Papillomavirus DNA not detected in canine lobular orbital adenoma and normal conjunctival tissue.

BACKGROUND: Canine lobular orbital adenomas are benign tumors that arise from orbital glandular tissue and extend into the orbit, conjunctiva, and third eyelid. Surgical excision is challenging and recurrence rates are high following excision alone. Enucleation and exenteration reduces the likelihood of recurrence, but is a radical therapeutic option for an otherwise visual and comfortable eye. Human papillomavirus causes 4.5% of worldwide cancers in people and has been identified in up to 23% of benign salivary gland tumors. To date, the etiology of canine lobular orbital adenomas has not been established and it is reasonable to consider canine papillomaviruses as an associative agent with benign glandular tumors in dogs. Identification of the underlying etiology of these tumors may help establish treatment or preventative measures. The purpose of this study was to evaluate conjunctival and orbital tissue of phenotypically normal dogs and tissue from canine lobular orbital adenomas for the presence of papillomavirus DNA. RESULTS: Thirty seven canine lobular orbital adenoma samples (36 formalin fixed paraffin embedded (FFPE) tissue samples from 33 dogs and one freshly collected sample) were evaluated via polymerase chain reaction for the presence of papillomavirus DNA. Conjunctival tissue samples, from 10 dogs with normal ocular examinations, excised immediately following euthanasia, were used as phenotypically normal controls. Three FFPE and one freshly collected tissue samples previously confirmed to be positive for papillomavirus DNA were used as positive controls. PCR products verified positive controls. Papillomavirus DNA was not detected in fresh conjunctival tissue of the phenotypically normal control dogs or in samples of fresh or FFPE canine lobular orbital adenoma tissue. CONCLUSIONS: An association between papillomavirus and the development of canine lobular orbital adenomas is unlikely. Further research is needed to evaluate if other viruses play a role in the pathogenesis of canine lobular orbital adenomas.

Hotspot Exon 15 Mutations in BRAF Are Uncommon in Feline Tumours.

BRAF is one of multiple RAF proteins responsible for the activation of the MAPK cell signalling cascade involved in cell growth, differentiation, and survival. A hotspot BRAFV600E mutation, in exon 15, was determined to be a driver in 100% hairy cell leukaemias, 50%-60% of human melanomas, 30%-50% of human thyroid carcinomas and 10%-20% of human colorectal carcinomas. The orthologous BRAFV595E mutation was seen in 67% and 80% of canine bladder transitional cell carcinomas and prostatic adenocarcinomas, respectively. Since veterinary and human cancers exploit similar pathways and BRAF is highly conserved across species, BRAF can be expected to be a driver in a feline cancer. Primers were developed to amplify exon 15 of feline BRAF. One hundred ninety-six feline tumours were analysed. Sanger sequencing of the 211 bp PCR amplicon was done. A BRAF mutation was found in one tumour, a cutaneous melanoma. The mutation was a BRAFV597E mutation, orthologous to the canine and human hotspot mutations. A common synonymous variant, BRAFT586T, was seen in 23% (47/196) of tumours. This variant was suspected to be a single nucleotide polymorphism. BRAF was not frequently mutated in common feline tumours or in tumour types that frequently harbour BRAF mutations in human and canine cancers. As is seen in canine cancer genomics, the mutational profile in feline tumours may not parallel the histologic equivalent in human oncology.

Single-cell T-cell receptor repertoire profiling in dogs.

Spontaneous cancers in companion dogs are robust models of human disease. Tracking tumor-specific immune responses in these models requires reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). scTCRseq and integration with scRNA data have not been demonstrated on companion dogs with cancer. Here, five healthy dogs, two dogs with T cell lymphoma and four dogs with melanoma are selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. Single-cell suspensions of PBMCs or lymph node aspirates are profiled using scRNA and dog-specific scTCRseq primers. In total, 77,809 V(D)J-expressing cells are detected, with an average of 3498 (348 - 5,971) unique clonotypes identified per sample. In total, 29/34, 40/40, 22/22 and 9/9 known functional TRAV, TRAJ, TRBV and TRBJ gene segments are observed respectively. Pseudogene or otherwise defective gene segments are also detected supporting re-annotation of several as functional. Healthy dogs exhibit highly diverse repertoires, T cell lymphomas exhibit clonal repertoires, and vaccine-treated melanoma dogs are dominated by a small number of highly abundant clonotypes. scRNA libraries define large clusters of V(D)J-expressing CD8+ and CD4 + T cells. Dominant clonotypes observed in melanoma PBMCs are predominantly CD8 + T cells, with activated phenotypes, suggesting possible anti-tumor T cell populations.

