Positive Circumferential Resection Margin in Rectal Cancer Is a Robust Predictor of Poor Long-term Prognosis With Clinicopathological Bias Between Groups Compensated by Propensity-score Matching Analysis.

BACKGROUND/AIM: Circumferential resection margin (CRM) is the most reliable predictor of local and distant recurrence in locally-advanced rectal cancer (LARC). The present study was conducted to compare the long-term outcomes between CRM (+) and (-) groups using propensity-score (PS) matching analysis to compensate for bias between groups. PATIENTS AND METHODS: Of 563 consecutive patients with Stage II/III rectal cancer who were treated surgically with curative-intent at Juntendo University Hospital between Jan 1989 and Mar 2018, 412 patients were enrolled retrospectively in the study. The patients were divided into a CRM (+) group (n=21; 5.1%) and a CRM (-) group (n=391; 94.9%). RESULTS: In the entire cohort, recurrence-free survival (RFS), local recurrence-free survival (LRFS), non-local recurrence-free survival (NLRFS), and cancer-specific survival (CSS) were significantly worse among patients in the CRM (+) group compared with those in the CRM (-) group. Univariate analysis demonstrated patients in the CRM (+) group had significantly larger primary tumors (p=0.02), more frequently had open surgery (p=0.009), had an abdominoperineal resection (APR) procedure (p=0.01) and a T4 primary tumor (p<0.0001). After PS matching analysis, in the propensity-matched cohort, RFS, LRFS, NLRFS and CSS were significantly worse among patients in the CRM (+) group compared with those in the CRM (-) group. CONCLUSION: PS matching analysis demonstrated that RFS, LRFS, NLRFS, and CSS were significantly worse among patients in the CRM (+) group compared with those in the CRM (-) group. The present results indicate that CRM (+) is a robust predictor of long-term outcome of LARC, independent of tumor size.

CHFR-Promoter-Methylation Status Is Predictive of Response to Irinotecan-based Systemic Chemotherapy in Advanced Colorectal Cancer.

BACKGROUND/AIM: We investigated whether promoter methylation of the checkpoint-with-forkhead-and-ring-finger-domains (CHFR) gene is a predictor of the efficacy of irinotecan-based systemic chemotherapy for advanced colorectal cancer (CRC) patients. MATERIALS AND METHODS: CHFR-promoter methylation was measured by quantitative methylation-specific PCR (qMSP). The histoculture drug response assay (HDRA) was used in vitro to analyze the correlation between CHFR-promoter methylation and the efficacy of the irinotecan-active-metabolite SN38 in colorectal-cancer tissues from 44 CRC patients. CHFR promoter-methylation was also analyzed for its correlation with clinical response to irinotecan-based systemic chemotherapy of 49 CRC patients. RESULTS: CHFR-promoter methylation significantly-positively correlated with inhibition of colon cancer by SN38 in the HDRA (p=0.002). CHFR-promoter methylation also significantly-positively correlated with clinical response to irinotecan-based systemic chemotherapy (p=0.04 for disease control). CHFR-promoter methylation also significantly-positively correlated (p=0.01) with increased progression-free survival for patients treated with irinotecan-containing FLOFIRI in combination with bevacizumab, the most-frequent regimen in the cohort. CONCLUSION: Sensitivity of advanced CRC patients to irinotecan-based systemic chemotherapy can be predicted by the extent of CHFR-promoter methylation.

Study of Purse-string Skin Closure Plus Negative-pressure Wound Therapy for Stoma Closure.

Background: Although purse-string skin closure (PSC) is an effective method for stoma closure considering wound infection, the period for scarring will be prolonged. The aim of this study was to assess whether negative-pressure wound therapy (NPWT) can reduce the wound-scarring period for PSC after stoma closure. Methods: Patients who underwent stoma closure between January 2015 and August 2020 at our department were retrospectively assessed. Patients in the control group received only PSC, and patients in the NPWT group received both PSC and NPWT using the VAC® or PICO®. The primary endpoint of this study was the short-term reduction ratio (RR). The RR is calculated by the length, width, and depth of the wound of the stoma closure site. The secondary endpoints were scarring period and wound-related complications such as surgical site infection, dermatitis, bleeding, enterocutaneous fistula, and ventral hernia. Results: Of the 53 patients included in this study, 21 had their stoma closed by PSC and 32 had their stoma closed by PSC plus NPWT. No significant differences were observed in patient characteristics or peri-operative states. The RR in the NPWT group was significantly smaller than that in the PSC group at 7 postoperative days (p=0.04). There was no difference in scarring period between the two groups (p=0.11).The rates of postoperative wound-related complications were similar in the two groups (control group: 4 (19%), NPWT group: 7 (21.9%), p=1.0). Conclusions: Our study suggests that PSC plus NPWT might be more effective for wound healing after stoma closure than only PSC.

