A prospective, observational cohort study was performed in the ICU at Hôpital Pontchaillou from September 15, 2010 to March 15, 2011. This ICU is a mixed 21-bed ICU admitting mostly medical patients in a 1,950-bed teaching hospital. In 2006, 31% of the patients admitted to this ICU were identified as at-risk drinkers, based on National Institute on Alcohol Abuse and Alcoholism (NIAAA) criteria 24 25 . Nonaplasic, medical, adult patients with an ICU stay of 3 days or more were eligible for the study if their admission was not due to acute alcohol consumption. We excluded pregnant women, patients declared to be deprived of their liberty by judicial or administrative decisions, patients who did not require blood sampling, and postoperative patients. The study was approved by the hospital’s Institutional Review Board. This noninterventional study did not require patient consent according to French law; however, information about the study was provided to the patient or their closest relative, who was informed that they had the option of refusing to contribute their samples or information to the study.

Assessments to determine alcohol consumption and categorization as at-risk or not-at-risk drinkers were similar to those used in a previous study 26 . Patients and/or their closest relatives were interviewed about medical history, dietary, and lifestyle habits. We systematically sought to determine the onset and duration of drinking and the average daily alcohol consumption. Whenever possible, information given by patients was confirmed by interviews with family members or family physicians.

At-risk and not-at-risk drinkers were classified according to criteria defined by the NIAAA. An at-risk drinker was defined as someone who had >14 drinks per week or more than 4 drinks per occasion for men aged ≤65 years, and as 7 drinks per week or more than 3 drinks per occasion for all women or men aged >65 years. Not-at-risk drinkers comprised abstainers (those who never drank alcohol) and moderate drinkers (2 or fewer drinks per day for men aged ≤65 years, and 1 drink or no drinks per day for all women or men aged >65 years) 25 27 28 . Patients with alcoholic cirrhosis were classified as not-at-risk drinkers when they had stopped their alcohol consumption 12 months or more before ICU admission.

Two intensivists and two specialists of infectious diseases retrospectively reviewed medical records and classified patients as not having systemic inflammatory response syndrome (SIRS) or sepsis, or as having SIRS, sepsis, severe sepsis, or septic shock at the time of admission to the ICU according to the consensus definitions 29 . Infection was considered as being hospital-acquired if it was diagnosed after 48 hours of hospital stay and was not incubating at admission.

Dental hygiene was grossly assessed by the same physician (AGa) for all patients and arbitrarily considered as “poor” when there was visual evidence of at least two untreated caries at examination. A tooth was classified as carious if there was evidence of cavity. Patients with body mass index <18.5 kg/m² were defined as underweight 30 .

Upon admission the following data were recorded: age, gender, body mass index, Simplified Acute Physiology Score II, Sepsis-related Organ Failure Assessment, presence of alcoholic liver cirrhosis, and, when available, serum levels of γ-glutamyl transferase (GGT), mean corpuscular volume (MCV), aspartate aminotransferase, and alanine aminotransferase. Current smoking also was considered.

In addition to the five types of circulating WBC classically differentiated by standard cytology (i.e., neutrophils, lymphocytes, monocytes, eosinophils, and basophils), we took the opportunity of routine flow cytometric evaluation (Hematoflow, Beckman Coulter, Miami, FL) using autogating software (Cytodiff CXP, Beckman Coulter) provided by the clinical hematology laboratory of our hospital to differentiate B lymphocytes, cytotoxic T lymphocytes, noncytotoxic T lymphocytes, natural killer lymphocytes, CD16-positive (CD16⁺) and CD16-negative (CD16^–) monocytes, and immature granulocytes. Blood samples were performed at the time of ICU admission. Details of the flow cytometer used, data management, and routine application of flow cytometry have been published elsewhere 31 . The antibody combination used included fluorescein isothiocyanate conjugated CD36 (clone F16.152), phycoerythrin (PE), conjugated CD2 (clone 39C1.5), PE conjugated CRTH2 (clone BM16), PE-Texas Red conjugated CD16 (clone 3 G8), and PE-cyanine 7 conjugated CD45 (clone J.33). Twenty-eight healthy subjects served as a control group.

