THESIS ABSTRACT
USE OF OSELTAMIVIR IN THE TREATMENT OF CANINE PARVOVIRAL
ENTERITIS
Michelle R. Savigny
Master of Science, May 10, 2008
(DVM, Texas A&M University, 2004)
(B.A., State University of New York at Buffalo, 2000)
Directed by Douglass K. Macintire
Despite the availability of an effective vaccine, canine parvovirus (CPV) enteritis remains a significant cause of disease in veterinary medicine. Appropriate treatment of the disease can result in a favorable survival rate; however, lack of treatment is almost universally fatal. Unfortunately, the treatment tends to be costly to the client, often times deterring medical care. There exists a need for an effective treatment option that will ameliorate disease morbidity in addition to hospitalization time, thereby decreasing client cost. Oseltamivir, a neuraminidase inhibitor antiviral drug, has resounding anecdotal support for achieving this goal. However, scientific validation at this point is lacking.
This study, using a prospective, blinded, randomized, placebo controlled clinical trial, aimed to investigate the effects of oseltamivir as an adjunct to standard treatment of CPV enteritis. Results show the only significant difference between groups to be in weight change during hospitalization in the oseltamivir group versus placebo.
TABLE OF CONTENTS
LIST OF TABLES .......................................................................................................viii
LIST OF FIGURES........................................................................................................ix
CHAPTER I. INTRODUCTION....................................................................................1
CHAPTER II. LITERATURE REVIEW........................................................................3
STATEMENT OF RESEARCH OBJECTIVE ..............................................................12
CHAPTER III. MATERIALS AND METHODS..........................................................12
Study Design......................................................................................................12
Standard Treatment ............................................................................................13
Oseltamivir Treatment .......................................................................................15
Monitoring/Data Acquisition..............................................................................16
Clinical Scoring System.....................................................................................17
Statistical Analysis.............................................................................................18
CHAPTER IV. RESULTS............................................................................................18
CHAPTER V. DISCUSSION AND CONCLUSIONS..................................................23
Findings .............................................................................................................23
Limitations.........................................................................................................28
Conclusions .......................................................................................................30
FOOTNOTES ...............................................................................................................32
REFERENCES..............................................................................................................33
vi ii
LIST OF TABLES
Table 1. Clinical Scoring System .................................................................................37
Table 2. White Blood Cell Counts ................................................................................38
TLIST OF FIGURES
Figure 1. Weight change over time for individual dogs .................................................39
Figure 2. Averaged white blood cell count over time ....................................................40
Figure 3. Box plot of cumulative total scores by day.....................................................41
INTRODUCTION
Canine parvovirus (CPV) is a single-stranded DNA virus that was first discovered in 1978.1 It is a hardy, highly contagious virus that remains a cause of significant disease process in young dogs. It is estimated that over one million dogs are affected each year in the United States2 despite the availability of an effective vaccine.
CPV infects and replicates in rapidly dividing cells, most notably the lymphoid organs, latter myeloid progenitor cells in the bone marrow, and intestinal epithelial cells.
Replication results in cell destruction, causing a clinical disease characterized by severe vomiting, hemorrhagic diarrhea, dehydration and neutropenia. This disease is almost universally fatal without treatment, with reported survival rates of only 9% in an experimental model.3 Treatment increases this figure significantly, with reported survival rates ranging from 64-95%.2, 4-10 Care given in a university setting may be associated with increased survival rates of 96-100%2, 8, versus 67-75%2, 7 at local day clinics. It is postulated that this increase may be due to the 24 hour care available at these universities, as well as the likelihood for more intensive treatments such as plasma or colloid therapy.2
Therapy for CPV enteritis remains mainly supportive and aimed at controlling the clinical signs of disease. Multiple more directed therapies have been studied, such as human recombinant granulocyte colony stimulating factor8, 9, 11, equine antiendotoxin7, 12, recombinant bactericidal/permeability-increasing protein2, and interferon omega13, 14 with variable or disappointing results. Early enteral nutrition has been the only modality to date to show promise for a shortened recovery time and decreased disease morbidity associated with CPV enteritis.10 However, the need still remains for a therapeutic agent hat will help ameliorate the morbidity and mortality of this disease, and in doing so also decrease the cost of treatment. While the survival rate with aggressive treatment is very good, financial constraints often result in suboptimal treatment with lower survival, or even euthanasia. This fact demonstrates the clear need for an agent to help make the appropriate treatment for CPV enteritis a more financially feasible option for many owners or shelters.
