A double dose of infectious disease: Histoplasmosis in a fox hound puppy

In late June and early July of 2012, a kennel of about 40 American-English cross fox hounds had an outbreak of Rocky Mountain spotted fever (RMSF) as demonstrated by rising titers that went through all hounds, including seven 8-week-old puppies. The puppies were housed with the bitch in a barn in a stall separate from the kennels of the adults. All seven puppies became ill (fever of unknown origin) and were hospitalized. 

Two male puppies succumbed to the RMSF on day 2. The remaining five puppies were hospitalized for five days and then treated with doxycycline for two weeks starting on day 2. The surviving puppies continued to grow and develop normally. 

About seven weeks later, the owners of the five remaining puppies noticed that an intact female puppy (now 15 weeks old) began to suffer from intermittent anorexia, vomiting and fever (105 F [40.6 C]). Six days later, the puppy presented with mild diarrhea. The owners started the puppy on an antiemetic and metronidazole that they had on hand. The puppy was losing weight, sleeping more and not interacting as much with its litter mates. Five days later, after failure to improve, the puppy was brought to the veterinary hospital.

On physical examination, the puppy was thin, weighing 37.6 lb (17.1 kg). It was alert and had pale mucous membranes and a temperature of 102.2 F (39 C). Blood work (Table 1) showed a normocytic normochromic nonregenerative anemia, thrombocytopenia and a prolonged activated clotting time. The results of a cage-side ELISA for Anaplasma species, Borrelia burgdorferi, Ehrlichia species and Dirofilaria immitis were negative. Treatment was initiated with doxycycline (5.8 mg/kg b.i.d. orally), ciprofloxacin (29 mg/kg b.i.d. orally), vitamin K1 (1.4 mg/kg b.i.d. subcutaneously for the first dose and then orally) and force-feeding. 

Diagnosis and treatment

Over the next two days, the puppy remained pale and anorectic with an undulating fever. It responded when stimulated but mainly slept during the day. The hematocrit remained 21%. The results of a Coombs test and parvovirus test were negative. The puppy continued to be force fed and had dark and soft stools. Epoetin alfa injections (98.6 U/kg subcutaneously) were started and given every three days. 

On the fourth day, the puppy was bright, alert and reactive. Its mucous membranes were still pale, and it had a hematocrit of 27.8%, white blood cell (WBC) count of 4.43 k/µl, platelet count of 52 k/µl and temperature of 105.2 F (40.7 C). A round, thin, clear cell wall organism with a basophilic nucleus was observed microscopically on a blood smear stained with Wright's stain, which was identified as Histoplasma species (Figure 1). 

1. A peripheral blood smear containing Histoplasma capsulatum fungemia yeast organisms free floating in the blood (arrow) (Wright's stain; 100X oil immersion).

A blood sample was cultured on Sabouraud dextrose agar (SDA) and a 5% sheep blood agar plate to confirm histoplasmosis. As we do before all transfusions, the puppy was pretreated with diphenhydramine (2.33 mg/kg), famotidine (0.46 mg/kg) and dexamethasone (0.26 mg/kg) intravenously and then transfused with fresh whole blood.

The day after transfusion, there was an increase in the hematocrit and platelets (33% and 128 k/µl, respectively) with a WBC count of 5.06 k/µl, a temperature of 102.8 F (39.3 C) and weight of 34.6 lb (15.7 kg). After discussing the guarded prognosis and cost of medications with the owners, the following treatment plan was agreed upon: 10 mg (0.6 mg/kg) prednisone once a day for 10 days, then reduced to 10 mg every other day, and 6.4 mg/kg fluconazole twice a day. 

Although the use of prednisone can be controversial, it was used at a low dose in this case to control pyrexia, stimulate appetite and reduce lethargy. Fluconazole was to be administered for a minimum of three to six months or longer, if indicated. The epoetin alfa given every three days was continued, but all antibiotics were discontinued. 

Outcome

The owners elected on day 6 to take the puppy home and monitor its temperature, treatment and force-feeding. They returned in three days for a repeat CBC and epoetin alfa injection. At the follow-up appointment, the owners reported that the puppy was eating some chicken breast, ground beef and treats but was still being force fed. The examination revealed the puppy was paler than after the blood transfusion and had lost a considerable amount of muscle mass. The owners stated that the puppy was more lethargic but still interacted when stimulated. The hematocrit was 26.4%, platelet count was 130 k/µl, temperature was 102.9 F (39.4 C) and WBC count was 18.15 k/µl, which was increased compared with the results three days prior.

The owners were informed that a blood transfusion was not indicated at this time and to continue the epoetin alfa injection, prednisone, fluconazole and force-feeding, with laboratory values repeated in three days. The owners called two days later and reported that the puppy had become blind and continued to have a fever. Ocular involvement is a clinical sign of disseminated disease with poor recovery. The owners elected to euthanize the puppy humanely because of the poor prognosis. 

The blood sample on the blood agar plate grew in seven days, and budding yeast cells were observed microscopically. The blood sample on the SDA took five weeks to grow, with smooth macroconidia and small, round to teardrop microconidia along the hyphae being observed microscopically, thereby confirming the Histoplasma capsulatum fungemia diagnosis. 

Discussion

Histoplasmosis capsulatum, a dimorphic soil-borne fungus, is endemic throughout the Mississippi and Ohio River valleys.1-3 Dogs usually acquire Histoplasma species microconidia from inhalation, which is presumably how this hound puppy become infected.4,5 In this case, it is possible that the puppy's immune system had been challenged and then possibly compromised because of the RMSF infection seven weeks prior. This may have facilitated an acute systemic histoplasmosis, leading to rapid deterioration of health. 

