Evaluation of Plasma Procalcitonin Concentrations in Healthy Foals and Foals Affected by Septic Systemic Inflammatory Response Syndrome

Accepted Manuscript Evaluation of plasma Procalcitonin concentrations in healthy foals and foals affected by septic Systemic Inflammatory Response Syndrome Francesca Bonelli, Valentina Meucci, Thomas Divers, Rolfe Radcliffe, Eduard JoseCunilleras, Michele Corazza, Grazia Guidi, Rosalba Tognetti, Carolina Castagnetti, Luigi Intorre, Micaela Sgorbini PII:

S0737-0806(15)00414-1

DOI:

10.1016/j.jevs.2015.06.007

Reference:

YJEVS 1888

To appear in:

Journal of Equine Veterinary Science

Received Date: 3 February 2015 Revised Date:

27 March 2015

Accepted Date: 11 June 2015

Please cite this article as: Bonelli F, Meucci V, Divers T, Radcliffe R, Jose-Cunilleras E, Corazza M, Guidi G, Tognetti R, Castagnetti C, Intorre L, Sgorbini M, Evaluation of plasma Procalcitonin concentrations in healthy foals and foals affected by septic Systemic Inflammatory Response Syndrome, Journal of Equine Veterinary Science (2015), doi: 10.1016/j.jevs.2015.06.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Evaluation of plasma Procalcitonin concentrations in healthy foals and foals affected by

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septic Systemic Inflammatory Response Syndrome

3 Francesca Bonellia, Valentina Meuccia, Thomas Diversb, Rolfe Radcliffeb, Eduard Jose-

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Cunillerasc, Michele Corazzaa, Grazia Guidia, Rosalba Tognettia, Carolina Castagnettid, Luigi

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Intorrea, Micaela Sgorbinia

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a

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Veterinary Medicine, Cornell University, Ithaca, NY; cDepartment of Animal Medicine and

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Surgery, Universitat Autonoma de Barcelona, Bellaterra, Spain; dDepartment of Veterinary

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Department of Veterinary Sciences, University of Pisa, San Piero a Grado, Italy; bCollege of

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Medical Sciences, University of Bologna, Ozzano dell’Emila, Bologna, Italy.

11 Francesca Bonelli,

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Department of Veterinary Sciences, University of Pisa

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via Livornese snc, San Piero a Grado (PI)

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56122 - Italy

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tel +390502210117

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fax +390502210182

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[email protected]

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ACCEPTED MANUSCRIPT Abstract.

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The aim of this work was to evaluate procalcitonin concentrations in healthy foals and in

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foals with septic Systemic Inflammatory Response Syndrome (SIRS). Plasma procalcitonin

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(PCT) concentrations were evaluated in 51 foals and SIRS scale was calculated. Foals were

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divided into control group (no septic SIRS criteria met) and septic SIRS group (SIRS score ≥

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2 plus evidence of sepsis or localized infection). PCT concentrations were evaluated with a

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commercial ELISA kit. The data were expressed as mean and standard deviation. A T-test

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was performed between healthy and septic SIRS groups. A ROC curve was carried out.

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Finally, correlation analysis between PCT concentration and SIRS scale was performed by

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using Pearson test. The PCT concentrations in control and septic SIRS groups were

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30.0±33.1 and 178.9±76.0 pg/ml, respectively. The T-test showed differences between

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control group and septic SIRS group (p<0.0001). A positive linear correlation between PCT

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concentration and SIRS scale was observed (r=0.73, r2=0.53, p<0.0001). The ROC curve was

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statistically significant (p<0.0001) and the best cut-off value to determine septic SIRS was

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73.04 pg/ml (87.5% sensitivity, 97.1% specificity and a likelihood ratio of 30.6). Overall, the

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results showed an increase in plasma PCT concentrations in septic SIRS foals. A cut-off

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between control and septic SIRS groups was obtained.

