Impact of microscopic extrathyroidal extension on differentiated thyroid cancer post-surgical risk of recurrence: a retrospective analysis.

PURPOSE: In the last edition of the American Joint Committee on Cancer (AJCC) staging system, differentiated thyroid cancers (DTC) showing microscopic extrathyroidal extension (mETE) are considered comparable to intrathyroidal cancers for their clinical behavior and prognosis. The aim of the study is to evaluate the impact of this updated assessment of T, when applied to the postoperative recurrence risk stratification, according to the American Thyroid Association Guidelines (ATA-RR). METHODS: One-hundred DTC patients who underwent total thyroidectomy were retrospectively evaluated. The downstaging of mETE was introduced in the definition of T, and the updated classification defined as modified ATA-RR (ATAm-RR). For each patient, post-surgical basal and stimulated thyroglobulin (Tg) levels, neck ultrasound (US) and post-ablative 131-I whole body scan (WBS) reports were considered. The predictive performance (PP) of disease recurrence was calculated both for each single parameter, as well as for all of them. RESULTS: According to ATAm-RR classification, 19/100 patients (19%) were downstaged. ATA-RR proved a significant PP for disease recurrence (DR) (sensitivity 75.0%, specificity 63.0%, p = 0.023). However, ATAm-RR performed slightly better due to an increased specificity (sensitivity 75.0%, specificity 83.7%, p < 0.001). For both classifications, the PP was optimal when all the above-mentioned predictive parameters were considered. CONCLUSION: Our results suggest that the new assessment of T considering mETE resulted in a downgrading of ATA-RR class in a significant number of patients. This provides a better PP for disease recurrence, and the best PP was obtained when considering the whole predictive variables together.

Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR.

The aim of the present work was to develop two new independent SYBR Green I-based real-time PCR assays for both detection and quantification of betanodavirus, an RNA virus that infects several species of marine teleost fish causing massive mortalities in larvae and juveniles. The assays utilized two pairs of primers targeting highly conserved regions of both the RNA molecules forming the betanodavirus genome: RNA1 encoding the RNA-dependent RNA polymerase (RdRP) and RNA2 encoding the coat protein (CP). The specificity of amplifications was monitored by the melting analysis and agarose gel electrophoresis of the amplified products. The applicability of these assays was confirmed with 21 betanodavirus strains, covering all the four main clades. In addition, a BLAST (NCBI) search with the primer sequences showed no genomic cross-reactivity with other viruses. The new assays were able to quantify concentrations of betanodavirus genes ranging from 10(1) to 10(8) copies per reaction. The intra-assay coefficients of variation (CV) of threshold cycle (Ct) values of the assays were 1.5% and 1.4% for CP and RdRP RNAs, respectively. The inter-assay CVs of Ct values were 2.3% and 2.4% for CP and RdRP RNAs, respectively. Moreover, regression analysis showed a significant correlation (R2>0.97) between genome number, as determined by real-time PCR assays and the corresponding virus titer expressed as TCID50/ml of two different betanodavirus strains propagated in cell culture. The two assays were compared with a previously established one-step RT-PCR assay and with the classical virus isolation test and found to be more sensitive. In conclusion, the developed real-time RT-PCR assays are a reliable, specific and sensitive tool for the quantitative diagnosis of betanodavirus.

Sequence comparison and phylogenetic analysis of fish nodaviruses based on the coat protein gene.

We have amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced a 605-bp fragment covering the variable region of the coat protein gene of fish nodaviruses infecting European sea bass, Dicentrarchus labrax (n = 24), and shi drum, Umbrina cirrosa (n = 2), in the Mediterranean basin. Nine new isolates were identified and their sequences were combined with sequences in the literature to produce three different data sets. The first set, based on amino acid sequences, was used to verify the monophyly of fish nodaviruses. The second and third data sets, based on nucleic acids, were used to resolve the phylogenetic relationships between closely related fish nodaviruses. Phylogenetic analyses were performed according to the maximum parsimony and neighbor-joining methods. Our results support the monophyly of fish nodaviruses. Moreover, they confirm the subdivision of fish nodaviruses into four main clusters, in agreement with the previously suggested phylogeny of the genus Piscinodavirus, that was based on a smaller number of sequences and an alternative phylogenetic approach [14]. All the Mediterranean isolates were clustered in the group of the red-spotted grouper nervous necrosis virus and appear to have a restricted geographic distribution, except for one sequence-type (10 samples) that is widespread throughout the basin.

Viral encephalopathy and retinopathy of farmed marine fish species in Italy.

Viral encephalopathy and retinopathy, otherwise known as fish encephalitis or viral nervous necrosis (VNN), is an emerging problem in several farmed marine fish species in various geographic areas all over the world. Since summer 1995, heavy losses affecting mainly juvenile and adult sea bass (Dicentrarchus labrax) have been observed in several on-growing facilities in Italy. Dying fish show abnormal swimming behaviour and, at temperatures higher than 20-22 degrees C, mortality rates range between 15 and 50%. Neither significant external nor internal gross pathological signs, except frequent abnormal swim bladder hyperinflation, were detected. Histological investigations reveal vacuolations in the grey matter of the brain and spinal cord and in the granular layers of the retina. Serial tissue sections examined by an immunohistochemical method carried out with antisera against fish nodaviruses showed a positive reaction. Additionally, spherical virus-like particles 22-25 nm in diameter were detected by electron microscopy in negative stained preparations of brain tissues, and the same samples gave a positive reverse transcription-polymerase chain reaction (RT-PCR) for the T4 region of the fish nodavirus gene. These results indicate that both juvenile and adult sea bass subject to mass mortality in Italy since summer 1995 are infected with a fish nodavirus and strongly suggest that the identified virus is the cause of the observed mortality.