Clinical features, MRI findings and outcome of a primary extranodal B-cell lymphoma affecting the tympanic bulla treated with chemotherapy alone.

Case summary: A 2-year-old neutered female feline leukaemia virus (FeLV)-negative domestic shorthair cat was referred with an acute onset of vestibular signs. A clinical examination identified evidence of otitis externa of the right ear and enlargement of the right mandibular lymph node. MRI revealed predominantly T2 and FLAIR hyperintense and contrast-enhancing lesions affecting the right external ear canal, tympanic bulla and nasopharyngeal regions with intracranial extension. Cytology and culture and sensitivity samples collected from the middle ear via myringotomy revealed a population of intermediate to large lymphocytes consistent with lymphoma and mixed Staphylococcus chromogenes and Pasteurella species infection. PCR for antigen receptor rearrangements on the ear cytology was consistent with a B-cell rearrangement. A primary extranodal B-cell lymphoma affecting the tympanic bulla and other sites with secondary septic otitis media and interna was diagnosed. After the improvement of clinical conditions after corticosteroid, antibiotic and chemotherapy treatment, the cat was alive 22 months after diagnosis without recurrence of clinical signs. Relevance and novel information: This is the first report of a primary extranodal B-cell lymphoma affecting the tympanic bulla with suspected involvement of the nasopharynx and cranial vault treated with chemotherapy alone in the veterinary literature. Although very rare, B-cell lymphoma should be included in the differentials for diseases affecting the inner and/or middle ear and extending intracranially in cats. Chemotherapy represents a non-invasive treatment modality with a survival of up to 22 months appearing possible.

Analysis of annual distributions of hemoglobin A2 values as a method to test for HbA2 standardization.

BACKGROUND AND AIMS: The monitoring of yearly distributions of HbA2 measured has been indicated as a reliable indicator of worldwide standardization. MATERIALS AND METHODS: Measurements/year of HbA2 have been collected over three consecutive years in 15 Italian laboratories each using the same analytical method over three years period. HbA2 distributions, cleaned of replicated measurements, were compared by the overlapping area of the raw probability density functions expressed by coefficient eta (η), and by comparing the reference intervals for the central part of each distribution estimated by the indirect method refineR using the R package "refineR". RESULTS: According to the overlapping areas analysis the distributions/year of the data provided by 4 centers able to perform at least 1000 measurements/year were similar in 2 consecutive years. Moreover, the reference intervals provided by 2 centers using the same analytical methods in two separate locations over the three consecutive years, were very similar. The highest overlap (99.7 %) was observed in one center over two consecutive years. The overlapping areas were very high (93.6-95.7%) in 8 out of 9 inter-comparisons. CONCLUSION: Despite the limitations of this study the yearly distribution of the HbA2 measured in various centers appears a reliable tool to test HbA2 standardization over different centers using different analytical methods.

Progressive Thinning of Retinal Nerve Fiber Layer/Ganglion Cell Layer (RNFL/GCL) as Biomarker and Pharmacological Target of Diabetic Retinopathy.

Diabetes-driven retinal neurodegeneration has recently been shown to be involved in the initial phases of diabetic retinopathy, raising the possibility of setting up a preventive strategy based on early retinal neuroprotection. To make this possible, it is crucial to identify a biomarker for early retinal neurodegeneration. To this end, in this study, we verified and confirmed that, in the Akita mouse model of diabetes, the thinning of the retinal nerve fiber layer/ganglion cell layer (the RNFL/GCL-the layer that contains the retinal ganglion cells) precedes the death of these same cells, suggesting that this dysfunction is a possible biomarker of retinal neurodegeneration. We then confirmed the validity of this assumption by starting a neuroprotective treatment (based on nerve growth factor eye drops) in concert with the first demonstration of RNFL/GCL thinning. In this way, it was possible not only to avoid the loss of retinal ganglion cells but also to prevent the subsequent development of the microvascular stage of diabetic retinopathy. In conclusion, in the case of diabetes, the thinning of the RNFL/GCL appears to be both a valid biomarker and a pharmacological target of diabetic retinopathy; it precedes the development of vascular dysfunctions and represents the ideal starting point for prevention.