Also, because neutrophils play an important role as primary phagocytes, neutrophil CD64 expression, a diagnostic marker for infection and sepsis 32 , was measured on the blood sample used for cytometry using a Leuko64 kit (Trillium Diagnostic, Brewer, ME) containing fluorescent beads, CD64, and CD163 antibodies analyzed with a FC500 flow cytometer.

The main study endpoint was to compare circulating subsets of WBC identified by flow cytometry and neutrophil CD64 indexes between at-risk drinkers and not-at-risk drinkers, whether they presented with infection at admission to the ICU or not. The secondary endpoint was to assess the influence of coexisting infection on subsets of WBC and neutrophil CD64 indexes in at-risk and not-at-risk drinkers.

Proportions were compared by using the Chi-square test or Fisher’s exact test when required. Continuous variables were expressed as median values and interquartile ranges. The Mann-Whitney U test was used for comparisons between at-risk and not-at-risk drinkers. Four groups of patients were distinguished: not-at-risk drinkers with no infection; not-at-risk drinkers with infection; at-risk drinkers with no infection; and at-risk drinkers with infection.

Because the distributions of circulating WBC and neutrophil CD64 indexes were not normal in the four groups of patients or in the groups distinguished according to the severity of infection, the nonparametric Kruskal-Wallis test followed by Dunn’s multiple comparison post test were consequently used to evaluate the differences in circulating WBC counts and neutrophil CD64 indexes between patient groups in post-hoc analyses.

Forward multiple regression analyses were performed to determine whether at-risk drinking was an independent predictor of circulating B lymphocytes, noncytotoxic T lymphocytes, cytotoxic T lymphocytes, and CD16^– monocytes. In addition to infection and at-risk drinking, current smoking and poor dental state were entered into the model, because their proportions differed significantly between groups in the univariate analysis. Tests were two-sided, and P < 0.05 was considered to be statistically significant. For reasons of clarity, figures only show the subsets of WBC with significant variations after comparisons using the Kruskal-Wallis test.

During the study period, 385 admissions had an ICU stay of 3 days or more. Sixty-three patients were not included in the study for the following reasons: 41 patients were admitted immediately after surgery (mainly liver transplantation); 3 patients were readmitted; 4 patients suffered from postchemotherapy aplasia; and 11 patients were admitted after acute alcohol consumption with a blood alcohol level >1 g/dL. In addition, nine patients were missed, and technical problems occurred in the laboratory for three patients. Among the 281 patients who were evaluated, 103 (37%) were classified as at-risk drinkers, of whom 3 patients (3%) had been at-risk drinkers for <5 years, 20 (19%) for 5–10 years, and 80 (78%) for >10 years. No patient was admitted with the diagnosis of acute alcoholic hepatitis.

At-risk drinkers were predominantly males, were more frequently current smokers, and were more likely to have poor dental hygiene compared with not-at-risk drinkers (Table 1). Not-at-risk and at-risk drinkers also differed significantly for biomarkers of alcoholism. The only significant difference in circulating WBC between at-risk and not-at-risk drinkers was a lower number of B lymphocytes in at-risk drinkers (P = 0.002). To assess the effect of severe alcohol abuse in at-risk drinkers, we distinguished the patients with a daily intake of five or more drinks per day from those with a daily intake fewer than five drinks per day. The median number of circulating B lymphocytes was 0.117 × 10⁹/ L (0.03-0.14) in patients consuming ≥5 drinks per day and 0.145 × 10⁹/ L (0.08-0.18) in patients consuming <5 drinks par day (P = 0.04 after comparison).

ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyl transferase; IQR, interquartile ranges; MCV, mean corpuscular volume; NK, natural killer; SAPS II, Simplified Acute Physiology Score; SIRS, systemic inflammatory response syndrome; SOFA, sepsis-related organ failure assessment; ULN, upper limit of normal; WBC, white blood cells.

