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Departments of Ophthalmology, Medicine
Pathology, The Johns Hopkins University School of Medicine
Department of Epidemiology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland
Background.
The amount of cytomegalovirus (CMV) DNA in the blood (CMV load) may be a marker for detection of resistant CMV.
Methods.
A total of 165 patients with AIDS and CMV retinitis had CMV load measurements (plasma and leukocyte) and cultures performed every 3 months; these measurements were correlated with CMV resistance to antiviral drugs and CMV retinitis progression (from masked readings of retinal photographs).
Results.
Detectable plasma and leukocyte CMV loads were associated with CMV retinitis progression (odds ratios [OR], 6.3; P < .0001 and OR, 6.6; P < .0001, respectively), phenotypic resistance (OR, 6.1; P < .0001 and OR, 23.4; P = .0002, respectively), and genotypic resistance (OR, 17.5; P < .0001 and OR, 51.6; P = .0004, respectively). The sensitivity, specificity, and positive and negative predictive values of plasma CMV loads were 0.47, 0.86, 0.36, and 0.91, respectively, for progression and 0.59, 0.81, 0.07, and 0.99, respectively, for resistance; those of leukocyte CMV loads were 0.52, 0.83, 0.35, and 0.91, respectively, for progression and 0.82, 0.78, 0.08, and 0.99, respectively, for resistance. A detectable plasma CMV load at the time of diagnosis of CMV retinitis was associated with mortality (median survival time, 13.6 vs. 29.7 months; P = .007).
Conclusions.
CMV load has limited clinical utility, because of its low positive predictive value. Its high negative predictive value for occurrence of resistant CMV suggests that it may have utility as a screening tool to exclude resistance.
Cytomegalovirus (CMV) retinitis is among the most frequent opportunistic infections in patients with AIDS [13]. Although highly active antiretroviral therapy (HAART) has resulted in an 80% decline in the incidence of CMV retinitis, this decline has leveled off, and new cases continue to occur [17]. Unless immune reconstitution occurs, long-term suppressive anti-CMV therapy is required to prevent relapse of retinitis, since, in immunocompromised patients, relapse occurs promptly after discontinuation of anti-CMV therapy [8, 9]. Although CMV retinitis may occur in patients with AIDS who are naive to HAART, the large majority of patients with CMV retinitis are experienced with HAART and either have not responded to it or could not tolerate it. As such, most patients with CMV retinitis will require long-term suppressive anti-CMV therapy and will be at risk of harboring resistant CMV [10].
Resistance to ganciclovir or foscarnet has been reported to occur at the rate of 25%/person-year, with similar rates reported for both drugs [1113]. Phenotypic resistance is measured by the ability of CMV to grow in the presence of an anti-CMV drug, by either a plaque reduction assay or a DNA hybridization assay, and typically is expressed as the IC50. However, standard phenotypic-resistance testing requires sufficient amounts of CMV from culture specimens and may take 6 weeks to perform, limiting its clinical utility. Genotypic resistance is the presence of a mutation known to confer a resistant phenotype. For CMV, resistance to ganciclovir occurs because of mutations either in the UL97 gene, a phosphotransferase, or in both the UL97 and UL54 genes, the latter of which is the CMV DNA polymerase [1422]. For foscarnet, resistance occurs because of mutations in the UL54 gene, but these mutations are in different regions from those for ganciclovir resistance [13, 2124]. To sequence CMV, either an isolate from culture specimens or a polymerase chain reaction (PCR)amplified gene from a blood specimen is required. For ganciclovir and foscarnet, there is good to excellent genotype-phenotype agreement [13, 14] and excellent agreement between the virus detected in the blood and that detected in the eye [25]. Since resistance to anti-CMV agents is associated with poor outcomes [13, 26], techniques to rapidly identify patients who harbor resistant CMV are needed.
CMV load refers to the amount of viral DNA (or RNA) in the blood and can be measured by several methods, the most frequently used of which is PCR [27]. Detectable CMV load in patients with AIDS predicts the occurrence of CMV disease, is associated with increased mortality in the pre-HAART era [2833] and in the HAART era [34] and with the ability to isolate CMV from blood cultures in patients with CMV retinitis, and may correlate with CMV retinitis progression [27]. As such, it has the potential to be a marker for patients who harbor resistant CMV.
