Evidence‐Based Medicine: The Design and Interpretation of Noninferiority Clinical Trials in Veterinary Medicine

Noninferiority trials are clinical studies designed to demonstrate that an investigational drug is at least as effective as an established treatment within a predetermined margin. They are conducted, in part, because of ethical concerns of administering a placebo to veterinary patients when an established effective treatment exists. The use of noninferiority trial designs has become more common in veterinary medicine with the increasing number of established veterinary therapeutics and the desire to eliminate potential pain or distress in a placebo‐controlled study. Selecting the appropriate active control and an a priori noninferiority margin between the investigational and active control drug are unique and critical design factors for noninferiority studies. Without reliable historical knowledge of the disease response in the absence of treatment and of the response to the selected active control drug, proper design and interpretation of a noninferiority trial is not possible. Despite the appeal of conducting noninferiority trials to eliminate ethical concerns of placebo‐controlled studies, there are real limitations and possible ethical conundrums associated with noninferiority trials. The consequences of incorrect study conclusions because of poor noninferiority trial design need careful attention. Alternative trial designs to typical noninferiority studies exist, but these too have limitations and must also be carefully considered.     

Effect of N‐acetyl‐L‐cysteine Supplementation in Semen Extenders on Semen Quality and Reactive Oxygen Species of Chilled Canine Spermatozoa

The objective of this study was to evaluate the quality of chilled dog semen processed with extenders containing various concentrations of N‐acetyl‐L‐cysteine (NAC). Ejaculates from five dogs were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF‐movement), viability, acrosomal integrity and by the hypo‐osmotic swelling test (HOST). In addition, superoxide anion (O₂^‐?) production, hydroxyl radicals (OH^?) and total reactive oxygen species (tROS) were determined. The pool was divided into five aliquots, which were diluted to a final concentration of 66.66 × 10⁶ spermatozoa/ml with Tris‐glucose‐egg yolk extender containing one of the following concentrations of NAC (0, 0.5, 1, 2.5 or 5 mm ). The semen aliquots were chilled and preserved at 4°C. Semen quality was evaluated after rewarming at 72 h. Sperm motility was significantly higher with the 0.5 mm concentration compared with the control group (p = 0.001). Rapid steady forward movement was higher with the 0.5 and 1 mm concentrations compared with the control and 5 mm group (p < 0.001). Viability and HOST percentages were not significantly altered. Compared with the control, the 5 mm concentration showed significantly reduced percentages of spermatozoa with normal acrosomes (p = 0.049). None of the ROS values at 72 h were significantly affected by the presence of NAC in semen extenders, although all NAC concentrations showed lower O₂^‐? and OH^? values compared with the control. Only the concentrations of 1 and 5 mm inhibited the significant increase of tROS values after 72 h, compared with the fresh semen value. In conclusion, NAC supplementation of semen extenders is beneficial to semen motility of canine spermatozoa during chilling with the 0.5 mm concentration being the most effective, although no significant ROS inhibition was observed at 72 h.     

Canine cutaneous epitheliotropic T‐cell lymphoma: a review

Cutaneous epitheliotropic T‐cell lymphoma in the dog is a rare neoplastic condition with unknown aetiology. The dermatitis is characterized by infiltration of neoplastic T lymphocytes with a specific tropism for the epidermis and the adnexal structures. The different clinical and histological forms (mycosis fungoides, pagetoid reticulosis and Sézary syndrome) are reviewed. The disease in the dog resembles the human syndrome, but in 80% of the canine cases, neoplastic cells are CD4^?/CD8⁺ versus CD4⁺/CD8^? in 90% of the human patients. Prognosis is poor with a survival time from few months to 2 years. Treatments frequently have a low efficacy. New protocols using lomustine may improve the poor prognosis of the disease.     

Evidence‐based considerations for control of Parascaris spp. infections in horses

Parascaris spp. infection is virtually ubiquitous in young foals and worm burdens can achieve high numbers. The most important disease manifestation is impaction of the small intestine, which occurs in a small proportion of infected foals but is associated with a guarded prognosis for survival. Control of Parascaris spp. is complicated by emerging resistance to currently available anthelmintic drug classes. Resistance to macrocyclic lactones has been reported worldwide and a few studies have also documented signs of resistance to pyrantel salts and benzimidazoles. Foals generally develop immunity to Parascaris spp. parasites around age 6 months, but a proportion of weanlings and yearlings can harbour smaller burdens at age 8–10 months. Older horses have occasionally been reported with substantial ascarid burdens as well. Qualitative detection of ascarid eggs has good diagnostic value whereas an actual egg count does not correlate well with the size of the worm burden. A recent investigation documented the applicability of a transabdominal ultrasound technique for semiquantitatively monitoring ascarid burdens in foals. Control of ascarids is complicated by the limited drug classes available for treating this parasite, and by the fact that foals are often concurrently infected with strongyles. In many cases, none of the 3 available anthelmintic classes are simultaneously effective against both parasite groups, so close monitoring is required to select the most appropriate anthelmintic in each case.     

