Reactivation of multiple viruses in patients with sepsis

Abstract

A current controversy is whether patients with sepsis progress to an immunosuppressed state. We hypothesized that reactivation of latent viruses occurred with prolonged sepsis thereby providing evidence of clinically-relevant immunosuppression and potentially providing a means to serially-monitor patients' immune status. Secondly, if viral loads are markedly elevated, they may contribute to morbidity and mortality. This study determined if reactivation of herpesviruses, polyomaviruses, and the anellovirus TTV occurred in sepsis and correlated with severity. Serial whole blood and plasma samples from 560 critically-ill septic, 161 critically-ill non-septic, and 164 healthy age-matched patients were analyzed by quantitative-polymerase-chain-reaction for cytomegalovirus (CMV), Epstein-Barr (EBV), herpes-simplex (HSV), human herpes virus-6 (HHV-6), and TTV. Polyomaviruses BK and JC were quantitated in urine. Detectable virus was analyzed with respect to secondary fungal and opportunistic bacterial infections, ICU duration, severity of illness, and survival. Patients with protracted sepsis had markedly increased frequency of detectable virus. Cumulative viral DNA detection rates in blood were: CMV (24.2%), EBV (53.2%), HSV (14.1%), HHV-6 (10.4%), and TTV (77.5%). 42.7% of septic patients had presence of two or more viruses. The 50% detection rate for herpesviruses was 5-8 days after sepsis onset. A small subgroup of septic patients had markedly elevated viral loads (>104-106 DNA copies/ml blood) for CMV, EBV, and HSV. Excluding TTV, DNAemia was uncommon in critically-ill non-septic patients and in age-matched healthy controls. Compared to septic patients without DNAemia, septic patients with viremia had increased fungal and opportunistic bacterial infections. Patients with detectable CMV in plasma had higher 90-day mortality compared to CMV-negative patients; p<0.05. Reactivation of latent viruses is common with prolonged sepsis, with frequencies similar to those occurring in transplant patients on immunosuppressive therapy and consistent with development of an immunosuppressive state. Whether reactivated latent viruses contribute to morbidity and mortality in sepsis remains unknown.

Figure 1. Viral levels in septic and control patients.

The maximum viral load for each patient is displayed. (Figure 1A) Only data from septic patients are displayed for CMV, EBV, HSV, and HHV-6 because viral levels in control patients for these viruses were at or below the limit of quantitation. (Figure 1B) For JC and BK, data are from urine samples of septic and critically-ill non-septic (CINS) patients. (Figure 1C) The maximum viral load for TTV is displayed for septic, CINS, and healthy control pre-operative elective-surgery (HC) patients. The horizontal line in each graph represents the geometric mean for the virus level.

Figure 2. Correlation of viral loads among the individual viruses.

Populations were established based upon viral DNA loads; each of these populations was examined for presence or absence of other viruses. The number of patients in each of the groups is defined as the following: Negative  =  no detectable virus, Low  =  less than lower limit of quantitation (lloq), and High  =  greater than lloq. The negative, low, and high values for CMV are N = 274, 34, and 37 septic patients, respectively. Negative, low, and high values for EBV are N = 247, 213, and 61 septic patients, respectively; for HSV comparable values are N = 465, 38, 18, septic patients, respectively). For HHV-6, Negative = no detectable virus (n = 485 patients), Positive = detectable virus (n = 36 patients); *p<0.05, **p<0.01, ***p<0.001. These results show that as the blood viral load of one particular virus increases, there is a corresponding increase in the prevalence of the other herpes family members.

Figure 3. Peak viral detection rate and time course of viral detection.

The percentage of patients who tested positive in blood for particular viruses during the course of sepsis (limited to 30 days) is displayed in two formats. Day 0 represents the day that the patient fulfilled sepsis criteria . Figure 3A represents all septic patients positive for viral reactivation divided by the total number of septic patients who were tested on or before the same day. Figure 3B represents only those septic patients who were negative for the particular viruses and who ultimately became positive during their septic course. The % represents the increase in the number of septic patients who convert from virus negative to virus positive status. *TTV was tested only in plasma (see Methods S1).

Figure 4. Impact of viral reactivation on fungal and opportunistic bacterial infections.

Septic patients with CMV detected in either blood or plasma had increased fungal infections compared to CMV negative patients; only results for plasma are shown and are significant, p<0.001. Similarly, patients who had EBV detected in blood had increased fungal infections compared to viral negative patients, p = 0.05. Patients who were HSV positive in blood had increased opportunistic bacterial infections due to Stenotrophomonas, Acinetobacter, or Enterococcus compared to viral negative patients, p<0.05. Censored subject (vertical hash marks) represent patients who were either discharged from the ICU or who died without events. Analysis was performed using all events but plot was truncated at 60 days for clarity. N = 35 patients with fungal infections, n = 86 patients with Stenotrophomonas, Acinetobacter, or Enterococcus infections.

Figure 5. Patients with viral reactivation have increased ICU length of stays.

The average number of days spent in the ICU for septic patients with versus without viremia was determined. Septic patients who were positive for CVM, EBV, TTV, HSV, and HHV-6 had longer ICU stays compared to comparable patients who were viral negative. There was no impact of urine JC or BK positivity on ICU length of stay. Values were compared by student's t test.

Figure 6. Impact of CMV and EBV on sepsis mortality.

Septic patients who were CMV positive in plasma had increased 90 day mortality compared to CMV negative patients, p<0.05. Surprisingly, patients who were EBV positive in whole blood (but not plasma) had decreased 90 day mortality compared to EBV negative patients, p<0.001. Data analyzed by Kaplan Meier.

Figure 7. Impact of CMV and TTV viral loads on sepsis mortality.

The relationship between CMV and TTV viral load in blood and 90 day mortality is displayed. There was a non-statistically significant increase in mortality due to sepsis with increasing CMV viral levels in blood. (Note that septic patients who were CMV positive in plasma did have increased mortality compared to CMV negative patients - see Figure 6). Compared to septic patients who were TTV negative, patients with the highest quartile viral load for TTV (Q4) had a trend toward increased 90 day mortality (p = 0.06).

Figure 8. Effect of EBV load on survival.

EBV in whole blood (but not plasma) was associated with a decrease in sepsis mortality. This protective effect of EBV DNAemia tended to lessen with increased viral burden although the effect was not statistically significant.