-1) leads to high levels of cell-free virions in plasma within days following infection(1). Intensive study of HIV-1 plasma viremia has contributed to the current understanding of HIV-1 pathogenesis and has guided the development of effective antiretroviral therapy (ART). In chronically57
infected untreated patients, the amount of virus in plasma reflects the number of productively infected cells and is a strong independent predictor of HIV-1 disease progression to AIDS and death(2, 3). Following the discovery of potent antiretroviral drugs, the decay dynamics of plasma viremia and productively infected cells was possible down to the limit of detection of available HIV-1 RNA assays(4-6). More sensitive qualitative assays revealed the persistence of viremia on combination antiretroviral therapy (ART) (7), indicating that ART alone does not eliminate plasma viremia.
Further studies using a two-step quantitative real-time polymerase chain reaction (qRT-PCR) assay with single copy sensitivity targeting HIV-1 gag (gSCA)(8), revealed that plasma viremia between 1 and 3 copies of HIV-1 RNA per milliliter of plasma persists in most patients independent of the ART regimen used to suppress viremia(9). Additional studies revealed three phases of plasma HIV-1 RNA decay, reflective of HIV70 infected populations of cells with different half-lives, followed by a fourth phase of decay with an infinite half-life during which HIV-1 RNA levels average 1 copy per milliliter for at least 7 years following initiation of ART(10). Whether the persistent viremia on ART represents infectious virus remains unknown, but in some instances, viral sequences in plasma may match sequences of infectious virus recovered from resting CD4+ T cells(11). gSCA has also been used in a number of settings to evaluate whether clinical interventions can affect residual viremia. Several investigators have used gSCA as an endpoint in ART intensification studies to evaluate whether addition of raltegravir or other antiretroviral agents to suppressive ART would reduce persistent low-level plasma virema, and found that there was no effect on plasma viremia(12-14).
In a recent study, incomplete adherence to ART was also found to be associated with higher levels of residual viremia(15). Another study evaluated cellular HIV-1 RNA and plasma viremia following in vivo administration of vorinostat (SAHA), and found that cellular HIV-1 RNA was increased without a concomitant increase in plasma viremia as measured with gSCA(16). Levels of plasma viremia as measured by gSCA have also been evaluated as a potential biomarker of the size of the infectious reservoir in resting CD4+ T cells(17). Despite its widespread use, the original gSCA assay has limitations. First, the extraction method to isolate HIV-1 RNA published in 2003 may not maximize the recovery of nucleic acid from plasma. Second, the qRT-PCR primers and probe were designed to bind a conserved region of gag from HIV-1 subtype B variants from the United States based on sequences available in 2001-2002, but new sequences in the Los Alamos database indicate that there are more highly conserved regions elsewhere in the HIV-1 genome. Third, the volume of plasma extracted in the original description of the assay was limited to 7 milliliters. In theory, sensitivity could be improved by assaying larger plasma volumes, although this has not been shown. We sought to address these limitations of gSCA by improving nucleic acid recovery, designing new primers and probe in a more conserved region of the HIV-1 genome, and increasing the volume of plasma assayed to potentially reduce censoring of data below the limit of detection, thereby improving the ability to detect an effect of interventions on residual viremia.
Discussion
We have developed improved HIV-1 single-copy assays with improved sensitivity to quantify the persistence of plasma viremia in patients on suppressive ART. The original single-copy assay targeting HIV-1 gag sequences (gSCA) has been successfully applied in many studies to gain insight into the persistence of viremia on ART, although inefficient amplification of viral sequences in 15-30% of patients and consequent false negative results have been limitations. We have evaluated these limitations and modified the assay to improve amplification efficiency and lower the limit of detection.
First, we found that isolation of nucleic acid by sequential GuHCl/proteinase K incubation and GuSCN addition, followed by precipitation of nucleic acid recovered more RCAS and HIV-1 RNA in test panels. The higher recovery observed is likely due to the use of GuHCl/proteinase K during the initial incubation, after the virions have been pelleted, rather than using the original extraction method with water and proteinase K. Use of the chaotropic agent GuHCl with proteinase K likely increases the digestion of proteins in protein/nucleic acid complexes as compared with proteinase K in water alone. GuHCl and EDTA also likely inactivate RNases.
