Introduction
Patients infected with HIV are often coinfected with other viruses. Coinfection with the hepatitis B virus (HBV), hepatitis C virus (HCV) or human T cell lymphotrophic virus type I (HTLV-I) appears to increase the mortality rate among HIV-infected patients, whereas coinfection with hepatitis G virus is associated with a reduced mortality rate [1-7].
In 1999, a new virus (SEN) was isolated from the serum of a HIV-positive patient who used intravenous drugs and had post-transfusion hepatitis of unknown aetiology. Eight different strains of SENV, named SENV-A to SEN-H, were identified and provisionally classified as members of the Circoviridae family [8]. Only SENV-D and SENV-H seem to cause post-transfusion hepatitis [9].
The present study examines whether coinfection with SENV influences mortality in HIV-positive patients.
Methods
Participants
The study enrolled and analysed retrospectively 217 HIV-infected patients who attended the outpatient clinic between January 1997 and July 1997 and were not lost during follow-up. The end of follow-up was November 2003. The stage of disease was classified in accordance with the European modification of the staging system of the Centers of Disease Control and Prevention (CDC).
Cumulative survival was calculated from two different starting points to the date of last follow-up during the present study: from the date of the first documented positive HIV test and from the date when the patient entered the present study and a blood sample was drawn for SENV testing.
If patients received highly active antiretroviral therapy (HAART) at the date of blood sampling for SENV testing, the standard medication included two nucleoside reverse transcriptase inhibitors and either a non-nucleoside reverse transcriptase inhibitor or a protease inhibitor. Prophylaxis against Pneumocystis carinii pneumonia included treatment with trimethoprim-sulfamethoxazole or pentamidine inhalation for patients with CD4 cell counts of < 200 × 106 cells/l or patients with a history of Pneumocystis carinii pneumonia.
The control group consisted of 122 healthy blood donors (90 men and 32 women; mean age 37 ± 13 years).
Virus detection and quantification
SENV-D and SENV-H were detected as described previously [10].
HIV RNA was analysed quantitatively using a commercial signal amplification assay (Quantiplex 2.0, Chiron Diagnostics, Fenwald, Germany). The sensitivity of the assay was determined as < 500 Eq/ml.
A commercial assay for HCV antibodies (HCV Version 3.0, Abbott, Wiesbaden, Germany) was used.
Statistical analysis
Results are presented as mean ± SD. A Fisher's exact test was used for the comparison of categorical variables, and the Mann-Whitney test was used for the comparison of continuous variables. The significance level was set at 0.05, and all P values were two tailed. Cumulative survival was assessed by Kaplan-Meier analysis. Equality of survival distributions was evaluated by the log rank test. A multivariate Cox regression analysis was performed that included categories for SENV-H DNA level, sex, age (< 40 years), HAART, HIV RNA (detectable/not detectable), CD4 cells (≤ 200/> 200 × 106 cells/l), CDC stage (stage A and B versus stage C), and HCV antibody status (positive/negative).
All statistic analyses were performed with the use of SPSS version 11.5 (Munich, Germany).
Results
The characteristics of the patients according to their SENV status at enrolment are summarized in Table 1.
SENV-H and SENV-D were detected in 56 of 217 (25.8%) HIV-positive patients compared with 12 of 122 (9.8%) healthy blood donors (P < 0.001). Two HIV-positive patients were infected with SENV-D (0.9%), 50 with SENV-H (23%) and four with both strains (1.9%), compared with three (2.5%), nine (7.4%) and none, respectively, in the control cohort. The mean SENV-H DNA was higher in the HIV-positive group but this trend was not statistically significant (85 114 ± 593 771 versus 530 ± 963 copies/ml; P = 0.7); because of the large SD of the SENV-H DNA in the HIV-positive group, the difference between the groups did not reach statistical significance.