Oseltamivira (Tamiflu) is a neuraminidase inhibitor originally designed to treat human influenza virus.15 It has recently also shown efficacy in the treatment of avian influenza. Oseltamivir inhibits the viral neuraminidase enzyme, and thus prevents the cleavage of sialic acid residues. This cleavage is necessary for liberating newly formed virons from the host cell, as well as for preventing the aggregation of viral particles. Both the viron liberation and decreased aggregation are mechanisms necessary for the spread and dissemination of the virus throughout the host in order to further infection. The neuraminidase enzyme is also needed for the virus to cleave the sialic acid residues
in mucin to allow for penetration of this protective layer and infection of respiratory epithelial tissue. Anecdotal reports of oseltamivir use in veterinary medicine claim that it is associated with a less severe form of disease and a quicker recovery when administered to dogs with CPV enteritis. However, unlike the influenza virus, CPV does not rely on neuraminidase for effective replication. Therefore, any beneficial effects that may be present would not be due to a direct antiviral action. Human studies have shown a ignificant decrease in the development of bacterial infections secondary to influenza when oseltamivir is used.15-21 This effect is believed to be due to a decrease in bacterial
permeation through the mucin layer of the respiratory epithelial cells. An extension of this is that oseltamivir could also have a similar inhibitory effect on bacterial permeation through the mucin layer of the gut epithelial cells. This would decrease bacterial translocation, resulting in a potentially lower incidence of endotoxemia, sepsis, systemic inflammatory response and eventual organ failure that is thought to be the main mechanism behind the mortality of CPV enteritis. Thus, oseltamivir could have an effect on the disease process of CPV enteritis in a non-viral dependent manner. In one report, 90% of dogs that died from CPV enteritis had Escherichia coli cultured from their liver
and lungs.22 ARDS-like changes in the lungs of these dogs on histopathologic exam was also present, demonstrating the possibility for organ failure secondary to SIRS development.22
This study was designed to investigate the influence of oseltamivir when added to standard treatment on the disease process of CPV enteritis, as well as to monitor for significant adverse side effects associated with its use. Our hypothesis was that oseltamivir would help to ameliorate the disease morbidity and mortality associated with naturally occurring CPV enteritis, thereby decreasing hospitalization time, the need for colloid and other adjunctive therapies, and thus the cost associated with treatment.
LITERATURE REVIEW
Canine parvovirus (CPV) is a single-stranded, non-enveloped DNA virus that
infects and destroys rapidly dividing cells of the host. It is a highly contagious and hardy
virus, resistant to environmental conditions and many cleaning agents. The virus is
transmitted via the fecal-oral route. It primarily affects young dogs six weeks to six
months old, with the majority of older dogs showing immunity from either natural
infection or vaccination. CPV has a propensity to infect rapidly dividing cells. These
cells in dogs over eight weeks old include those of lymphoid organs, the latter myeloid
progenitor cells in the bone marrow, and intestinal epithelial cells. Rarely, the lungs,
liver and/or kidney can be infected. Dogs younger than eight weeks or pups in utero can
also have the virus infect myocardial cells, resulting in a myocarditis that can lead to
acute death or chronic heart failure symptoms with death by six months of age.1, 11, 23
Once infected, CPV localizes to the lymphoid tissue of the oropharanyx where it
replicates before entering the bloodstream. Viremia will usually occur two to three days
post-infection, and is manifested by a slight anorexia and fever. Dogs recover from this
stage briefly before progressing into clinical enteritis, typically between days five and six
post-infection.11 Clinical signs of enteritis include anorexia, vomiting, diarrhea and
severe dehydration. Severity of signs is related to age, stress level (other disease
processes, weaning), immune status and presence of intestinal parasites. These signs will
usually pinnacle around day six to seven post-infection.11
CPV infects and destroys the epithelial cells of the intestinal crypts. These cells
are responsible for the regeneration and differentiation into the villous epithelial cells.
Therefore, without the intestinal crypt cells, there is marked villous blunting, atrophy and
even necrosis, as well as malabsorption of nutrients with resultant diarrhea. Intestinal
inflammation accompanies this destruction and can lead to the breakdown of the gutblood
barrier. Translocation of bacteria from the gut lumen into the bloodstream, along
with an impaired immune response, can result in bacteremia. Bacteria are most often
gram negative, releasing endotoxin upon their destruction. Endotoxemia can then lead to
systemic inflammatory response syndrome through the activation of various cytokine
mediators, progressing into multiple organ dysfunction syndrome and death.
