Mefenoxam, Itraconazole, and Terbinafine Combination Therapy for Management of Pythiosis in Dogs (Six Cases)
ABSTRACT
Pythium insidiosum is an oomycete that encysts in the skin or gastrointestinal tract, leading to pythiosis. Pythiosis is reported in tropical and subtropical climates, affecting dogs and rarely cats. Surgical resection is the treatment of choice; however, cases present late in the disease and lesions are often nonresectable. Medical management is typically unsuccessful, with uncommon exceptions; however, mefenoxam, an agricultural fungicide, has in vitro efficacy against P insidiosum. We describe the use of mefenoxam, itraconazole, and terbinafine (MIT) in five dogs with gastrointestinal pythiosis and one dog with cutaneous pythiosis. Two of the gastrointestinal cases had disease extending to surgical margins and received MIT: resolution of clinical signs and seronegativity occurred after 189–193 days. Another case underwent surgical resection and MIT. The dog improved but subsequently developed a rectal mass, which responded to addition of prednisone and immunotherapy. Two cases were treated with MIT alone, and response varied. Efficacy of MIT in cutaneous pythiosis could not be determined. MIT may result in improved survival and seronegativity in dogs with incompletely resected gastrointestinal pythiosis. Mefenoxam is EPA registered, and extralabel use under the Animal Medicinal Drug Use Clarification Act does not apply. Additional research is recommended before use.
Introduction
Pythium insidiosum is an aquatic oomycete with a biflagellate zoospore infectious stage. The zoospore encysts in either damaged skin or the gastrointestinal mucosa, leading to two distinct clinical syndromes.1 Common clinical signs of the cutaneous form include ulcerative nodular lesions with draining tracts, whereas clinical signs of the gastrointestinal form include vomiting, progressive weight loss, anorexia, and diarrhea. Surgical resection with wide margins is the treatment of choice for pythiosis; however, many animals are not diagnosed until late in the disease, and lesions are often inoperable, leading to a poor prognosis. Medical management options are limited, as oomycetes lack cell membrane ergosterol, a key target of many antifungal medications.2 However, itraconazole and terbinafine are commonly used, with limited success, for medical management of pythiosis.3–5 The addition of anti-inflammatory doses of corticosteroids has also been investigated, with a positive outcome in a limited number of cases.6
Mefenoxam is an agricultural fungicide traditionally used to control oomycetes in plants.7–9 Mefenoxam has been shown to markedly inhibit radial growth of P insidiosum in vitro, whereas in comparison, terbinafine and itraconazole have only limited effect on the radial growth of P insidiosum.10 A single case report has documented the use of mefenoxam in a dog with gastrointestinal pythiosis that did not undergo surgical resection.5 In that report, minimal adverse effects were noted; however, the contribution of mefenoxam to disease control was unclear as clinical improvement was observed prior to addition of mefenoxam. Additional reports of clinical outcomes following the use of mefenoxam are therefore required to help guide clinical decision making. To our knowledge, this case series is the first to document the use of mefenoxam in a group of dogs with pythiosis. The purpose of our study was to evaluate the efficacy of combination therapy with mefenoxam, itraconazole, and terbinafine (MIT) in patients with and without attempted surgical management of pythiosis.
Materials and Methods
Dogs with gastrointestinal or cutaneous pythiosis were identified by searching the medical records at the authors’ institution between June 2018 and June 2019 for the term “mefenoxam.” Criteria for inclusion were a diagnosis of either cutaneous or gastrointestinal pythiosis based on serology, cytology, and (when available) histopathology, in addition to the use of mefenoxam. All owners signed an informed consent form prior to mefenoxam use that documented that they understood that the compound was an agricultural fungicide and that it had not been evaluated in well-controlled clinical trials for administration to animals or humans. The informed consent form also documented that the owner understood that alternatives were available and that mefenoxam was prescribed after detailed discussion with the owner. The form also stated that owners were aware that unknown risks and complications may be associated with the use of mefenoxam. Information extracted from the medical records included signalment, clinical signs, physical examination findings, clinicopathological data, abdominal ultrasound and computed tomography reports, and anti–P insidiosum antibody serology titers in addition to cytology and histopathology reports. Anti–P insidiosum antibody titers were considered positive if greater than 40%, as recommended by the reference laboratory. Both medical and surgical treatments and response to therapy were recorded. If recheck appointments were performed at the primary care veterinarian, medical records pertaining to those visits and any telephone conversations with the veterinarian and/or owner were also evaluated.
