Genome-Wide Single Nucleotide Polymorphism Typing Method for Identification of Bacillus anthracis Species and Strains among B. cereus Group Species ▿ †

As an issue of biosecurity, species-specific genetic markers have been well characterized. However, Bacillus anthracis strain-specific information is currently not sufficient for traceability to identify the origin of the strain. By using genome-wide screening using short read mapping, we identified strain-specific single nucleotide polymorphisms (SNPs) among B. anthracis strains including Japanese isolates, and we further developed a simplified 80-tag SNP typing method for the primary investigation of traceability. These 80-tag SNPs were selected from 2,965 SNPs on the chromosome and the pXO1 and pXO2 plasmids from a total of 19 B. anthracis strains, including the available genome sequences of 17 strains in the GenBank database and 2 Japanese isolates that were sequenced in this study. Phylogenetic analysis based on 80-tag SNP typing showed a higher resolution power to discriminate 12 Japanese isolates rather than the 25 loci identified by multiple-locus variable-number tandem-repeat analysis (MLVA). In addition, the 80-tag PCR testing enabled the discrimination of B. anthracis from other B. cereus group species, helping to identify whether a suspected sample originates from the intentional release of a bioterrorism agent or environmental contamination with a virulent agent. In conclusion, 80-tag SNP typing can be a rapid and sufficient test for the primary investigation of strain origin. Subsequent whole-genome sequencing will reveal apparent strain-specific genetic markers for traceability of strains following an anthrax outbreak

Successful conversion surgery for unresectable gastric cancer with giant para-aortic lymph node metastasis after downsizing chemotherapy with S-1 and oxaliplatin: a case report

Abstract Background Although patients with stage IV gastric cancer who respond well to systemic chemotherapy can be treated with gastrectomy, the prognosis of patients with unresectable gastric cancer with para-aortic lymph node metastasis is poor. We herein report a case of remnant gastric cancer with para-aortic lymph node metastasis that was treated with potentially curative conversion surgery after showing a complete response to chemotherapy with S-1 and oxaliplatin (SOX). Case presentation An 81-year-old man was diagnosed with type 3 remnant gastric cancer with giant para-aortic lymph node metastasis, and he received SOX chemotherapy. After three courses of SOX chemotherapy, the primary tumor and para-aortic lymph node metastases markedly reduced in size, indicating a partial response. Because conversion surgery was possible, the patient underwent total remnant gastrectomy with D2 and para-aortic lymph node dissection. Histological examination revealed no residual cancer cells in the resected stomach and lymph nodes. The patient was diagnosed with a complete pathological response and was discharged on postoperative day 24. Currently, 1 year after surgery, the patient is alive and has not shown any tumor recurrence. Conclusion To the best of our knowledge, this is the first case of advanced remnant gastric cancer with giant para-aortic lymph node metastasis that showed a pathological complete response and favorable outcome after SOX chemotherapy

Staging of gastric cancer with the Clinical Stage Prediction score

Abstract Background Chemotherapy with or without surgery is the first-line treatment for stage III/IV gastric cancer, while surgery is the first-line treatment for stage I/II gastric cancer. Accordingly, it is important to distinguish between stage III/IV and stage I/II gastric cancer, but clinical staging is less accurate than pathological staging. This study was performed to develop a clinical score that could distinguish stage III/IV gastric cancer from stage I/II gastric cancer. Methods We reviewed 2722 patients who underwent gastrectomy at our hospital from January 1996 to December 2015. As pretreatment factors potentially related to tumor stage, we assessed age, sex, tumor markers, tumor diameter, tumor location, tumor histology, and macroscopic type. Factors showing significance on multivariate analysis were used to develop the Clinical Stage Prediction score (CSP score), and a cutoff value for the score was determined by receiver operating characteristics analysis. Results According to multivariate analysis, clinical factors associated with stage III/IV disease were elevation of the carcinoembryonic antigen level, tumor diameter ≥ 60 mm, circumferential gastric involvement, esophageal infiltration, mucinous adenocarcinoma, and macroscopic types 2–4. The CSP score was obtained by weighting these factors according to the non-standardized β-coefficient. Receiver operating characteristics analysis indicated that the optimum cutoff value of the CSP score was 17 points. Among 1042 patients with a CSP score ≥ 17 points, 820 patients (78.7%) had stage III/IV gastric cancer. Conversely, among 1680 patients with a CSP score < 17 points, 1547 patients (92.1%) had stage I/II gastric cancer. When discrimination of stage III/IV gastric cancer from stage I/II gastric cancer by the CSP score was assessed, the sensitivity was 78.7%, specificity was 92.1%, positive predictive value was 86.0%, and negative predictive value was 87.5%. Conclusions The CSP score can be helpful for differentiating stage III/IV gastric cancer from stage I/II gastric cancer based on pretreatment clinical factors

