Cytomegalovirus (CMV) infection was one of the most important opportunistic infections in HIV-infected patients before the introduction of highly active antiretroviral therapy (HAART). With the introduction of HAART in 1996, life expectancy and quality of life increased dramatically with the persistent suppression of HIV viremia and, in parallel with persistent immune reconstitution, decreased CMV replication and incidence of CMV disease in HIV-infected patients were observed . This was illustrated by cohort study data indicating that the risk of developing CMV disease is highest during the initial months of therapy . Although HAART has greatly controlled end organ disease caused by CMV, CMV viremia is still strongly associated with death in AIDS patients . Also, a substantial number of HIV-infected patients at risk for CMV disease do not receive HAART because of nonadherence or intolerance to prescribed regimens  and CMV disease may recur despite immune reconstitution in selected cases . In addition, evidence is growing that CMV glycoprotein B (gB) genotypes may also be relevant for the risk of CMV disease . To study this, 132 antiretroviral naive patients starting HAART were selected for a 2-year follow-up study in the Netherlands in the period 1997–2004.
Material and methods
The study was conducted in the HIV-outpatient clinic of the Maastricht University Medical Centre, Maastricht (the Netherlands). Within an observational clinical cohort of HIV-1 seropositive individuals participating in the Dutch HIV-monitoring Foundation , all antiretroviral naive patients starting HAART were selected for a 2-year follow-up study in the period January 1997 until September 2004. In addition to HIV-1 viral load, CMV DNA was measured retrospectively before treatment and at least every 3 months during the first 2 years of HAART treatment. Patients infected with HIV-2 were excluded. In total, 1348 frozen samples (−80°C) of 132 patients were available for retrospective CMV DNA measurement.
CMV and HIV-1 quantification: HIV-1 viral loads were determined using the commercially available COBAS Amplicor system and COBAS Amplicor HIV monitor reagents (Roche Diagnostics, Almere, The Netherlands). For CMV quantification a routine singleplex PCR was performed. In short, real-time PCR was performed on a ABI 7000 (Applied Biosystems, Foster City, California, USA) using ABsolute QPCR mastermix (ABgene, Epsom, UK) and primers and Taqman probe sequences obtained from the literature. Using plasmid based probability studies together with daily standard curve analysis; the detection limit of this CMV DNA PCR was determined as 100 copies/ml for reproducible quantitative CMV DNA results. For practical use, results less than 100 copies/ml including less reproducible results in the range 20–99 copies/ml were considered negative.
CMV genotyping: Plasma samples with more than 100 CMV DNA copies/ml were analyzed for CMV glycoprotein B (gB) genotyping using two different methods [8,9]. Analysis of the PCR products was performed by using gel electrophoresis. Details of our comparison of the two methods were presented previously . In short, both methods are suited for CMV gB genotyping and results of samples that were positively genotyped by both methods were all in agreement.
Of 132 patients included in this study, 105 (80%) patients presented with fewer than 100 CMV DNA copies/ml in all plasma samples during the whole follow-up period. Twenty-seven patients (20%) presented with more than 100 CMV DNA copies/ml in at least one plasma sample during the 2-year follow-up period. In seven patients with CD4 cell count below 100 × 106/l and CMV IgG positivity before HAART, the decrease in HIV-1 viral loads was accompanied by a temporary increase in CMV DNA during the first months of HAART. For more details of four illustrative patients, see Fig. 1.
Of 1348 plasma samples, 1298 samples (96%) were negative for CMV DNA, meaning that less than 100 CMV DNA copies per ml plasma were detected. Fifty plasma samples (3.7%) were positive for CMV DNA (range 104–32088 copies/ml) with 100–1000, 1000–10 000 and 10 000–100 000 copies/ml in 37, nine and four samples respectively. CMV DNA more than 1000 copies/ml were found only in samples with detectable HIV-1 viral load (Table 1). Similarly, CMV DNA more than 1000 copies/ml were found only in samples with CD4 cell counts less than 250 × 106 cells/l (Table 1). Of all CMV positive samples, 33 (66%) were found in the first 3 months of HAART including 12 samples with CMV DNA more than 1000 copies/ml. Thereafter, the remaining 17 positive samples were equally distributed throughout the whole remaining period including 16 samples in the range 100–1000 copies/ml (e.g. 104–688 copies/ml) and one isolated positive sample (at day 650) with 8580 copies/ml (Table 1).
In 27 CMV DNA positive patients, all plasma samples with more than 100 CMV DNA copies/ml were analyzed for CMV gB typing. CMV gB typing was successful for 19 patients including four patients with triple CMV infection (1× gB1 + 2 + 3, 1× gB1 + 3 + 4, 2× gB2 + 3 + 4), seven patients with double CMV infection (3× gB1 + 2, 3× gB2 + 3, 1× gB3 + 4) and eight patients in which only one gB type (8x gB3) could be detected. The most prevalent genotype was gB3 (84%; 16 out of 19 patients) followed by gB2 (47%), gB1 (26%) and gB4 (21%). In patients triplly or doublly infected by CMV, peak CMV DNA (mean, 7293 copies/ml; range, 250–32088) was higher than in patients with single CMV infection (mean, 1394 copies/ml; range, 171–8580).