Results
Animals
Five client-owned dogs, three males (intact) and two females (spayed), were diagnosed with gastrointestinal pythiosis, and one female (intact) dog was diagnosed with cutaneous pythiosis. Complete case details can be found in Table 1.
History, Clinical Signs, and Physical Examination Findings
In dogs with gastrointestinal pythiosis, the median duration of clinical signs prior to referral was 4 wk (range, 3 wk to 6 mo). The presenting clinical signs are reported in Table 1. The median body condition score was 4/9 (range, 2 to 5/9). Other physical examination findings included melena (n = 3), abdominal discomfort (n = 2), dehydration (n = 2), and a palpable abdominal mass (n = 1). The patient with cutaneous pythiosis originally presented to the primary care veterinarian for a wound over the left scapula, which was resected. Histopathology was not submitted at that time, and 35 days later, the dog presented to the authors’ institution for progressive lethargy and significant deterioration of the wound. The wound was reported to have become progressively larger over time and had developed serosanguinous discharge. At the time of presentation, there was a large (16.4 × 7.8 cm) ulcerative and nodular wound extending from the cranial angle of the scapula dorsally to the level of the proximal humerus ventrally. The lateral margins of the wound spanned the entire scapula.
Diagnostic Investigations
All six patients had anti–P insidiosum antibody titersa performed at the time of presentation to the authors’ institution. Two patients had repeat titers performed during treatment. Multiple diagnostic imaging technologies were used in this case series, including radiographs (n = 4), ultrasound (n = 5), computed tomography (n = 3), and esophagogastroduodenoscopy (n = 1). Complete case details including titers and lesion localization can be found in Table 1.
Method of Diagnosis
Four dogs were diagnosed with pythiosis based on a combination of a positive anti–P insidiosum antibody titer and histopathology. Histopathological lesions were characterized by severe eosinophilic and pyogranulomatous or granulomatous inflammation in all cases. Histological samples were collected surgically. Gomori’s methenamine silver (GMS) stain was used in all cases to assist in detection of fungal hyphae. Intralesional broad, irregular, and poorly septate fungal hyphae were considered to be consistent with P insidiosum. One dog was diagnosed with presumptive gastrointestinal pythiosis based on results of abdominal imaging, a positive anti–P insidiosum antibody titer, and cytological examination of GMS-stained fine-needle aspirates of an intestinal mass. One dog was diagnosed with suspected gastrointestinal pythiosis based on abdominal imaging findings and a positive anti–P insidiosum antibody titer. Histopathological confirmation was recommended in both of these cases; however, the medical records note that surgery and histopathological confirmation were declined.
Gastrointestinal Pythiosis: Medical and Surgical Treatment Pursued
Surgical resection was pursued in three dogs: Billroth I (n = 1), Billroth II (n = 1), and a resection and anastomosis of duodenal and jejunal tissue (n = 1). Postoperative histopathology revealed that two dogs (both cases of gastric pythiosis) had fungal hyphae extending to surgical margins, whereas one dog (with the duodenal and jejunal mass) had normal tissue at the proximal and distal ends of the resected tissue. The extent of the surgical margin was not recorded in this dog and is unknown. Both dogs with hyphae extending to surgical margins were started on medical therapy, as was the dog in whom the extent of surgical margins was not recorded. The two dogs that did not undergo surgery were also started on medical therapy. Medical therapy consisted of mefenoxamb, itraconazole, and terbinafine. Various formulations of itraconazole and terbinafine were used. One dog received ancillary therapy consisting of six doses of Pythium immunotherapyc and prednisone at 0.58 mg/kg/day (Table 1).
