Evaluation and comparison of serum procalcitonin and heparin-binding protein levels as biomarkers of bacterial infection in cats

Abstract

Objectives

As bacterial infection can lead to sepsis and high mortality, early and easy diagnosis of sepsis can improve survival. In cats, the diagnosis of systemic bacterial infection is quite challenging, and, usually, non-specific markers for inflammation are employed. In humans, procalcitonin, heparin-binding protein and absolute neutrophil count are biomarkers that are studied in bacterial infections and sepsis owing to their high sensitivity and specificity.

Methods

A total of 56 cats were categorised into 16 healthy cats and 40 bacterially infected cats, diagnosed by various examinations. In all cats, serum procalcitonin and heparin-binding protein levels were measured using ELISA and an absolute neutrophil count was performed.

Results

The median values of procalcitonin levels and absolute neutrophil count were significantly higher in the infection group than in the normal group, but heparin-binding protein levels were not. A procalcitonin level >366 pg/ml was a better biomarker of bacterial infection than heparin-binding protein and absolute neutrophil count (sensitivity: 67.5%; specificity: 93.8%). Procalcitonin was not correlated with heparin-binding protein (r = 0.213, P = 0.115) and absolute neutrophil count (r = 0.393, P = 0.003).

Conclusions and relevance

High procalcitonin levels in cats were associated with bacterial infection. Hence, procalcitonin could be a valuable marker for diagnosing bacterial infections in cats.

Keywords

Bacterial infection biomarker heparin-binding protein inflammation procalcitonin

Introduction

Bacterial infections are common in cats.¹ Frequently diagnosed bacterial diseases in cats include sinusitis, conjunctivitis, cholangitis, pyothorax, subcutaneous abscess, gastroenteritis and cystitis. Depending on the severity of the infection and the host defences, a local bacterial infection could progress to life-threatening conditions, such as systemic inflammatory response syndrome (SIRS), sepsis, septic shock and multiorgan dysfunction syndrome (MODS), which result in significantly high mortality.^2,3 As cats in such devastating states tend to present with non-specific clinical features, unlike dogs, these conditions are challenging to diagnose, and the tools for diagnosing them are limited.^3,4 Only a small portion of the cat population shows leukocyte changes (23–30%) and clinical signs, including tachypnoea (52.6%), bradycardia (28.1%), fever (48.6%) and hypothermia (14.3%).⁴ These subtle changes and limited diagnostic tools in cats can lead to increased mortality if treatment is delayed. The immediate detection of bacterial involvement and decision-making for antibiotic treatment is critical in such emergencies.

Procalcitonin (PCT) and heparin-binding protein (HBP) have been studied and used as acute phase mar-kers for identifying bacterial infection, sepsis and septic shock in human medicine.^5–11 PCT is a precursor of calcito-nin that is released from the C cell of the thyroid and other organs when the host gets infected by bacterial pathogens. It is detectable from 2 h after infection and peaks at 12–24 h.^5–11 PCT is well known for higher specificity and sensitivity vs other biomarkers and capacity for differentiating bacterial infections from non-infectious inflammations than other acute phase proteins (APPs) such as C-reactive protein (CRP) and interleukin in human medicine.^12–16 In one study, it was found that CRP slowly increased after exposure to endotoxin in vivo, while PCT levels in humans concurrently infected with viruses and bacteria decreased in response to appropriate antibiotic treatment. During this period, the body temperature was still high, suggesting that PCT is a promising indicator for infection, as well as therapeutic monitoring.⁸ This is important so that the prognosis of patients with bacterial infections can be predicted. Furthermore, as the quantitative evaluation of PCT can be performed clinically and is already well researched, the protocol from antibiotic guidelines has been used as a valuable strategy in emergency human medicine.^17–21

HBP is a cationic antimicrobial glycoprotein that is released from activated neutrophils and has a variety of roles against bacterial infection. As a multifunctional inflammatory mediator, it has a specific chemotactic property for monocytes, as it induces vascular leakage.^22–24 Thus, it has been proven that high plasma levels of HBP correlate with bacterial infection and sepsis in human medicine.²⁵ PCT and HBP levels are superior to other markers, such as CRP and leukocyte count, in detecting bacterial infection in critical conditions.^15,16,25

In veterinary medicine, there is limited literature available for PCT and HBP.^26–31 Most of the studies are about the evaluation of PCT in relation to inflammatory conditions and sepsis in dogs and domestic animals.^26–31 To our knowledge, PCT and HBP in feline patients have not been assessed. Therefore, this study aims to investigate PCT and HBP levels in both healthy and diseased cats with bacterial infections, compare blood PCT, HBP and absolute neutrophil count (ANC) between the two groups, and determine their sensitivity and specificity as diagnostic biomarkers for bacterial infection.

