Fusarium Wilt of Gerbera Caused by a Fusarium sp. in Brazil.

In 2006, gerbera (Gerbera jamesonii) plants, cvs. Basic, Xena, and Olimpia grown for cut flower production in two greenhouse farms in the region of Guarapuava, Paraná, Brazil, exhibited symptoms of a wilt disease. Affected plants (approximately 20, 60, and 50% on cvs. Basic, Xena, and Olimpia, respectively) were stunted and developed yellow leaves unilaterally with initially brown and eventually black streaks in the vascular system. The first symptoms occurred 2 months after transplanting during the hottest period of the summer with an average air temperature of 27°C. Vascular streaks in the yellow leaves were continuous with a brown discoloration in the vascular system of the crown and upper taproot. Occasionally, the leaves of affected plants turned red. A Fusarium sp. was consistently and readily isolated onto a Fusarium-selective medium from symptomatic vascular tissue sampled from the crown of infected plants. Colonies were identified as Fusarium oxysporum on the basis of colony and conidia morphology (1) after subculturing on potato dextrose agar. Since F. oxysporum f. sp. chrysanthemi has been previously reported on Chrysanthemum morifolium, Argyranthemum frutescens, and gerbera (4), pathogenicity tests were carried out by using one monoconidial isolate obtained from wilted plants and one Italian isolate (F. oxysporum f. sp. chrysanthemi MASS 6). The isolates of F. oxysporum were grown in casein hydrolysate in shake culture (90 rpm) for 10 days at 25°C with 12 h of fluorescent light per day. Healthy rooted plants of 30-, 20-, and 45-day-old C. morifolium (cv. Captiva), A. frutescens (cv. Stella 2000), and gerbera (cvs. Jaska, Dalma, and Excellence), respectively, were inoculated by separately dipping roots into a conidial suspension (5 × 107 conidia/ml) of the two isolates of F. oxysporum. Plants were transplanted (one plant per pot) into pots (3.5 liter vol). Noninoculated plants served as control treatments. Plants (15 per treatment) were grown in a glasshouse at an average day temperature of 32°C and night temperature of 23°C (minimum 21°C and maximum 43°C). Wilt symptoms and discoloration of the vascular system in roots, crown, and petioles developed within 29 days on C. morifolium, 26 days on A. frutescens, and 14 days on gerbera. Noninoculated plants remained healthy. F. oxysporum was consistently reisolated from infected plants. The pathogenicity test was carried out twice. Gerbera wilt caused by F. oxysporum f. sp. chrysanthemi was recently reported in Italy (2) and Spain (3). Currently, the wilt of gerbera in the area of Paraná is limited to two farms. To our knowledge, this is the first report of the disease in Brazil as well as in South America. References: (1) C. Booth. Fusarium. CMI, Kew, UK, 1977. (2) A. Garibaldi et al. Plant Dis. 88:311, 2004. (3) A. Garibaldi et al. Plant Dis. 91:638, 2007. (4) A. Minuto et al. J. Phytopathol. 155:373, 2007.

Improved differentiation of Mycobacterium tuberculosis strains, including many Beijing genotype strains, using a new combination of variable number of tandem repeats loci.