Characterization of rat TOM40, a central component of the preprotein translocase of the mitochondrial outer membrane.

We cloned a 38-kDa rat mitochondrial outer membrane protein (OM38) with structural homology to the central component of preprotein translocase of the fungal mitochondrial outer membrane, Tom40. Although it has no predictable alpha-helical transmembrane segments, OM38 is resistant to alkaline carbonate extraction and is inaccessible to proteases and polyclonal antibodies added from outside the mitochondria, suggesting that it is embedded in the membrane, probably in a beta-barrel structure, as has been similarly speculated for fungal Tom40. Immunoprecipitation demonstrated that OM38 is associated with the major import receptors rTOM20 and rTOM22, and several other unidentified components with molecular masses of 5-10 kDa in digitonin-solubilized membrane: OM10, OM7.5, and OM5. Blue native polyacrylamide gel electrophoresis revealed that OM38 is a component of a approximately 400-kDa complex, firmly associating with rTOM22 and loosely associating with rTOM20. The preprotein in transit to the matrix interacted with the TOM complex containing OM38, and immunodepletion of OM38 resulted in the loss of preprotein import activity of the detergent-solubilized and reconstituted outer membrane vesicles. Taken together, these results indicate that OM38 is a structural and functional homolog of fungal Tom40 and functions as a component of the preprotein import machinery of the rat mitochondrial outer membrane.

[Antimicrobial susceptibility testing for Mycobacterium tuberculosis by the bioluminescence assay of mycobacterial ATP using filamentous cell treatment].

The antimicrobial susceptibility testing for Mycobacterium tuberculosis by the bioluminescence assay of adenosine triphosphate(ATP) derived from living mycobacteria was improved introducing filamentous cell treatment(FCT) reported for beta-lactam susceptibility test of Pseudomonas aeruginosa by Hattori. Before ATP extraction, bacterial cells were treated with the FCT reagent for 30 minutes at room temperature. Adenosine phosphate deaminase in the FCT reagent simultaneously digested the extracted ATP and released ATP in a liquid culture of M. tuberculosis H37Rv and the RLU level was decreased markedly. Using this improved ATP method, we determined the ATP contents of M. tuberculosis inoculated into Middle-brook 7H9 broth medium with or without drugs. In ethambutol(EB) susceptibility, the ATP method reported previously, showed false-resistance when judged within 7 days. To eliminate false-resistance in EB susceptibility we applied the modified ATP method with FCT treatment to strains determined EB susceptible by reference methods. Using this modified ATP method, we could judge EB susceptibility of 5 ATCC reference strains within 3 days, and these of 15 clinical isolates of M. tuberculosis within 5 days. And all the results obtained were coincident between the ATP method and the reference methods. The reproducibility of this modified ATP method was evaluated with six ATCC reference strains at the concentrations of 0.1 microgram/ml of isoniazid(INH), 2.0 micrograms/ml of rifampicin(RFP), 2.5 micrograms/ml of EB, 2.0 micrograms/ml of streptomycin(SM), and 5.0 micrograms/ml of kanamycin(KM). The test was repeated six times. Reduction of ATP contents were observed in susceptible strains but not in resistant ones within 3 days of cultivation and susceptibilities to drugs could be determined within 3 days at every time when combined FCT to the ATP method. And highly reproducible results were obtained. It is strongly suggested that this modified method is simple, rapid, highly reproducible and nonradiometric, and could be used for the assessment of drug susceptibility for M. tuberculosis.

[Antimicrobial susceptibility testing for Mycobacterium tuberculosis by measuring mycobacterial adenosine triphosphate].

