The prognostic value of microalbuminuria in puppies with canine parvoviral enteritis

Abstract Increased urine albumin concentration (UALB) or urine albumin-to-creatinine ratio (UACR) at admission has been associated with systemic disease and increased morbidity and mortality in critically ill canine patients. The objective of this study was to assess the prognostic value of UALB and UACR for the survival, as well as for the development and duration of systemic inflammatory response syndrome (SIRS) in puppies with canine parvoviral enteritis (CPVE). Unvaccinated puppies, aged 1-12 months with confirmed CPVE, hospitalized for ≥5 days were included. Urine was collected at admission via cystocentesis; albumin was measured immunoturbidimetrically and creatinine spectrophotometrically. The presence of SIRS was daily evaluated. Statistical analysis was conducted using R language. Twenty-six dogs were enrolled; 12/26 (46%) developed SIRS during hospitalization, while 5/26 (19%) died. A significant correlation was found between UALB and UACR (ϱ=0.868, p<0.001). The dogs with SIRS had higher median UALB [0.5 (0-12.7) mg/dL] and UACR [4.2 (0-2,093) mg/g] compared to dogs without SIRS [UALB= 0.1 (0-0.8) mg/dL, UACR= 1.6 (0-5.6) mg/g], but the differences were non-significant (p>0.05). SIRS duration was significantly correlated with UACR (ϱ=0.427, p=0.030), but not with UALB (ϱ=0.386, p=0.052). The non-survivors had higher median UALB [0.6 (0.1-12.7) mg/dL] and UACR [19.6 (0.7-2,093) mg/g] compared to survivors [UALB= 0.2 (0-1.5) mg/dL, UACR= 2.3 (0-16.9) mg/g], but the differences were non-significant (p>0.05). UACR appears to be a prognostic indicator of SIRS duration in puppies with CPVE. However, a large-scale study is warranted to confirm the usefulness of UALB and UACR for clinical risk assessment in puppies with CPVE.


INTRODUCTION
Canine parvoviral enteritis (CPVE) is one of the most common infectious disorders and a signifi cant cause of morbidity and mortality in dogs younger than 6 months [1]. The etiological agent, canine parvovirus (CPV)-2, is a small, non-enveloped DNA virus with tropism to rapidly dividing cells, which can naturally infect almost all Canidae species [1].
Dogs with CPVE often develop systemic infl ammatory response syndrome (SIRS), which can eventually lead to multiple organ dysfunction syndrome and death [2]. Studies in human medicine have shown that SIRS and sepsis are characterized by massive production of pro-infl ammatory cytokines and activation of coagulation cascade, subsequently leading to increased vascular permeability [3,4]. The latter may be associated with increased urinary excretion of plasma proteins [5,6]. Increased urine albumin concentration (UALB) or urine albumin-to-creatinine ratio (UACR) at admission has been associated with systemic disease in dogs and cats [7,8] and increased morbidity and mortality in critically ill human and canine patients [9,10].
Our hypothesis was that UALB and UACR measured at admission, act as prognostic indicators of the outcome, SIRS development, and SIRS duration in puppies with CPVE. Therefore, the aim of this study was to assess the prognostic value of UALB and UACR for the survival, as well as the development and duration of SIRS in puppies with CPVE.

MA TERIALS AND METHODS
The present study took place at the Clinic of Companion Animals, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Greece. The inclusion criteria were the following: a) age between 1 to 12 months; b) no previous treatment and no vaccination against CPV during the preceding 2 weeks of admission; c) clinical signs compatible with CPVE; d) absence of clinical or diagnostic imaging fi ndings indicative of foreign body, intussusception, or other morphological abnormalities of the gastrointestinal tract; e) positive ELISA fecal test for CPV antigen (Canine Parvovirus Antigen Test Kit, IDEXX Laboratories, Westbrook, USA) and positive PCR for CPV using a previously described method [11]; f) negative ELISA fecal test for Giardia antigen (Giardia Antigen Test Kit, IDEXX Laboratories, Westbrook, USA); g) negative fecal parasitological examination (fecal zinc sulphate fl otation) for intestinal parasites and protozoa performed at admission and every 48h; h) hospitalization for at least 5 days; i) owner's written consent.
In a ll dogs included in the present study, a complete physical examination was performed 3 times a day. Blood samples were collected daily via jugular venipuncture into K 3 EDTA tubes (Potassium -EDTA, Vet Collect tubes, IDEXX, UK) and a complete blood count was performed on the automated hematology analyzer Advia 120 (Siemens Healthcare Diagnostics, Deerfi eld, USA). Urine was collected at admission via cystocentesis; albumin concentration was measured using a previously validated method [12], while creatinine concentration was measured spectrophotometrically. Systemic infl ammatory response syndrome was defi ned as the presence of at least three of the following criteria: heart rate >140 beats/min, respiratory rate >30 breaths/min, body temperature >39.2 o C or <37.8 o C, and total white blood cell count >17,000 cells/μL or <6,000/μL. The a forementioned criteria were daily evaluated for each dog during the hospitalization period.
The da ta distribution was assessed using the Shapiro-Wilk test. The exact Wilcoxon rank-sum test was used for the median comparison between two independent groups, while the Spearman's rank correlation coeffi cient was employed for the determination of the correlation between two variables. Receiver operator characteristics (ROC) analysis was carried out to assess the performance of UACR in predicting survival. The statistical analyses were conducted using R statistical language (R Foundation for Statistical Computing, Vienna, Austria).
Informed consent: informed consent has been obtained for client-owned animals included in this study.