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Key words: foal, marker, procalcitonin, sepsis, Systemic Inflammatory Response Syndrome.

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ACCEPTED MANUSCRIPT 1. Introduction

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The term Systemic Inflammatory Response Syndrome (SIRS) refers to a clinical condition

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that represents the culmination of the activation of a complex network of acute endogenous

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mediators, the inflammatory cytokines, leading to an uncontrolled, malignant and widespread

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inflammation. This process can be associated with many different factors, including bacterial

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and viral infections, hypoxia, burns, trauma, and immunologic reactions [1,2].

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The management of septic SIRS associated with bacterial infection, traditionally called

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sepsis, is still one of the most challenging problems for veterinarians in equine neonatal

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medicine [2,3]. Despite the substantial advances made in the medical management of this

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condition, the mortality rate in foals remains high [2,3]. This is due to several reasons, such

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as the multi-factorial nature, the delayed identification of the disease and the rapid

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progression of septic SIRS to septic shock and death [3].

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The most commonly used methods of detecting septic SIRS, such as blood culture, clinical

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signs and CBC count may be not rapid enough to allow immediate identification of sepsis

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[2,4].

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In human medicine, procalcitonin (PCT) seems to be an early marker of sepsis, both for adult

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and pediatric patients [5,6]. The aims of this work were: 1) to evaluate the PCT concentration

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in healthy and septic SIRS foals in order to verify differences; 2) to obtain a cut-off which

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could be used as a tool for the diagnosis of septic SIRS.

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2. Material and Methods

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2.1.Animals

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The present in vivo multicentric experimental trial in clinical setting was approved by the

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Institutional Animal Care and Use Committee of the University of Pisa, University of

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ACCEPTED MANUSCRIPT Barcelona and Cornell University. A total of 51 foals were included in the study. Sixteen

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were healthy Standardbred foals aged between 1 and 30 days, used as a control group, born

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on the same stud farm and underwent similar management conditions. The following

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inclusion criteria were set for the “control group”: (1) normal gestation time (>320 days) [7];

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(2) unassisted delivery; (3) mares treated against gastrointestinal parasites and vaccinated for

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equine influenza, tetanus, and Equine Herpes Virus-1 according to the guidelines of the

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American Association of Equine Practitioners Infectious Disease Committee [8]; (4) Apgar

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Score ≥7 within 5 minutes of birth [9]; (5) good passive transfer of immunity at 24 hours of

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agea (Immunoglobulin G ≥800 mg/dl) [10]; (6) righting reflex immediately after birth and

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sucking reflex within 10 minutes, sternal recumbence within 5 minutes, standing position

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within 60 minutes, first suckling within 180 minutes [10]; and (7) normal at physical

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examination at all sampling times. Samples and data were also collected from 35 sick client-

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owned foals, referred to three different veterinary teaching hospitals providing secondary

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health care. It was not possible to collect a complete history in all the foals at admission.

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Twenty out of 35 (57%) were fillies and 15/35 (43%) were colts, aged between 24 hours to 1

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month of life (median age of 6.2±8.5 days at admission). The foals were of different breeds:

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Standardbred (n = 11), Thoroughbred (n = 6), Italian working horse (n = 3), Paint Horse (n =

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3), Quarter Horse (n = 3), Shetland ponies (n = 3), Italian saddle horse (n = 2), Pure Spanish

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Horse (n = 2), Arabian (n = 1), Morgan (n = 1). The owner’s written consent was obtained for

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plasma collection for all the foals included in this study.