Sixty-five (58%) of the 112 not-at-risk drinkers with infection received antibiotics before admission to the ICU, whereas 35 (70%) of the 50 at-risk drinkers with infection received antibiotics (P = 0.15 after comparison). The median duration of symptoms was suggestive that infection before admission did not significantly differ between groups (3 days (range, 2–8) in not-at-risk drinkers versus 3 days (range, 2–5) in at-risk drinkers, P = 0.19). The two groups of patients were similar with respect to the site of infection the pathogens involved, and systemic inflammatory response (Table 1). Twenty at-risk drinkers (19%) and 33 not-at-risk drinkers (18%) died in the ICU.

The influence of infection in not-at-risk and at-risk drinkers is documented in Table 2. B lymphocytes, noncytotoxic T lymphocytes, cytotoxic T lymphocytes, and CD16^– monocytes varied significantly when compared between the five groups of patients (see P values listed in the right most column). Post-hoc comparisons showed that for B lymphocytes, noncytotoxic and cytotoxic T lymphocytes, and CD16^– counts obtained for at-risk drinkers were similar to those in not-at-risk drinkers with infection and were significantly lower than those in not-at-risk drinkers with no infection (Table 2). Neutrophil CD64 indexes varied significantly between groups and clearly variations were related to infection and not to previous alcohol consumption. Indeed, proportions of patients with a neutrophil CD64 index >2 were lower than 15% in at-risk and not-at-risk drinkers without infection and approximately 55% in at-risk and not-at-risk drinkers with infection (Table 2).

IQR, interquartile range.

^†Global comparisons with Kruskal-Wallis test between the four groups of patients.

*P < 0.05 after post-hoc comparisons between control group and not-at-risk drinkers with no infection, not-at-risk drinkers with infection, at-risk drinkers with no infection and at-risk drinkers with infection.

**P < 0 .05 after post-hoc comparisons between not-at-risk drinkers with no infection and not-at-risk drinkers with infection, at-risk drinkers with no infection and at-risk drinkers with infection.

***P < 0 .05 after post-hoc comparisons with not-at-risk drinkers with no infection and at-risk drinkers with no infection.

At-risk drinking and infection were not found to be independent predictors of circulating B lymphocytes, cytotoxic T lymphocytes, and CD16^– monocytes after multiregression analysis. On the other hand, both at-risk drinking (β-coefficient = −0.174, standard error of β-coefficient = 0.07, P = 0.01) and infection (β-coefficient = −0.167, standard error of β-coefficient = 0.06, P = 0.01) were independently associated with noncytotoxic lymphocyte counts but not previous treatment with antibiotics (β-coefficient = −0.161, standard error of β-coefficient = 0.11, P = 0.17), current smoking (β-coefficient = −0.154, standard error of β-coefficient = 0.2, P = 0.24), and poor dental state (β-coefficient = −0.174, standard error of β-coefficient = 0.12, P = 0.15).

In the group of at-risk drinkers, none of the subsets of circulating WBC counts varied significantly after comparisons between patients with no SIRS or sepsis, patients with SIRS, patients with sepsis, and those with severe sepsis or septic shock (Figure 1), indicating that the severity of infection did not have an impact on WBC counts in at-risk drinkers. Conversely, in not-at-risk drinkers, neutrophils, B lymphocytes, and cytotoxic and noncytotoxic T lymphocytes (such as CD16^– and CD16⁺ monocytes) varied significantly according to infection severity.

Neutrophil CD64 index varied significantly by severity of infection in both at-risk and not-at-risk drinkers, being obviously higher in those with severe sepsis or septic shock in both patient groups (Figure 2).

– At-risk drinkers had a lower number of B lymphocytes at admission to the ICU.

– At-risk drinkers exhibited less variation in circulating WBC in response to infection than nonalcoholic patients.

– CD64 expression did not appear to be affected by chronic alcohol exposure.