Cytomegalovirus (CMV) disease, especially retinitis, is a common opportunistic complication of advanced HIV infection. The incidence increases markedly when the CD4 cell count falls below 100×106cells/l [1-3], and before the use of protease inhibitors, at least 20% of patients with counts below that level will develop CMV-related disease over a 2year period . Data from several studies show that methods for the detection of CMV viraemia, such as polymerase chain reaction (PCR) or the detection of pp65 antigenaemia, could identify those patients at highest risk of developing CMV disease and also those patients at risk of decreased survival [5-8].
However, the immunological and clinical benefit observed with the widespread use of protease inhibitor-containing therapy has changed the natural history of CMV disease . Preliminary data suggest an important reduction in the incidence of opportunistic infections, including CMV retinitis, for patients treated with highly active antiretroviral therapy (HAART) . Moreover, observational studies show cases of CMV disease in patients with more than 100×106cells/l, a rare presentation before protease inhibitors were introduced into the clinical management of HIV patients . However, little information exists on the incidence, risk factors, and clinical presentation of CMV disease for patients receiving HAART. In addition, no data are available on the evolution of CMV markers during protease inhibitor therapy, or on the association of CMV viraemia and outcome in a large cohort of HIV-infected patients in the era of HAART.
Therefore, the aim of this study was to estimate the incidence of CMV retinitis and to determine risk factors for the development of the disease in a cohort of HIV-infected patients with a CD4 cell count below 100×106cells/l, and who initiated protease inhibitor-containing therapy.
Patients and methods
Patients were enrolled in the cohort at 10 AIDS care units participating in a multicentre group for the study of CMV disease in AIDS. Patients with HIV infection were enrolled if they had a CD4 cell count <100×106cells/l, positive CMV immunoglobulin G serology, and were to initiate antiretroviral therapy containing a protease inhibitor. Patients were evaluated in this study for whom baseline and follow-up plasma samples were available. The first patient was included in February 1996 and the last patient in April 1997. Those patients with past or present CMV disease, or using anti-CMV therapy, were excluded.
A complete medical history and physical examination, CD4 cell count, and HIV RNA estimate were performed at baseline, 1 month, 3 months, and every 3 months thereafter. Plasma samples for CMV detection was collected at the same time points during the first 6 months of therapy at each of the participating study centres. CD4 cell count was assessed by flow cytometry, and HIV RNA was determined using PCR (Amplicor HIV Monitor, Roche Molecular Systems, Branchburg, New Jersey, USA). CMV viraemia was detected by a commercially available qualitative PCR technique (Amplicor CMV, Roche Diagnóstica, Madrid, Spain). CMV DNA quantification was also performed on the positive samples using a quantitative CMV microwell plate assay, developed by Roche Molecular Systems .
In each follow-up visit, patients were evaluated for signs or symptoms of CMV retinitis. Those with visual signs or symptoms were referred to an ophthalmologist experienced in the diagnosis of CMV retinitis. Retinal disease due to CMV was diagnosed in the presence of white and granular lesions associated with haemorrhage that usually follows the vascular distribution producing large areas of necrosis. Funduscopic retinal photographs were not required.
Screening ophthalmological examinations were not required at baseline or during follow-up for asymptomatic patients.
Differences in demographic details according to initial PCR status were tested using the Mann-Whitney U test for quantitative variables and the χ2 test for qualitative variables. Time to development of disease was estimated using the Kaplan-Meier method, and the relationships between PCR status and time to CMV retinitis were compared by log-rank test, with patient follow-up censored at the time of death or on the 31 May 1998 if the patient had not developed CMV disease. A Cox proportional hazards model was developed to determine the influence of the baseline covariates on the development of CMV retinitis.
During the study period, 418 HIV-infected patients with CD4 cell counts below 100×106cells/l initiated protease inhibitor therapy. Baseline plasmas from 172 patients with a positive IgG serology against CMV were available, and were evaluated for CMV DNA load by qualitative PCR. Overall, 11% (18 out of 172) of the patients were CMV PCR-positive at study entry. Baseline characteristics of the patients according to initial PCR status were similar. Most patients had been heavily pre-treated with nucleoside analogues (86%), and had experienced a prior AIDS-defining condition (76%). Median CD4 cell count at entry was 28×106cells/l, and median HIV RNA was 5.12log10copies/ml.