The Cytomegalovirus Retinitis and Viral Resistance (CRVR) study is a prospective cohort study of the occurrence of resistant CMV, the molecular biology of resistance, and the clinical implications of resistance in patients with AIDS and CMV retinitis [35]. In the present study, the CRVR Research Group evaluated the role that CMV load plays in the follow-up of patients with CMV retinitis.
PATIENTS AND METHODS
Patients.
The CRVR study enrolled patients with both AIDS and previously untreated CMV retinitis, at 1 of 3 clinical centers: The Johns Hopkins University School of Medicine, the Northwestern University Medical School, and the University of Miami School of Medicine. Patients were treated for CMV retinitis in accordance with the clinician's judgment, and treatments were administered in a standardized fashion [35]. Patients treated with the ganciclovir implant typically also received either oral ganciclovir or valganciclovir. Because of the time lag in obtaining virologic test results, treatment decisions were made on the basis of clinical judgmentthat is, without the knowledge of resistance testing and CMV load results. Patients were seen monthly for ophthalmologic examinations, at which time standardized fundus photographs were taken. One set of photographs was sent to the CRVR Fundus Photograph Reading Center (FPRC) at the University of Wisconsin, Madison, where evaluation of CMV retinitis progression was performed by graders masked to the results of laboratory tests and to treatment. The definition of CMV retinitis progression was the standard one used for clinical trials and epidemiologic studies of CMV retinitisnamely, the movement of a border of a CMV lesion 750 m along a front 750 m in size or the occurrence of a new lesion greater than or equal to one-quarter disc area in size. Treatment decisions were made without the knowledge of the results of the FPRC gradings [35].
Susceptibility testing.
Before the initiation of therapy, blood and urine specimens for CMV isolation were collected from all patients. Follow-up cultures were performed at 1 and 3 months after enrollment and every 3 months thereafter. Culture specimens were processed locally at each clinical center, as described elsewhere [35, 36]. All CMV isolates were tested for viral susceptibility at the virology laboratory at The Johns Hopkins Hospital by use of either a DNA hybridization assay (Hybriwix Probe System/CMV Susceptibility Test Kit; Diagnostic Hybrids) or a plaque reduction assay [35, 37, 38]. Previous data have shown good to excellent correlation between the 2 techniques [11, 13, 39]. For ganciclovir, an isolate was defined as resistant if the IC50 was >6 mol/L [11, 40, 41]; for foscarnet, an isolate was defined as resistant if the IC50 was >600 mol/L [13]. If there was no growth of CMV from a culture (negative CMV culture), the patient was assumed to harbor a sensitive virus.
All CMV isolates were sequenced for mutations in either the UL97 or the UL54 gene at the virology laboratory at The Johns Hopkins Hospital, as described elsewhere [1315]. Patients were classified as harboring a virus with genotypic resistance to ganciclovir if an appropriate mutation in the UL97 gene was detected, and they were classified as harboring a virus with genotypic resistance to foscarnet if an appropriate mutation in the UL54 gene was detected. Because all UL54 mutations contributing to high-level ganciclovir resistance occurred in the presence of a UL97 mutation, only UL97 mutations were considered for classification of an isolate as susceptible or resistant to ganciclovir [14, 15, 25]. Because the genotype-phenotype correlation and the thresholds for defining an isolate as cidofovir resistant are less well defined [12], only ganciclovir and foscarnet resistance were evaluated in the present study.
CMV load testing.
At the time that cultures were obtained, a blood specimen was obtained for measurement of the amount of CMV DNA (CMV load) in plasma and in leukocytes. Specimens were prepared locally at each clinical center and were shipped to the virology laboratory at The Johns Hopkins Hospital for CMV load testing. Quantification of the CMV load was performed by use of the COBAS Amplicor CMV Monitor test system (Roche Diagnostics), in accordance with the manufacturer's instructions, as described elsewhere [27]. Briefly, after manual nucleic acid extraction, the samples were amplified, detected, and quantified on an automated COBAS Amplicor instrument. This assay quantifies CMV DNA by coamplifying a 365-bp fragment of the CMV polymerase gene in the presence of a known amount of quantitation standard (QS). The QS has primer-binding sites identical to those of the target sequence but with a modified probe-binding site to enable differentiation of the QS-specific amplicon from the target amplicon. The dynamic range for quantification was 3 log10 units; the lower limit of detection was 400 copies. Plasma CMV loads are expressed as copies per milliliter, and leukocyte CMV loads are expressed as copies per 106 leukocytes [27].