Development of microsatellite markers for the invasive mosquito Aedes koreicus.

BACKGROUND: Aedes koreicus is a mosquito species native to East Asia which has recently invaded several countries in Europe. In Italy, this mosquito was first detected in the North-East in 2011 and is now widely distributed in the entire northern part of the country. The development of specific genetic markers, such as microsatellites, is necessary to uncover the dispersal routes of this mosquito from its native areas and, eventually, to plan future control interventions. METHODS: Available raw sequences of genomic DNA of Ae. koreicus were screened in silico using BLASTn to identify possible microsatellite-containing sequences. Specific primer pairs were then designed, and their efficiency was determined through polymerase chain reaction (PCR) on 32 individuals of Ae. koreicus collected in Italy. PCR conditions were optimised in three multiplex reactions. Genotyping of individual mosquitoes was performed on both single and multiplex PCR reactions. Finally, analysis of intra-population variation was performed to assess the level of polymorphism of the markers. RESULTS: Mosquito genotyping provided consistent results in both single and multiplex reactions. Out of the 31 microsatellite markers identified in the Ae. koreicus genome raw sequences, 11 were polymorphic in the examined mosquito samples. CONCLUSIONS: The results show that the 11 microsatellite markers developed here hold potential for investigating the genetic structure of Ae. koreicus populations. These markers could thus represent a novel and useful tool to infer the routes of invasion of this mosquito species into Europe and other non-native areas.

Reference intervals for glycated albumin during physiological pregnancy of Europid women: Evidences from a prospective observational study.

BACKGROUND AND AIMS: Glycated albumin (GA) may assess glycometabolic control over a short period of time respect to HbA1c, and its use to screen for gestational diabetes in pregnancy has been suggested. To this regard few data on reference intervals (RI) for GA on Europid women have been collected, only from cross-sectional investigations. Aim of this work has been to collect trimester-specific RI for GA in physiological pregnancies, following a longitudinal prospective study. METHODS: Forty-five healthy pregnant Europid women have been enrolled for whom a GDM screening test was scheduled at 24-28 weeks, in 5 different Italian centers. Only those negative to the OGTT were included. The women had 4 successive visits at 6-10 weeks of gestation, at 16-18 weeks, at 24-28 weeks and at the end of pregnancy. ALT, AST, total bilirubin, C-reactive protein, cholinesterase, creatinine, GGT, glycated albumin, iron, total serum proteins, transferrin were measured in duplicate on aliquots of serum samples by a central laboratory. RESULTS: The RI (2.5-97.5 percentiles) for GA were 11.1-14.8 % (I visit), 10.9-15.6 % (II visit), 10.6-14.1 % (III visit) and 10.7-14.3 % (IV visit). The RI of other biomarkers confirmed previously published data. The RI for serum cholinesterase we present are novel, and were 5049-9906 U/L (Iv), 4212-8965 U/L (IIv), 3518-8470 U/L (IIIv) and 3945-8727 U/L (IVv). CONCLUSIONS: Trimester-specific RI are important for using GA and serum cholinesterase in pregnancy. However, considering the high inter-individual variability of both markers, the use of longitudinal interpretations of the individual variations of both proteins during pregnancy should be preferred.

First report of two Asian invasive mosquito species, Aedes japonicus and Aedes koreicus, in Piedmont, northwest Italy.

INTRODUCTION: Aedes japonicus japonicus and Aedes koreicus are two invasive mosquitoes recently reported in various parts of Europe, including areas very close to Piedmont where, since 2012, specific tools have been implemented to allow the early detection of invasive mosquitoes, through the surveillance of the main points of entry. RESULTS: Thanks to the regional surveillance system, Ae. j. japonicus was intercepted in Piedmont for the first time in 2019, in the northernmost part of the region and now it is reported in six provinces. Ae. koreicus was intercepted for the first time in 2012 in three provinces. DISCUSSION AND CONCLUSION: The spread of these two invasive mosquitoes in Europe is still ongoing. Where Aedes albopictus is abundant, probably their presence goes undetected, therefore, it is crucial to begin surveillance early in the season. Due to their competence for several arboviruses and tolerance to the cold temperatures Ae. j. japonicus and Ae. koreicus represent a further concern for Public Health. A longer seasonal period for surveillance and response to mosquito-borne diseases, as well as a shift up of these activities to previously uncovered altitudes are indeed needed.