The antimicrobial susceptibility test for Mycobacterium tuberculosis H37Rv and 43 clinical isolates was performed using a bioluminescence assay by measuring the content of adenosine triphosphate (ATP) derived from mycobacteria. The drugs tested were isoniazid (INH), rifampicin (RFP), ethambutol (EB), streptomycin (SM), and kanamycin (KM). The ATP contents of M. tuberculosis incubated in the Middle-brook 7H9 broth medium containing antituberculous agents were measured at days of 0, 1, 3, 5, 7 and 10. A reduction of ATP content, indicating growth inhibition, was observed in susceptible strains within 5 to 10 days of incubation. Optimal concentrations to distinguish between susceptible and resistant strains were determined as being INH 0.20, RFP 0.50, EB 5.0, SM 4.0, KM 6.0 g/ml. The agreements of ATP method (evaluated at 10 days) with Vite Spectrum and MIC determinations were 81.4% and 100%, respectively. Susceptibilities to most drugs, except for EB, could be determined within 7 days. This method is simple, rapid, nonradiometric, and can be used for drug susceptibility.

[Basic evaluation for new antimicrobial susceptibility testing of Mycobacterium tuberculosis by bioluminescence assay of mycobacterial ATP].

It has been reported that the number of living bacteria is correlated to their ATP contents. Based on this, ATP measurement was applied to the susceptibility test for Mycobacterium tuberculosis to antimicrobial agents. ATP was extracted from the bacterial suspension prepared from M. tuberculosis H37Rv grown on 1% Ogawa medium and measured by bioluminescent assay. The highest relative light units (RLU) was obtained when ATP was extracted with the reagent supplied by Kikkoman Inc. (Chiba, Japan) at 100 degrees C for 3 minutes. The amounts of ATP recovered was constant at 100 degrees C for 8 minutes. The ATP contents correlated well with the number of bacteria expressed as CFU. The ATP contents of M. tuberculosis H37Rv inoculated into the Middlebrook 7H9 broth medium containing antituberculous agents were measured at days of 0, 3, 5 and 7. The control culture showed the time-dependent increase in the RLU values, while cultures supplemented with antimicrobial agents reduced their ATP contents concomitant with the concentrations of drugs. The growth of tubercle bacilli was expressed as RLU ratio, the ratio of RLU in the drug-containing cultures to those in drug-free ones. RLU ratio of 0.5 or less was determined as sensitive and the ratio of more than 0.5 as resistant to drugs. The inoculum size of bacteria did not affect the days giving RLU ratio below 0.5 or 0.3. Within 7 days, susceptibilities to drugs could be determined. In conclusion, this test is simple, rapid, sensitive and highly reproducible and useful for the assessment of susceptibility.

[Evaluation of a newly developed broth microdilution test method to determine minimum inhibitory concentrations (MICs) of antimicrobial agents for mycobacteria].

We developed a new broth microdilution antimycobacterial susceptibility test for determination of minimum inhibitory concentration (MICs) using an air-dried microplate containing serially diluted antimicrobial agents and the modified Middlebrook 7H9 broth. The eight agents included were streptomycin (SM), isoniazid (INH), rifampicin (RFP), ethambutol (EB), kanamycin (KM), levofloxacin (LVFX), sparfloxacin (SPFX) and clarithromycin (CAM). Serial dilutions of the agents (128 micrograms/ml to 0.125 micrograms/ml) were prepared in microplates, and were reconstituted by inoculation of 0.2ml of cell suspensions (approximately 3 x 10(5) cells/ml). The test plates were incubated at 36 degrees C in 5% CO2, and the growth endpoints were read visually after 5-day, 7-day and 10-day incubations. Four ATCC reference strains, Mycobacterium tuberculosis, M. avium, M. kansasii and M. intracellulare, were repeatedly tested at three sites. Of 480 determination against eight agents, 455 (94.8%), 470 (97.9%) and 455 (94.8%) of the MICs read after 5-day, 7-day and 10-day incubations fell within 3log2 dilutions, respectively. The MICs gradually elevated during the incubation, however those of 7-day incubation were highly precise and easily determined. A total of 160 clinical isolates of M. tuberculosis and 114 of nontuberculous mycobacteria were tested against eight agents. As for the primary drugs (SM, INH, RFP and EB), most isolates of M. tuberculosis were highly susceptible with MIC90, < or = micrograms/ml. Both LVFX and SPFX were also active. The MICs against nontuberculous mycobacteria distributed in a wide range, and the activities of RFP, LVFX, SPFX and CAM were more potent. These results demonstrate this newly developed test method to be a practical, rapid, quantitative and nonradiometric alternative for the determination of MICs in clinical mycobacteriology laboratories.