DISCUSSION
In the present study, the prognostic value of UALB and UACR, measured at admission, for survival, SIRS development, and SIRS duration was investigated. Increased UALB or UACR at admission has been previously documented in dogs and cats with systemic disease [7,8] and have been related to increased morbidity and mortality in critically ill human and canine patients [9,10]. To the best of our knowledge, the present study is the fi rst attempt to investigate the prognostic value of UALB and UACR for the survival, as well as the development and duration of SIRS in puppies naturally infected with CPVE.
Canine parvoviral enteritis is one of the most important infectious diseases of puppies due to its high prevalence and high mortality rate [1]. The intestinal tract damage by the CPV-2 and the compromised immune system increase the risk of SIRS due to the hematogenous spread of intestinal bacterial toxins and pro-infl ammatory cytokines, such as tumour necrosis factor [13]. In fact, SIRS is commonly documented in dogs with CPVE and can lead to multiple organ dysfunction syndrome and death [2].
Urine albumin was very well correlated with UACR; however, our results suggest that the latter may be a better prognostic indicator in puppies with CPVE. Urine albumin was devoid of any prognostic value in puppies naturally infected with CPVE. On the other hand, a moderate positive correlation between UACR at admission and 120 SIRS duration was demonstrated, indicating that UACR might be useful in predicting the duration of SIRS in hospitalized puppies with CPVE. Altered urinary protein excretion has been previously documented in dogs with SIRS [14]; however, to our knowledge, this is the fi rst study reporting an association between UACR at admission with the duration of SIRS in the hospitalization period.
The early phase of acute infl ammation is characterized by increased vascular permeability, which allows the transmigration of neutrophils and the leakage of plasma proteins, including albumin. In human medicine, there are several experimental and clinical studies suggesting the increased vascular permeability as the mechanism that lies behind the association between microalbuminuria and SIRS. Specifi cally, increased transcapillary leakage rate of radiolabelled albumin has been found in human patients with sepsis, neoplasia, or after cardiac surgery [5]. Systemic endothelial dysfunction has also been associated with albumin transcapillary leakage rate and urine albumin in patients with diabetes or hypertension [15]. Furthermore, in a clinical setting, it has been documented that UALB was associated with the severity of infl ammatory events [16].
Although, UALB and UACR were higher in dogs with negative outcome compared to dogs with positive outcome, the observed differences were proven to be statistically non-signifi cant. Nonetheless, it is worthy of mentioning that UACR was signifi cantly higher in non-survivors at 0.10 signifi cance level compared to survivors. Thus, the results of this pilot study should be interpreted cautiously, especially in the context of a small sample size. The substandard AUC for UACR was similar to that previously reported in critically ill dogs [10]. The calculated optimal cut-off value of 18.3 mg/g is characterized by excellent specifi city (100%), but lacks a substantial sensitivity (60%).
In conclusion, our results are suggestive of the potentially adjunct role of UACR in predicting SIRS duration and survival in puppies with CPVE. Nevertheless, this study shares a limitation with other pilot studies, which is the small sample size. However, it could be a trigger for a large-scale prospective study, which is warranted to confi dently determine the usefulness of microalbuminuria for clinical risk assessment in puppies with CPVE.

Authors' contributions:
OIL did the statistical analysis, wrote the manuscript, and was involved in the routine laboratory work (e.g. hematology analysis) of this study. SN was responsible for the study design and was involvedd as a clinician (clinical examinations, sampling and management of the patients) in the study. CJJ did the measurement of urine analysis of this study. TK and RT were involved as clinicians (clinical examinations, sampling and management of the patients) in the study. PZ was involved in the routine laboratory work (e.g. biochemical analysis) and was the coordinator of the study. All authors have approved the fi nal version of the manuscript.

Declaration of confl icting interests:
The author(s) declared no potential confl icts of interest with respect to the research, authorship, and/or publication of this article.