Second, to improve amplification efficiency, we targeted a more conserved region of the HIV-1 genome. The high rate of nucleotide substitution in HIV-1 has been extensively studied(30), and contributes to difficulties with sensitivity of nucleic acid based diagnostics for HIV-1(31). Using sequences in the Los Alamos database, we uncovered a region at the 3' end of the pol gene that was more highly conserved than gag and that was also amenable to primer and probe design for qRT-PCR. Selection and use of this integrase region of the HIV-1 genome for qRT-PCR was effective, in that we eliminated primer/probe mismatches in the test panel of 10 viremic patients.
With the improved performance of iSCA for samples from patients with viremia detectable by Roche TM2.0, we proceeded with a head-to-head comparison of iSCA versus gSCA in samples from patients on suppressive ART, and generally with HIV-1 RNA <20 copies/mL by Roche TM2.0. In the suppressed patients, iSCA again had better performance than gSCA as evidenced by: i) detecting viremia in more patients (17 of 25 detectable by iSCA versus 13 of 25 detectable by gSCA with 8 of 12 not detected by gSCA detected by iSCA); ii) measuring ≥3-fold higher HIV-1 RNA values in 10 of 25 with iSCA versus 5 of 25 with gSCA; and iii) trending towards measuring a higher mean level of residual viremia (2.0 cps/mL for iSCA vs.1.3 cps/mL for gSCA). The median levels of HIV-1 RNA detected in samples from suppressed patients were low enough that stochastic variation in the number of virions between samples may have increased the variability in each assay. Other limitations of our study are the relatively small number of patients evaluated and the likelihood that only subtype B samples were tested.Nevertheless, the combination of a new extraction method and new primers and probe improved assay performance for iSCA compared with gSCA.
Finally, we assessed whether the sensitivity of iSCA could be improved by increasing the volume of plasma assayed. The typical limit of detection for gSCA/iSCA is 0.6 copies of HIV-1 RNA per milliliter of plasma, assuming that 3.0 mL of plasma have been assayed. It is possible that patients have levels of residual viremia that are below this limit of detection and thus score as undetectable by iSCA using the standard volume of plasma. Our data support this hypothesis, as we have shown that 4 of 5 patients with undetectable viremia by standard iSCA had detectable viremia by Mega-iSCA. Mega- iSCA is ≥10-fold more sensitive than iSCA, and will be useful for future evaluations of residual viremia and changes in residual viremia following interventions. Plasma volumes of 20-30 mL can be easily obtained from large-volume blood draws (75-120 mL), which is less blood volume than that required for an assay of inducible HIV-1 reservoirs from resting CD4+ T cells(32). Furthermore, the same whole blood sample can be used for both assays.
Two studies have reported a positive correlation between the levels of persistent viremia measured by gSCA and infectious virus recovery from resting CD4+ T cells (17, 33), but another study found no relationship between gSCA and infectious virus recovery(32). The correlation between gSCA and levels of infectious virus suggests that HIV-1 RNA in the plasma may originate from stochastic reactivation of latently infected cells, and that changes in levels of HIV-1 RNA in plasma following an intervention may be reflective of changes in the size of the latent reservoir. Future studies should seek to further elucidate the relationship between persistent plasma viremia and the infectious virus reservoir.
In summary, we have developed and validated an improved single-copy assay (iSCA) with an improved ability to detect and quantify low levels of plasma viremia. iSCA will undoubtedly be useful for future studies, such as an endpoint of clinical trials with interventions aimed at reducing the size of the HIV-1 reservoir. As the field of HIV-1 cure research continues to evolve, the ability to accurately and precisely quantify residual plasma viremia will be important to evaluate whether novel treatments have caused a change in viremia and whether HIV-1 RNA in plasma remains detectable by the most sensitive means possible after potentially curative interventions.
|
|
| |
| |
|
|
|