Histoplasmosis can cause gastrointestinal signs such as vomiting and diarrhea in dogs, which can lead to lethargy and weight loss.3 In addition, nonregenerative anemia and thrombocytopenia4-6 are commonly seen. Clinicians should include histoplasmosis as a differential diagnosis in dogs with these clinical and laboratory signs, especially if they are in or have visited an endemic area recently. 

Diagnosis of H. capsulatum can be difficult because of the clinical similarities with other organisms.5 Impression smears from fine-needle aspirates are commonly used when diagnosing lesions.3 The most direct way to isolate and identify the organisms is by culturing blood. However, the organism is slow-growing; it can take up to four weeks for the fungus to grow on SDA7,8 as observed in cultures in this study. Lactophenol cotton blue stain was used in identification of the fungus grown on SDA. 

An ELISA is not considered a reliable diagnostic test because of antibodies cross-reacting between H. capsulatum and Blastomyces dermatitidis.3,4,8 Additionally, it is difficult to distinguish whether circulating antibodies are due to exposure or active infection. Antigen detection by nested polymerase chain reaction in the urine, blood and tissue is now performed in dogs to diagnose histoplasmosis.3 There are no published data regarding the sensitivity and specificity of this test in dogs, but it is thought to be similar to the results in cats (94%) as well as people (95% to 99%).8-10 

The owners of the puppy had the remaining littermates tested for histoplasmosis with an enzyme immunoassay that detects a galactomannan antigen located in the cell wall of H. capsulatum (Mira Vista Diagnostics). Urine was collected, and initially all four littermates had negative results (results below 0.4 to 0.8 ng/ml). Upon retesting three weeks later, two of the four littermates had positive test results at low levels, most likely from exposure through inhalation because of being in the same environment as the ill puppy. Currently, all four littermates are healthy and have no clinical signs of active histoplasmosis. 

There are several antifungal medications that have been recommended for histoplasmosis treatment. Itraconazole is considered the treatment of choice for infections in dogs,3,4,8 but it is expensive. Fluconazole can penetrate more efficiently in the central nervous system and eye, has fewer side effects than itraconazole and costs less. Amphotericin B is considered the gold standard when treating many systemic fungal infections. However, it is not used in most cases of histoplasmosis because of its significant nephrotoxicity. It is recommended that treatment continue for one to two months after resolution of clinical signs.5,8 The owners in this case elected to go with fluconazole because of the decreased side effects and because it is considerably less expensive then the other two antifungal choices.

There is currently no vaccine available for histoplasmosis, so early detection and treatment is imperative for a possible positive outcome. There is no practical way to prevent the organism in the environment. Clinicians should be suspicious of fungal infections in young dogs in endemic regions if the dogs display signs of anorexia, lethargy, diarrhea, fever and nonregenerative anemia that do not respond to initial antibiotic therapy and continue to show no clinical improvement chronically. Although the outcome was grave, this case demonstrates the value of careful examination of a peripheral blood smear that identified H. capsulatum and directed the appropriate treatment plan. 

Acknowledgments

The authors thank Drs. Robin Allison and Andrew Hanzlicek for their contributions in identification and treatment, respectively, and Jay Bullard, SI(ASCP) for his assistance in the laboratory.

References

1. Wheat LJ, Kauffman CA. Histoplasmosis. Infect Dis Clin North Am 2003;17(1):1-19.

2. Center for Disease Control and Prevention. Histoplasmosis. Available at http://www.cdc.gov/fungal/diseases/histoplasmosis/. Accessed April 28, 2015. 

3. Brömel C, Greene CE. Histoplasmosis. In: Infectious diseases of the dog and cat. 4th ed. St. Louis: Elsevier, 2012;614-621.

4. Brömel C, Sykes JE. Histoplasmosis in dogs and cats. Clin Tech Small Anim Pract 2005;20(4):227-232.

5. Tyre E, Eisenbart D, Foley P, et al. Histoplasmosis in a dog from New Brunswick. Can Vet J 2007;48(7):734-736.

6. Mitchell M, Stark DR. Disseminated canine histoplasmosis: a clinical survey of 24 cases in Texas. Can Vet J 1980;21(3):95-100.

7. Kern ME, Blevins KS. Organisms causing systemic mycoses. In: Medical mycology a self-instructional text. 2nd ed. Philadelphia: FA Davis Co, 1997;193-203.

8. Mira Vista Diagnostics. Histoplasmosis in animals. Available at http://miravistalabs.com/wp-content/uploads/2014/09/MVD-Histoplasmosis-Animals-Veterinary.pdf. Accessed May 26, 2015.

9. Connolly PA, Durkin MM, LeMonte AM, et al. Detection of histoplasma antigen by a quantitative enzyme immunoassay. Clin Vaccine Immunol 2007;14(12):1587-1591.

10. Cook AC, Cunningham LY, Cowell AK, et al. Clinical evaluation of urine Histoplasma capsulatum antigen measurement in cats with suspected disseminated histoplasmosis. J Feline Med Surg 2012;14(8):512-515.

Sallie A. Ruskoski, PhD, MT(ASCP)

Department of Health Professions

Northeastern State University

Broken Arrow, OK 74014

Joseph D. Landers, DVM

Heritage Veterinary Hospital

4011 S. 79th East Ave.

Tulsa, OK 74145