Materials and methods

Animals

From March 2017 to April 2018, 56 client-owned cats that were admitted to the Veterinary Medical Teaching Hospital and a referral animal hospital were chosen; their medical records were reviewed and their blood was sam-pled with their owners’ consent. All patients were categorised into two groups; the control group (n = 16) and the diseased group with bacterial infection (n = 40). A total of 16 clinically healthy cats on a regular examination were included in the normal group, and 40 cats with acute and chronic signs, diagnosed with or suspected to have bacterial infections, were included in the diseased group. The following were conducted for the patients and grouping was performed using the results: physical examination, blood examination (complete blood count [Advia 2120; Siemens Healthcare Diagnostics] and biochemistry profile [Mindray BS-200; Bio-Medical Electronics]), diagnostic imaging test, bacterial culture, cytology and PCR (NEODIN and IDEXX; Veterinary Diagnostic Laboratory).

Serum sampling

Except for cats with an emergency status, all cats were fasted at least 8 h before sampling. Blood samples were obtained in a serum separation tube (Greiner Bio-One) via venepuncture at admission and centrifuged at 3000 g for 20 mins. Serum samples were aliquoted in each microcentrifuge tube separately, stored instantly at −70°C and thawed just before use. The process of sample collection was approved by the Institutional Animal Care and Use Committee at Chungnam National University (CNU-01147).

Measurement of feline PCT and HBP

The PCT concentration for the two groups was measured by a commercial ELISA kit (MBS073354; MyBioSource), and HBP concentration was also measured by a commercial ELISA kit (MBS093922; MyBioSource). Intra- and interassay variabilities of these two commercial kits were <15% according to the manufacturer’s information. Serum samples of all 56 cats were assayed in duplicate, and the absorbance was detected at a wavelength of 405 nm (Infinite M200; Tecan).

Statistical analysis

GraphPad Prism (version 6.01) and SPSS (version 22; IBM) were used for statistical analysis. The Shapiro–Wilk normality test was used for assessing the normality of distribution. For comparisons of serum PCT and HBP levels and ANC among the two groups, a Mann–Whitney U test was performed. Correlations between each of the markers (PCT, HBP and ANC) were analysed by Spearman analysis. To evaluate the sensitivity and specificity of biomarkers and their capability for diagnosing cases of bacterial infection, a receiver–operating characteristic (ROC) curve was illustrated, and the area under the ROC curves (AUC) was assessed for each parameter. Each AUC index represented excellent (0.9–1), good (0.80–0.90), fair (0.70–0.80), poor (0.60–0.70) or bad (0.05–0.60) ability. A P value <0.05 was considered to be statistically significant in all analyses.

Results

Clinical characteristics

The 56 cats in the two groups consisted of various breeds, but the majority of them were domestic shorthair (n = 32). The rest included Persian (n = 6), Siamese (n = 5), Turkish Angora (n = 4), Norwegian Forest Cat (n = 2), Scottish Fold (n = 2), American Curl (n = 1), American Shorthair (n = 1), Maine Coon (n = 1), Munchkin (n = 1) and Russian Blue (n = 1). The median age was about 5 years (range 3 months to 17 years). The sex of the cats in this study included six intact males, 25 castrated males, six entire females and 19 spayed females. A total of 44 diseases were diagnosed as bacterial infections and the most common sites of bacterial infections were the respiratory (n = 16) and gastrointestinal (n = 10) systems (Table 1). The serum PCT, HBP levels and ANC of healthy and diseased cats with bacterial infections were assessed. The median PCT levels (healthy cats, 286.025 pg/ml; diseased cats, 426.93 pg/ml [P <0.0001]) and ANC (healthy cats, 6.5 × 10³/µl; diseased cats, 11.845 × 10³/µl [P = 0.0091]) between the two groups were significantly different, but HBP levels (healthy cats, 0.237 ng/ml; diseased cats, 0.667 ng/ml [P = 0.2897]) were not (Figure 1). PCT had no correlation with HBP (r = 0.213, P = 0.115) and ANC (r = 0.393, P = 0.003) (Table 2).