Quantitative elastography of liver fibrosis and spleen stiffness in chronic hepatitis B carriers: comparison of shear-wave elastography and transient elastography with liver biopsy correlation.

PURPOSE: To document utility of shear-wave (SW) elastography for assessing liver fibrosis in chronic hepatitis B and to compare its performance with that of transient elastography. MATERIALS AND METHODS: Ethics committee approved the study, and informed consent was obtained. Patients with liver biopsy correlation (n = 226) and healthy patients (n = 171) were analyzed. Results of SW elastography of liver, SW elastography of spleen, and transient elastography of liver were compared and correlated according to METAVIR scores. Areas under the receiver operating characteristic curve (AUCs), binary logistic regression, and Delong test were used. RESULTS: AUC for SW elastography of liver, transient elastography of liver, and SW elastography of spleen was, respectively, 0.86, 0.80, and 0.81 for fibrosis (≥ F1 stage); 0.88, 0.78, and 0.82 for moderate fibrosis (≥ F2 stage); 0.93, 0.83, and 0.83 for severe fibrosis (≥ F3 stage); and 0.98, 0.92, and 0.84 for cirrhosis (F4 stage). SW elastography of liver showed significantly higher accuracy than transient elastography of liver and SW elastography of spleen in all fibrosis stages (P = .01-.04). SW elastography of spleen showed similar accuracy with transient elastography of liver (P = .21-.99). Combination SW elastography of liver and SW elastography of spleen to predict fibrosis staging showed diagnostic accuracy not further improved compared with SW elastography of liver alone (similar AUC; ≥ F1, P = .87; ≥ F2, P = .81; ≥ F3, P = .84; ≥ F4, P = .88). SW elastography of liver had higher successful rate than transient elastography of liver (98.9% vs 89.6%). Prevalence of discordance in at least two stages with liver histologic staging was 10.2% (23 of 226) for SW elastography of liver and 28.2% (58 of 206) for SW elastography of spleen. CONCLUSION: SW elastography provides more accurate correlation of liver elasticity with liver fibrosis stage compared with transient elastography, especially in identification of stage F2 or greater.

Hookworm dermatitis due to Uncinaria stenocephala in a dog from Saskatchewan.

Uncinaria stenocephala hookworm dermatitis (uncinariosis) was diagnosed on fecal examination and macerated skin biopsy in a 1.5-year-old greyhound dog from Saskatchewan. This is the first reported case in Canada. Treatment with moxidectin cleared gastrointestinal and dermal infections.

Dermatite de l'ankylostomiase causée parUncinaria stenocephalachez un chien de la Saskatchewan. La dermatite de l'ankylostomiase à Uncinaria stenocephala (uncinariose) a été diagnostiquée à l'examen fécal et lors d'une biopsie de la peau macérée chez un chien Greyhound âgé de 1 an et demi provenant de la Saskatchewan. Il s'agit du premier cas signalé au Canada. Le traitement à la moxidectine a guéri les infections gastro-intestinales et cutanées.(Traduit par Isabelle Vallières).

Prevalence of non-alcoholic fatty liver disease and advanced fibrosis in Hong Kong Chinese: a population study using proton-magnetic resonance spectroscopy and transient elastography.

