healthmedicinet

Screening

The study was carried out in a tertiary care hospital in Singapore over a three-month
period (November 2010 and January to February 2011). Patients above 18 years of age,
who were admitted to Singapore General Hospital (SGH) surgical intensive care unit
(SICU) for at least 48 h, were prospectively identified from Electronic Health Records
(Eclipsys Sunrise, Allscripts Corp., Chicago, IL). The following patients were excluded
from the study: aged younger than 18; neutropenic (defined as absolute neutrophil
count 0.5 × 10
⁹
/L); pregnant/lactating; or were colonized/had documented or possible fungal infections
as diagnosed by an infectious diseases specialist (including fungal infections other
than Candida spp.) prior to SICU admission .

This study was approved by the SingHealth Centralized Institutional Review Board (IRB
no. 2010/494/D) and waiver of consent was granted.

Surveillance cultures

Surveillance cultures for Candida spp. were obtained from eligible patients, with the first samples taken on the third day
post ICU admission, and weekly thereafter until discharge from the ICU or death. This
corresponded to the days which BDG levels were determined. The following sites were
cultured for fungal microorganisms: i) rectum (i.e. rectal swabs or feces), ii) urine (if any), iii) respiratory tract (e.g. bronchoalveolar lavage specimens), iv) wound (if any), and v) blood. Results were
considered positive in the presence of Candida growth in the culture medium. The different Candida isolates were further speciated at the microbiology laboratory.

All included patients would be eventually classified into three groups with respect
to their fungal status: 1) neither colonized nor infected, 2) Candida species colonization without IC, and 3) Candida species colonization with proven IC.

Colonization was considered unifocal when Candida species were isolated from one site and considered multifocal when Candida species were simultaneously isolated from various noncontiguous foci, even if two
different Candida species were isolated.

Diagnosis of Candida infections

Proven IC will require one of the following criteria: 1) Histologically confirmed
candidiasis from a specimen obtained by needle aspiration or biopsy from a normally
sterile site (other than mucous membranes); or 2) Isolation of Candida species in a sample obtained by a sterile procedure (including a freshly placed [24 h ago]
drain) from a normally sterile site in a patient with consistent clinical manifestations;
or 3) documentation of one or more blood culture(s) that yielded a Candida species in a patient with consistent clinical manifestations 7].

Candidal peritonitis is defined as the isolation of Candida species in a peritoneal sample obtained by laparotomy, including perforation of an
abdominal organ, dehiscence of an intestinal suture with peritonitis, severe acute
pancreatitis, or presence of peritoneal catheter for dialysis. Catheter-related candidemia
will be considered in patients who have an intravascular device and one or more positive
cultures of blood samples obtained from the peripheral vein, clinical manifestations
of infection (e.g., fever, chills, and/or hypotension), and no apparent source for bloodstream infection
(with the exception of the catheter), as well as a positive catheter culture. Candiduria
is defined as the presence of at least 10
⁴
colony-forming units/mL of the same Candida species.

The decision to treat a patient with antifungal drugs during the course of this study
was entirely dependent on the attending physician’s clinical judgment. For all study
patients on antifungal agents, details of therapy as well as clinical and microbiological
information were collected.

BDG assay

Serum samples were collected from all eligible patients on (i) the third day of ICU
admission and (ii) once a week there after until ICU discharge, death or occurrence
of IC.

Blood samples (15 mL) were collected in three tubes without anticoagulant, centrifuged
at 1800 rpm for 10 min, separated into aliquots, and stored at ?80 °C until analysis.
Detection of BDG was performed using Fungitell assays (Associates of Cape Cod Incorporated,
MA, US) in duplicates by the serology and immunology laboratory in our institution
according to the manufacturer’s instructions. Negative controls (blood from healthy
volunteers) were included. Results were regarded as valid if both duplicates were
in the same category (both positive and negative) and duplicates did not differ by
more than 20 %. Detection of positive BDG is defined as BDG level of???80 pg/ ?L.

Data collection

The following variables were recorded for each patient: age, gender, date of ICU admission,
dates of ICU and hospital discharge, reason for ICU admission, underlying diseases,
concomitant infections, number and sites of Candida colonization and risk factors (treatment with corticosteroids with a daily dose ?20 mg
prednisolone for at least 2 weeks, use of broad spectrum antibiotics or antimicrobials
within 10 days before the study, use of mechanical ventilation before the day of inclusion
in the study, and urinary catheter in place on the day of inclusion). Type of surgery
(abdominal vs non-abdominal, elective vs urgent) and the number of major procedures
performed before and during ICU stay were registered. Acute Physiology and Chronic
Health Evaluation (APACHE) II and Charlson comorbidity index were also recorded at
ICU admission.

Candida score and Candida colonization index

Candida score (CS) and colonization index (CI) were calculated on the days whenever BDG levels
were determined. According to Pittet’s definitions, the CI was defined as the ratio
of the number of distinct non-blood body sites colonized by Candida species to the total number of body sites cultured 8]. Patients with CI???0.5 were considered heavily colonized. The CS was calculated
as follows (variables coded as absent?=?0, present?=?1): total parenteral nutrition?×?1,
plus surgery?×?1, plus multifocal Candida colonization?×?1, plus severe sepsis?×?2. A CS???3 accurately selected patients at
high risk for IC.

Anti-fungal susceptibility testing

Minimum inhibitory concentrations (MICs) of amphotericin B, fluconazole, voriconazole,
anidulafungin, caspofungin and micafungin were tested for each non-duplicate isolate
using the Etest method performed in accordance to manufacturer’s recommendations (bioMérieux,
Marcy l’Etoile, France). Briefly, Etests were carried out on plates containing RPMI
agar supplemented with 2 % glucose and buffered to pH 7.0 with morpholinepropanesulfonic
acid (Sigma-Aldrich, St Louis, MO, US). The inoculum (0.5 McFarland-adjusted cell
suspension in 0.85 % NaCl) was swabbed in three directions on the entire RPMI-agar
plate, and the E-test strip was applied after excess moisture had been absorbed into
the agar. The plates were incubated at 35 °C, and MICs were read after 24 h.

MICs were interpreted in accordance to Clinical and Laboratory Standards Institute
(CLSI) species-specific clinical breakpoints, where available 8]. Epidemiological cut-off values were used when breakpoints are not proposed by CLSI.

Statistical analysis

Categorical variables were expressed as frequencies and percentages, and continuous
variables as medians and range. Kruskal Wallis and Chi-square (or Fisher’s exact)
tests were used to analyze continuous and categorical variables, respectively. Sensitivity
and specificity were calculated for BDG, Candida score and CI. Receiver operator curves
(ROC) was used to compare sensitivity and specificity between assays.