Under the microscope with ACAHF grant recipient - Dr Claire Sharp

01 Apr 2019
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The Australian Companion Animal Health Foundation (ACAHF) was founded in 1989. It is a not-for-profit trust of the AVA and is administered through the ASAV office. Its purpose is to help fund veterinary clinical research relating to the causes, prevention, treatment and cure of diseases affecting companion animals, especially dogs, cats and other pets relevant to small animal veterinary practice.

The Foundation raises money through donations from veterinarians and companies associated with the pet industry, corporate sponsorship and through the ACAHF Memorial Donation Scheme.

Every year the ACAHF awards grants to deserving companion animal research projects around Australia.

For this edition of Companion, Heather Vaile talks to grant recipient, Dr Claire Sharp, a senior lecturer in the School of Veterinary Medicine at Murdoch University in Western Australia.

Claire teaches in courses such as Emergency and Critical Care and Small Animal Medicine for preclinical veterinary students and she also has a clinical teaching role in small animal emergency and critical care rotation, and critical care streaming. In addition to this work, she is involved in supervising master’s students in courses such as Veterinary Case Management.

Her clinical interests include the management of sepsis, respiratory distress, trauma, gastric dilatation and volvulus, along with many other emergency and critical care (ECC) diseases.

Claire also has a wide range of research interests in ECC medicine and more, including biomarkers for sepsis and cancer, transfusion medicine, virology, dog genetics, trauma and coagulation.

Since taking up her current role at Murdoch University, Claire has been awarded ACAHF grants for two fascinating research projects which she hopes will help small animal vets to treat critically ill canine patients more successfully in the future.


Can you tell us a little bit about your current role and how long you’ve been doing it?

I am currently a senior lecturer at Murdoch University and head of the Emergency and Critical Care section in The Animal Hospital at the university. I have been in the role since mid-2015 when I returned from more than a decade working in the United States. My role is a balance between clinical work in the ICU and ER, teaching veterinary students, interns and residents, and research.

Have you noticed any differences between ECC veterinary environments in Australia and those in the US?

The ECC veterinary environment is a little different in Australia vs. the US. Small animal emergency and critical care is a long-established specialty in the US with more than 500 specialists, and dozens of specialty training programs at universities and large private practices around the country. In contrast, ECC in its own right is a newer specialty in Australia, with less than 20 specialists nationwide. Traditionally in Australia, emergency medicine has been the realm of dedicated ER vets, and critical care has been done by anaesthesiologists and vets with a special interest in the field. The role of ECC specialists is now being increasingly recognised in Australia and as such the specialty is growing rapidly. It is great to be involved in the Australian and New Zealand College of Veterinary Scientists ECC Chapter during this period of rapid growth.

When did you first develop an interest in management of sepsis in canine patients (among other things)?

I first developed an interest in the management of septic dogs (and cats) as an intern and later junior resident in emergency and critical care at the University of Missouri. Sepsis fascinated me because it wasn’t always an obvious diagnosis, but could be such a devastating syndrome. Sepsis also frustrated me because sometimes despite doing everything right – antibiotics, control of the source of the infection, high-level ICU care – and I still couldn’t save some of my sepsis patients. The desire to be better at caring for patients with sepsis, and particularly those with sepsis-induced organ dysfunctions, has been my main motivator for research in this field. Additionally, I was fortunate that I had wonderful mentors during my residency that supported me through my master’s research on sepsis.

Are there any reliable statistics available in terms of what percentage of ECC canine patients develop sepsis?

Unfortunately, we know very little about the prevalence of sepsis in dogs presenting to veterinary hospitals, but I believe it is more common that we give it credit for. I think part of the reason that we don’t appreciate how commonly sepsis occurs is that we (as a veterinary profession) often don’t use the term sepsis – rather we refer to the specific underlying cause of sepsis like pneumonia, pyelonephritis, pyometra, parvovirus, or septic peritonitis. When we consider all of these together, as well as other causes of sepsis, the syndrome actually encompasses quite a number of the patients that we see in an ECC setting.