Leukopenia is considered a characteristic and often diagnostic quality of CPV
infection. However, this finding is reported to be evident in as few as 33% of cases when
diarrhea first developes.6 Most pronounced of the decreased white blood cells is the
neutrophil portion. This neutropenia is postulated to be due to a combination of
significant intestinal tract inflammation acting as a sink for the cells, rapid depletion of
bone marrow stores, and infection of the granulocyte precursors in the bone marrow.
Interestingly, it appears that the CPV spares the early hematopoietic precursors and
destroys mainly the later stages. Neutrophil nadir was found to coincide with the days
when clinical signs were at their worst at roughly day seven to eight post-infection. In
addition, neutrophil recovery coincides with clinical recovery at approximately days eight
through 12.11 A rebound neutrophilia is often observed as granulopoiesis is stimulated
with a decreasing peripheral consumption of mature neutrophils and a decreasing central
destruction of their progenitor cells. Hypoproteinemia, in particular hypoalbuminemia, is
another common clinicopathologic abnormality associated with CPV enteritis. This is a
result of a combination of factors, including intestinal loss, decreased synthesis as a
negative acute phase protein and decreased nutritional intake. Other laboratory
abnormalities vary, and can include anemia, azotemia, or increased liver enzymes.
Maternal antibodies can offer protection to pups from CPV for the first few weeks
of life. The period of protection varies, depending on maternal antibody level, amount of colostrum ingested, and size of the litter. The titer of the pup will equal approximately
50-60% of the bitch’s titer at whelping, meaning that proper vaccination of the mother is
essential to conferring protection to her pups.23 Maternal antibodies have a half-life of
around 10 days in the pup, and can interfere with proper immune response to
vaccination.23 The primary cause for vaccine failure is interference by maternal
antibodies. The high titer (higher amount of virus per dose), low passage (fewer times
virus grown on tissue culture) vaccines tend to be more effective despite maternal
antibody levels.23 A schedule of vaccination starting at six weeks old with boosters every
three weeks until 12-16 weeks old is the current recommendation to avoid interference
and provide proper protection to the pup. Proper vaccination is highly recommended, as
unvaccinated dogs have been found to be 12.7 times more likely to develop CPV enteritis
when compared to vaccinated dogs.24 Local antibody activity is responsible for binding
intestinal CPV to prevent further virus shedding in the feces. Shedding will typically
occur until between days three and 10 post infection. However, it is the systemic
humoral immune response or the circulating antibodies that confer protection as they
inactivate the virus in the blood before it has a chance to invade intestinal, lymphoid or
bone marrow cells.
Treatment of CPV consists principally of supportive care. Maintaining hydration
is the staple of this supportive care. Balanced electrolyte solutions supplemented as
needed with potassium and dextrose are the primary fluid type of choice. Colloidal
solutions can be used when indicated to support colloidal osmotic pressure. Antibiotic
use is an area where opinions vary, although their use is generally accepted once the
neutrophil count falls below 1500 cells/ul. Antibiotics targeting enteric bacteria are recommended, such as a combination of ampicillin and enrofloxacin. A lower dose of
5mg/kg q12 hours and use for a limited time (i.e. less than 5 days if possible) are
precautions taken to help avoid the side effect of cartilage deformities in these young
dogs. Antiemetics such as a constant rate infusion of metaclopramide and/or
chlorpromazine are frequently used to help control the vomiting associated with the
disease. The concept of starvation to rest the gut is being challenged lately. A recent
study found that dogs fed through a nasoesophageal tube soon after admission and
through vomiting episodes showed a significantly more rapid clinical improvement
without major adverse side effects.10 These dogs were found to have a quicker recovery
of appetite and normal attitude as well as recovery from vomiting and diarrhea when
compared to the food restricted group.
Several other potential treatments have been tested, often with conflicting or
disappointing results. Treatment with equine LPS endotoxin antiserum has shown
varying results. One source reports that its use decreased mortality from 48% to 17%.12
Another study found that its use was associated with an increased risk of death in puppies
less than 16 weeks.7 Recombinant human granulocyte colony stimulating factor (rhGCSF)
has been shown to help reverse neutropenia associated with radiation therapy and
drugs as well as cyclic neutropenia in dogs. However, its use to treat the neutropenia of
CPV infection has not shown significant benefit in recovery time or neutrophil
response.8, 9 Bactericidal/permeability-increasing protein (BPI) is an enzyme located in
the granules of polymorphonuclear cells. It is cytotoxic to gram negative bacteria by
increasing membrane permeability, and will also bind to free LPS to block its effects.