Topical nerve growth factor prevents neurodegenerative and vascular stages of diabetic retinopathy.

Specific and effective preventive treatment for diabetic retinopathy (DR) is presently unavailable, mostly because the early stages of the complication have been, until recently, poorly understood. The recent demonstration that the vascular phase of DR is preceded and possibly caused by the neurodegeneration of retinal ganglion cells suggests that DR could, at least theoretically, be prevented through an early neuroprotective approach. The aims of our study were to clarify the natural history of diabetes-driven retinal neurodegeneration and to verify the possibility to prevent DR using topical nerve growth factor (NGF). The results of the study show that retinal neurodegeneration, characterized by the loss of retinal ganglion cells represents a relatively early phenomenon of diabetes (between 5 and 16 weeks of age), which tends to be self-limiting in the long run. Neurodegeneration is followed by the development of DR-related vascular dysfunctions, as confirmed by the development of acellular capillaries and the loss of retinal pericytes. Both retinal neurodegeneration and subsequent vascular dysfunction can be successfully prevented by topical NGF administration. These findings suggest that: 1) The first stage of DR consists in a self-limiting retinal neurodegeneration 2) The demonstrated effectiveness of topical NGF in the prevention of DR could be rapidly translated into clinical practice.

Assessing the distribution of invasive Asian mosquitoes in Northern Italy and modelling the potential spread of Aedes koreicus in Europe.

In the last decade, Aedes koreicus and Aedes japonicus japonicus mosquitoes, which are competent vectors for various arboviruses of public health relevance, colonised Italy and other European countries. Nevertheless, information about their current and potential distribution is partial. Accordingly, in this study four regions of Northern Italy (Lombardy, Liguria, Piedmont and Aosta Valley) were surveyed during 2021 for the presence of eggs, larvae and pupae of these two invasive species. We found evidence for a widespread presence of Ae. koreicus in pre-Alpine territories of Lombardy and Piedmont. Larvae from the invasive subspecies of Ae. j. japonicus were also collected in the same geographic areas, though they were less frequent. Occurrence data from this study and results from previous monitoring campaigns were used to generate a Maxent model for the prediction of habitat suitability for Ae. koreicus mosquitoes in Northern Italy and the rest of Europe. Peri-urban areas located in proximity to forests, pastures and vineyards were revealed as highly suitable environments for colonisation by this invasive species. Maps of the potential distribution also suggest the presence of further suitable areas in currently uncolonized countries. We conclude that this invasive mosquito species has the potential for a broad expansion at the European level in the coming decades.

Re-thinking diabetic nephropathy: Microalbuminuria is just a piece of the diagnostic puzzle.

The decline of the estimated glomerular filtration rate (eGFR) and the presence of albuminuria are the typical hallmarks of kidney disease arising as one of the most frequent diabetic complications over a long period of time, generally known as diabetic nephropathy or diabetes kidney disease (DKD). However, a decline in the renal function may occur in diabetic patients for other reasons unrelated to glycemic control, and this condition is known as non-diabetic kidney disease (NDKD). In this opinion paper we will review these conditions, and we outline the importance of other investigations, such as kidney biopsy and the measurement of novel biomarkers, in order to identify the disease progression early, and to allow a timely intervention. We will also focus on the actual limits of the quantitative measurements of albumin in urine, especially with regards to potential interferences due to the treatment of patients with statins.

Modified prepubic urethrostomy with body wall tunneling: Description of technique and long-term outcome in eight male cats.