OBJECTIVE: Knowledge of the epidemiology of non-alcoholic fatty liver disease (NAFLD) is incomplete because liver biopsy cannot be performed on the general population to assess disease severity. New non-invasive tests allow accurate and safe assessment in healthy individuals. The aim of this study was to examine the prevalence of NAFLD and advanced fibrosis in the general Hong Kong Chinese population. METHODS: Subjects were recruited from the community by random selection from the government census database. Liver fat and fibrosis were assessed by proton-magnetic resonance spectroscopy and transient elastography, respectively. RESULTS: Overall, 264 of 922 (28.6%) subjects had intrahepatic triglyceride content ≥5%. Excluding 12 subjects with significant alcohol consumption, the population prevalence of NAFLD was 27.3% (95% CI 24.5% to 30.2%). Each component of the metabolic syndrome increased the risk of fatty liver in a dose-dependent manner (prevalence of 4.5% in subjects without any component and 80.0% in those with all five components). 8 (3.7%) patients with fatty liver had liver stiffness ≥9.6 kPa, a level suggestive of advanced fibrosis. Body mass index and alanine aminotransferase level were independent factors associated with liver stiffness. Together with other clinical prediction scores, the estimated prevalence of advanced fibrosis in patients with fatty liver in the community was <10%. Compared with non-drinkers, modest drinkers (<10 g per day) did not have higher risk of fatty liver after adjustment for demographic and metabolic factors. The liver stiffness was 4.7±1.9 kPa in modest drinkers and 4.6±1.7 kPa in non-drinkers (p=0.54). CONCLUSION: NAFLD is found in over a quarter of the general adult Chinese population, but the proportion of patients with advanced fibrosis is low. Modest alcohol consumption does not increase the risk of fatty liver or liver fibrosis.

A multi-institutional epidemiologic study evaluating environmental risk factors for feline oral squamous cell carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is an aggressive cancer in domestic cats that has no effective treatment option when advanced. Preventative or early diagnostic measures are thus crucial. FOSCC is also a model for human head and neck SCC (HNSCC); strong risk factors in HNSCC include exposure to alcohol, tobacco, areca nut, and high-risk human papillomavirus. Previous studies have identified flea collar and tobacco smoke exposure, feeding canned tuna, canned cat food and cat foods with chemical additives, living in a rural environment, and having outdoor access as risk factors for FOSCC but there was no overlap in the risk factors between studies. In our study, risks for FOSCC were evaluated in an online epidemiologic survey study in 67 cats with FOSCC and 129 control cats. Clumping clay cat litter and flea collar use were significant risk factors for FOSCC on multiple logistic regression with odds ratios of 1.66 (95% CI 1.20-2.30) and 4.48 (95% CI 1.46-13.75) respectively. Crystalline silica is a carcinogen that may be present in all clay cat litters and tetrachlorvinphos is a carcinogen that is present in the most commonly used flea collars in our study. We recommend further investigation into the association between FOSCC and clay-based litter and/or flea collars containing tetrachlorvinphos.

Metagenomic Analysis of DNA Viruses with Targeted Sequence Capture of Canine Lobular Orbital Adenomas and Normal Conjunctiva.

Our study aims are: (1) to evaluate phenotypically normal canine conjunctival and orbital tissue and tissue from canine lobular orbital adenomas (CLOAs) for the presence of viral genomic material and (2) phylogenetically classify detected DNA viruses to determine if a DNA virus is associated with CLOAs. A total of 31 formalin fixed paraffin embedded CLOA tissue samples, 4 papillomas or sarcoid, and 10 fresh clinically normal conjunctival tissues were included in this study. Genomic DNA was isolated from all samples and sequencing libraries were prepared. The libraries were molecularly indexed and pooled and viral DNA was enriched via targeted sequence capture utilizing ViroCap. The libraries were sequenced on the Illumina HiSeq platform and compared to known viral DNA reference genomes to identify viral DNA. Carnivore parvovirus was identified in 6.4% and 20% of CLOA tissue and normal conjunctival samples, respectively. This study showed that conjunctival tissue from healthy dogs and CLOAs uncommonly harbor DNA viruses, and no DNA virus was associated with these tumors. Further studies are needed to evaluate the etiologic cause of CLOAs.