Gastrointestinal Pythiosis: Response to Combined Medical and Surgical Therapy
Patients that underwent combined surgical and MIT therapy (three dogs) had a follow-up duration of 287–443 days. All three patients were alive at the time of last follow-up and had complete resolution of clinical signs. Two patients had repeat serology performed (Table 1). The third patient did not have repeat serology performed but was doing well at the time of last follow-up, with no clinical signs of pythiosis. The third patient, however, was prescribed ancillary therapies, consisting of six doses of Pythium immunotherapy in addition to oral prednisone at 0.58 mg/kg/day following discovery of tenesmus and a new mass on rectal palpation 28 days after the resection and anastomosis surgery. Biopsies of the rectal mass were not performed. The rectal mass and tenesmus resolved 63 days after initiation of ancillary therapy. Prior medical therapy was continued. Two of the three dogs had MIT therapy discontinued (Table 1). One of these patients had an additional serology sample performed 132 days after discontinuing MIT therapy, and the serology was negative. The second patient was doing well at the time of last follow-up; however, repeat serology was not performed after discontinuation of MIT.
Gastrointestinal Pythiosis: Response to Medical Therapy Alone
Two dogs underwent MIT therapy alone following a diagnosis of gastrointestinal pythiosis based on clinical signs, imaging findings, and a positive anti–P insidiosum serology titer. Cytology was also performed on an intestinal mass in one of the two dogs and revealed hyphae consistent with P insidiosum. Histopathological confirmation was not performed in these patients. Both patients were prescribed MIT (doses recorded in Table 1). One patient had resolution of clinical signs as reported by the owner; however, repeat serology and imaging were not performed to confirm remission. MIT therapy was subsequently discontinued by one owner because of financial limitations. The exact timing of when medical therapy was discontinued could not be determined via evaluation of the medical records or telephone conversation with the owner. The owner did, however, report that the dog was doing well at the time of last follow-up (210 days after initiation of MIT). The second dog had a large caudal abdominal mass and multiple segments of small intestine affected on abdominal ultrasonography. Medical therapy was initiated, and surgery was not pursued. This patient unfortunately continued to decline and was euthanized 12 days after initiation of MIT.
Cutaneous Pythiosis: Medical and Surgical Treatments and Response to Therapy
The patient with cutaneous pythiosis underwent a left forelimb amputation, including removal of the scapula and regional lymph nodes (left prescapular and axillary lymph nodes). A mesh skin graft was placed owing to the size of the defect following removal of the limb. The excised tissue was submitted for histopathology, which revealed extensive zones of eosinophilic, pyogranulomatous, and fibrosing cellulitis with intralesional hyphal structures consistent with P insidiosum extending to surgical margins. Six days postoperatively, mucopurulent discharge was noted to be coming from the wound and the graft began to show signs of failure (white discoloration). At that time, a repeat culture was performed, which revealed growth of Pseudomonas aeruginosa, Proteus mirabilis, and Staphylococcus intermedius. The dog was prescribed chloramphenicol (29.5 mg/kg per os q 8 hr). Twenty-two days following the forelimb amputation, an area of dehiscence was noted and a revision surgery was performed. Vancomycin gel was infused into a pocked above the wound, based on the results of a prior culture and consultation with a board-certified veterinary pharmacologist. Thirty-seven days following the original surgery, a second revision surgery was performed owing to further wound dehiscence. During this procedure, amikacin gel was placed in the wound. Seven days after the second revision surgery, the patient was discharged from the hospital on ceftazidime (29.9 mg/kg subcutaneously q 8 hr) in response to a multidrug-resistant P aeruginosa infection. The dog was also discharged with MIT (see Table 1 for doses). Telephone conversation with the referring practice 48 days after referral revealed that the patient was doing well, with no evidence of palpable regrowth. Unfortunately, the patient was subsequently lost to follow-up, and long-term efficacy of MIT cannot be concluded.