Statistical analysis.
Time-dependent analyses were performed for the association of CMV load with CMV retinitis progression and with occurrence of resistant CMV, for the following reasons: (1) CMV retinitis progression occurs in nearly all patients treated with systemic anti-CMV therapy [1, 26, 42], because of the limited intraocular penetration of these drugs [43, 44], even without the occurrence of resistance; and (2) regardless of whether a patient harbors a resistant virus, treatment regimens and CMV load may change over time [15, 27]. Patient follow-up was divided into 3-month blocks centered on the collection of specimens for CMV culture and CMV load measurement. The resistance status of each interval was determined by whether the patient harbored a virus resistant to ganciclovir or foscarnet while receiving either of these drugs [13, 26]. Both phenotypic resistance and genotypic resistance were analyzed. Because immune reconstitution due to HAART can control CMV retinitis progression without anti-CMV therapy [9, 45, 46] and because the ganciclovir implant is associated with longer times to CMV retinitis progression than is systemic therapy [47, 48], both of these factors were included in the analysis of CMV retinitis progression. For the resistance analyses, implant use was not included, since it does not affect the detection of a resistant virus in the blood, but HAART was included. The associations of CMV load with CMV retinitis progression and occurrence of resistant CMV were analyzed by use of generalized-estimating-equations logistic regression [13, 26, 49]. Because resistance does not occur before treatment and because a detectable CMV load is present in the majority of untreated patients, only follow-up data were used for the analyses of these associations. The sensitivity, specificity, and positive and negative predictive values of CMV load for both CMV retinitis progression and occurrence of resistant CMV were calculated by use of thresholds of 400, 1000, 10,000, and 50,000 copies/mL for plasma and 400, 1000, 10,000, and 50,000 copies/106 leukocytes for leukocytes, and receiver operator curves were constructed [50]. Survival analyses were performed by use of the Kaplan-Meier method, and hazard ratios (HRs) for mortality were determined by use of Cox proportional hazards regression. The institutional review board at each participating institution approved the study, and all participants gave written, informed consent.
RESULTS
CMV retinitis progression.
CMV retinitis progression occurred at a rate of 0.60/person-year. Risk factors for CMV retinitis progression are shown in table 2. Both plasma CMV loads >400 copies/mL (odds ratio [OR], 6.3; P < .0001) and leukocyte CMV loads >400 copies/106 leukocytes (OR, 6.6; P < .0001) were associated with an increased risk of CMV retinitis progression. The ganciclovir implant was associated with a 50%60% reduction in the odds of CMV retinitis progression. Although there was an association of detectable CMV load with CMV retinitis progression, the sensitivity, specificity, and positive and negative predictive values for CMV retinitis progression (table 3)in particular, the sensitivity and positive predictive valuewere limited for both plasma and leukocyte CMV loads. To determine whether different thresholds of CMV load resulted in better performance of the test, we analyzed sensitivity and specificity at the higher thresholds (figure 2). None of these thresholds markedly improved the performance of CMV loads for prediction of CMV retinitis progression. At the highest thresholds (50,000 copies/mL and 50,000 copies/106 leukocytes), the sensitivity, specificity, and positive and negative predictive values were 0.12, 0.98, 0.13, and 0.98, respectively, for plasma and 0.24, 0.97, 0.17, and 0.98, respectively, for leukocytes.
Resistant CMV.