On initiation of protease inhibitor therapy, a rapid improvement of CD4 cell count and HIV load was observed. By the third month, median CD4 cell count was 97×106cells/l (range, 6-640) and HIV RNA was 3.62log10copies/ml (range, 2.3-6.4). At the same time, and excluding those patients who developed CMV disease, the number of patients who were initially CMV PCR-positive decreased from 11% at study entry to 7% (11 out of 151) at the end of the first month of protease inhibitor therapy, to 2% (two out of 99) at the end of the third month, and to 0% (of 69 patients) at the end of the sixth month of therapy. Seven per cent of patients (nine out of 137) who were CMV PCR-negative at baseline had PCR-positive results at the end of the first month of protease inhibitor therapy. However, by the third month of treatment their CMV PCR results were once again negative.
In a median follow-up of 24 months (range, 13-26), the cumulative incidence of CMV retinitis was 5% at 1 year and 6% at 2 years. Most cases of disease were observed for patients who had a baseline CD4 cell count below 50×106cells/l, and occurred during the first 90 days after the initiation of protease inhibitor-containing therapy (six out of nine patients, 67%). A detailed analysis of the nine cases of CMV retinitis showed that the disease developed in three patients with CD4 cell counts above 50×106cells/l, and in one patient with a count above 200×106cells/l (Table 1).
A positive CMV PCR result at baseline was significantly associated with the development of CMV retinitis (P<0.001, log-rank test; Fig. 1). Thus, the 12 months Kaplan-Meier CMV retinitis event rate was 38% in patients who were CMV PCR-positive, compared with 2% for those who were CMV PCR-negative. In a Cox proportional hazards model, a positive CMV PCR result at study entry was also significantly associated with the development of CMV retinitis (relative hazard, 4.41; 95% confidence interval 2.12-8.93; P<0.001). There was no significant association with CMV disease risk for CD4 lymphocyte count, HIV load, protease inhibitor used, previous AIDS diagnosis, or prior antiretroviral treatment (Table 2). The association between a positive baseline CMV PCR result and CMV retinitis was not observed for those patients with transient positive CMV PCR results at the first month of protease inhibitor therapy (P=0.23; log-rank test), and no patient developed CMV retinitis. Quantitative analysis of patients who were initially CMV PCR-positive showed that mean CMV load at entry was significantly higher in patients who went on to develop CMV retinitis than those who did not (3700 versus 384copies/ml; P=0.002). Moreover, 75% of patients with a CMV load higher than 1000copies/ml developed the disease. When combined with a CD4 cell count below 50×106cells/l, this cut-off value identified 100% of the patients who went on to develop CMV disease. Characteristically, mean CMV load in patients with transient CMV PCR positivity was low (559 copies/ml, range 275-1256).
Despite the strong association between CMV PCR status and development of CMV retinitis, clinical, virological and immunological outcome was similar for patients with or without initial CMV viraemia. After 15 months on protease inhibitor therapy, median CD4 cell count (240 versus 245×106cells/l; P=0.91) and HIV RNA (3.01 versus 3.32log10copies/ml; P=0.46) were similar and independent of initial CMV PCR status. During follow-up, 13 patients died, three of them shortly after the diagnosis of CMV retinitis. Causes of death were non-Hodgkin lymphoma in three cases, MAC disease in two, chronic hepatitis in four, and gram-negative sepsis, interstitial pneumonia, disseminated tuberculosis and central nervous system lymphoma in one each. No death was attributed to CMV disease. There was no statistically significant difference in mortality rate according to initial CMV viraemia (P=0.87; log-rank test).
This study shows an important reduction in the incidence of CMV retinitis for patients who initiated protease inhibitor therapy. For patients with CD4 cell counts below 100×106cells/l at study entry, the cumulative incidence after 2 years was 6%. This is significantly lower than the 20-28% at 1 year reported in recent studies for a similar population of patients not receiving protease inhibitor therapy [13,14]. Furthermore, the incidence in this study is lower than in a study of CMV prophylaxis in patients receiving oral ganciclovir that reported a CMV event rate of 14% at 1 year . This impressive benefit appears to be attributable to the improvement of the patient‚s immunological status while on protease inhibitor therapy.
Most cases of CMV retinitis in our study were observed during the first three months of therapy. During this period, the increase in CD4 cell count is considered to be insufficient to provide adequate protection [16,17]. Indeed, recent data demonstrate that disruptions in the T-cell receptor repertoire observed in advanced HIV disease may not be corrected in the first weeks following initiation of protease inhibitor therapy . Evidence from previous studies also suggests that patients in our study may have already seeded their retinas with CMV, or may have had an asymptomatic peripheral retinitis, before initiating protease inhibitor therapy. However, this fact has been found in a minority of patients . Furthermore, when patients with more than three months of protease inhibitor therapy are considered separately, the cumulative incidence of CMV retinitis in our study was 2% at 2 years, a figure similar to that found in CMV PCR-negative patients receiving prophylactic oral ganciclovir .