Genomewide Screening for Novel Genetic Variations Associated with Ciprofloxacin Resistance in Bacillus anthracis▿ †

Fluoroquinolone (FQ) resistance of Bacillus anthracis is a serious concern in the fields of biodefense and bioterrorism since FQs are very effective antibiotics and are recommended as first-line treatment against this lethal bacterium. In this study, we obtained 2 strains of B. anthracis showing resistance or intermediate resistance to ciprofloxacin (CIP) by a stepwise selection procedure with increasing CIP concentrations. Fifteen genetic variations were identified between the parental and CIP-resistant strains by next-generation sequencing. Nonsynonymous mutations in the quinolone resistance-determining region (QRDR) of type II DNA topoisomerase were identified in the resistant strain but not in the intermediate-resistant strain. The GBAA0834 (TetR-type transcriptional regulator) locus was also revealed to be a novel “mutation hot spot” that leads to the increased expression of multidrug efflux systems for CIP resistance. As an initial step of CIP resistance in B. anthracis, such disruptive mutations of GBAA0834 appear to be more easily acquired than those in an essential gene, such as that encoding type II DNA topoisomerase. Such an intermediate-resistant phenotype could increase a cell population under CIP-selective pressure and might promote the emergence of highly resistant isolates. Our findings reveal, in addition to QRDR, crucial genetic targets for the investigation of intermediate resistance of B. anthracis to FQs

Orchiectomy due to delayed severe scrotal hematocele after laparoscopic transabdominal preperitoneal repair for a giant inguinoscrotal hernia: a case report

Abstract Background A giant inguinoscrotal hernia is a rare inguinal hernia that extends below the midpoint of the inner thigh while standing. Although reports of laparoscopic surgery for giant inguinoscrotal hernias have increased, the risk of delayed hematocele has not yet been clarified. Case presentation A 68-year-old man was evaluated for a left giant inguinoscrotal hernia, and laparoscopic transabdominal preperitoneal repair (TAPP) was performed. In the procedure, the distal hernia sac was not resected. The postoperative course was uneventful for 3 months postsurgery, after which he complained of giant scrotal swelling, which gradually grew to 13 cm. It did not improve with several punctures and caused dysuria because of increased pressure on the urethra. Thus, reoperation was performed 9 months after surgery. The hematocele consisted of a thickened hernia sac, which was tightly adhered to the spermatic cord and testicle. The hernia sac including the hematocele was removed from the scrotum through an anterior approach, preserving the spermatic cord and testicle. On the third postoperative day, an orchiectomy was performed due to poor testicular perfusion caused by spermatic cord injury. There was no hematocele or hernia at the 3-year follow-up. The remnant sac after laparoscopic TAPP for a giant inguinoscrotal hernia possibly caused refractory hematocele. Additionally, the removal of the hernia sac, including hematocele, from the spermatic cord and testicle has a risk of inducing injury, leading to orchiectomy. Conclusion Surgeons should be aware of the possibility of delayed refractory hematoceles after laparoscopic TAPP for giant inguinoscrotal hernias when the hernia sac is not resected