Genomic landscape and gene expression profiles of feline oral squamous cell carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is a cancer of the squamous cell lining in the oral cavity and represents up to 80% of all oral cancers in cats, with a poor prognosis. We have used whole exome sequencing (WES) and RNA sequencing of the tumor to discover somatic mutations and gene expression changes that may be associated with FOSCC occurrence. FOSCC offers a potential comparative model to study human head and neck squamous cell carcinoma (HNSCC) due to its similar spontaneous formation, and morphological and histological features. In this first study using WES to identify somatic mutations in feline cancer, we have identified tumor-associated gene mutations in six cats with FOSCC and found some overlap with identified recurrently mutated genes observed in HNSCC. Four samples each had mutations in TP53, a common mutation in all cancers, but each was unique. Mutations in other cellular growth control genes were also found such as KAT2B and ARID1A. Enrichment analysis of FOSCC gene expression profiles suggests a molecular similarity to human OSCC as well, including alterations in epithelial to mesenchymal transition and IL6/JAK/STAT pathways. In this preliminary study, we present exome and transcriptome results that further our understanding of FOSCC.

Hepatitis B virus infection and fatty liver in the general population.

BACKGROUND & AIMS: In animal studies, expression of hepatitis B virus (HBV) proteins causes hepatic steatosis. We aimed to study the prevalence of fatty liver in people with and without HBV infection in the general population. METHODS: We performed a cross-sectional population study in Hong Kong Chinese. Intrahepatic triglyceride content (IHTG) was measured by proton-magnetic resonance spectroscopy. RESULTS: One thousand and thirteen subjects (91 HBV patients and 922 controls) were recruited. The median IHTG was 1.3% (0.2-33.3) in HBV patients and 2.1% (0-44.2) in controls (p <0.001). Excluding subjects with significant alcohol consumption, the prevalence of nonalcoholic fatty liver disease was 13.5% (95% confidence interval [CI] 6.4%, 20.6%) in HBV patients and 28.3% (95% CI 25.3%, 31.2%) in controls (p=0.003). The fatty liver prevalence differed in HBV patients and controls aged 40-59 years but was similar in those aged 60 years or above. After adjusting for demographic and metabolic factors, HBV infection remained an independent factor associated with lower risk of fatty liver (adjusted odds ratio 0.42; 95% CI 0.20, 0.88; p=0.022). HBV patients also had a lower prevalence of metabolic syndrome (11.0% vs. 20.2%; p=0.034), but the difference was mainly attributed to lower triglyceride levels. Among HBV patients, viral genotypes, HBV DNA level and hepatitis B e antigen status were not associated with fatty liver. CONCLUSIONS: HBV infection is associated with a lower prevalence of fatty liver, hypertriglyceridemia and metabolic syndrome. Viral replication may affect lipid metabolism and this warrants further studies.

Diagnosis of liver fibrosis and cirrhosis using liver stiffness measurement: comparison between M and XL probe of FibroScan®.

BACKGROUND & AIMS: Unreliable results of liver stiffness measurement are obtained in 16% of cases and are independently associated with body mass index (BMI) greater than 30 kg/m(2). A new FibroScan® probe (XL probe) was designed specifically for obese patients. The aim of this study was to evaluate the accuracy of liver stiffness measurement using M and XL probes of Fibroscan® for the diagnosis of fibrosis and cirrhosis in a large cohort of patients. METHODS: Consecutive patients undergoing liver biopsies for chronic liver disease were prospectively recruited. Liver stiffness measurement was performed within 1 week before liver biopsy using both M and XL probes of FibroScan®. RESULTS: A total of 286 patients were evaluated. A reliable liver stiffness measurement using M probe was obtained in 79.7% of cases. In the other 21.3%, liver stiffness measurement using XL probe was obtained in 56.9% of patients. A strong correlation was found between M and XL values, regardless of BMI. In all groups, median liver stiffness measurement using the XL probe was significantly lower than liver stiffness measurement using the M probe. By multivariate analysis, unsuccessful liver stiffness examination with M probe was independently associated with age >50 years and BMI >30 kg/m(2). By univariate analysis, only BMI >30 kg/m(2) was associated with unsuccessful liver stiffness measurement with XL probe. No significant difference was observed between the M and XL probes for the diagnosis of liver fibrosis. CONCLUSIONS: Liver stiffness measurement with either M or XL probe is possible in 91.2% of patients with comparable diagnostic accuracy. In clinical practice, the M probe could be used as first step for liver stiffness measurement. In case of no valid shot or unreliable measurement, the XL probe could be used. This result could be useful for the assessment of liver fibrosis in NAFLD and/or obese patients.