Work that I did while I was a faculty member at Tufts University suggested that the prevalence of sepsis in cats at hospital admission was 6.2 cases/100 admissions; while hospital-acquired sepsis developed in an additional 1.5 cases/100 hospital admissions.1 I believe that it is likely that the prevalence in dogs is similar, if not higher, and I will soon start a study to investigate sepsis epidemiology in dogs.

What’s the mortality rate?

Sepsis is a major cause of morbidity and is associated with 50-70 per cent mortality in veterinary patients. We also know that the mortality rate of sepsis is related to the magnitude of the associated organ dysfunctions; the more organ systems affected as a result of sepsis, the more likely a patient is to succumb to the condition.

What kind of canine patients are most susceptible to developing sepsis?

Sepsis can occur in dogs of any age, breed, or sex, but there are certain patterns of disease we recognise. Young dogs, for example, may develop sepsis from parvovirus gastroenteritis, or bacterial pneumonia secondary to an upper respiratory tract infection/infectious tracheobronchitis (now called canine infectious respiratory disease complex). At the other end of the spectrum, we also know that immune function deteriorates with age, and as such, geriatric patients are at increased risk of sepsis; secondary to conditions such as urinary tract infections. But some conditions occur irrespective of age – for example, septic peritonitis from a perforated gastrointestinal foreign body. Examples of patients with concurrent diseases and treatments that increase the risk of sepsis include cancer patients undergoing chemotherapy, patients with immune-mediated disease receiving immunosuppressive medications like prednisolone or ciclosporin, and those with diabetes mellitus. These things increase our index of suspicion of sepsis in a sick dog or cat patient presenting to the emergency room.

What made you decide you wanted to develop a specific tool to quantify biomarkers of sepsis in dogs?

While sepsis is obvious in some patients, like a dog with pyometra and purulent vaginal discharge or a cat with a cat bite abscess, it may be difficult to diagnose in others. In specialty practice we have many tools at our disposal, like ultrasound, CT, and inhouse clinical pathology, to aid us in making timely diagnoses, but we recognise that our colleagues in general practice face even greater diagnostic challenges with fewer resources at their fingertips. Making a prompt diagnosis of sepsis is vital for a successful outcome, as delays in diagnosis often lead to delays in initiation of effective treatment like antimicrobial therapy and source control surgery. Data in human medicine suggest that delays in initiation of appropriate antibiotics result in increased mortality in human patients, thus all efforts should be made to diagnose and treat sepsis expediently.

However, our profession is also under pressure to ensure judicious use of antimicrobials. Avoiding antibiotics in patients without infection is just as important as giving antibiotics to those with infections.

Having a test to help us rule out sepsis, for example, in a patient with a fever due to immune-mediated disease rather than infection, would improve our confidence in not prescribing antimicrobials.

A good biomarker for sepsis would also help us to know when an infection treated with antibiotics is eliminated, facilitating discontinuation of the antibiotics, and avoiding unnecessarily long antibiotic courses given ‘just in case’. In fact, this is the way that the biomarker procalcitonin (PCT) has shown most promise in human medicine – to guide the duration of antibiotic therapy.

How did you first hear about the ACAHF research grant opportunities?

When I returned to Australia, I joined a group of esteemed researchers at Murdoch University. My immediate colleague in ECC, Dr Lisa Smart (previously featured in Companion magazine), has previously received ACAHF funding for ECC-related projects, and thus recommended that I apply for funding. I have also been aware of the great work that ACAHF facilitates as an AVA and ASAV member and reader of Companion magazine for many years.

Could you tell us a little about each of the two studies that the ACAHF has helped to fund?

Our first study is called: Development of a mass spectrometry assay to quantify canine plasma procalcitonin and its application as a diagnostic biomarker for sepsis in dogs.

Sepsis can be difficult to differentiate from non-infectious causes of systemic inflammation such as pancreatitis, cancer, and autoimmune conditions. In human medicine, a blood test that measures a protein called procalcitonin is used to help diagnose sepsis, and to guide sepsis treatment (such as when to start and stop antibiotics). Early investigation of this blood test in dogs has been disappointing because the dog procalcitonin protein is quite different to the human protein.