Variable number of tandem repeats (VNTR) typing was done on 230 Mycobacterium tuberculosis strains, including 41 strains isolated from 17 groups of epidemiologically linked patients. By PCR amplification, 185 (80.4%) of the 230 strains were Beijing genotype strains. VNTR typing was performed using the 15 loci proposed as a standard set by Supply et al. [Supply, P., Allix, C., Lesjean, S., Cardoso-Oelemann, M., Rusch-Gerdes, S., Willery, E., Savine, E., de Haas, P., van Deutekom, H., Roring, S., Bifani, P., Kurepina, N., Kreiswirth, B., Sola, C., Rastogi, N., Vatin, V., Gutierrez, M.C., Fauville, M., Niemann, S., Skuce, R., Kremer, K., Locht, C., van Soolingen, D., 2006. Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J. Clin. Microbiol. 44, 4498-4510], and cluster analyses of these data were done. By the VNTR typing with the proposed 15 loci, strains having low similarity values by restriction fragment length polymorphism (RFLP) analysis were clustered. Use of a supplemental9 loci, proposed as a high-resolution tool, with the 15 loci showed that strains with low similarity by RFLP analysis were still clustered. Twelve VNTR loci were selected based on previously reported discriminatory index (DI) values and used with the proposed 15 loci for better differentiation by VNTR typing. When eight loci with higher DI values were used with the 15 loci, there were no clusters, including strains with low RFLP similarity. The15 loci and eight additional loci decreased the numbers of clustered strains isolated from epidemiologically unlinked patients significantly compared to using only the 15 loci. Among all tested loci, obvious differences of DI values were observed for 8 loci (miru10, miru16, miru39, Mtub29, Mtub30, QUB11a, QUB26, and QUB1895) of RD105 lineage strains compared to those of other lineage strains. These results suggest that the proposed VNTR typing method cannot be used as a routine epidemiological tool in areas where Beijing genotype strains are prevalent. Several VNTR loci should be added to the proposed method based on differences in polymorphism of VNTR loci among Beijing genotype lineages.

Optimal settings of fingerprint-type analysing computer software for the analysis of enterohaemorrhagic Escherichia coli pulsed-field gel electrophoresis patterns.

Settings of fingerprint-type analysing computer software were optimized for analysis of enterohaemorrhagic Escherichia coli (EHEC) pulsed-field gel electrophoresis (PFGE) patterns. Under the lowest values of parameters, maximum value of similarities calculated using the Dice coefficient were obtained between PFGE patterns from one EHEC strain on the same gel when reference lanes for calibration of distortions during electrophoresis were set to every fourth lane. PFGE patterns of 15 EHEC strains on different gels were investigated. Similarity values calculated using the Pearson product-moment correlation coefficient (Pearson correlation) were significantly higher than those using the Dice coefficient with optimal values of parameters determined by the program (P < 0.01). When PFGE patterns of 45 EHEC strains were analysed by the computer program, EHEC strains from one mass outbreak and three intra-family outbreaks were each clustered and the similarity values within the clusters were > 90% using Pearson correlation.

Whether or not prophylactic excision of the extrahepatic bile duct is appropriate for patients with pancreaticobiliary maljunction without bile duct dilatation.

BACKGROUND/AIMS: The standard treatment for patients with a pancreaticobiliary maljunction (PBM) without bile duct dilatation remains controversial. METHODOLOGY: We followed up 29 patients with such PBM who mainly underwent a cholecystectomy alone. The ages of the patients ranged from 3 to 76 years (average age 47.3 years) and the ratio of males to females was 8 vs. 21. When the diameter of the common bile duct was less than 10mm, such bile ducts were diagnosed to have no dilatation. The main clinical indications for surgery were cholecystolithiasis in 15 patients, choledocholithiasis in 3, cholecystocholedocholithiasis in 2, gallbladder polyp in 2, adenomyomatosis in 2, cholecystitis in 2, and protein plug in 1. RESULTS: The amylase levels of gallbladder bile in 20 patients ranged from 115 to 460,200 IU/mL (a mean of 191,698 IU/mL). One patient died of gastric cancer 182 months after surgery and two patients died of other diseases 153, 171 months after surgeries, respectively. The remaining 26 patients have all been doing well for 36 months to 326 months after surgery (a median follow-up period, 160.5 months). The 10- and 15-year survival rates were 100% and 89.7%. CONCLUSIONS: In conclusion, a prophylactic resection of the extrahepatic bile duct and biliary diversion could be unnecessary for patients with PBM without bile duct dilatation.

Late-onset biliary leakage after laparoscopic cholecystectomy using laparoscopic coagulating shears. Report of a case.