With the finding that 82% of CPV enteritis cases have endotoxemia, and that a relationship exists between increasing detectable tumor necrosis factor activity and
mortality,6 it was postulated that administration of a recombinant BPI (rBPI21) would
help improve survival. Unfortunately, rBPI21 treatment was not found to have a
significant effect on outcome, hospitalization time or plasma endotoxin levels in one
study.2 Interferons have antiviral effects that vary depending on dose, species and virus
type. A recombinant form of feline interferon omega (rFeIFN) has shown promise in
reducing severity of disease and mortality,13, 14 warranting further investigation
It is not necessarily the infection of the rapidly dividing cells and resultant
gastroenteritis that causes the high morbidity and mortality associated with CPV disease.
Gnotobiotic dogs infected with CPV were found to have only mild clinical signs.6 This is
highly suggestive that it is the bacterial translocation and resultant septicemia that is
significantly related to the morbidity and mortality of CPV enteritis. Endotoxemia with
subsequent systemic inflammatory response syndrome (SIRS) and multiple organ
dysfunction syndrome (MODS) are associated with a high mortality in and of themselves.
E. coli has been cultured from the lungs and liver in up to 90% of dogs that have died
from CPV,22 illustrating the high potential for bacteremia to develop as well as its
devastating consequences.
Oseltamivir is a drug that was designed to inhibit the human influenza A and B
viruses through inhibition of neuraminidase. Hemagglutinin (HA) and neuraminidase
(NA) are two viral surface proteins that interact with receptors containing terminal
neuraminic acid residues.15 The purpose of the HA is to mediate viral attachment to the
host cell membranes to allow entry and subsequent replication. NA has multiple
functions that allow for the replication of the virus as well as enhancing its virulence. First, NA cleaves the sialic acid residue (N-acetyl-neuraminic acid is synonomous with
sialic acid) of the cellular receptor binding newly formed virons.20 This frees the virons
from the host cell, and allows their systemic release. Secondly, NA prevents the
aggregation of the virons, allowing more effective systemic distribution with subsequent
cellular infectivity.20 Thirdly, in order to come in contact with and invade respiratory
epithelial cells, the influenza virus must first penetrate through the protective mucin layer
covering these cells. This mucin contains abundant sialic acid residues. NA cleaves
these residues, facilitating the viral infiltration through mucin to the epithelial cells which
the virus then invades and replicates within.20 Inhibition of NA will result in a direct
antiviral effect in viruses, such as the influenza virus, that contain the cell surface protein
NA. These effects include decreased replication, decreased viral release, increased viral
aggregation and decreased dissemination. In viruses that lack NA receptors such as CPV,
NA inhibition may prevent viral migration through mucin and subsequent invasion of
respiratory epithelial or gastrointestinal epithelial cells.
The above listed functions are accepted and proven effects of NA. In addition,
there currently are hypothesized effects of NA on the immune system itself. Secretory
IgA provides the body’s primary immunologic defense of mucosal surfaces such as the
respiratory or the gut epithelium. Under normal circumstances, sialic acid residues are
covalently bound to the hinge region of the IgA molecule. Removal of these residues
converts IgA into an appropriate ligand for the liver asialoglycoprotein receptor, which
effectively removes IgA from circulation and results in its clearance.20 Working on the
unproven assumption that there is an equilibrium between circulating and mucosal IgA,
the loss of IgA mediated by NA would result in impaired mucosal defenses. The IgA of mucosal surfaces is secreted by B cells under the influence of gamma-delta T cells. Both
of these cells contain sialic acid moieties on their surfaces.20 If cleaved by NA, the
lymphocyte homing would be altered from the mucosa to the bone marrow, resulting in a
localized immunosuppression for that mucosal surface. It is also believed that NA itself
may allow the increased production of certain cytokines,15 resulting in a proinflammatory
effect. Thus, inhibition of neuraminidase has the potential to improve
mucosal immunity by increasing the amount of mucosal IgA, decreasing the clearance of
circulating IgA, allowing normal function of the B and T cells and decreasing the
cytokine release associated with the proinflammatory response. All of these effects could
be beneficial to a dog with severe gastrointestinal inflammation secondary to CPV
infection.