Table 1

List of bacterial infection sites in diseased cats

Infected organ system	Number of cats
Upper respiratory system	10
Gastrointestinal system	10
Lower respiratory system	6
Oral cavity	6
Urinary system	5
Dermatological system	3
Hepatobiliary system	3
Reproductive system	1
Total	44
More than one system	4*

Number of cats that have diseases in more than one system

Figure 1

Comparison of the median level of procalcitonin (PCT), heparin-binding protein (HBP) and absolute neutrophil count (ANC). (a) PCT levels between healthy and diseased groups were significantly different (P <0.0001). (b) HBP levels between healthy and diseased groups had no significant difference (P = 0.2897). (c) ANCs between healthy and diseased groups had no significant difference (P = 0.0091)

Table 2

Correlation between procalcitonin (PCT), heparin-binding protein (HBP) and absolute neutrophil count (ANC)

	PCT	HBP	ANC
PCT	–	r = 0.213 P = 0.115	r = 0.393 P = 0.003
HBP	r = 0.213 P = 0.115	–	r = 0.373 P = 0.005
ANC	r = 0.393 P = 0.003	r = 0.373 P = 0.005	–

PCT, HBP and ANC for diagnosing bacterial infection

The capability of PTC as a biomarker for diagnosing bacterial infection was evaluated using ROC curves. The AUC of PCT was 0.867 (P <0.05), whereas the AUCs for HBP and PCT were 0.592 and 0.722, respectively. PCT, ANC and HBP had good, fair and poor capabilities, respectively, to differentiate between the diseased group with bacterial infection and the normal group (Figure 2). In the diagnosis of bacterial infection, PCT and ANC were more specific than HBP (Table 3).

Figure 2

Receiver–operating characteristic (ROC) curves for comparing the diagnostic capability of three biomarkers (procalcitonin [PCT], heparin-binding protein [HBP] and absolute neutrophil count [ANC]) for bacterial infection in cats

Table 3

Sensitivity, specificity and cut-off levels of procalcitonin (PCT), heparin-binding protein (HBP) and absolute neutrophil count (ANC) for diagnosing bacterial infections

	Sensitivity (%)	Specificity (%)	Cut-off
PCT (pg/ml)	67.5	93.75	>366
HBP (ng/ml)	85	50	>0.1275
ANC (10³/µl)	50	93.75	>12.05

Discussion

In the present study, the median serum PCT levels of diseased cats with bacterial infection were significantly higher than those of healthy cats; however, the median serum HBP level and the median ANC of the diseased cats were not different from those of the healthy group. Therefore, it was established that a high PCT concentration in cats is associated with bacterial infection, as we hypothesised. In a previous canine study, serum PCT levels in dogs were higher in the bacterial infection group than in the non-infection group.³² This study revealed that PCT levels could differentiate an infection group from an inflammatory group. Recent research on PCT in dogs with sepsis reported that plasma PCT concentration was significantly greater in patients with sepsis, similar to reports in previous human studies.^27,28 However, there is limited literature about diagnosing bacterial infection in feline patients, and there are no studies examining feline PCT and HBP.^27–29

Cats are more susceptible to secondary infections in specific immunocompromised conditions because of viral infection and progress to emergency conditions such as SIRS, sepsis and MODS, which are hard to identify and diagnose in the early phase.^1–4 From the results of this study, the AUC of each marker was as follows: 0.867 for PCT; 0.592 for HBP; and 0.722 for ANC. Based on these results, PCT and ANC were more specific than HBP, and PCT was more sensitive than ANC for diagnosing bacterial infections. Thus, we suggest that PCT can also be considered as a supportive diagnostic marker to differentiate bacterial infections and non-infectious inflammation in feline patients. Moreover, this finding could be useful for veterinary clinicians using other tests such as ANC, the grade of toxic neutrophil and serum amyloid A (sAA). Feline sAA, as APPs related to inflammatory conditions, has been clinically used in many clinics; however, it is not a specific tool for diagnosing bacterial infections.^33–36 Therefore, PCT would be a valuable diagnostic option in decision-making by feline practitioners on the use of antibiotics.