With the introduction of HAART in 1996, the overall incidence of opportunistic infections has declined and survival after an AIDS-defining event has improved. However, rates of CMV disease remain high for the first 3 months before declining . According to literature, the risk of CMV disease is highly correlated with the CMV DNA load in the blood. This is well studied in different groups of immunocompromised transplant recipients [12–14] with two threshold levels in use. CMV-seronegative patients are at risk for CMV disease with high CMV DNA loads above 10.000 copies/ml. CMV-seropositive patients (like most HIV-positive patients) are at risk for CMV disease with very high CMV DNA levels above 100 000 copies/ml. In our study, we focused on the measurement of quantitative CMV DNA loads in plasma (as marker of the risk for the development of CMV disease) during ongoing HAART treatment in correlation with the HIV-1 viral load and CD4 cell count at the same time. These parameters were analyzed in ART-naive patients, before and during the first 2 years of HAART treatment.
In this study of 132 HAART treated patients, 105 (80%) patients presented with undetectable CMV loads (<100 copies/ml) in all of their plasma samples during 2-year follow-up. This already is a first indication of a declining risk for CMV disease in HAART treated patients. In only 27 of 132 (20%) patients, was CMV DNA more than 100 copies/ml found in at least one plasma sample. Focusing on the total number of samples (n = 1348), in only 50 plasma samples (3.7%) CMV DNA more than 100 copies/ml was found: 37 in the range 100–1000 (low), 9 in the range 1000–10000 (moderate) and only four samples with high CMV DNA more than 10 000 (with a highest value of 32 088). In individuals with advanced HIV infection, Erice et al.  found an increased risk for CMV disease in patients with CMV-DNA levels of more than 100 000 copies/ml, although a substantial number of patients suffer from CMV disease with CMV-DNA levels significantly lower than this value [16,17]. In critically ill immunocompetent intensive care units, Limaye et al.  found CMV-DNA levels of more than 1000 copies/ml independently associated with hospitalization or death. According to our results, CMV DNA exceeding these different thresholds is unlikely (<1%) in HAART-treated HIV-patients. The combination of both findings, firstly that most HAART treated patients have undetectable CMV DNA levels in plasma, and secondly that high CMV DNA levels in plasma are very rare, illustrates a dramatic but beneficial decrease in risk for CMV disease during HAART. In practice, the remaining risk is almost limited to those patients who fulfil all three criteria of only during the first 3 months of HAART, only patients with CD4 cell levels less than 250 × 106 cells/l during HAART and only as long as HIV-1 viral load is still detectable (>50 copies/ml).
According to the literature, in addition to CMV disease characterized by high or very high CMV DNA plasma levels, some patients initiating antiretroviral therapy experience an immune reconstitution inflammatory syndrome (IRIS) [19,20]. Both noninfectious diseases and infections, including CMV, are associated with IRIS in HIV-infected patients [19,20]. The interval between onset of HAART and IRIS is highly variable, but is usually less than 8 weeks [21,22]. In our study, the timing of the rebound in CMV DNA during the first 3 months of HAART coincidences with the timing of IRIS in the literature. Also an initial lower CD4 cell count favors the possibility of IRIS in these patients. However, despite the fact that rebound CMV DNA can reach more than 1000 to more than 10 000 copies/ml, an eventual relationship between CMV DNAemia and IRIS is not well established and needs further research.
Previous studies have shown that the gB genotype is correlated with the course of CMV disease in bone marrow transplant recipients and AIDS patients [23–25] and infections with HCMV gB type 1 were previously correlated with a more favorable outcome than infections with gB types two to four when HIV-infected patients were analyzed [23,24]. However, both geographic and demographic differences between patients affect gB distribution and should be considered before associations of gB genotypes and virulence are made . In our study within HAART-treated patients in the Netherlands between 1997 and 2004, CMV gB3 was most prevalent with 16 strains (84%) isolated, followed by CMV gB2 (47%), gB1 (26%) and gB4 (21%). This is different from our previous finding that, even in the same hospital and in an included period (1997–1998), there was a clear predominance of 53% CMV gB1 strains and only 18% CMV gB3 strains in renal transplant recipients . Therefore, these two studies, including 135 HIV-1 patients and 69 renal transplant recipients  within the same hospital, clearly indicate that the distribution of CMV gB subtypes can differ, not only between different geographic regions, but also between different patient groups in the same geographic region. Preexisting immunity to one strain of CMV will not necessarily be protective against infection with another strain of the virus [28–32] as illustrated in our study encompassing multiple patients with PCR-proven double or even triple CMV infection with higher CMV DNA levels in plasma.
CMV disease during HAART is very unlikely as soon as the HIV-1 viral load becomes undetectable (<50 copies/ml) and/or CD4 cell levels are restored to more than 250 × 106 cells/l. Within Dutch HAART-treated patients, infection with CMV gB3 is most prevalent, but double or triple infection with other CMV gB strains is also common.
Contributors: VG, CB and AV contributed to the study concept and design. VG was the study coordinator and performed analysis and interpretation of data. VG, IL and AV actively collected data in the field. AV performed the clinical evaluation. VG and IL performed the virological evaluation. PW performed the CMV loads, HIV loads and CMV genotyping. CB led the writing of the paper, and all investigators participated in its final writing and editing. None of the authors had a personal or financial conflict of interest.
The results were orally presented in part at the ESCV 2008 Clinical Virology Annual Meeting, Saariselkä, Lapland, Finland, in March 2008.
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