Effects of the neuraminidase inhibitors beyond direct antiviral effects have been
appreciated in multiple studies. Bacterial complications following influenza virus
infection in children and adults are common. The incidence of acute otitis media
complicating influenza infection in children ranges between 21% to over 50%.12 Use of
oseltamivir has been reported to reduce the incidence of otitis media by 44% (children 1-
12 years) and 56% (children 1-5 years). 21 In adults, secondary complications were
reduced from 15% in the placebo group to 7% in the oseltamivir group in one study.25
Secondary bacterial pneumonias are responsible for an estimated 25% of influenzarelated
deaths19 in adults. Streptococcus pneumoniae is a bacteria commonly associated
with pneumonia secondary to influenza infection. A lethal synergism between the
influenza virus and S. pneumonia has been established,17 presumably accounting for the
high morbidity and mortality associated with the combination. It has been shown that the influenza virus promotes adherence of S. pneumonia to respiratory epithelium.17, 18 Viral
NA cleaves the sialic acid residues on the host respiratory epithelial cell, revealing
cryptic binding sites for the bacteria, 17, 18 thus facilitating colonization. Oseltamivir
reverses this effect in vitro.17
In a mouse model of influenza with secondary pneumococcal pneumonia,
oseltamivir use alone increased survival from 0% to 75%.18 This study also found that
oseltamivir use in addition to proper antibiotics resulted in 100% survival, compared to
0% in mice treated with antibiotics alone. Prophylactic use of oseltamivir resulted in
significantly less weight loss than placebo treated mice (prophylactic use, 5%; placebo,
25%).18 Prophylactic as well as delayed treatment use decreased the development of
pneumonia, increased survival and slowed the development and progression of disease in
those animals that did acquire pneumonia.18 In adults, oseltamivir use was found to
decrease the incidence of lower respiratory tract complications (LRTC) in influenza
patients overall by 55%.16 Patients considered at risk for secondary complications
showed a decrease in LRTC and antibiotic use by 34%, and in otherwise healthy patients
by 67%.16 In these studies, the beneficial effect of oseltamivir in preventing secondary
bacterial infections is most likely the result of NA inhibition preventing bacterial and
viral penetration of the mucin layer to allow bacteria to bind to epithelial cells.
Oseltamivir is labeled for use in children over 1 year old; use in children less than
1 year is discouraged based on fatality seen in animal toxicology studies in seven day old
rats with high doses of drug.21 Although the etiology of such adverse effects has not been
specifically identified, there is concern over lack of a fully developed blood brain barrier.
Side effects in children over 1 year are minimal, with vomiting being the only significant effect versus placebo.21 Similarly, nausea (12% vs 4%) and vomiting (10% versus 3%)
were found to be significant side effects over placebo in adults that subsided after the first
few days.27 These effects can be diminished with administration of a small amount of
food along with the drug. Studies in adult humans have shown an 80% bioavailability of
oseltamivir after oral dosing.21
STATEMENT OF RESEARCH OBJECTIVES
This study was designed to investigate the influence of oseltamivir when added to
standard treatment on the disease process of CPV enteritis, as well as to monitor for
significant adverse side effects associated with its use. Our hypothesis was that
oseltamivir would help to ameliorate the disease morbidity and mortality associated with
naturally occurring CPV enteritis, thereby decreasing hospitalization time, the need for
colloid and other adjunctive therapies, and thus the cost associated with treatment.
MATERIALS AND METHODS
Study Design
The present study was a prospective, randomized, placebo-controlled, blinded
clinical trial of the effects of oseltamivir in dogs with naturally occurring parvoviral
enteritis. Cases of CPV enteritis were recruited from the local area and animal shelter
between April 2005 and August 2006. A financial incentive was offered to the owners or
agent to allow their dogs to participate in the study. Inclusion criteria was simply a
positive CPV fecal antigen test, presence of appropriate clinical signs (vomiting, diarrhea,
lethargy, anorexia), and lack of any treatment initiated prior to presentation. Informed consent was received from all owners or agents prior to any treatment initiation at the
Auburn University Veterinary Teaching Hospital. In addition, all procedures were
reviewed and approved by the Animal Care and Use Committee of Auburn University.