Antiviral drug resistance in CMV occurred at the rate of 0.09/person-year. Phenotypic and genotypic resistance each occurred at the rate of 0.07/person-year, because of the occasional discordant result between phenotypic and genotypic measures of resistance. Plasma and leukocyte CMV loads (table 2) each were associated with phenotypic resistance (OR, 6.1; P = .0009 and OR, 23.4; P = .0002, respectively) and with genotypic resistance (OR, 17.5; P < .0001 and OR, 51.6; P = .0004, respectively). Although there was a strong association between detectable CMV load and occurrence of resistant CMV, as detected by either method, the sensitivity, specificity, and positive and negative predictive values of CMV load for occurrence of resistant CMV (table 3)in particular, the sensitivity and positive predictive valuewere limited for both plasma and leukocyte CMV loads. However, the negative predictive value of either plasma CMV load or leukocyte CMV load for either phenotypic or genotypic resistance was 0.99. To determine whether different thresholds of CMV load resulted in better performance of the test, we analyzed sensitivity and specificity at higher thresholds (figure 2). Although higher thresholds improved the specificity of both plasma CMV load and leukocyte CMV load for either measure of resistance, the cost was a drop in sensitivity, and, even at the highest thresholds, there were limited positive predictive values. At the threshold of 50,000 copies/mL, the positive predictive values for phenotypic and genotypic resistance were 0.13 and 0.21, respectively; at the threshold of 50,000 copies/106 leukocytes, the positive predictive values for phenotypic and genotypic resistance were 0.12 and 0.17, respectively.
DISCUSSION
These data demonstrate an association between CMV load and both CMV retinitis progression and the occurrence of resistant CMV. Despite the strong association between CMV load and these outcomes, the clinical utility of CMV load is limited, largely because of its low positive predictive value for these outcomes. Attempts to improve the performance of CMV load by use of quantitative measures and different thresholds did not result in substantially improved performance of the test. However, the negative predictive value of CMV load for occurrence of resistant CMV was excellent (0.99), suggesting that it might have clinical utility in exclusion of resistant CMV. Because CMV load testing can be completed, and the results known, within 24 h, whereas conventional CMV susceptibility testing takes weeks, it may be a useful screening tool for determination of whom to test for resistant CMV by use of more-conventional means.
Sequencing of the CMV UL97 gene from PCR-amplified blood specimens can be performed directly to detect CMV resistance to ganciclovir and also can be performed much more rapidly than can conventional CMV-susceptibility testing [18]. However, this approach may be more difficult for foscarnet or cidofovir, in which the mutations arise in the much larger UL54 gene. Although resistance-causing mutations in the UL54 gene are being catalogued [5154], because of the size of the UL54 gene, these methods are more labor intensive and more expensive than they are for the UL97 gene. Therefore, screening that uses CMV load to detect persons at risk of resistance, who would then undergo additional testing, and to exclude persons not at risk of resistance may be a useful strategy.
The limited performance of CMV load in the prediction of CMV retinitis progression might have been predicted, since most patients receiving systemic anti-CMV therapy suffer relapse of retinitis because of the limited intraocular penetration of systemic anti-CMV drugs, and not because of resistance [26, 43, 44]. Therefore, events occurring in the blood may be less relevant for CMV retinitis progression. However, there is an excellent correlation between detection of resistant virus in the blood and in the eye [25], and CMV load could have correlated well with the detection of resistant CMV in the blood. Nevertheless, CMV load was detectable in a higher proportion of patients than was resistance, and it identified patients who did not harbor resistant CMV. Leukocyte CMV load had somewhat better sensitivity than did plasma CMV load but had marginally lower specificity. However, both had poor positive predictive values and excellent negative predictive values.
CMV appears to worsen the prognosis of HIV infection. In populations of patients with lower rates of latent CMV infections (such as pediatric and transfusion-related HIV infection), latent CMV infection accelerates the disease process and shortens the time to progression to AIDS [5557]. In populations of patients with AIDS, both CMV disease and detectable CMV load are associated independently with increased mortality, even in the HAART era [34]. Data from the CRVR study demonstrate that detectable plasma CMV load is associated with increased mortality in patients with CMV retinitis. Mechanisms by which CMV may increase mortality include transactivation of HIV [58, 59], production of an interleukin (IL)10 homologue (which binds to the IL-10 receptor and down-regulates Th1 immune responses [60, 61]), production of chemokine receptors (which bind chemokines and inhibit the immune response [62, 63]), and interference with NK cell function (which decreases the host's ability to clear viruses [62, 63]).
A detectable plasma CMV load at the time of diagnosis of CMV retinitis was associated with an increased risk of mortality, as were higher levels of viral DNA in the plasma. However, for leukocyte CMV load, only the highest level (50,000 copies/106 leukocytes) was associated with an increased risk of mortality. Intermediate levels of leukocyte CMV load were not associated with an increased risk. The greater proportion of patients with a detectable leukocyte CMV load than with a detectable plasma CMV load and the lack of an increased risk of the detectable leukocyte CMV load <50,000 copies/106 leukocytes explain why a detectable leukocyte CMV load was not associated with an increased risk of mortality, compared with an undetectable leukocyte CMV load.