Documented Adverse Events
Potential adverse reactions were documented in two dogs. One dog (that had undergone a Billroth II procedure) developed marked increases in both alkaline phosphatase (ALP; 3453 U/L, reference interval: 11–140 U/L) and alanine aminotransferase (ALT; 1440 U/L, reference interval: 10–90 U/L) 19 days after initiation of MIT. The dose of itraconazole was reduced by 25% and amoxicillin clavulanic acid (15.1 mg/kg per os q 12 hr) was prescribed, in case of potential bacterial hepatitis. Repeat bloodwork performed 16 days later revealed a lower ALP (1177 U/L) and ALT (737 U/L) concentration. Repeat bloodwork performed 45 days after itraconazole dose reduction revealed that the ALP (460 U/L) and ALT (232 U/L) had reduced further. The ALP and ALT were within normal limits at the time of last follow-up. One dog (that underwent a Billroth I procedure) developed a mild increase in ALP (199 U/L) and ALT (166 U/L) 22 days after initiation of medical therapy. The itraconazole dose was reduced by 25%. Both the ALP and ALT returned to within the reference interval 32 days later. Medical record review failed to document any episodes of poor tolerance following initiation of medical therapy; however, one patient continued to clinically deteriorate, despite medical therapy, and the contribution of disease progression versus medication administration cannot be definitively determined.
Discussion
This case series is the first to document the use of a combination treatment protocol, including mefenoxam, for the management of pythiosis in a group of dogs. To the authors’ knowledge, this is also the first report of mefenoxam in the management of cutaneous pythiosis. Pythiosis is associated with a poor prognosis, with many dogs dying within 2–3 mo of onset of clinical signs.3,11,12 Surgical resection of affected tissue with wide margins is the treatment of choice; however, surgery is not always pursued owing to location, extent of disease, and financial limitations.13 Historically, medical therapy has been unrewarding; however, uncommon case reports have documented good response to combinations of marginal excision, medical therapy, and immunotherapy.2,4–6,13,14 These case reports, and the cases described here, suggest that additional research is required to determine if aggressive resection is required in all cases.
Commonly used medical therapy includes the use of itraconazole and terbinafine. Itraconazole is an azole antifungal medication that inhibits sterol 14∝-demethylase, which is involved in ergosterol synthesis.15 Terbinafine has an alternative mechanism and inhibits squalene epoxidase, which is involved in ergosterol and lanosterol synthesis.15 One potential cause of the poor success rate with traditional antifungal medications is that P insidiosum typically lacks ergosterol in its cell membrane.2
Mefenoxam is an agricultural fungicide that inhibits RNA polymerase.7–9 A safety study was performed prior to registration by the EPA and revealed minimal toxic effects in dogs and a “no-observable-effect” level at 8 mg/kg/day.16 Mefenoxam has been reported in a single previous case of gastrointestinal pythiosis in a dog; however, it was added after initiation of itraconazole and terbinafine, and therefore, the contribution of mefenoxam to disease resolution was difficult to determine.5 A prior study used radial growth to assess the in vitro drug susceptibility of P insidiosum and reported that mefenoxam had the most profound effect on growth and was more efficacious than traditional antifungal medications.10
In this case series, we document the use of a combination therapy consisting of MIT in five cases of gastrointestinal pythiosis in dogs. We also document the use of this combination treatment in a dog with cutaneous pythiosis, although the length of follow-up in this case limits interpretation of efficacy.
Both cases of gastric pythiosis that had attempted surgical resection followed by MIT were alive and well at the time of article submission, despite incomplete surgical margins. Both of these patients were diagnosed with pythiosis based on evaluation of abdominal imaging findings, a positive antibody serology, and evaluation of surgical histopathology samples. Both of these patients had repeat anti–P insidiosum antibody serology titers performed, and both subsequently became negative after 189–193 days. One of the two dogs had repeat abdominal ultrasound examinations performed by a radiology resident under the supervision of a board-certified veterinary radiologist, and there was no evidence of ultrasonographically detectable regrowth. It is, however, important to consider that microscopic or small macroscopic lesions may not be detected using ultrasonography. Both patients subsequently had medical therapy discontinued.