OBJECTIVE: To describe the procedure, complications, and long-term outcome of cats that underwent a modified prepubic urethrostomy (mPPU) technique for the management of proximal urethral obstructions. ANIMALS: Eight male cats. STUDY DESIGN: Short case series. METHODS: Medical records were reviewed for signalment, diagnostic investigation, details of the surgical procedure, and complications of cats that underwent mPPU. RESULTS: mPPU was well tolerated by all patients, and no intraoperative complications were reported. The duration of follow-up ranged from 13 to 84 months (median 19 months). Early postoperative skin scalding around the stoma associated with mild urinary incontinence during recumbency occurred and was self-limiting in all patients. Two cats required surgical revision at 5 and 6 months, respectively, due to a progressive weight gain and accumulation of abdominal fat around the stoma, causing a partial stomal obstruction. Resolution of clinical signs was reported in both patients. CONCLUSIONS: mPPU was easy to perform and offered favorable outcomes in this cohort of cats.

Should we be discouraging use of 'anticancer' supplements in dogs and cats with cancer?

Antonio Giuliano and colleagues argue that 'anticancer' supplements could be harmful and hinder chemotherapy and radiotherapy treatment, and so their use should be discouraged in cancer patients.

Mosquitoes of the Maculipennis complex in Northern Italy.

The correct identification of mosquito vectors is often hampered by the presence of morphologically indiscernible sibling species. The Maculipennis complex is one of these groups that include both malaria vectors of primary importance and species of low/negligible epidemiological relevance, of which distribution data in Italy are outdated. Our study was aimed at providing an updated distribution of Maculipennis complex in Northern Italy through the sampling and morphological/molecular identification of specimens from five regions. The most abundant species was Anopheles messeae (2032), followed by Anopheles maculipennis s.s. (418), Anopheles atroparvus (28) and Anopheles melanoon (13). Taking advantage of ITS2 barcoding, we were able to finely characterize tested mosquitoes, classifying all the Anopheles messeae specimens as Anopheles daciae, a taxon with debated rank to which we referred as species inquirenda (sp. inq.). The distribution of species was characterized by Ecological Niche Models (ENMs), fed by recorded points of presence. ENMs provided clues on the ecological preferences of the detected species, with An. daciae sp. inq. linked to stable breeding sites and An. maculipennis s.s. more associated to ephemeral breeding sites. We demonstrate that historical Anopheles malaria vectors are still present in Northern Italy.

Expression of Phosphorylated Signal Transducer and Activator of Transcription 3 and its Prognostic Significance in Canine Anal Sac Adenocarcinoma.

Prognostication in canine anal sac adenocarcinomas (ASACs) is difficult due to conflicting evidence regarding metastatic rates and median survival times (MSTs). The transcription factor signal transducer and activator of transcription 3 (STAT3) is a prognostic predictor in several human cancers. The aim of this retrospective study was to assess STAT3 expression in ASACs and to explore its association with clinical presentation and outcome. We hypothesized that STAT3 expression would distinguish tumours with early versus late metastasis. Records from The Queen's Veterinary School Hospital, Cambridge, UK, were searched for dogs diagnosed with ASAC from 2008 to 2019. Immunohistochemical expression of phosphorylated STAT3 (pSTAT3) was assessed in primary tumours (n = 57) and metastatic lymph nodes (n = 30) and MSTs were calculated for cases with low and high pSTAT3 expression. Of the 57 cases assessed, 27 presented with primary tumours but no metastasis and 30 with both primary and local metastatic disease. Most cases (50/57) expressed nuclear pSTAT3 within neoplastic cells in both primary tumour and metastatic lymph nodes. pSTAT3 expression was predominantly observed in neoplastic cells at the edges of neoplastic lobules, suggesting a potential role in invasion. There was no significant difference in pSTAT3 expression between cases metastatic at presentation and those that did not have detectable metastasis at presentation. There was no significant difference between the MSTs in cases with high and low pSTAT3 expression. Cases that presented with metastatic disease had shorter MSTs (395 days) than those with primary tumours alone (623 days). Although pSTAT3 is variably expressed in primary and metastatic ASAC cells, pSTAT3 did not provide prognostic information for canine ASAC.