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2.2. Clinical and clinic-pathological data

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All foals were submitted to a complete physical examination and blood collection for CBC

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count evaluation, blood culture and plasma PCT analysis at admission time; contextually,

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each foal was scored according to a modified SIRS scale, already used [11]. Foals needed

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ACCEPTED MANUSCRIPT only manual restraint for all the procedures carried out. Blood samples for CBC and PCT

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concentrations were collected from the jugular vein and divided in two aliquots: a 1 ml

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aliquot was collected in a dipotassium salt of ethylenediaminetetraacetic acid (K2EDTA) test

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tube and analyzed by a cell counterb within 5 minutes after collection. A second 2.5 ml

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aliquot was collected in heparinized-tubes and immediately centrifuged at 2100 RCF for 10

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minutes. The harvested plasma was placed in sterile tubes, frozen at -18°C and analyzed in a

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single batch within 3 months. A sample for blood culture was also collected aseptically. The

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validity of the commercial culture systemc has been confirmed by others [12,13].

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2.3. PCT evaluation

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PCT concentrations were determined with a commercial kit for equine specie.d The intra-

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assay coefficient of variation was determined from 10 replicates of equine plasma samples

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containing low and high PCT concentrations. These samples were obtained by addition of

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standard PCT in equine blank samples. The inter-assay coefficient of variation was

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determined from values obtained by repeating the analysis of duplicate samples with low and

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high PCT concentrations in 5 different assays. According to the manufacturer, this assay has

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a calculated limit of detection of 10 pg/ml. To establish the detection limit for equine PCT,

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we performed repeated PCT measurements (inter-assay and intra-assay) using equine

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samples with low PCT concentrations (<10.0 pg/ml). Samples were measured in 10 replicates

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in a single assay and in 5 different assays. Mean PCT values with a coefficient of variation

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below 15% were considered valid.

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2.4.Septic SIRS evaluation

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The diagnosis of septic SIRS was based on meeting two or more of the following criteria:

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leukocytosis or leukopenia (peripheral white cell count >12.5 x 109/l or <4 x 109/l) or >10%

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immature (‘band’) neutrophils, hyper or hypothermia (rectal temperature >39.2°C or

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ACCEPTED MANUSCRIPT <37.2°C), tachycardia (heart rate >120 beats/min), tachypnea (respiratory rate >30

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breaths/min); and evidence of sepsis or localized infection [11].

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2.5.Statistical analysis

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The mean and standard deviation were calculated for PCT concentrations both for control

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group and septic SIRS group. A Kolmogorov-Smirnov test was applied to verify the data

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distribution. The results showed a Gaussian distribution, thus a T-test for parametric unpaired

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data was performed to verify significant differences in PCT concentrations between control

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and septic SIRS groups. The Receiver Operating Characteristic (ROC) analysis was

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performed to obtain specificity and sensitivity of the test at various cut-off values with a

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confidence interval of 95%. The likelihood ratio was also calculated for each cut-off values.

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Finally, Pearson test was used to evaluate the correlation between PCT concentration and

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SIRS scale. The significance level was set at P <0.05. A commercial statistical software

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package was usede.

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3. Results

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All foals included in the control group showed no signs of SIRS, while all the 35 sick foals

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met ≥2 SIRS criteria plus evidence of sepsis or localized infection [11]. In particular, 18 out

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of 35 (51.4%) foals were considered septic based on a positive admission blood culture and

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17 out of 35 (48.6%) were considered septic based on microbiological evidence of localized

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infection. The localized infections were: diarrhea diagnosed in 7/17 (41.2%) foals (4/7 foals

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were positive for Clostridium spp. [14,15]; 3/7 were positive for Salmonella spp. [16]),

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pneumonia in 6/17 (35.4%) foals (positive tracheal wash culture), polyarthritis in 2/17

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subjects (11.7%) (total protein concentration, total nucleated cell count and cytological

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ACCEPTED MANUSCRIPT examination of synovial fluid [17]), both pneumonia and polyarthritis diagnosed in 2/17

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(11.7%) subjects.