This study has several limitations. First, like other biomarkers, the results should be evaluated carefully owing to the biological half-lives of each APP. PCT also tends to increase under specific conditions regardless of bacterial infection. Therefore, PCT results should be interpreted considering other underlying and concurrent diseases of the patient by the patient’s clinician. For instance, it has been reported that PCT is likely to increase in cases of cardiogenic shock, severe renal dysfunction, liver dysfunction and end-stage tumour disease in humans.²⁰ Also, the PCT concentration of neonates after birth peaks on days 1 and 2.^18,20 Second, in cases where bacterial infection was strongly suspected but sampling could not be performed, a cat having good response to antibiotic treatment was tentatively diagnosed as having a bacterial infection in this study. It was possible that there were cats that improved but were not excluded because the observed response was to symptomatic treatment and not necessarily bacterial infection. Third, we performed a small-scale study because of the difficulty in recruiting feline patients with infection and even more with critical conditions. Large-scale studies are also needed to improve test accuracy and establish a reference interval. Moreover, studies on the relationship between PCT and various systemic conditions in cats would be needed in the future. In addition to the well-known specificity and sensitivity of PCT, its measurement and relations to specific conditions has been well studied in human medicine; PCT-guided antibiotic protocols would also help provide appropriate drugs and prognosis in feline patients through serial measurements.^18,20

Conclusions

Serum PCT level would be a valuable biomarker in diagnosing bacterial infection, but serum HBP level may not be worthwhile for detecting bacterial infection in cats. PCT could support optimised antibiotic treatment for cats with bacterial infection, thus helping to reduce the overuse and misuse of antibiotics.

Footnotes

Acknowledgements

The authors wish to thank the Royal Animal Medical Center (Seoul, Republic of Korea) for providing sites for sampling.

Accepted: 24 August 2020

Author note

This manuscript was written based on the master’s thesis of Jae-Geum Cho. This study was presented as a poster at the American College of Veterinary Internal Medicine Forum.

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This research was carried out with the support of the Cooperative Research Program of Center for Companion Animal Research (Project No PJ014045022020): Rural Development Administration, Republic of Korea.

Ethical approval

This work involved the use of non-experimental animals (owned or unowned) and procedures that differed from established internationally recognised high standards (‘best practice’) of veterinary clinical care for the individual patient. The study therefore had ethical approval from an established committee as stated in the manuscript.

Informed consent

Informed consent (either verbal or written) was obtained from the owner or legal custodian of all animal(s) described in this work (either experimental or non-experimental animals) for the procedure(s) undertaken (either prospective or retrospective studies). No animals or humans are identifiable within this publication, and therefore additional informed consent for publication was not required.

ORCID iD

Ye-In Oh

Kyoung-Won Seo

References

Greene

CE.

Infectious diseases of the dog and cat.

4th ed. St Louis, MO: Elsevier Health Sciences, 2013.

Babyak

Sharp

CR.

Epidemiology of systemic inflammatory response syndrome and sepsis in cats hospitalized in a veterinary teaching hospital.

J Am Vet Med Assoc 2016; 249: 65–71.

Brady

Otto

Van Winkle

, et al. Severe sepsis in cats: 29 cases (1986–1998). J Am Vet Med Assoc 2000; 217: 531–535.

Greiner

Wolf

Hartmann

A retrospective study of the clinical presentation of 140 dogs and 39 cats with bacteraemia.

J Small Anim Pract 2008; 49: 378–383.

Whicher

Bienvenu

Monneret

Procalcitonin as an acute phase marker.

Ann Clin Biochem 2001; 38: 483–493.

Gendrel

Bohuon

Procalcitonin, a marker of bacterial infection.

Infection 1997; 25: 133–134.

Oczenski

Fitzgerald

Schwarz

Procalcitonin: a new parameter for the diagnosis of bacterial infection in the peri-operative period.

Eur J Anaesthesiol 1998; 15: 202–209.

Gendrel

Bohuon

Procalcitonin as a marker of bacterial infection.

Pediatr Infect Dis J 2000; 19: 679–688.

Ferriere

Procalcitonin: a new marker for bacterial infections.

Ann Biol Clin 2000; 58: 49–59.

10.

Jin

Khan

AI.

Procalcitonin: uses in the clinical laboratory for the diagnosis of sepsis.

Lab Medicine 2010; 41: 173–177.

11.

Al-Nawas

Shah

PM.

Procalcitonin, a new diagnostic and prognostic marker for severe infections.

Clin Microbiol Infect 1998; 4: 237–241.

12.

Ruokonen

Ilkka

Niskanen

, et al. Procalcitonin and neopterin as indicators of infection in critically ill patients. Acta Anaesthesiol Scand 2002; 46: 398–404.

13.

Jones

Fiechtl

Brown

, et al. Procalcitonin test in the diagnosis of bacteremia: a meta-analysis. Ann Emerg Med 2007; 50: 34–41.

14.