A possible role of coarse fractionated radiotherapy in the management of gingival squamous cell carcinoma in dogs: A retrospective study of 21 cases from two referral centers in the UK.

Surgery with or without the addition of radiotherapy is the treatment of choice for canine oral squamous cell carcinoma (SCC). Fractionated radiotherapy alone is also effective in the long-term control of the disease, however coarse fractionated radiotherapy (CF-RT) for gingival SCC has not been extensively reported. The aim of this study was to describe side effects, clinical response, and median survival time (MST) of dogs with gingival SCC treated with CF-RT in the palliative and adjuvant setting. Twenty-one cases from two referral centres in the UK treated with CF-RT for gingival SCC between July 2013 and June 2019 were retrospectively evaluated. Of the 21 dogs, 11 developed mild acute adverse effects. Oral mucositis was the most common radiation induced toxicity. Three dogs developed chronic severe adverse effects (oro-nasal fistula, bone necrosis and gum recession). Overall clinical response rate was 77% in dogs receiving palliative treatment with MST of 365 days (60-1,095 days). MST was not reached for dogs treated in the adjuvant setting with a mean of 466 days (121-730 days). In cases of advanced gross disease CF-RT might have a role in short term palliation of clinical signs. However, it carries a significant risk of late toxicity for cases with unexpectedly long survival times and further investigations are required to identify an optimal CF-RT protocol. Randomized controlled trials are needed to confirm the role of CF-RT as adjuvant treatment of incompletely resected gingival SCC.

A roadmap for the standardization of hemoglobin A2.

BACKGROUND: Standardization of laboratory tests can be a long process, and this is the case with regards to the methods used to measure hemoglobin A2 (HbA2), an important marker for beta-thalassemia and other thalassemic conditions. The IFCC standardization project started in 2004, and it took at least 15 years before developing a reference measurement procedure, defining and producing calibrators and certified reference materials. METHODS: A series of steps have to be undertaken in order to promote the standardization in the field, a process involving a number of stakeholders (manufacturers, scientific societies, national health bodies, laboratory professionals, clinicians). In this work we describe some possible process indicators, in order to assure that the standardization will have internal and external validity and be effective for a long time. These indicators concern the inter-method studies, elaboration of External Quality Assessment Schemes, and the evaluation of the yearly distributions of HbA2 measurements collected in selected laboratories. RESULTS: Preliminary results are reported concerning the yearly distributions of HbA2, collected in two different locations, and using different analytical methods. Median yearly values were found very constant over the years, but different between methods. On the other side, results obtained on the same specimens using two different techniques, proved that results by capillary electrophoresis in 2 out of the 3 years of observation, were significantly lower than those by HPLC. CONCLUSION: In this document we report what has been done so far, and what has to be done to achieve the standardization of the measurement of HbA2 worldwide.

Capture-based Next-Generation Sequencing Improves the Identification of Immunoglobulin/T-Cell Receptor Clonal Markers and Gene Mutations in Adult Acute Lymphoblastic Leukemia Patients Lacking Molecular Probes.

The monitoring of minimal residual disease (MRD) in Philadelphia-negative acute lymphoblastic leukemia (ALL) requires the identification at diagnosis of immunoglobulin/T-cell receptor (Ig/TCR) rearrangements as clonality markers. Aiming to simplify and possibly improve the patients' initial screening, we designed a capture-based next-generation sequencing (NGS) panel combining the Ig/TCR rearrangement detection with the profiling of relevant leukemia-related genes. The validation of the assay on well-characterized samples allowed us to identify all the known Ig/TCR rearrangements as well as additional clonalities, including rare rearrangements characterized by uncommon combinations of variable, diversity, and joining (V-D-J) gene segments, oligoclonal rearrangements, and low represented clones. Upon validation, the capture NGS approach allowed us to identify Ig/TCR clonal markers in 87% of a retrospective cohort (MRD-unknown within the Northern Italy Leukemia Group (NILG)-ALL 09/00 clinical trial) and in 83% of newly-diagnosed ALL cases in which conventional method failed, thus proving its prospective applicability. Finally, we identified gene variants in 94.7% of patients analyzed for mutational status with the same implemented capture assay. The prospective application of this technology could simplify clonality assessment and improve standard assay development for leukemia monitoring, as well as provide information about the mutational status of selected leukemia-related genes, potentially representing new prognostic elements, MRD markers, and targets for specific therapies.