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The mean PCT concentration was 30.0±33.1 pg/ml in the control group and 178.9±76.1

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pg/ml in the septic SIRS group, respectively (Fig. 1). Statistical analysis showed differences

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between the two groups (p<0.0001). A positive linear correlation between PCT concentration

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and SIRS scale was observed (r=0.73, r2=0.53, p<0.0001) (Fig. 2). The ROC curve was

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statistically significant (p<0.0001) (Fig. 3) and the best cut-off value to determine septic

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SIRS was 73.04 pg/ml, with 87.5% sensitivity (62% to 98%), 97.1% specificity (85% to

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99%) and a likelihood ratio of 30.6 (Table 1).

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144 4. Discussion

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In 1991 a consensus conference was held by the American College of Chest Physicians

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(ACCP) and the Society of Critical Care Medicine (SCCM) to create a set of definitions that

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could be applied to patients with sepsis and its “sequelae” [18]. Sepsis has been defined as

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the systemic inflammatory response syndrome (SIRS) to infection, manifested by two or

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more of the following conditions: leukocytosis or leukopenia or >10% immature (‘band’)

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neutrophils, hyper or hypothermia, tachycardia, tachypnea, plus evidence of sepsis or

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localized infection [18]. These criteria have also been applied in equine species for a

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redefinition of sepsis in foals and endotoxemia in adult horses, and a SIRS scale was

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validated for both [11,19].

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PCT originates from the CALC-I gene transcription and calcitonin-mRNA translation [6]. In

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healthy patients, the transcription of the CALC-I gene is restricted to neuroendocrine cells in

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the thyroid gland and the lung and healthy individuals have very low serum concentrations

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[6]. During infection, particularly a systemic infection such as sepsis, the expression of the

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ACCEPTED MANUSCRIPT CALC-I gene is up regulated in many tissues and cell types in hamsters [20], dogs [21] and

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horses [22], as well as in human patients and PCT is released into the circulation from many

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tissues and cell types in the body [23]. In human medicine PCT seems to be a strong and

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early marker of sepsis because its concentration rises rapidly, within 3-6 h, especially in

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response to bacterial infection. When the infection and/or host immune response is

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controlled, circulating PCT levels decrease by half within 24 h [6,20]. Given the potential

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usefulness of PCT as a diagnostic and prognostic marker, the aims of this study were to

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evaluate differences between the plasma PCT concentration in healthy and septic SIRS foals

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and to obtain a cut-off value that could help with the diagnosis of septic SIRS. There appear

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to be no prior publications with data concerning the evaluation of PCT concentrations in

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healthy and septic SIRS foals. Pusterla et al. (2006) [24] evaluated the PCT gene expression

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in blood of healthy and sick foals using real-time PCR and did not find differences in PCT

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gene expression between the two populations [24], while in our study differences have been

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found in PCT concentration between septic SIRS and healthy foals. These dissimilarities

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might be due to the different target investigated: PCT gene expression [24] vs plasma PCT

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concentration.

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Moreover, a recent paper by Rieger and colleagues (2014) evaluated plasmatic PCT levels in

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in 29 horses (24 healthy horses and 5 septic horses) [25]. The mean plasma PCT

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concentration reported in this study, both for control and septic SIRS groups, were lower than

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those reported in Rieger and colleagues for healthy and septic adult horses [25]. The

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dissimilarity may be due to the different PCT ELISA kit used: in our study an horse PCT

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ELISA kit assay for PCT determination was used, while Rieger and colleagues (2014) [25]

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used an ELISA assay in which human antibodies were directed against equine PCT.

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ACCEPTED MANUSCRIPT The mean PCT values reported in this study, both for the control and septic SIRS group, were

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lower than those reported for healthy and SIRS newborns [5] and children [26], and higher

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than reported by Yilmaz and colleagues in dogs [27]. These dissimilarities could be related to

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the different species studied and different assay used.

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The control group showed a lower PCT concentration than the septic SIRS one. Our results

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are in line with earlier studies performed in equine, canine and human species [6,25,27,28].