Glycerol,Methyl‐Formamide and Dimethyl‐Formamide in Canine Semen Cryopreservation

The aim of the present work was to compare the efficiency of methyl‐formamide (MF), dimethyl‐formamide (DF) and glycerol (GL) as cryoprotectants in canine semen cryopreservation. For the experiment, pooled semen was submitted to one of the three cryoprotectants, with a final concentration of 3% in egg yolk–TRIS extender. Semen was subjectively evaluated for total and progressive motility, vigour and morphology. Sperm membrane functional integrity was assessed by hypo‐osmotic swelling test (HOST), and longevity was assessed using the thermoresistance test (TRT). Fresh semen showed normal physical and morphological characteristics. After thawing, differences were observed between semen frozen using GL and DF, regarding total and progressive motility and vigour (p < 0.05), but not between MF and GL or MF and DF. Means for total motility, progressive motility, vigour and morphologically normal spermatozoa were, respectively, 69.0 ± 5.4%, 61.0 ± 7.4%, 2.9 ± 0.5 and 57.1 ± 5.0% for GL; 59.0 ± 8.9%, 50.0 ± 10.0%, 2.5 ± 0.7 and 66.9 ± 7.7% for MF; and 44.0 ± 21.0%, 37.0 ± 19.8%, 2.1 ± 0.6 and 61.1 ± 5.5% for DF. On HOST, GL was superior (p < 0.05) to MF and DF (57.8 ± 12.4%, 35.8 ± 18.4% and 34.4 ± 9.4%, respectively). During the TRT, both GL and MF were superior to DF, with no differences between GL and MF. In conclusion, the use of MF as cryoprotectant showed results similar to GL, and can be considered as an alternative in canine semen cryopreservation. Further studies testing different concentrations of MF may improve its effects on cryopreservation of canine semen.     

Nemaline Rods in Canine Myopathies: 4 Case Reports and Literature Review

The diagnosis of nemaline rod myopathy (NM) is based on the presence of numerous pathognomonic rods within a fresh frozen muscle biopsy specimen. Three forms of congenital NM have been described in humans, and rods have been found to occur in various other conditions. A similar myopathy was described in 1986 in a family of cats. In this report, we describe a case of congenital NM in a 10-month-old Border Collie, an adult-onset NM in an 11-year-old Schipperke, and 2 acquired myopathies with nemaline rods in adult dogs associated with hypothyroidism and Cushing's syndrome. Common clinical features included exercise intolerance, abnormal electromyography, and the presence of nemaline rods in fresh, frozen, and glutaraldehyde-fixed biopsies from proximal appendicular limb muscles. Staining of cryostat sections of muscle biopsy specimens by the modified Gomori trichrome technique disclosed numerous rod bodies that were localized to type 1 fibers by the histochemical adenosine triphosphatase reaction. Accumulation of rods also was demonstrated by electron microscopy in 2 of the cases with localized enlargement and streaming of Z lines. Documentation of NM in a young Border Collie and the adult-onset form in the Schipperke alerts clinicians to the existence of this disorder in these breeds.     

Canine Reproduction: I. Reaction of Animals to Collection of Semen and Insemination Procedures

Two methods of semen collection were attempted on ten untrained dogs of several breeds. Manual manipulation as described proved most reliable for untrained dogs. Electro-ejaculation was attempted unsucessfully with twelve additional dogs.

Two methods of insemination were compared. Equipment found most satisfactory is described and a modification suggested.