Reitman

Pisk

Gates

III , et al. Serial procalcitonin levels to detect bacteremia in febrile neutropenia. Clin Pediatr (Phila) 2012; 51: 1175–1183.

15.

Luzzani

Polati

Dorizzi

, et al. Comparison of procalcitonin and C-reactive protein as markers of sepsis. Crit Care Med 2003; 31: 1737–1741.

16.

Hatherill

Tibby

Sykes

, et al. Diagnostic markers of infection: comparison of procalcitonin with C reactive protein and leucocyte count. Arch Dis Child 1999; 81: 417–421.

17.

Georgopoulou

A-P

Savva

Giamarellos-Bourboulis

, et al. Early changes of procalcitonin may advise about prognosis and appropriateness of antimicrobial therapy in sepsis. J Crit Care 2011; 26: 331. DOI: 10.1016/j.jcrc.2010.07.012.

18.

Meisner

Update on procalcitonin measurements.

Ann Lab Med 2014; 34: 263–273.

19.

Gong

Wang

Relationship of serum procalcitonin levels to severity and prognosis in pediatric bacterial meningitis.

Clin Pediatr 2015; 54: 1141–1144.

20.

Yap

TC.

The use of procalcitonin in clinical practice.

Proc Singapore Healthc 2014; 23: 33–37.

21.

Bishop

Bon

Trienski

, et al. Effect of introducing procalcitonin on antimicrobial therapy duration in patients with sepsis and/or pneumonia in the intensive care unit. Ann Pharmacother 2014; 48: 577–583.

22.

Linder

Soehnlein

Åkesson

Roles of heparin-binding protein in bacterial infections.

J Innate Immun 2010; 2: 431–438.

23.

Tapper

Karlsson

Mörgelin

, et al. Secretion of heparin-binding protein from human neutrophils is determined by its localization in azurophilic granules and secretory vesicles. Blood 2002; 99: 1785–1793.

24.

Soehnlein

Lindbom

Neutrophil-derived azurocidin alarms the immune system.

J Leukoc Biols 2009; 85: 344–351.

25.

Linder

Arnold

Boyd

, et al. Heparin-binding protein measurement improves the prediction of severe infection with organ dysfunction in the emergency department. Crit Care Med 2015; 43: 2378–2386. DOI: 10.1097/CCM.0000000000001265.

26.

Floras

Holowaychuk

Hodgins

, et al. Investigation of a commercial ELISA for the detection of canine procalcitonin. J Vet Intern Med 2014; 28: 599–602.

27.

Troia

Giunti

Goggs

Plasma procalcitonin concentrations predict organ dysfunction and outcome in dogs with sepsis.

BMC Vet Res 2018; 14: 111. DOI: 10.1186/s12917-018-1427-y.

28.

Goggs

Milloway

Troia

, et al. Plasma procalcitonin concentrations are increased in dogs with sepsis. Vet Rec Open 2018; 5: e000255. DOI: 10.1136/vetreco-2017-000255.

29.

Brkljačić

Torti

Pleadin

, et al. The concentrations of the inflammatory markers the amino-terminal portion of C-type pronatriuretic peptide and procalcitonin in canine babesiosis caused by Babesia canis. Vet Arhiv 2014; 84: 575–589.

30.

Bonelli

Meucci

Divers

, et al. Plasma procalcitonin concentration in healthy horses and horses affected by systemic inflammatory response syndrome. J Vet Intern Med 2015; 29: 1689–1691.

31.

Bonelli

Meucci

Divers

, et al. Evaluation of plasma procalcitonin concentrations in healthy foals and foals affected by septic systemic inflammatory response syndrome. J Equine Vet Sci 2015; 35: 645–649.

32.

Seo

Lee

Song

, et al. Evaluating of serum procalcitonin as a biomarker in patients with inflammatory disease. Korean Soc Vet Clin Med 2015; 32: 56–57.

33.

Paltrinieri

The feline acute phase reaction.

Vet J 2008; 177: 26–35.

34.

Kann

Seddon

Henning

, et al. Acute phase proteins in healthy and sick cats. Res Vet Sci 2012; 93: 649–654.

35.

Sasaki

Khatlani

, et al. Evaluation of feline serum amyloid A (SAA) as an inflammatory marker. J Vet Med Sci 2003; 65: 545–548.

36.

Tamamoto

Ohno

Takahashi

, et al. Serum amyloid A as a prognostic marker in cats with various diseases. J Vet Diagn Invest 2013; 25: 428–432.