Bordon et al.
Outcomes & inflammatory responses in bacteremic pneumococcal pneumonia
DATA SUPPLEMENT
Bacteremic Pneumococcal Pneumonia: Clinical Outcomes and
Inflammatory Response
MATERIALS AND METHODS FOR FUNCTIONAL STUDIES
Study Design
This was a pilot prospective observational study of hospitalized patients with pneumococcal
CAP at the University of Louisville Hospital and the Louisville’s Veteran Administration
Hospital from 01/04/2011 to 01/08/2012. The University of Louisville Human Subjects Program
Protection Office and the Robley Rex Veterans Affairs Medical Center Institutional Review
Boards approved this study prior to any data collection (Approvals #: 07.0182 and 0009,
respectively).
Inclusion Criteria/Criteria for CAP: CAP was defined based on evidence of a new pulmonary
infiltrate at chest radiograph associated with at least one of the following: 1) new or increased
cough; 2) fever or hypothermia; and 3) leukocytosis, left shift, or leucopenia. Patients with CAP
were included in this study following previous written consent. The inclusion and exclusion
criteria and full case report forms for this study can be found at the Community Acquired
Pneumonia Organization study site at www.caposite.com.
Exclusion Criteria: Patients were excluded if suffering from an unstable psychiatric or
psychological condition rendering the subject unlikely to be cooperative or to complete the study
requirements, or if they had a medical history that, in the investigator’s opinion, precluded
subject compliance with the protocol. Patients that received corticosteroids were excluded from
this study.
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Bordon et al.
Outcomes & inflammatory responses in bacteremic pneumococcal pneumonia
Study groups: The study group included only pneumococcal CAP (CAP) patients.
Pneumococcal CAP diagnosis was based on sputum or blood cultures positive for Streptococcus
pneumoniae and/or positive urine pneumococcus antigen. The identification of microorganisms
and susceptibility testing were performed according to standard methods as described in the main
text of the manuscript. Out of a total of ten patients, four had positive blood cultures (all samples
taken on admission) whereas the other six were diagnosed based on positive urinary
pneumococcal antigen.
Healthy Control Group: In order to compare results of the plasma cytokines and neutrophil
functional assays from CAP patients with those of healthy individuals, blood samples were also
obtained from a control group (n=12) of healthy adult donors (approved by the University of
Louisville’s IRB #191.06).
Plasma samples
Blood samples were obtained on the day of admission and on every other day during the
patients’ stay at the hospital. Venous blood was collected using sodium citrate Vacutainer tubes.
Following centrifugation at 300 x g for 10 min, the plasma was separated by aspiration, aliquoted
and stored frozen at -80oC until assayed.
Cytokine measurements
The concentrations of interleukin (IL)-1β, IL-1 Receptor Antagonist (IL-1RA), IL-6, CXCL8
(IL-8), IL-10, IL-12p40, IL-17, interferon (IFN)γ, tumor necrosis factor (TNF)α, CXCL10 (IP10) in plasma samples were determined using Milliplex MAP Multiplex kits (EMD Millipore,
Billerica, MA). Following thawing, plasma and processed sputum samples were centrifuged at
10,000xg for 5 minutes and the supernatants used in the assay according to the manufacturer’s
instructions.
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Bordon et al.
Outcomes & inflammatory responses in bacteremic pneumococcal pneumonia
Neutrophil functional studies
Neutrophil functional assays were performed using whole blood samples. Basal or formylmethionyl-leucyl-phenylalanine (fMLF)-stimulated exocytosis were determined by measuring
the plasma membrane expression of secretory vesicles (CD35) and specific granules (CD66b)
(antibodies E11 and G10F5, respectively, both from Biolegend, San Diego, CA) on a
FACSCalibur instrument (Becton Dickinson, Franklin Lakes, NJ). Following antibody
treatment, red cells were lysed with BD red cell lysis buffer (Beckton Dickinson), followed by
two washes with 0.05% sodium azide, and fixation in 1% paraformaldehyde before analysis.
Phagocytosis and phagocytosis-stimulated respiratory burst activity were measured using a flow
cytometric assay based on the production of H2O2 stimulated by the phagocytosis of
Staphylococcus aureus, as previously described [10]. Briefly, neutrophils (2 x 106 cells/ml) were
incubated with 2’,7’-dichlorofluorescein diacetate (0.5 µM final concentration) for 10 min at
37oC. Then, 50 µl aliquots of the cell suspension were sampled before and 10 min after the
addition of opsonized, propidium iodidide-labelled Staphylococcus aureus (at a final
concentration of 108 bacteria/ml). After fixation in 1% paraformaldehyde, the cells were then
analyzed for phagocytosis and H2O2 production by flow cytometry on a FACSCalibur
instrument.
Statistics
Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA)
and R version 2.15 (www.r-project.org). All cytokine and neutrophil data are presented as the
medians ± SEM.
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Bordon et al.
Outcomes & inflammatory responses in bacteremic pneumococcal pneumonia
Data supplement FIGURE 1a. Kaplan–Meier curve for length of hospital stay (LOS) for
non-ICU patients among non-bacteremic and bacteremic patients with S. pneumoniae
community-acquired pneumonia.
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Bordon et al.
Outcomes & inflammatory responses in bacteremic pneumococcal pneumonia
Data supplement FIGURE 1b. Kaplan–Meier curve for length of hospital stay (LOS) for
ICU patients among non-bacteremic and bacteremic patients with S. Pneumoniae
community-acquired pneumonia.
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Bordon et al.
