Impact of acyclovir use on survival of patients with ventilator-associated pneumonia and high load herpes simplex virus replication

We retrospectively identified all adult ICU patients who were on ventilator support, received a diagnosis of VAP and PCR testing on clinical grounds (as determined jointly by clinicians and clinical microbiologists, based on elevated C-reactive protein, leukocytosis, purulent tracheal secretions, infiltrates on chest X-ray, or typical signs in bronchoscopy), and had a viral load of > 10³ HSV-1/2 copies/mL by PCR in respiratory specimens (excluding oropharyngeal swabs from herpes-suspected lesions) within the period of January 1, 2013, through April 1, 2018. We defined the time point of VAP diagnosis as the first radiological demonstration of pulmonary infiltrates. In those patients without clear-cut infiltrates, a diagnosis of VAP was made based on bronchoscopy findings. Neutropenic patients (neutrophils < 1500/μL) were excluded. Response to antibiotic treatment was assessed during regular daily ICU rounds, usually 48 h after initiation; responding patients were excluded from the analysis. Quantitative HSV-1/2 PCR was performed after automated DNA extraction by COBAS AmpliPrep (Roche) with the RealStar HSV-PCR kit 1.0 (AltonaDiagnostics) on a Rotor-Gene-Q thermocycler (Qiagen) according to the manufacturer’s recommendations. Additional CMV testing was performed as required by the treating physician. Quantitative CMV PCR was performed with the COBAS AmpliPrep/COBAS TaqMan CMV test (Roche) according to the manufacturer’s recommendations.

Day 0 (d0) was defined as the date of first detection of significant HSV-1/2 replication (low load, 10³–10⁵ copies/mL; high load, > 10⁵ copies/mL) in untreated patients or as the date of acyclovir treatment start for patients receiving acyclovir. Data were evaluated from d-4 up to d+14.

The Charlson Comorbidity Index was determined on d0 based on the treating physician’s discharge letters [18]. The “Acute Physiology, Age, Chronic Health Evaluation II” (APACHE II) score and “Sequential Organ Failure Assessment” (SOFA) score were calculated on d0 and with the most abnormal values in first 24 h after ICU admission, as described [19, 20]. The Glasgow Coma Score was used as documented by responsible ICU staff in the patient charts.

Chest X-rays and lung computed tomography (CT) were taken based on individual clinical necessity as assessed by treating ICU physicians. The standardized Lung Injury Score (LIS) and the simplified version of the Clinical Pulmonary Infection Score (CPIS) were calculated as described [21, 22]. Imaging results from all patients with a high viral load were re-analyzed in a standardized way to quantitatively assess appearance and distribution of pulmonary infiltrates over time. Based on the descriptive radiologic part of the LIS score, we assigned points to each of the six lung fields (upper/middle/lower, left, and right, respectively), with 1 point = “no infiltrates,” 2 points = “discrete/questionable infiltrate,” and 3 points = “distinct infiltrate.” We evaluated our score by re-analyzing all radiographs using the descriptive radiologic part of the LIS score with essentially identical results (Additional file 1: Figure S3).

The clinical and laboratory parameters listed in Table 1 and Figs. 1, 2, 3, and 4 were extracted from the hospital information systems (Hydmedia G5 and Orbis, Agfa), from the laboratory information system (Swisslab, Nexus AG, Donaueschingen, Germany), or manually from handwritten ICU charts at different time points.

Norepinephrine use was recorded as micrograms/kilogram body weight/minute. The following parameters were calculated: partial pressure arterial oxygen and fraction of inspired oxygen (PaO₂/FiO₂) ratio, alveolar-arterial O₂ gradient ([677 mmHg × FiO₂ − (PaCO₂/0.8)] − PaO₂), and lung compliance (tidal volume/[plateau pressure − positive endexpiratory pressure]) [23, 24]. In cases with more than one recorded value per day in the chart, we used the 24-h arithmetic mean for analysis.

Statistical calculations were performed in R (version 3.5.0) [25] with the following external packages: exactRankTests, survival, survminer, ggplot2, and PMCMRplus [26‐30]. The nonparametric Mann-Whitney U test and Fisher’s exact test were used because data was not normally distributed. The Kaplan-Meier curves and the log-rank test were used to analyze ICU survival. We then applied a multivariable Cox regression model for the effect of acyclovir on ICU survival time, adjusted for age, sex, and SOFA score at d0. The Cox regression model was tested for its proportional hazard assumption via the Schoenfeld residuals tests. Additional models and a propensity score analysis were calculated as shown in Additional file 1: Table S2. The propensity score was determined in a logistic regression model with acyclovir treatment as response variable and age, sex, and SOFA score as predictor covariates.

Daily medians of norepinephrine doses and PaO₂/Fio₂ ratios were analyzed for all patients without missing values within 7 days from d0 using the nonparametric Friedman test. This was followed by post hoc analysis by the Friedman-Conover test with p value adjustment by false discovery rate (fdr).

The radiographic score and the descriptive part of the LIS score were analyzed by the Wilcoxon signed-rank test comparing the last available X-ray or CT before d0 and the maximum change within a time span of 3 to 15 days.