Liver stiffness measurement using XL probe in patients with nonalcoholic fatty liver disease.

OBJECTIVES: Liver stiffness measurement (LSM) by transient elastography is a noninvasive test of liver fibrosis, but cannot be performed in a significant proportion of obese patients. The aim of this study was to evaluate the performance of the new XL probe in patients with nonalcoholic fatty liver disease (NAFLD). METHODS: Liver biopsy and paired LSM by both the original M probe and XL probe were performed on 193 consecutive NAFLD patients in France and Hong Kong. RESULTS: Compared with M probe, XL probe was more likely to achieve 10 valid measurements (95% vs. 81%; P<0.001) and a success rate of over 60% (90% vs. 74%; P<0.001). The areas under receiver operating characteristics curves of XL probe for F2, F3, and F4 disease were 0.80, 0.85, and 0.91, respectively. XL probe tended to generate lower LSM than M probe in the same patient. At a cutoff of 7.2 kPa, the sensitivity, specificity, positive, and negative predictive values for F3 or greater disease were 78%, 78%, 60%, and 89%, respectively. Discordance of at least two stages between XL probe and histology was observed in 16 (9%) patients. Body mass index (BMI) over 35 kg/m(2) was independently associated with discordance (adjusted odds ratio 9.09; 95% confidence interval 1.10-75.43). Reliable measurements by XL probe were obtained in 75% of the overall population and 65% of patients with BMI over 30 kg/m(2). CONCLUSIONS: LSM by XL probe can be performed successfully in most NAFLD patients, but obesity is associated with less accurate and reliable measurements.

Genome wide exploration of the methylome in aggressive B-cell lymphoma in Golden Retrievers reveals a conserved hypermethylome.

Few recurrent DNA mutations are seen in aggressive canine B cell lymphomas (cBCL), suggesting other frequent drivers. The methylated island recovery assay (MIRA-seq) or methylated CpG-binding domain sequencing (MBD-seq) was used to define the genome-wide methylation profiles in aggressive cBCL in Golden Retrievers to determine if cBCL can be better defined by epigenetic changes than by DNA mutations. DNA hypermethylation patterns were relatively homogenous within cBCL samples in Golden Retrievers, in different breeds and in geographical regions. Aberrant hypermethylation is thus suspected to be a central and early event in cBCL lymphomagenesis. Distinct subgroups within cBCL in Golden Retrievers were not identified with DNA methylation profiles. In comparison, the methylome profile of human DLBCL (hDLBCL) is relatively heterogeneous. Only moderate similarity between hDLBCL and cBCL was seen and cBCL likely cannot be accurately classified into the subtypes seen in hDLBCL. Genes with hypermethylated regions in the promoter-TSS-first exon of cBCL compared to normal B cells often also had additional hyper- and hypomethylated regions distributed throughout the gene suggesting non-randomized repeat targeting of key genes by epigenetic mechanisms. The prevalence of hypermethylation in transcription factor families in aggressive cBCL may represent a fundamental step in lymphomagenesis.

Entecavir treatment in patients with severe acute exacerbation of chronic hepatitis B.

BACKGROUND & AIMS: Severe acute exacerbation of chronic hepatitis B is a unique clinical presentation with significant morbidity and mortality. Lamivudine was used in most previous studies, but the drug was limited by the development of resistance. Our objective is to study the safety and efficacy of entecavir in patients with severe acute exacerbation. METHODS: Consecutive patients with severe acute exacerbation of chronic hepatitis B were recruited from 1998 to 2009. All patients had serum alanine aminotransferase and bilirubin increased beyond 10 and 3 times the upper limit of normal, respectively. The primary endpoint was overall mortality at week 48. Virological and biochemical responses were also studied. RESULTS: Thirty-six patients and 117 patients were treated with entecavir and lamivudine, respectively. By week 48, 7 (19%) patients in the entecavir group and 5 (4%) patients in the lamivudine group died (adjusted hazard ratio 5.1, 95% confidence interval 1.5-17.2, p=0.010). Similarly, the entecavir group had higher liver-related mortality (adjusted hazard ratio 4.0, 95% confidence interval 1.0-15.7, p=0.044). Despite a lower prevalence of cirrhosis, more patients in the entecavir group developed prolonged jaundice, hepatic encephalopathy, and ascites. Entecavir resulted in more rapid and complete viral suppression, with 71% of patients achieving undetectable hepatitis B virus (HBV) DNA at week 48, compared to 40% in the lamivudine group (p=0.007). However, rapid HBV DNA reduction at week 4 was associated with prolonged jaundice. CONCLUSIONS: Entecavir treatment is associated with increased short-term mortality in patients with severe acute exacerbation of chronic hepatitis B but achieves better virological response in the long run.