Dogs were excluded if they lacked a positive fecal antigen test for CPV, if treatment had
been administered prior to presentation, or if consent was not granted. Based on these
criteria, only one dog that was eligible during the trial period was not included due to
lack of owner consent. All dogs that entered the study completed the trial.
Randomization was achieved via assignment of numbers with predetermined
treatment designation in blocks of 5. Assignment was allocated by hospital personnel not
directly involved in the trial, and these assignments were uncovered to the investigators
only at the conclusion of the study. Power analysis was not performed as part of the
design of the study, but post-hoc analysis revealed that 47 dogs would be needed in each
group to give the study a power of 0.80 at a significance level of 0.05.
Standard Treatment
A treatment protocol was designed to standardize therapy between the two groups
with the exception of oseltamivir administration to the treatment group and a placebo to
the control group. No deviations were made from this protocol for any of the patients.
An intravenous catheter was placed in all dogs, either a peripheral cephalic catheter or
central venous catheter in a jugular vein. Intravenous fluids consisting of a balanced
electrolyte solution b, c were administered at an initial rate to replace an estimated
dehydration deficit over a minimum of 2 hours and a maximum of 4 hours. In the case
were it was determined that shock was present, the same intravenous fluids were bolused
at a dose of 10-30ml/kg over approximately 20 minutes. Boluses were repeated until it was assessed that the state of shock had been resolved, and the remaining fluid deficit
estimated to replace dehydration was administered as above. No patient required the
administration of colloidal therapy to treat a state of shock. Assessment of hydration and
perfusion status, as well as fluid rates were decided upon by a single investigator (MRS)
for all dogs. After rehydration, the hydration status was reassessed via physical
parameters and the fluid rate adjusted as deemed appropriate. Fluids were supplemented
with potassium chlorided and/or dextrosee as needed based on laboratory results.
All dogs received intravenous (IV) antibiotics. Ampicillinf was administered at
22mg/kg (11mg/lb) IV every 8 hours and enrofloxacing at 5mg/kg (2.5mg/lb) IV every 12
hours. A metoclopromideh constant rate infusion was also initiated in all patients at a rate
of 1-2mg/kg/day (0.5-1mg/lb/day). If vomiting persisted at a rate of more than twice per
12 hour period despite a maximum rate of metoclopramide, chlorpromazinei was added at
0.5mg/kg (0.25mg/lb) subcutaneously every 8 hours. Pyrantel pamoatej (10mg/kg
[5mg/lb] per os) was administered to all dogs within the first 3 days of arrival to eradicate
intestinal parasites. If the total solids by refractometery fell below 3.5g/dl, Hetastarch
6%k was infused at a rate of 10-20ml/kg/day (5-10ml/lb). If anemia, defined as a PCV of
less than or equal to 20%, was present, a fresh whole blood transfusion from an available
donor was given at 10-20ml/kg (5-10ml/lb) over 4 hours. Packed red blood cell
availability was very limited at the time of the first required transfusion. Fresh whole
blood was collected from a donor dog and administered in its place. In order to minimize
differences in treatment between dogs, when the second transfusion became necessary,
although packed red cells were available, fresh whole blood from the previous donor was
again administered. If vomiting episodes were less than 4 per 12 hour period, water and a bland dietl were offered. If either appeared to induce nausea, they were immediately
removed from the animal. Voluntary eating was allowed through mild vomiting but food
was not forced upon any dog. True client cost for the treatment administered to the dogs
was not calculated; this figure can only be inferred from the intensity of treatments
(additional antiemetics, colloid or transfusion therapy, increased length of
hospitalization) required for each dog between the groups.
Discontinuation of intravenous fluids was at the discretion of a single investigator
(MRS). This was accomplished primarily when the animal was no longer vomiting and
consistently eating and drinking sufficiently to maintain hydration. Once fluids were
discontinued, all patients were monitored for at least 12 hours before discharge from the
hospital in order to ensure no relapse of clinical signs. The day of discharge was
assigned as the day when it was deemed the dog was healthy enough to be discharged. If
the dog stayed in the hospital for non-medical reasons, these extra days were not included
when figuring length of hospitalization. In those animals that did not survive, the day of
death was considered an endpoint equivalent to the day of discharge for the purpose of
data analysis. Post-mortem exams were performed on all non-surviving dogs.