Our study aims to develop a blood test specific to the dog procalcitonin protein that can then be evaluated in sick dogs, to see if it helps with diagnosing sepsis.

The second study is entitled: Evaluating changes in haemostatic factor activity and the risk of bacterial contamination associated with prolonged storage of canine liquid plasma.

In the setting of high-volume veterinary emergency clinics, dogs often present with life-threatening bleeding, necessitating rapid and immediate transfusion of blood products. Transfusion medicine in both humans and companion animals is an evolving science, with ongoing research designed to ensure that our clinical practice best meets the needs of these patients. Optimising transfusion management in turn will help ensure the best outcomes for these patients, since bleeding complications are a common reason for morbidity and mortality.

Historically, transfusions were provided as fresh whole blood, given immediately from the donor to the recipient. As transfusion medicine has developed, so too has the use of component therapy and our ability to store blood components for longer periods of time. Component therapy involves separating fresh whole blood into its components; most commonly packed red blood cells (pRBCs) that are stored refrigerated for up to 42 days, and plasma. Component therapy facilitates targeted transfusion therapy; patients that only require red cells can be administered pRBCs, and patients that only require plasma can be administered a plasma product. Not only does this facilitate optimal use of a precious resource, it also reduces the risk of certain transfusion reactions, such as volume overload.

Plasma is typically stored as fresh frozen plasma (FFP); a process which involves separating the plasma from the pRBCs and freezing it within eight hours of collection. Freezing plasma has traditionally been considered the best method to preserve clotting factors, and prevent bacterial contamination. Current practice in veterinary medicine is to maintain plasma frozen (as FFP for up to one year, or frozen plasma for more than one year) and thaw just prior to transfusion.

The disadvantage of storing plasma as a frozen product is the lag time to administration. Canine FFP takes 30-45 minutes to thaw in a 37°C water bath before it can be administered. This time delay, from initiation of thawing to transfusion, increases the risk of ongoing blood loss and haemorrhagic shock in dogs with massive haemorrhage.

To circumvent this problem in human hospitals, blood banks now stock liquid plasma (refrigerated at 4°C) to ensure availability for immediate transfusion; however, use of liquid plasma is still in its infancy in veterinary medicine. While stocking liquid plasma would allow immediate transfusion in unstable patients, there is a need to ensure both efficacy and safety of this product prior to routine use in veterinary medicine. Additionally, a shorter ‘shelf-life’ of liquid plasma needs to be considered with regard to optimal resource utilisation.

The objective of our study is to evaluate the activity of clotting factors in dog plasma units stored at 4°C (liquid plasma) for up to 28 days after collection (i.e. NEVERFROZEN liquid plasma), or after thawing from fresh frozen plasma (THAWED liquid plasma). If clotting factors are found to be stable in these products, it will allow vets to store plasma in this fashion, giving us more options for transfusion to bleeding patients.

What stage are you at with each project now?

We have made good progress with both projects. Our sepsis project has been able to identify dog procalcitonin with an advanced laboratory technique called mass spectroscopy; it has been more challenging to detect PCT with other types of methods such as immunoassays. Our work continues to optimise these assays and apply them to blood samples from dogs with sepsis.

Our plasma project has been a little more straightforward in that the laboratory methods are tried and true. We are currently measuring clotting factor concentrations in our liquid plasma products and should have some results from these analyses in the next few months.

What sort of time frame are you working on for completion?

Both projects as funded should be completed by the end of the year and we are hoping to receive further grant funding for continued investigation of sepsis and blood products to carry on from these projects.

Are both studies team projects, and if so, who else is working on them with you?

Both studies are absolutely team projects; it is difficult to get good research without a team of people working towards a common goal, and I am very fortunate to have some amazing collaborators.

The sepsis project is being undertaken by Dr Laura Brookes, for whom this work forms part of her PhD project, under the supervision of myself, veterinary clinical pathologist Dr Gabriele Rossi, DVM, PhD, ECVCP, esteemed sepsis researcher Dr Andrew Currie, PhD, BSc (Hons), and mass spectroscopy expert Dr Joel Gummer, PhD, BSc (Hons). This multidisciplinary team provides us with great opportunities to do some cutting-edge research.