Factors associated with unreliable liver stiffness measurement and its failure with transient elastography in the Chinese population.

BACKGROUND AND AIM: Liver stiffness measurement (LSM) with transient elastography is a non-invasive and reliable test for liver fibrosis. However a small proportion of patients may have unreliable LSM or LSM failure. The aim of the present study was to investigate the factors associated with unreliable LSM or LSM failure in Chinese patients. METHODS: We prospectively recruited liver patients for LSM. Unreliable LSM was defined as < 10 valid shots, an interquartile range (IQR)/LSM > 30%, or a success rate < 60%. LSM failure was defined as zero valid shots. RESULTS: Among 3205 patients with LSM, 371 (11.6%) and 88 (2.7%) had unreliable LSM and LSM failure, respectively. The rates started to increase when body mass index (BMI) ≥ 28.0 kg/m(2) . Comparing patients with BMI ≥ 28.0-29.9 kg/m(2) versus those with BMI ≥ 30.0 kg/m², the rates of unreliable LSM (16.4% vs 18.9%; P = 0.62) and LSM failure (11.8% vs 17.8%; P = 0.16) were similar. BMI ≥ 28.0 kg/m² was the most important factor associated with unreliable LSM (odds ratio [OR] = 2.9, 95% confidence interval [CI] = 2.1-3.9, P < 0.0001) and LSM failure (OR = 10.1, 95% CI = 6.4-14.2, P < 0.0001). Central obesity, defined as waist circumference > 80 cm in women and > 90 cm in men, was another independent risk factor of unreliable LSM (OR = 1.3, 95% CI = 1.0-1.6, P = 0.04) and LSM failure (OR = 5.8, 95% CI = 2.9-11.5, P < 0.0001). CONCLUSION: BMI ≥ 28.0 kg/m² and central obesity were the independent risk factors of unreliable LSM and LSM failure in Chinese, and these rates were significantly higher in patients with extreme BMI.

Unraveling the chaotic genomic landscape of primary and metastatic canine appendicular osteosarcoma with current sequencing technologies and bioinformatic approaches.

Osteosarcoma is a rare disease in children but is one of the most common cancers in adult large breed dogs. The mutational landscape of both the primary and pulmonary metastatic tumor in two dogs with appendicular osteosarcoma (OSA) was comprehensively evaluated using an automated whole genome sequencing, exome and RNA-seq pipeline that was adapted for this study for use in dogs. Chromosomal lesions were the most common type of mutation. The mutational landscape varied substantially between dogs but the lesions within the same patient were similar. Copy number neutral loss of heterozygosity in mutant TP53 was the most significant driver mutation and involved a large region in the middle of chromosome 5. Canine and human OSA is characterized by loss of cell cycle checkpoint integrity and DNA damage response pathways. Mutational profiling of individual patients with canine OSA would be recommended prior to targeted therapy, given the heterogeneity seen in our study and previous studies.

A virome sequencing approach to feline oral squamous cell carcinoma to evaluate viral causative factors.