CD4 cell count declined to < 200 × 106 cells/l at the end of the follow up in 4 of the 35 SENV-positive patients compared with 5 of the 85 SENV-negative patients (P = 0.44). HIV RNA at the end of the follow up had declined to below the limit of detection in 13 of 46 SENV-positive patients compared with 34 of 137 SENV-negative patients (P = 0.69). Survival was significant associated with CD4 cell decline to < 200 × 106 cells/l (P = 0.001) and HIV RNA decline to below the limit of detection (P = 0.023).
Of the 56 SENV-positive patients, 10 (18%) died during the follow-up study period, compared with 38 of 161 (23.6%) SENV-negative patients. Figure 1a shows the Kaplan-Meier survival analysis (log rank test for the study period: P = 0.38; log rank test for the period from first diagnosis of HIV infection to the end of follow-up: P = 0.38).
A significant difference in survival was found after dividing the 54 SENV-H-positive patients (50 positive only for SENV-H and four for SENV-H and SENV-D) into two groups depending on a SENV-H DNA cut off at 530 copies/ml (16 with > 530 copies/ml and 38 with < 530 copies/ml). This level was chosen because it reflected the mean SENV-H DNA level in HIV-negative controls. There was no significant difference between these groups in age, sex, CD4 cell count, CD8 cell count, CDC stage, HCV status, HAART and duration of HIV infection. During the period from blood sampling to the end of follow-up, eight (50%) of the patients with SENV-H DNA level > 530 copies/ml had died compared with two (5%) in the group with SENV-H DNA level < 530 copies/ml (P < 0.001). Figure 1b shows the Kaplan-Meier survival analysis for the different time periods. Mean survival during the study period to the end of follow-up was 58 ± 7 months [95% confidence interval (CI), 45-71] for the high SENV-H group and 81 ± 2 months (95% CI, 78-84) for the low SEN-H group (P < 0.0001, log rank test). This difference was also significant for the period from diagnosis of HIV infection to the end of follow-up during the study: 156 ± 14 months (95% CI, 130-183) with SENV-H DNA level > 530 copies/ml and 257 ± 9 months (95% CI, 240-275) with SENV-H DNA level < 530 copies/ml (P = 0.0008, log rank test). To control for the effect of age, HAART, sex, HIV RNA, CD4 cell count (above or below 200 × 106 cells/l), CDC stage and HCV status, a multivariate Cox regression analysis was performed. Only high SENV-H DNA levels were associated with a shorter survival in both analysed periods (actual study period: P = 0.003; period from diagnosis of HIV infection to the end of study follow-up: P = 0.023).
Among HIV-positive patients, the survival of patients with either high or low SENV-H DNA was compared with that of SENV-negative patients.
The characteristics of the patients with SENV-H DNA > 530 copies/ml were not significantly different from those of SENV-negative patients. During the period from blood sampling to end of follow-up, mean survival in the SENV-negative group was 71 ± 2 months (95% CI, 68-75) and in the SENV-H-positive group it was 58 ± 7 months (95% CI, 45-71) (P = 0.018, log rank test). This difference was also significant for the period from diagnosis of HIV infection to the end of study follow-up [207 ± 7 months (95% CI, 194-221) versus 156 ± 14 months (95% CI, 130-183); P = 0.032, log rank test]. To control for effects of age, HAART, sex, HIV RNA, CD4 cell count (above or below 200 × 106 cells/l), CDC stage and HCV status, a multivariate Cox regression analysis was performed including these factors. For the study period, shorter survival was significantly correlated with high SENV-H DNA level (P = 0.027), low CD4 cell count (<200 × 106 cells/l; P = 0.005), and HCV coinfection (P = 0.044). The analysis of the period from diagnosis of HIV infection to end of study follow-up showed a significant association of survival with SENV-H DNA (P = 0.033) and low CD4 cell count (P = 0.009).
A second multivariate Cox regression analysis compared the HIV-positive SENV-negative group with the HIV-positive patients with low SENV-H DNA levels. There was reduced mortality among the SENV-H-positive patients for the time from blood sampling to end of follow up (P = 0.027; odds ratio, 0.42; 95% CI, 0.05-0.833), but this effect did not reach statistical significance for the time period from diagnosis of HIV infection to end of follow up (P = 0.053; odds ratio, 0.24; 95% CI, 0.056-1.021). In the multivariate analysis, CD4 cell count and HCV status had a greater influence on the mortality than SENV-H coinfection.