The results of this study are strikingly similar to recent findings for the incidence of other opportunistic infections such as Mycobacterium avium complex infection or cryptosporidiosis, in severely immunosuppressed patients taking protease inhibitors . However, we found a lower incidence of patients with CMV viraemia at baseline in our study compared with similar cohorts of patients in other studies (11 versus 28-45%) [7,13]. This lower number of CMV PCR-positive results can be explained by differences in the sensitivity of PCR methods. In a comparison of several CMV markers, Boivin et al. described a sensitivity of 65% for plasma-based PCR (Amplicor CMV; Roche Molecular Systems, Branchburg, New Jersey, USA) and 100% for leukocyte-based PCR assays . However, the positive predictive value of the plasma-based PCR was higher than the leukocyte-based assay (64 versus 40%). Therefore, despite having fewer CMV PCR-positive patients in our study, we believe that the low incidence of CMV retinitis could be attributed to the initiation of protease inhibitor therapy.
As with previous studies, our data shows a 4.4-fold increased risk of developing CMV disease for patients who had CMV viraemia [5-8,14]. A positive CMV PCR result was also a better predictor for the risk of developing CMV disease than CD4 lymphocyte counts. In addition, quantitative PCR identified those patients at highest risk for CMV retinitis, a group in which it is worth performing controlled trials of pre-emptive therapy. This study provides important data that may alter the current prevention and management of CMV retinitis. Firstly, our study confirms the progressive decrease of CMV load after the initiation of antiretroviral therapy containing protease inhibitors, reflected by the increase in negative qualitative CMV PCR results in patients with low CMV load . The results also suggest that an increase in CD4 cell count may be sufficient to avoid the development of CMV disease, allowing prophylaxis or pre-emptive therapy to be reserved for those high-risk patients in the short time period after the initiation of protease inhibitor therapy. Secondly, our study suggests that a transient positivity of CMV PCR, with low CMV load, in the first few weeks after the initiation of therapy is not necessarily indicative of increased risk of disease. The cause of this transient positivity is not known, and one hypothesis could be that it reflects the ongoing immune restoration. Finally, at variance with previous reports, patients in our study who were initially CMV PCR-positive and who did not develop CMV disease, had a similar clinical, immunological, and virological response while on therapy to those who were CMV PCR-negative. These data indicate that the risk of death directly related to CMV viraemia in persons with advanced AIDS declines after the initiation of protease inhibitor therapy, and change the previously-reported role of CMV viraemia as an independent predictor of poor prognosis [5,7]. It is not known if the survival benefit seen in our study is directly related to immune restoration or if it is secondary to the reduction of CMV viraemia.
In summary, our study indicates that protease inhibitor therapy is effective in reducing the incidence of CMV viraemia and disease in HIV-infected patients with a CD4 cell count below 100×106cells/l at the time of therapy initiation. The results also offer a new approach to the management of CMV disease in HIV-positive patients, where prevention strategies could be implemented during a short period after the initiation of protease inhibitor therapy. Furthermore, the significant improvement in survival for patients who were initially viraemic supports the fundamental role of protease inhibitors in this immunocompromised population. Finally, controlled trials of CMV prophylaxis or pre-emptive therapy should be conducted in patients who have a high CMV load and who are therefore at high risk of CMV retinitis. These studies should also take into account the contribution of protease inhibitor therapy to the reduction of CMV viraemia.
We are indebted to M. Garrido and I. Diaz, from Roche Pharmaceuticals, for their assistance and support. Also, thanks to R. Manjiry and C. Boucher for the determination of CMV load.
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Grupo de Estudio de CMV y SIDA
In addition to the authors, the following investigators are members of the CMV-AIDS study group. Hospital Ntra.Sra.de Aránzazu (San Sebastian): J.A. Iribarren, F. Rodriguez-Arrondo, M.A. von Wichmann, M. Dolores de Juan. Hospital de Cruces (Bilbao): M. Montejo. Hospital Germán Trías i Pujol (Badalona): B. Clotet. Hospital Miguel Servet (Zaragoza): P. Arazo. Hospital Provincial La Rioja (Logroño): J.A. Oteo. Hospital Ramón y Cajal (Madrid): M.J. Perez-Elías, A. Antela, F. Dronda. Hospital Reina Sofía (Tudela, Navarra): M.T. Rubio. Hospital San Millan (Logroño): P. Labarga. Hospital de Teruel (Teruel): M. Yuyol. Hospital Txagorritxu (Vitoria): J.M. Agud.
Keywords:© 1999 Lippincott Williams & Wilkins, Inc.
Cytomegalovirus; retinitis; opportunistic infections; protease inhibitor; HIV