Oseltamivir Treatment
Dogs assigned to the treatment group received oseltamivir at 2mg/kg (1mg/lb) per
os every 12 hours. An equivalent volume of a placebo, consisting of a standard
suspension agent with color additive, was administered per os every 12 hours to dogs in
the control group. Prior experience with oseltamivir by the authors has shown that dogs
often react to its taste and frequently vomit shortly after administration. Dilution of the
oseltamivir with water (1:1) prior to administration seemed to decrease these reactions; therefore, in this study, both oseltamivir and placebo were diluted out as above to lessen
the risk of adverse reaction to the oseltamivir. In addition, in dogs receiving
chlorpromazine for protracted vomiting, it was attempted to dose this medication 30-60
minutes before administration of the oseltamivir or placebo to minimize the risk of
vomiting either suspension as often as coincidental timing of the drugs allowed (typically
for the morning treatment). Given its propensity for gastric side effects (vomiting,
nausea),15 if significant adverse effects were associated with the administration of
oseltamivir to these patients despite these precautions, it should have been reflected in the
clinical scoring system employed.
Monitoring/Data Acquisition
Historical data, including previous vaccination against CPV and the duration of
clinical signs as noted by the owner or caretaker (rounded to the closest 12 hour period of
time), was obtained when this information was available at study entry. Signalment for
each dog was also recorded, as was its estimated percent dehydration on entry. Baseline
bloodwork was evaluated for all dogs on entry, consisting of a complete blood count
(CBC), a spun packed cell volume (PCV), total solids (TS) via refractometery, serum
electrolyte concentrations (sodium, potassium and chloride), blood glucose concentration
and blood lactate concentrationm. These values were monitored daily for all dogs. For
white blood cell values, the following variables were evaluated: initial total white blood
cell (WBC), neutrophil (neut) and lymphocyte (lymph) counts, the absolute nadir value
and the day of hospitalization (with day of presentation being day 1) on which the nadir
occurred for each of the WBC, neutrophil and lymphocyte counts, and the number of
days for which the counts for each value were significantly decreased, as defined by a WBC less than 3,000 cells/ul, neutrophils less than 2,000 cells/ul and lymphocytes less
than 1,000 cells/ul. Body weight was recorded twice daily, with the same scale used to
monitor each dog during their stay for consistency. In addition, vital parameters (heart
rate, respiratory rate, rectal temperature, mucous membrane color and capillary refill
time) were assessed a minimum of twice daily, as were hydration status and mentation.
Days on which dogs demonstrated SIRS criteria were also calculated as a percentage of
days of their total stay (days with positive SIRS criteria/total days in hospital x 100%).
SIRS was defined as to presence of at least 2 of the following 4 criteria: 1) temperature
greater than 102.5°F (39.2°C) or less than 100.0°F (37.8°C), 2)heart rate greater than 140
beats per minute (bpm), 3) respiratory rate greater than 40/minute or 4) total white blood
cell count greater than 19,000 cells/ul or less than 6,000 cells /ul.
Clinical Scoring System
A previously published clinical scoring system10 was used to evaluate 4 clinical
attributes of each patient: attitude, appetite, vomiting and feces. A score of 0 represented
a clinically normal parameter, with increasing severity of signs as the score increased up
to a maximum of 3 for each variable (Table 1). Scores were assigned twice daily, to
encompass the previous 12 hour period, and were assigned by the same investigator for
all dogs (MRS). The clinical scores were totaled for each dog per day for each of the 4
categories (attitude, appetite, vomit, feces), with a possible score ranging from 0 (normal)
to 6 (most severely affected) per 24 hour period. This total consisted of the combined
scores (ranging from 0 to 3) for each 12 hour periods for that day, assigned to each
category. In addition, a cumulative score consisting of a total across all 4 categories was calculated for each dog per day, with a possible score ranging from 0 (normal) to 24
(most severely affected).
STATISTICAL ANALYSIS
The Shapiro-Wilks test was used to evaluate the distribution of continuous
variables. Median (minimum, maximum) was used to describe continuous variables not
normally distributed and mean (+/- standard deviation [SD]) was used to describe
normally distributed variables. The Wilcoxon ranksum test was used to compare not
normally distributed continuous variables while the t test was used for normally
distributed variables. Categorical variables were described using percent and the Fisher’s
exact test was used to compare these variables between the treatment and control groups.
For all comparisons, a p-value <0 .05="" all="" analyses="" considered="" p="" significant.="" statistical="" was="">were performed using a statistical software programn.
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