Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories.

To date, a plenty of techniques for the detection of JAK2V617F is used over different laboratories, with substantial differences in specificity and sensitivity. Therefore, to provide reliable and comparable results, the standardization of molecular techniques is mandatory.A network of 19 centers was established to 1) evaluate the inter- and intra-laboratory variability in JAK2V617F quantification, 2) identify the most robust assay for the standardization of the molecular test and 3) allow consistent interpretation of individual patient analysis results. The study was conceived in 3 different rounds, in which all centers had to blindly test DNA samples with different JAK2V617F allele burden (AB) using both quantitative and qualitative assays.The positivity of samples with an AB < 1% was not detected by qualitative assays. Conversely, laboratories performing the quantitative approach were able to determine the expected JAK2V617F AB. Quantitative results were reliable across all mutation loads with moderate variability at low AB (0.1 and 1%; CV = 0.46 and 0.77, respectively). Remarkably, all laboratories clearly distinguished between the 0.1 and 1% mutated samples.In conclusion, a qualitative approach is not sensitive enough to detect the JAK2V617F mutation, especially at low AB. On the contrary, the ipsogen JAK2 MutaQuant CE-IVD kit resulted in a high, efficient and sensitive quantification detection of all mutation loads. This study sets the basis for the standardization of molecular techniques for JAK2V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2V617F.

TET2 mutations in Ph-negative myeloproliferative neoplasms: identification of three novel mutations and relationship with clinical and laboratory findings.

High-throughput DNA sequence analysis was used to screen for TET2 mutations in peripheral blood derived DNA from 97 patients with BCR-ABL-negative myeloproliferative neoplasms (MPNs). Overall six mutations in the coding region of the gene were identified in 7 patients with an overall mutational frequency of 7.2%. In polycythemia vera patients (n = 25) 2 mutations were identified (8%), and in those with essential thrombocythemia (n = 55) 2 mutations (3.6%); in those with unclassifiable MPN (n = 8) 3 mutations (37.5%). No primary myelofibrosis patients (n = 6) harboured TET2 mutations. Three unreported mutations were identified (p.P177fs, p.C1298del, and p.P411del), the first two in patients with unclassifiable MPN, the last in a patient with essential thrombocythemia. On multivariate analysis the diagnosis of an unclassifiable MPN was significantly related to the presence of TET2 mutations (P = 0.02; OR: 2.81; 95% CI 1.11-7.06). We conclude that TET2 mutations occur in both JAK2 V617F-positive and -negative MPNs and are more frequent in MPN-U patients. This could represent the biological link between the different classes of myeloid malignancies.

Multidrug-resistant Escherichia fergusonii: a case of acute cystitis.

We report a case in which Escherichia fergusonii, an emerging pathogen in various types of infections, was associated with cystitis in a 52-year-old woman. The offending strain was found to be multidrug resistant. Despite in vitro activity, beta-lactam treatment failed because of a lack of patient compliance with therapy. The work confirms the pathogenic potential of E. fergusonii.

Beta-lactam failure in treatment of two group G Streptococcus dysgalactiae subsp. equisimilis Pharyngitis patients.

We present two cases of exudative pharyngitis due to Streptococcus dysgalactiae subsp. equisimilis, Lancefield group G. While the participation of this organism as an agent of pharyngitis is well documented, we focus on failure of beta-lactam therapy, a phenomenon that is well described for pharyngitis due to Streptococcus pyogenes. Therefore, these case reports add to our knowledge of pharyngitis caused by non-S. pyogenes streptococci.