Role of fructosamine-3-kinase in protecting against the onset of microvascular and macrovascular complications in patients with T2DM.

INTRODUCTION: Microangiopathic and macroangiopathic complications are the main cause of morbidity and mortality in the diabetic population. Numerous publications have highlighted the role of glycation in the onset of complications of diabetes. In this context, the detection of fructosamine-3-kinase (FN3K)-an enzyme capable of counteracting the effect of hyperglycemia by intervening in protein glycation-has attracted great interest. Several studies have linked FN3K genetic variability to its enzymatic activity and glycated hemoglobin (HbA1c) levels. Here, we investigated the role of FN3K polymorphisms in the development of microvascular and macrovascular complications of diabetes. RESEARCH DESIGN AND METHODS: The anthropometric and biochemical parameters, and any medical history of microangiopathic and macroangiopathic complications, were documented in a sample of 80 subjects with type 2 diabetes. All subjects were screened for FN3K gene and analyzed for the combination of three polymorphisms known to be associated with its enzymatic activity (rs3859206 and rs2256339 in the promoter region and rs1056534 in exon 6). RESULTS: The combination of allelic variants of FN3K polymorphisms resulted in 13 distinct genotypic variants within the cohort. Comparison between genotypes showed no significant differences in terms of demographic, anthropometric and biochemical parameters, risk markers and long-term complications, except for a higher age and vitamin E levels associated with the genotype presenting GG at position -385, TT at position -232, and CC at c.900 A. Evaluating the microangiopathic and macroangiopathic complications as a whole, we found that they appeared significantly less present in this genotype compared with all other genotypes (p=0.0306). CONCLUSIONS: The group of patients carrying the favorable allele for the three polymorphisms of the FN3K gene revealed less severe microangiopathy and macroangiopathy, suggesting a protective role of this genotype against the onset of the complications of diabetes.

The analytical performance of laboratory plasma glucose and HbA1c measurements are largely acceptable.

AIMS: Poor comparability between laboratory results may have a strong impact on clinical decisions. The aim of this study was to assess the quality of glucose and HbA1c measurements in a large cohort of laboratories in various countries, in order to evaluate whether the current state of these very basic laboratory examinations can be considered to be adequate with respect to the clinical needs in the management of glucose control in diabetic patients. METHODS: External quality assessment schemes and proficiency testing surveys performed in 2017 in several European and American laboratories were analyzed in order to estimate the percentage of laboratories reaching the desired quality criteria based on the allowable total error in accordance with various international recommendations. RESULTS: In 2017 more than 95% of laboratories met the allowable total error for measuring HbA1c, and 92-94% of the studied laboratories met the target for glucose measurement. CONCLUSIONS: The analytical quality for measuring glycated hemoglobin and glucose at laboratory level is generally acceptable, and accreditation to the ISO 15189:2012 standard is a robust guarantee that the laboratory meets the required criteria of acceptability. Several pre-analytical factors which may explain the discrepancies between the measured HbA1c and that estimated from other indicators of glucose control have to be taken into account, by focusing more on the pre-analytical than the analytical phase. In the case of glucose, special attention should be paid to the use of the correct anticoagulant, in order to avoid false negative results.

Standardization of the HbA2 Assay.

BACKGROUND: A project for the standardization of HbA2 was launched by the IFCC back in 2004. MATERIALS AND METHODS: In this work we report on the state-of-the-art of the project on standardization of HbA2. Data obtained from various EQAS studies, and from previous experimental evaluations, are presented. RESULTS: We have proven that biases between various commercial methods are still currently significant. We have also shown that calibration by commutable control materials may halve the inter-method variability. CONCLUSIONS: The foundation of the reference system for HbA2, together with a brief preliminary presentation of the proposed primary reference measurement procedure based on ID-MS are outlined.