We tested respiratory secretions of 425 ICU patients for HSV-1/2 replication by PCR. Of these, 126 (29.6%) tested positive for at least 10³ HSV-1 copies/mL. Only one patient had additional HSV-2 replication (Fig. 1). All but two patients had clinical signs of pneumonia (elevated C-reactive protein, leukocytosis, purulent tracheal secretions, pulmonary infiltrates, or typical signs in bronchoscopy). Strikingly, 20/50 (40%) low viral load patients responded to culture-guided antibiotic treatment, in contrast to 8/67 (12%) of high viral load patients (p < 0.001). These antibiotic responders as well as the two patients without clinical signs of pneumonia were excluded from analysis, so that 89 patients with antibiotic refractory VAP remained: 30 with low viral load (10³–10⁵ copies/mL) and 59 with high viral load (> 10⁵ copies/mL). Additional file 1: Table S1 shows microbiology culture results and antibiotics received by these patients; all were treated with at least one antibiotic active against the bacteria identified. A significantly higher proportion of high load patients received antiviral treatment (83% vs 57%, p = 0.005, Fig. 1).

The time from VAP diagnosis (detection of infiltrates) to HSV detection was not significantly different between untreated and acyclovir treated patients (Table 1), nor did it correlate with viral load (Additional file 1: Figure S1).

In general, tracheobronchial secretions tended to have higher HSV viral loads than bronchoalveolar lavages, although this difference reached statistical significance only in the entire cohort (p = 0.026, Additional file 1: Figure S2a); it was not significant in the subgroups of high and low load patients, respectively (Additional file 1: Figure S2b, c).

As shown in Table 1, antiviral-treated patients had higher HSV loads (median 1.1 × 10⁶ vs 1.3 × 10⁴, p < 0.001) especially in bronchoalveolar lavage fluid (median 1.1 × 10⁶ vs 0.9 × 10⁴, p < 0.001) and longer total ICU and hospital stays (mean 26 vs 16 days, p = 0.009, and 41 vs 26 days, p = 0.004, respectively) than untreated patients. During the entire ICU stay, they had longer antibiotic treatment (18 vs 14.5 days, p = 0.046); had a greater number of different antibiotic classes (5.0 vs 3.5, p = 0.025), particularly ceftazidime (18% vs 0%, p = 0.031); and were intubated longer (13 vs 9 days, p = 0.017). On the other hand, treated patients had less chronic obstructive pulmonary disease (20% vs 50%, p = 0.008) and required lower norepinephrine doses on d0 than untreated patients (median 0.001 vs 0.101 μg/kg body weight/min, p = 0.009).

Among the subgroup with high viral load, those receiving antiviral treatment had higher HSV loads (median 3.1 × 10⁶ vs 2.8 × 10⁵, p < 0.001) and longer total ICU and hospital stays (26 vs 15 days, p = 0.006, and 42 vs 24 days, p = 0.008, respectively) than untreated patients. They also received longer antibiotic courses (median 17 vs 12 days, p = 0.045) than untreated patients. None of these differences were significant in the subgroup with low viral load.

In the entire cohort of 89 patients with detectable respiratory HSV replication, acyclovir had no significant impact on ICU survival (Fig. 2a). Upon subgroup analysis, however, patients with high viral load who received acyclovir had significantly longer median ICU survival compared to patients without antiviral treatment (22 vs 8 days, p = 0.014; Fig. 2c), while no significant difference was demonstrated in low viral load patients (Fig. 2b). To correct for possible confounding, a multiple Cox regression model was adjusted for age, sex, and SOFA score (Fig. 3). The Cox model key assumption of proportional hazards was not violated as tested with the Schoenfeld residuals tests. In all patient groups, higher SOFA scores were consistently associated with increased hazard ratios for ICU death (Fig. 3a–c). The impact of acyclovir on survival remained significant only in high viral load patients (hazard ratio for ICU death 0.31, 95% CI 0.11–0.92, p = 0.035; Fig. 3c). Additional Cox models adjusted for different covariates (SOFA, APACHE II, COPD, HSV viral load as continuous variable) and a propensity score analysis gave essentially identical results (see Additional file 1: Table S2). Absolute ICU mortality, however, did not differ between untreated and acyclovir treated patients in the entire cohort (12/24 vs 26/65, p = 0.344), the low viral load subgroup (6/14 vs 6/16, p = 1), or the high viral subgroup (6/10 vs 20/49, p = 0.311).

Overall, 66 of 89 patients were tested for CMV replication together with HSV. CMV replication could be detected in 14/66 (21%) patients but had no effect on median ICU survival (Additional file 1: Figure S4).

Upon antiviral treatment (Fig. 4), high viral load patients improved in terms of circulatory support (from day 0 to 6 decrease in mean norepinephrine doses from 0.05 to 0.02 μg/kg body weight/min, fdr-adjusted p = 0.049; Fig. 4a) and pulmonary oxygenation (median PaO₂/FiO₂ ratio increased during treatment from 187 on day 3 to 241 on day 7, fdr-adjusted p = 0.02; Fig. 4b). Pulmonary and circulatory function of untreated patients remained unchanged. Finally, we evaluated all chest X-rays and CTs from high viral load patients. Scores of the first radiograph were slightly worse in patients who subsequently received antiviral treatment than in patients who did not (median 11 vs 8 points, p = 0.10; Fig. 4c), but they improved significantly during the course of treatment (11 vs 10 points, p < 0.001). The radiographic score did not change in untreated patients for whom at least two sequential X-rays were available for analysis.