     

Canine inflamed nonepitheliotropic cutaneous T‐cell lymphoma: a diagnostic conundrum

Background –  Cutaneous T‐cell lymphoma (CTCL) in dogs is a heterogeneous disease complex, which consists of nonepitheliotropic (NE) and epitheliotropic forms. These lymphomas are readily recognized by the presence of dominant populations of cytologically atypical lymphocytes. Objective –  The objective of this study was to introduce the key features of inflamed NE‐CTCL, which is easily confused with reactive, inflammatory histiocytic disease. Animals –  Twenty‐four dogs (mean age 7.5 years) presented with inflamed NE‐CTCL. Lesions presented as nodules, plaques or masses. An initial diagnosis of cutaneous reactive histiocytosis (11 dogs) or histiocytic neoplasia (three dogs) was made by primary pathologists. Methods –  Lesions were assessed by histology and immunohistochemistry to detect canine leukocyte antigens. Lesional genomic DNA was extracted and gene rearrangement analysis of the T‐cell receptor γ locus was assessed. Results –  The cutaneous lesions consisted of pleocellular infiltration of the dermis with variable extension into the subcutis. The lesions often surrounded vessels and adnexae. Epitheliotropism was minimal or lacking. Small lymphocytes, plasma cells and intermediate to large, cytologically atypical lymphocytes were scattered between prominent histiocytic infiltrates. Atypical lymphocytes often had marked variation in the intensity of CD3 expression. Molecular clonality analysis of the T‐cell receptor γ locus revealed clonal expansion of T cells in 22 of 23 dogs tested. Conclusion –  The recognition of inflamed NE‐CTCL and its differentiation from cutaneous reactive histiocytosis depends on careful assessment of lymphocyte morphology and immunostaining patterns. Confirmation of the diagnosis is best accomplished by T‐cell antigen receptor gene rearrangement analysis.     

Canine mammary mixed tumours: immunohistochemical expressions of EGFR and HER‐2

Background Benign mixed tumours (BMTs) are frequently found in the mammary glands of female dogs, but the factors determining malignant transformation in these tumours are unknown. Objective To evaluate the expression of the oncoproteins, human epidermal growth factor receptor 2 (HER‐2) and epidermal growth factor receptor (EGFR), in 46 carcinomas in BMTs (CBMTs) and to verify their possible association with the malignancy of the tumours. Methods Immunohistochemical expression was analysed in benign and malignant components separately, and then compared with 74 cases of BMTs. Results Among the CBMTs, positivity for HER‐2 was found in the benign histological component of 4.3% (2/46), in the malignant epithelial non‐invasive component of 14.8% (4/27) and in the malignant invasive epithelial component of 13.6% (6/44) of cases. Two of the 24 (8.3%) BMTs were positive for HER‐2. There was no relationship between HER‐2 and the tumour components. There was no significant difference between BMTs and CBMTs. Positivity for EGFR was found in the benign component of 17.4% (8/46) of the CBMTs, in the malignant epithelial non‐invasive component of 40.7% (11/27%) and in the invasive epithelial malignant component of 45.4% (20/44). EGFR positivity was significantly associated with the invasive component of CBMTs. Conclusion EGFR may contribute to malignant epithelial transformation of BMTs. In contrast, HER‐2 overexpression may not be associated with the acquisition of a malignant epithelial phenotype.     

Use of Contrast‐Enhanced Ultrasonography in Chronic Pathologic Canine Testes

Contrast‐enhanced ultrasound with sulphur hexafluoride microbubbles was performed in seven healthy dogs without a history of reproductive pathology and with histologically confirmed normal testes and in 42 dogs with chronic scrotal anomalies. All dogs underwent orchiectomy and histological examination. Enhancement patterns and perfusion parameters (peak intensity and regional blood flow) of testes of healthy dogs and testes with chronic lesions were compared. Fourteen non‐pathologic and 60 pathologic testes were considered. Forty testes were neoplastic (24 interstitial cell tumours, 9 seminomas, 7 Sertoli cell tumours), 20 were non‐neoplastic (16 testicular degenerations, 2 chronic orchitis, 1 testicular atrophy, 1 interstitial cell hyperplasia). In healthy dogs, the contrast medium flow had a rapid homogeneous wash‐in and wash‐out, with a short peak phase. With contrast ultrasound, testes that were inhomogeneous with a hyperenhancing pattern were associated with neoplasia (sensitivity: 87.5%, specificity: 100%). Lesions with persistent inner vessels and a hypo‐to‐isoechoic background were significantly associated with seminomas (sensitivity: 77.8%, specificity: 100%). Testes with non‐neoplastic lesions were characterized by a scant/moderate homogeneous enhancement. Perfusion parameters were higher in neoplastic lesions. Contrast ultrasound was a feasible diagnostic tool in the assessment of testicular lesions, with hyperenhancement being an important feature in the diagnosis of malignancy.