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The cut-off value (73.04 pg/mL) is lower compared to findings reported for men, both adults

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[6] and neonates/children [29]; this difference might be related to the species studied. To the

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authors’ knowledge, there have been no reports concerning plasmatic PCT cut-off values for

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septic SIRS diagnosis in veterinary medicine.

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Most recent studies in human medicine on this item evaluated the use of PCT as diagnostic

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marker to discriminate SIRS of non-infectious origin vs SIRS of infectious [30,31].

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Moreover, the use of PCT has been recently studied to guide antibiotic therapy in order to

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shorten antimicrobial treatment [31-33]. Our results showed a positive correlation between

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PCT concentration and SIRS scale, suggesting that PCT could be a laboratory expression of

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septic SIRS in foals. These findings are in line with literature in human medicine concerning

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the use of PCT as a diagnostic marker of septic SIRS.

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In conclusion plasma PCT concentration seems to be a good marker of septic SIRS in equine

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foals. A limit of our study could be the incomplete history collected in some foal cases,

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missing complete information about previous antimicrobial therapy administration. Further

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studies could be useful to investigate the use of PCT in shortening antimicrobial therapy as

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already reported in humans [31-33].

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Acknowledgements

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This study was entirely supported by funds from the University of Pisa.

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Sources and manufacturers

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a

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b

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c

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d

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e

SNAP Foal, IDEXX, USA. ProCyte Dx ™, IDEXX, USA

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OXOID SIGNAL® Blood Culture System, Oxoid, USA.

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Horse Procalcitonin ELISA kit, MyBiosource.com, USA.

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Graph Pad Prism 6, USA.

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PCT (pg/ml)

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Fig.1 PCT concentration levels in control group (n=16 foals) and septic SIRS group (n=35

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foals); **** p<0.0001.

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SIRS scale

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PCT (pg/ml)

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Fig. 2 Linear correlation analysis between PCT concentrations (pg/ml) and SIRS scale in

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septic SIRS group (n=35 foals).

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80 60 40 20 0 20 40 60 100% - Specificity%

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Sensitivity%

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Fig. 3 Receiver Operating Characteristic (ROC) curve for the defined septic SIRS analysis

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performed to obtain specificity and sensitivity of the test at various cut-off values with a

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confidence interval of 95%.

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< 27.72

62 (35-85)

97 (85-100)

21.87

69 (41-89)

97 (85-100)

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Specificity (%) mean value (95% confidence interval) 97 (85-100)

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Cut-off value < 26.60

Sensitivity (%) mean value (95% confidence interval) 56 (30-80)

Likelihood ratio 19.69

75 (48-93)

97 (85-100)

26.25

< 55.60

81 (54-96)

97 (85-100)

28.44

< 73.04

87 (62-98)

97 (85-100)

30.62

< 77.63

87 (62-98)

94 (81-100)

15.31

< 89.10

87 (62-98)

91 (77-98)

10.21

< 97.81

87 (62-98)

86 (70-95)

6.12

< 99.45

94 (70-100)

86 (70-95)

6.56

< 103.8

100 (79-100)

86 (70-95)

7.00

< 108.0

100 (79-100)

83 (66-93)

5.83

< 110.8

100 (79-100)

80 (63-91)

5.00

AC C

< 38.75

313

Table 1. ROC analysis data as assessed for the definition of an optimum cut-off value for

314

plasmatic PCT concentration for diagnosis of septic SIRS (n=51 foals).

16

ACCEPTED MANUSCRIPT 1

Highlights

2




3

We evaluated plasma Procalcitonin (PCT) concentration in a population of healthy and septic SIRS foals.




PCT concentration was higher in septic SIRS foals than in healthy group.

5




The positive correlation between PCT concentration and septic SIRS scale suggests that

6

TE D

M AN U

SC

PCT could be used as marker for septic SIRS in foals.

EP




AC C

7

PCT could be a laboratory expression of septic SIRS.

RI PT

4