There was no significant correlation between CD4 cell count and SENV-H DNA level (r = -0.l8 by Pearson correlation; P = 0.18).
Discussion
It is known that coinfections with HBV, HCV or HTLV-I have adverse effects on the survival of HIV infected patients [11-13], while coinfection with hepatitis G virus seems to improve survival.
We found a higher prevalence of SENV in HIV-positive patients compared with healthy controls. This has also been reported for other viruses such as HBV and HCV. As in other studies, survival in our study depended significantly on CD4 cell decline and detectable HIV RNA under HAART during follow-up.
Quiros-Roldan et al. [14] found no adverse effects of SENV coinfection in HIV-positive patients; only SENV positivity was investigated and SENV DNA was not determined quantitatively. This result for coinfection with SENV per se was confirmed in our study. However, detailed analysis led to the important finding that high viral load of SENV-H DNA (> 530 copies/ml) was significantly associated with poor survival. This adverse effect on survival reached statistical significance for both the time periods investigated. Immune suppression, low CD4 cell counts at baseline, AIDS at baseline, decline of CD4 cell counts during follow-up and use of HAART are known as independent survival factors [15-18]. A multivariate Cox regression analysis was performed including sex, age, HCV antibody status, CD4 cell count, CDC stage, HAART and HIV RNA to study the effect of these factors on survival. This analysis identified only high SENV-H DNA levels as a significant independent prognostic factor for both the time periods analysed. This was confirmed with a Kaplan-Meier analysis, showing an adverse effect on survival for high SENV-H DNA levels. In addition to high SENV-H DNA levels, survival was significantly dependent on CD4 cell count and the presence of HCV antibodies in the comparison of SENV-positive patients with high SENV-H levels compared with the SENV-negative patients. Other known factors associated with survival did not reach statistical significance in this analysis. Therefore, in this study, high viral load of SENV DNA was a stronger predictor of poor survival than the well-established clinical markers used routinely (e.g., CD4 cell count, HIV RNA and CDC stage) [15,19].
The comparison of SENV-negative patients with SENV-H-positive patients with low virus load showed reduced mortality in the patients with low SENV-H viral loads, but the observation was only significant for the period from blood sampling to end of follow-up. This effect did not reach statistical significance for the period from diagnosis of HIV infection to end of follow-up. However, it is not clear whether the association between SENV viral load and survival reflected a causal relationship or whether high SENV DNA levels were only a surrogate marker for immunodeficiency, which may affect outcome, but is not reliably reflected by the CD4 cell count or HIV virus load. However, the present observations should lead to prospective studies investigating the effect of treatment of SENV infection in these patients. It may be that SENV is sensitive to interferon-α [10]. An effective therapy of SENV with interferon-α could lead to improvement of survival in coinfected HIV-positive individuals.
Data about HIV-positive patients coinfected with SENV are too sparse to allow an explanation for how SENV coinfection might influence HIV-positive patients.
The negative influence of high viral loads of SENV could be similar to HBV. It is known that virulence of HBV depends on HBV load. Patients with a low virus load show no or rare elevated transaminases and inflammation. In the present study, there was no difference in baseline CD4 cells between SENV-positive and SENV-negative patients but any explanation of how SENV could influence HIV progression independent of the CD4 cell count can only be speculative. One possible reason could be that high viral loads of SENV is a pathogenetic factor having an impact independent from the immune status disease.
In summary, we found a significantly higher prevalence of SENV in HIV-positive patients than in healthy blood donors. High SENV-H viral load was more predictive for poor survival than other well-established markers of prognosis in HIV-infected individuals in this study. Further studies are needed to clarify the mechanisms by which SENV infection affects survival in HIV-positive patients and whether treatment of SENV infection would improve survival.
Note: Abdurrahman Sagir and Ortwin Adams contributed equally to the work in this paper.
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