Although plasma and leukocyte CMV loads are highly correlated [7], leukocyte CMV load is detectable in a greater proportion of patients than is plasma CMV load and correlates less well with positive CMV cultures [27]. Because CMV is latent in leukocytes, leukocyte CMV loads may be be detectable at lower levels of CMV replication than plasma CMV loads, the detection of which requires viral replication to have shed a sufficient amount of virus into plasma. Therefore, plasma CMV load may be a better marker for higher CMV burden, explaining why detectable plasma CMV load was associated with mortality whereas leukocyte CMV load was associated with mortality only at high copy numbers.
Although the present study was prospective and had a moderately large sample size, there are limitations to it. The event rate for resistance was lower than previously reported rates from the pre-HAART era [1113], resulting in wide 95% confidence intervals around several estimates and in possible type II errors. However, the detected associations had substantial ORs and statistical significance. The occasional discordance between phenotypic and genotypic measures of resistance seen in the present study has been described elsewhere and results from technical limitations of the current technology and the occasional presence of mixed populations of sensitive and resistant CMV [1315, 23, 64]. With conventional cell culture susceptibility testing, detection of a resistant subpopulation requires its presence at >25% of the total population, whereas a mutant virus can be detected genotypically at 10% of the total population [13, 14]. Furthermore, there may be a selection bias introduced by in vitro susceptibility testing when subpopulations have different replication properties [14]. However, analyses of CMV resistance using both measures arrived at similar results for CMV load, suggesting that technical issues did not influence the conclusions.
In conclusion, our data suggest that, in patients with AIDS and CMV retinitis, CMV load is associated with CMV retinitis progression and occurrence of resistant CMV. The clinical utility of CMV load for prediction of CMV retinitis progression is limited by its low sensitivity and positive predictive value, and that for prediction of resistance is limited by its low positive predictive value. The negative predictive value of CMV load for resistance is excellent, and measurement of CMV load may have clinical utility in rapidly excluding CMV resistance and in identifying patients for more-detailed resistance testing. CMV burden, as measured by CMV load, is associated with increased mortality.
THE CYTOMEGALOVIRUS RETINITIS AND VIRAL RESISTANCE STUDY GROUP
Clinical centers.
The Johns Hopkins University School of Medicine, Baltimore, Maryland: Douglas A. Jabs (principal investigator), John G. Bartlett, Stephen G. Bolton, Diane M. Brown, Lisa M. Brune, J. P. Dunn, John H. Kempen, Laura G. Neisser, Richard D. Semba, and Jennifer E. Thorne (current members) and Paul A. Latkany, Susan M. LaSalvia, Tracey Miller, Earline Nanan, Quan Dong Nguyen, Eva Rorer, George Peters, Qazi Faquir, and Armando Oliver (former members); Northwestern University Medical School, Chicago, Illinois: David V. Weinberg, Alice T. Lyon, Annie Muana, and Lori Kaminski; University of Miami, Miami, Florida: Janet L. Davis, Elias Mavofrides, and Elizabeth Fuentes (current members) and Elizabeth Cruz, Tina A. Rhee, and Patricia Vera (former members).
Data center.
The Johns Hopkins University School of Medicine and Bloomberg School of Public Health, Baltimore, Maryland: Barbara K. Martin, Michelle O. Ricks, and Lynn M. Hutt (current members) and Cheryl Enger, Shirley Quaskey, and Judy Southall (former members).
Flow cytometry laboratory.
The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland: Joseph B. Margolick and Fred Menendez.
Fundus Photograph Reading Center.
University of Wisconsin, Madison: Matthew D. Davis, Larry Hubbard, Jane Armstrong, Dolores Hurlburt, Sheri Glaeser, Jeff Joyce, Linda Kastorff, Nancy Robinson, and Marilyn Vanderhoof-Young (current members) and Judy Brickbauer (former member).
Virology laboratory.
The Johns Hopkins Medical Institutions, Baltimore, Maryland: J. Brooks Jackson, Michael Forman, Linda Gluck, and Avareena Schools-Cropper (current members) and Tamica Hamlin, Huiling Hu, and Alicja Rylka (former members).
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