The plasma project is also a group effort performed through the Comparative Health Research Group (CHRG) at Murdoch University. This project will form the basis of Dr Weiqin Chee’s master’s thesis during his ECC residency program. The other members of our research team are all Diplomates of the American College of Veterinary Emergency and Critical Care with a clinical interest in coagulation and blood banking: Drs Lisa Smart, Melissa Claus, and Corrin Boyd. Our blood bank coordinator, Michelle Rouffignac, RVN is the other key player in the success of this project.

Have you experienced any setbacks or obstacles along the way?

Yes, absolutely, research like this always presents unique challenges that can be hard to anticipate until the project has commenced. Our sepsis project has perhaps been the most challenging, as many of the commercially available reagents designed to detect dog procalcitonin don’t work the way they are meant to. This has meant that we have had to go back to the drawing board and develop our own reagents to detect the protein. Fortunately, our multidisciplinary team gives us a lot of brain power and experience to work out a plan B, C, D, and E, if plan A doesn’t work!

Can you give a brief description of the key ‘hands on’ elements of the projects?

Both projects involve some ‘hands on’ work with animals and some in the lab.

For the sepsis project, there are two components occurring concurrently. Firstly, collecting blood samples from dogs with sepsis, and non-infectious forms of systemic inflammation, and storing them for later testing. The second component is done in the laboratory. Dr Laura Brookes has used a variety of laboratory methods to purify the dog procalcitonin protein, both from dog thyroid tissue, and from dog blood. Protein purification involves separating out different protein components of the blood until just the protein of interest, in this case procalcitonin, is left. Once the protein has been purified it can then be used to develop laboratory tests to quantify exactly how much protein is present. Finally, once we have a test that can accurately measure the amount of procalcitonin protein, we will use it to measure the PCT concentration in the blood samples from our sick dogs.

For the plasma project, the first step is to obtain blood donations from our blood donor dogs. These are dogs that live in the community with their families, and come in every few months to donate. Following collection from the donor, the blood is separated into red blood cells which go into the blood bank, and plasma that is used for the study. The plasma is stored in the fridge, and samples taken at set intervals to measure clotting factors. A very sophisticated machine is used in our coagulation laboratory to measures the concentration of the various dog clotting factors, and statistical methods are then used to show how those clotting factor concentrations change over time. In addition, because one of the main risks of protracted refrigerated storage of plasma is bacterial contamination, we are sending samples from the plasma bags for bacterial culture to make sure that they stay sterile.

Once you’ve finishing your projects, do you have any plans to publish your findings online or perhaps in an academic/ veterinary journal?

Yes indeed; it is our intention to publish the results of both projects in veterinary journals. We also intend to present the results at scientific meetings so as to ensure our findings are disseminated amongst the veterinary community, and to seek feedback from our peers.

Do you think your findings might help companion animal vets to treat critically ill canine patients more successfully?

Absolutely. The sepsis project is one step of a larger project, and it is some time away from primetime clinical use. For this project, our next step will be obtaining additional funding to further improve our ability to test for dog procalcitonin.

The plasma project, in contrast, should yield findings that can immediately be translated into clinical practice. This study will tell us exactly how long clotting factors are stable in dog liquid plasma, and allow us to provide recommendations for exactly how long veterinarians can store dog plasma products in the liquid form.

Is there anything else you’d like to mention?

I am extremely grateful for the support of the ACAHF for these two projects. As a veterinary clinical researcher, it is wonderful to be able to obtain funding for clinically relevant research projects within Australia to help us tackle important issues in veterinary medicine. I am hopeful too that ACAHF will continue to support our research team as we aim to advance companion animal practice with a focus on emergency and critical care conditions.


References

  1. Babyak J, Sharp CR, Epidemiology of systemic inflammatory response syndrome and sepsis in cats hospitalised in a veterinary teaching hospital, J Am Vet Med Assoc. 2016 Jul 1;249(1):65-71. doi: 10.2460/ javma.249.1.65.

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