[Determination of pyrazinamide susceptibility for Mycobacterium tuberculosis by use of Middlebrook culture media and comparison with results of pyrazinamidase test].

We developed a new test method to determine pyrazinamide (PZA) susceptibility for Mycobacterium tuberculosis in an acidified Middlebrook 7H9 broth (pH6.0), and evaluated in comparison with the agar proportion method of the National Committee for Clinical Laboratory Standards (NCCLS) M24-T and with pyrazinamidase assay. The test method is based on a culture in 4 ml of the modified Middlebrook 7H9 broth containing 100, 200 and 400 micrograms PZA/ml, respectively. First, the cell suspension was adjusted to a McFarland #1 turbidity, and then diluted 1:10. After mixing, 0.1 ml of the diluted cell suspension was inoculated and incubated at 36 +/- 1 degrees C in an ambient air. After 7 day-incubation, the test broth was read in comparison with the growth control. When a significant growth at 100 micrograms PZA/ml or an attenuated growth at 100 micrograms PZA/ml but a significant growth at 400 micrograms PZA/ml were observed, the test isolate was interpreted as being PZA-resistant. When PZA-susceptible and PZA-resistant ATCC reference strains were repeatedly tested, the results obtained were highly precise and accurate. A total of 65 clinical isolates were tested, the results indicating 95.4% of agreements with the agar proportion method and 90.8% with pyrazinamidase assay. There found six discrepant results of 13 resistant isolates; three were susceptible by the agar proportion and all the six were positive by pyrazinamidase assay. Accordingly, we can conclude that, in place of radiometric Bactec System, our newly developed test method is an accurate, practical, rapid and nonradiometric alternative to determine PZA susceptibility for M. tuberculosis in clinical mycobacteriology laboratories.

[Determination of antimycobacterial activities of fluoroquinolones against clinical isolates of Mycobacterium tuberculosis: comparative determination with egg-based Ogawa and agar-based Middlebrook 7H10 media].

The minimum inhibitory concentrations (MICs) to the fluoroquinolones, ofloxacin (OFLX), ciprofloxacin (CPFX), sparfloxacin (SPFX), norfloxacin (NFLX), balofloxacin (BLFX) and CS-940, were determined in 100 clinical isolates of Mycobacterium tuberculosis. The MICs were determined with 1% egg-based Ogawa or agar-based Middlebrook 7H10 and each of them supplemented with oxidation-reduction color dye, 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC) by using the microculture technique. The MICs determined with Ogawa medium were approximately two- to four-fold higher when compared to those determined with Middlebrook agar medium. The supplement with STC slightly increased the MICs, probably as a result of easily recognizing small initial colonies. Among the six fluoroquinolones, CS-940 and SPFX showed the greatest antimycobacterial activities with inhibition of 50% of all the isolates at the concentrations between 0.25 to 0.5 microgram/ml. OFLX, CPFX and BLFX followed in potency at 0.5 to 2.0 micrograms /ml. NFLX was less potent requiring 8 to 16 micrograms/ml to inhibit 50% of the isolates.

[Detection of rifampicin-resistant strains of Mycobacterium tuberculosis by a non-radioactive PCR-SSCP method].

PCR-SSCP method to detect genetic mutations in rpoB gene as a marker of rifampicin-resistance was developed by Telenti et al., and we have modified it applying non-radioactive PhastSystem for more practical use in the detection of rifampicin-resistance of Mycobacterium tuberculosis. PCR products amplified with the primers specific to rpoB gene using extracted DNA from 89 strains of M. tuberculosis were sequenced and the amino acid sequences were morphism was determined by the PhastSystem. The bands were stained by silver staining. Among 89 strains of M. tuberculosis, 43 were confirmed as rifampicin-resistant (RFPr) and 46 were rifampicin-sensitive (RFPs) by the culture on the drug-containing media. All of the 43 RFPr strains had one or more mutations in the DNA sequence of rpoB gene, while none of the RFPs strains had such mutations. However, by PCR-SSCP, only 20 out of 43 RFPr strains showed clear differences in the band pattern of electrophoresis from that of RFPs strains. Other 23 RFPr strains had only slight differences in the band pattern of the PCR-SSCP from that of RFPs strains. But it was noticed that the main bands of RFPr strains were distinguishable from the main bands of RFPs strains even their patterns were similar. Thus, it is possible to apply a non-radioactive PCR-SSCP for the detection of rifampicin resistance of M. tuberculosis with further improvement of the condition of gel electrophoresis or staining techniques.