One patient that began MIT following an intestinal resection and anastomosis surgery had initial resolution of clinical signs. However, 28 days postoperatively, the dog began having tenesmus and was evaluated by the primary care veterinarian, who palpated a rectal mass. The mass had not been noted on previous physical examinations. No additional diagnostics were performed, and the dog was prescribed six doses of Pythium immunotherapy and oral prednisone at 0.58 mg/kg/day.
Dogs with gastrointestinal pythiosis treated with medical therapy alone had fewer follow-up data available. The reason that surgical therapy was declined in each case was not explicitly stated; however, potential reasons may include financial constraints, the invasive nature of attempted surgical resection, presumed inability to resect the mass(es), and concern for quality of life. One dog continued to decline, despite initiation of medical therapy, and was euthanized 12 days later. Given that the time to seronegativity following initiation of medical therapy was 181–193, and that the patient was euthanized after 12 days owing to a poor quality of life, it is likely that the patient did not receive MIT therapy for a long-enough period of time to determine if MIT alone would have been efficacious in this patient, if sufficient time was allowed. The second patient was reported to be doing well at the time of last follow-up; however, no repeat imaging or serology was performed in this patient, which limits determination of clinical efficacy.
In this case series, we also documented the use of mefenoxam in a case of cutaneous pythiosis. The follow-up period for this case was only 48 days. However, no adverse effects were noted, and there was no palpable regrowth at the time of last recheck. Additional studies with longer follow-up periods are required to determine the efficacy of MIT in cutaneous pythiosis.
Pythium immunotherapy and prednisone were used in one patient. Pythium immunotherapy contains Pythium exoantigens and cytoplasmic antigens and shifts the host immune response from a T-helper type II response to a T-helper type I immune response, associated with a decrease in immunoglobulin E and interleukin 4 and an increase in interleukin 2.17 These immunoglobulin and cytokine changes have not been documented in dogs, and the lack of such changes may be associated with the typically poor response of dogs to immunotherapy. Pythium immunotherapy and prednisone were prescribed by the primary care veterinarian in response to detection of a mass on rectal examination. The mass and associated tenesmus resolved after addition of the immunotherapy and prednisone; however, MIT was continued. It is unclear in this case whether the mass was due to pythiosis, and it is also unknown whether the mass responded to ancillary therapy or responded to a more prolonged period of treatment with MIT. However, given the results of a recent case report, anti-inflammatory prednisone was likely a contributing factor.6
Two patients developed a mild to moderate elevation in both ALT and ALP following surgery (Billroth I or Billroth II) and initiation of MIT therapy. These cases were suspected to be associated with a dose-dependent hepatoxicity from itraconazole, or with an ascending bacterial hepatitis in the patient that underwent a Billroth II procedure.18–20 As both patients responded to a 25% dose decrease in itraconazole and/or the addition of amoxicillin clavulanic acid, no additional diagnostics were performed. Additional potential differentials for the elevation in ALP and ALT include a self-limiting reactive hepatopathy from an underlying transient enteropathy or transient pancreatitis. Further consideration should be given to an adverse effect from mefenoxam; however, this is considered unlikely given that the liver enzyme elevations resolved with no change in mefenoxam therapy.
The dose of mefenoxam used in this case series was based on a previous case report and a safety study.5,16 However, to the authors’ knowledge, there have been no studies that determine the oral bioavailability of mefenoxam in dogs, and it is therefore unknown if mefenoxam reaches tissue concentrations equivalent to those successfully used in the previous in vitro study.10 Additional studies are therefore required to determine the oral bioavailability of mefenoxam in dogs and to determine if the dose used in this case series reaches therapeutic concentrations, or whether further dose optimization is required. Terbinafine was used at a wide variety of doses (5.9–29.3 mg/kg); this likely reflects the wide range of terbinafine doses reported in prior literature.1,13,21 Additional studies should be performed to determine the optimal dose of terbinafine for canine pythiosis.