Feline oral squamous cell carcinoma (FOSCC) may be the best naturally-occurring model of human head and neck squamous cell carcinoma (HNSCC). HNSCC can be broadly divided into human papillomavirus (HPV)-negative cancers and HPV-positive cancers where HPV is the causative agent. Previous studies in FOSCC have used both species-specific and species-nonspecific PCR primers that may be insensitive to the detection of PVs and other viruses that may be divergent from known sequences. ViroCap is a targeted capture and next generation sequencing tool that was designed to identify all known vertebrate DNA and RNA viruses. In this study we used a metagenomic approach using ViroCap for DNA viruses in 20 FOSCC, 9 normal feline oral mucosal, and 8 suspected PV positive control samples. We tested the hypothesis that viruses would be enriched in FOSCC compared to normal oral mucosa. The virome of the FOSCC and normal feline oral mucosa consisted of feline foamy virus in 7/20 and 2/9 (35% and 22%), feline torque teno virus in 2/20 and 0/9 (10% and 0%), alphaherpesvirus in 2/10 and 0/9 (10% and 0%), FIV (0% and 22%), Epstein-Barr virus in 1/20 and 0/9 (5% and 0%) and feline papillomavirus in 1/20 and 0/9 samples (5% and 0% respectively). Felis catus papillomavirus-3 was found in 1 of 20 FOSCC samples. A virus was not associated consistently with FOSCC. If PVs have a role in FOSCC it is at most a supplementary or uncommon role. FOSCC appears most closely related to HPV-negative HNSCC. Future research on FOSCC should focus on identifying genetic and environmental causes.

Evaluation of a Campylobacter fetus subspecies venerealis real-time quantitative polymerase chain reaction for direct analysis of bovine preputial samples.

The detection and subspeciation of Campylobacter fetus subsp. venerealis (CFV) from veterinary samples is important for both clinical and economic reasons. Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, a venereal disease that can lead to serious reproductive problems in cattle, and strict international regulations require animals and animal products to be CFV-free for trade. This study evaluated methods reported in the literature for CFV detection and reports the translation of an extensively tested CFV-specific polymerase chain reaction (PCR) primer set; including the VenSF/VenSR primers and a real-time, quantitative PCR (qPCR) platform using SYBR Green chemistry. Three methods of preputial sample preparation for direct qPCR were evaluated and a heat lysis DNA extraction method was shown to allow for CFV detection at the level of approximately one cell equivalent per reaction (or 1.0 × 10(3) CFU/mL) from prepuce. The optimized sample preparation and qPCR protocols were then used to evaluate 3 western Canadian bull cohorts, which included 377 bulls, for CFV. The qPCR assay detected 11 positive bulls for the CFV-specific parA gene target. DNA sequence data confirmed the identity of the amplified product and revealed that positive samples were comprised of 2 sequence types; one identical to previously reported CFV parA gene sequences and one with a 9% sequence divergence. These results add valuable information towards our understanding of an important CFV subspeciation target and offer a significantly improved format for an internationally recognized PCR test.

Prediction of off-treatment response to lamivudine by serum hepatitis B surface antigen quantification in hepatitis B e antigen-negative patients.

BACKGROUND: The timing of antiviral therapy cessation in hepatitis B e antigen (HBeAg)-negative patients is controversial. Here, we aimed to investigate the role of HBV DNA and hepatitis B surface antigen (HBsAg) monitoring to predict off-treatment sustained response. METHODS: A total of 53 HBeAg-negative chronic hepatitis B patients who received lamivudine for 34 ±23 (range 12-76) months and had lamivudine stopped for 47 ±35 months were studied. Primary outcome was sustained response, defined as HBV DNA≤200 IU/ml, at 12 months post-treatment (SR-12). RESULTS: A total of 9 (17%) patients achieved SR-12. HBV DNA at baseline, month 6 and end of treatment had no association with SR-12. HBsAg levels tended to decrease more significantly during treatment among SR-12 responders. At the end of treatment, both HBsAg ≤2 log IU/ml and reduction by >1 log from baseline had sensitivity, specificity, positive and negative predictive values for SR-12 of 78%, 96%, 78% and 96%, respectively. All 5 patients with HBsAg≤2 log IU/ml and reduction >1 log at the end of treatment achieved SR-12 and all 40 patients with HBsAg>2 log IU/ml and reduction ≤1 log did not have SR-12. The cumulative probability of sustained response and HBsAg clearance at 5 years among patients with HBsAg≤2 log IU/ml were 88% and 72%, respectively, that among patients with HBsAg reduction >1 log were 74% and 61%, respectively. CONCLUSIONS: Monitoring of HBsAg level can guide the timing of stopping lamivudine in HBeAg-negative chronic hepatitis B.