Multicenter evaluation of a colorimetric microplate antimycobacterial susceptibility test: comparative study with the NCCLS M24-P.

A colorimetric test method using the microplate culture technique for the determination of susceptibility of Mycobacterium tuberculosis against antimycobacterial agents was developed and evaluated by the multicenter study. The test method utilizes an oxidation-reduction dye, 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC), as an indicator of mycobacterial growth. When compared to the presently available test method, some modifications were also included; lower inoculum density (10-fold dilution), inclusion of an inoculum diluted 1:100 as a growth control, and the preparation of inoculum preincubated in Middlebrook 7H9 broth and spectrophotometrically adjusted to McFarland #1 turbidity. The test method evaluated was highly precise and reliable to detect antimycobacterial resistances when the ATCC reference strains were tested. Also, the interpretations of the test result were highly comparable to those determined by the method of NCCLS M24-P, the % agreements ranging from 76.1% (ethambutol) to 91.3% (streptomycin). The test results were also comparable to those determined by Ogawa media; > 90% agreed with susceptible, intermediate, or resistant. The appearance of mycobacterial colonies on the test media was easily read, and the test results were more comparable to those of NCCLS M24-P. With these results, it can be concluded that the colorimetric microplate susceptibility test method described will be more suitable for clinical mycobacteriology laboratories.

[Colorimetric broth microdilution for antifungal susceptibility testing].

A colorimetric broth microdilution modification of the National Committee for Clinical Laboratory Standards (NCCLS) for antifungal susceptibility testing was developed and evaluated. The test method modification includes; air-dried microdilution trays, in which serial two-fold dilutions of three antifungal agents, amphotericin B (AMPH), flucytosine (5-FC) and fluconazole (FCZ) were first prepared and evaporated, then reconstituted by adding 100 microliters of yeast inocula, and an oxidation-reduction color indicator (sodium resazurin, Sigma) added to RPMI 1640 medium buffered to pH7.0 with 0.165M morpholinepropanesulfonic acid. The trays were incubated in air at 35 degrees C and were inspected after 24 and 48hr of incubation. The MICs were defined as the lowest concentration of the respective agents; no color change (blue) for AMPH, and slight color change (blue to purple) for 5-FC and FCZ. The MICs of AMPH, 5-FC and FCZ were determined for four reference strains and 100 clinical isolates, in comparison with the NCCLS macrodilution method. The four reference strains were tested seven times each by macrodilution method (MACRO) and 14 times each against all three antifungal agents by microdilution method (MICRO). Overall, 100% of MICs determined by MACRO, 96% of those determined at 24hr incubation by MICRO, and 93% of those determined at 48hr incubation by MICRO fell within the 3-log2 dilutions, although 20 to 33% were out of the acceptable MIC ranges of the NCCLS M27-P proposal. Excellent reproducibility in determining growth endpoint by color change was also demonstrated, giving 99 to 100% agreements in duplicated dilutions. When the MICs determined for 100 clinical isolates against three antifungal agents were compared, those determined at 24hr incubation by MICRO gave 61.7% of agreement within the 3-log2 dilutions of the NCCLS MACRO, and those determined at 48hr incubation by MICRO were 86.2%. The MICRO read at 24hr trended to the lower MICs, except for 5-FC. While the MICs of MICRO read at 48hr were mostly comparable (78 to 97% agreement) to those of MACRO, especially against 5-FC (97%) and AMPH (85%), but the discrepant MICs of Candida tropicalis against FCZ were noted. With these results, it can be concluded that the resazurin colorimetric broth microdilution method is easy to perform and highly precise, and may provide MICs comparable to those determined by the reference NCCLS macrodilution method.