Mefenoxam is an EPA-registered fungicide and is not regulated by the FDA. Because the Animal Medicinal Drug Use Clarification Act is applicable only to drugs, use of mefenoxam does not fall within FDA extralabel drug use guidelines. Although enforcement discretion for veterinary extralabel use of some EPA pesticides exists, veterinarians using EPA-registered products such as mefenoxam should be aware that they are still subject to responsibilities outlined per the Federal Insecticide, Fungicide and Rodenticide Act.22 Interestingly, enilconazole, which is frequently used in veterinary practice to manage nasal aspergillosis, would fall under similar regulatory oversight as mefenoxam.23–27 The authors of this manuscript therefore suggest that informed owner consent and additional research are indicated prior to the routine clinical use of mefenoxam.
One limitation of this case series is that two dogs did not have histopathological confirmation of a presumptive diagnosis of pythiosis. Both of these patients, however, had clinical signs consistent with gastrointestinal pythiosis, and both had anti–P insidiosum antibody titers of 100%. In addition, one dog had cytological evidence of P insidiosum hyphae on GMS staining. The antibody enzyme-linked immunosorbent assay is, in fact, highly sensitive and specific for P insidiosum.6,13,28 A prior study documented that an enzyme-linked immunosorbent assay similar to the one used in this report had a 100% sensitivity and specificity for the diagnosis of pythiosis, when using the cutoff value of 40% used in this case series.28 Therefore, a positive titer in a population of animals thought to have a high prevalence of disease (i.e., dogs with consistent clinical signs and imaging findings in an endemic geographic location) is likely associated with a very low risk of a false-positive result.
Another limitation of this case series is a lack of complete follow-up in a number of cases, a limitation associated with the retrospective nature of this case series. Only two of the six (33%) patients had repeat anti–P insidiosum antibody titers performed, which may reduce the sensitivity of detection of disease relapse. It is, however, important to consider that five of six patients were asymptomatic at the time of last follow-up and had survival times superior to most of those previously reported (typically, 2–3 mo).3,11,12 The number of cases with documented long-term survival suggest a beneficial effect of combination medical therapy consisting of MIT in canine pythiosis. In the absence of follow-up serology, it is also difficult to determine the preferred duration of treatment in dogs with apparent clinical remission; however, in the two cases where serology was monitored, the duration of time to a negative serology was 189–193 days. Reversion to negative serology was noted in patients that received incomplete surgical resection followed by MIT.
As multiple treatments were prescribed concurrently, the contribution of each drug to resolution of pythiosis cannot be determined. A prospective randomized clinical trial with monitoring of clinical signs, adverse effects, clinicopathological data, imaging, and serology would be needed to fully determine the efficacy of mefenoxam (potentially in isolation versus as part of a combination protocol) and its contribution to disease resolution. Caution should therefore be taken when prescribing mefenoxam until such data are available. Mefenoxam is, however, readily available and appears safe in the management of canine pythiosis. The drug is therefore a potentially attractive addition to the therapeutic armamentarium for canine pythiosis.
Conclusion
This case series documents complete resolution of clinical signs and onset of seronegativity in two dogs undergoing MIT therapy following incomplete excision of gastric pythiosis. This case series also documents prolonged survival and resolution of clinical signs in a dog that underwent a resection and anastomosis for duodenal and jejunal pythiosis, that was treated with combination therapy in addition to Pythium immunotherapy and anti-inflammatory prednisone. When MIT therapy was used in the absence of attempted surgical correction, the prognosis was more varied, and additional study is required to determine efficacy in these cases. One case of cutaneous pythiosis was also presented; however, owing to a lack of long-term follow-up, conclusions regarding efficacy of MIT therapy in cutaneous pythiosis cannot be made. In all cases, MIT therapy was well tolerated with few adverse effects.
Contributor Notes
ALP (alkaline phosphatase); ALT (alanine aminotransferase); GMS (Gomori’s methenamine silver); MIT (mefenoxam, itraconazole, and terbinafine)