Outcomes & inflammatory responses in bacteremic pneumococcal pneumonia
Data supplement FIGURE 2. Neutrophil functional studies in bacteremic and nonbacteremic patients with pneumococcal CAP. Neutrophil function profiles in bacteremic
(open squares) and non-bacteremic (closed circles) pneumococcal CAP patients. Peripheral
blood neutrophils were assayed for basal and fMLF-stimulated expression of CD35 and CD66b,
as well as for phagocytosis and respiratory burst activity.
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Bordon et al.
Outcomes & inflammatory responses in bacteremic pneumococcal pneumonia
Data supplement TABLE 1. Demographic and clinical characteristics of S. pneumoniae
CAP patients with and without bacteremia used in inflammatory responses and biomarker
studies.
Bacteremia
No Bacteremia
Total
n =4
n=6
n = 10
Age> 65
1 (25)
4 (67)
5 (50)
Age, Median (IQR)
60 (22)
72 (21)
64 (22)
Male Gender
3 (75)
6 (100)
9 (90)
Nursing Home Resident
0 (0)
0 (0)
0 (0)
Smoking
1 (25)
3 (50)
4 (40)
Neurologic Diseases
1 (25)
0 (0)
1 (10)
Cerebrovascular Diseases
2 (50)
0 (0)
2 (20)
CHF
2 (50)
2 (33)
4 (40)
COPD
3 (75)
2 (33)
5 (50)
Diabetes mellitus
1 (25)
4 (67)
5 (50)
HIV
0 (0)
0 (0)
0 (0)
Acute Renal Diseases
0 (0)
0 (0)
0 (0)
Chronic Renal Diseases
0 (0)
2 (33)
2 (20)
Hepatic Diseases
0 (0)
0 (0)
0 (0)
Neoplastic Diseases
0 (0)
1 (17)
1 (10)
Hyperlipidemia
2 (50)
2 (33)
4 (40)
Statin Therapy
1 (25)
2 (33)
3 (30)
Immunosuppression
0 (0)
0 (0)
0 (0)
Variable
Demographics
Past Medical History
Vaccination
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Bordon et al.
Outcomes & inflammatory responses in bacteremic pneumococcal pneumonia
Previous Flu vaccine
1 (25)
4 (67)
5 (50)
Previous Pneumococcal vaccine
1 (25)
4 (67)
5 (40)
Altered Mental Status
0 (0)
1 (50)
1 (10)
Pleural Effusion
2 (50)
2 (33)
4 (40)
ICU Admission
4 (100)
2 (33)
6 (60)
Prior CAP
1 (25)
1 (17)
2 (20)
PSI ≥ 4
3 (75)
3 (50)
6 (60)
Macrolide Therapy
1 (25)
2 (33)
3 (30)
Early TCS (< 3 days)
2 (50)
6 (100)
8 (80)
In-Hospital Mortality
0 (0)
0 (0)
0 (0)
30-day Mortality
0 (0)
0 (0)
0 (0)
30-day Rehospitalization
0 (0)
0 (0)
0 (0)
Clinical Findings
TCS, Time to Clinical Stability; IQR, Interquartile; PSI, Pneumonia Severity Index; CAP, Community
Acquired Pneumonia. Results expressed as number (%) or median (interquartile).
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Outcomes & inflammatory responses in bacteremic pneumococcal pneumonia
Data supplement TABLE 2. Clinical and laboratory findings of S. pneumoniae CAP
patients with and without bacteremia used in inflammatory responses and biomarker
studies.
Bacteremia
No Bacteremia
Total
n =4
n=6
n = 10
98.4 (1.9)
98.8 (3.8)
98.6 (2.5)
Respiratory Rate
20 (5)
20 (9)
20 (5)
Heart Rate
80 (30)
94 (28)
93 (26)
Systolic BP
128 (71)
118 (61)
125 (53)
ABG PaO2
60.8 (35)
65.9 (16)
61.5 (11.3)
ABG Ph
7.4 (0.2)
7.5 (0.1)
7.5 (0.1)
Albumin
3.5 (1.6)
3.6 (0.9)
3.6 (0.9)
BUN
25 (41)
17 (22)
22 (19)
Glucose
109 (223)
114 (92)
112 (92)
BNP
524 (1003)
68 (127)
94 (502.7)
CRP
277 (368)
85 (84)
110 (322)
Procalcitonin
50 (14.3)
0.6 (1.0)
22 (53.2)
83 (7)
92 (13)
87 (12)
187 (51)
252 (82)
216 (74)
12.4 (12.3)
12.9 (6.7)
12.9 (6.7)
Na
133 (9)
136 (6)
134 (6)
Hematocrit
39 (11)
38 (5)
39 (5)
Hemoglobin
13.0 (4.2)
12.6 (2.5)
13.0 (2.5)
Variable
Clinical Findings
Temperature (oral)
Laboratory Findings
Neutrophils
Platelets
WBC
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Bordon et al.
Outcomes & inflammatory responses in bacteremic pneumococcal pneumonia
Time to Clinical Stability (TCS)
4 (5)
2 (0)
2 (1)
Length of hospital stay
4 (4)
4 (11)
4 (5)
Pneumonia severity index
4 (2)
4 (2)
4 (2)
Results expressed as median (interquartile range). ABG, Arterial Blood Gas; PaO2, partial oxygen
pressure; BNP, brain natriuretic peptide; BUN, blood urea nitrogen; CRP, C-reactive protein; WBC, white
cell blood count; RR, respiratory rate; HR, heart rate; BP, blood pressure; TCS, time to clinical stability;
LOS, length of stay; PSI, pneumonia severity index
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