We recently encountered a rare case of late-onset biliary leakage after laparoscopic cholecystectomy using laparoscopic coagulating shears (LCS). The patient was a 49-year-old Japanese man who had undergone a laparoscopic cholecystectomy at Hamamatsu Medical Center after a diagnosis of cholecystolithiasis associated with localized adenomyomatosis. The cystic duct and the cystic artery were closed using LCS instead of metal endoclips. The postoperative course was uneventful, and the patient was discharged on the 4th operative day. However, on the 7th day after the surgery, the patient developed severe upper abdominal pain and was readmitted to our center with the diagnosis of a late biliary leakage, which was confirmed by an endoscopic retrograde cholangiogram. We then treated the leak successfully with endoscopic nasobiliary drainage.

[Restoration of the habit of routine tooth brushing teeth in patients with severe dementia].

Purpose-related activities are often lost in severe dementia, including oral hygiene habits such as brushing teeth. The aim of this study is to investigate whether patients with severe dementia can be induced to develop an oral hygiene routine through an occupational therapy approach and whether routine training in oral hygiene habits can reduce dental or denture plaque. Six female inpatients in the same room, who had been given diagnosis of multiple cerebral infarction and dementia. The project design included 4 periods a, b1, b2, and c, a and c were observation periods, while b1 and b2 were general approach periods. In the approach periods we held meetings, and made the program for oral hygiene, planning and problem-solving. In the b2 period both direct intervention by the dentists, dental hygienists and occupational therapists reinforced the habit of brushing teeth in subjects and educated care approach guidance. No patient completely resumed routine habit of brushing teeth, but an improvement in brushings was found in four subjects, one case had no change, and in one case brushing deteriorated. The state of oral hygiene showed improvement in periods b1 and b2. Although the adherence of dental plaque decreased in period b2, it increased in period c. Despite the improvement in performance, sufficient improvement in oral disease prophylaxis was not recognized, suggesting the necessity of professional oral health care by dental staff, and of a disciplinary team approach.

[Increasing incidence and the mechanism of resistance of nalidixic acid resistant Shigella sonnei].

One hundred and fourteen Shigella sonnei strains obtained in 1991 to 2000 were tested for their susceptibilities to 12 antimicrobial agents. Nalidixic acid (NA) resistance was found in 2 of 15 strains (13.3%) in 1993, 2 of 8 strains (25%) in 1996, one of 5 strains (20%) in 1998, 7 of 21 strains (33.3%) in 1999 and 6 of 12 strains (50%) in 2000. The incidence of resistance to NA in S. sonnei strains increased significantly during this period. Among those 19 NA resistant strains, 11 strains were derived from patients with traveler's diarrhea and 8 strains were derived from patients who had not traveled abroad before the infection, namely domestic patients. PFGE analysis with Xba I revealed that all strains tested differentiated into two major clonal clusters, one cluster consisted of strains derived from patients who had traveled to India after 1993, and another cluster included strains derived from domestic patients. Mechanism of NA resistance was examined by sequencing the quinolone resistance-determining region (QRDR) of gyrA gene. Among 19 NA resistant strains tested, 11 strains presented a change at Ser-83 to Leu and 7 strains presented a change at Asp-87 to Try (5 strains) or Asn (2 strains), whereas 3 NA sensitive strains had no change in the region. These findings indicated that this mutation in gyrA plays an important role in acquisition of Nalidixic-acid resistance in clinical isolates of S. sonnei.

Gallbladder volvulus: experience of six consecutive cases at an institute.

Recently we treated three patients with gallbladder volvulus within a short period. All three patients were examined preoperatively using computed tomography (CT). The first two cases were not diagnosed accurately before laparotomy. A precise diagnosis was made for the third one prior to surgery, based on our former experiences. Typical images, with marked edema and thickened wall of the gallbladder volvulus were shown on CT. We discuss six consecutive cases of the disease experienced at Hamamatsu Medical Center (i.e., the three patients rofed above, and three who did not have preoperative CT).

[Characterization of enterohemorrhagic Escherichia coli O26 and development of its isolation media].

We studied 101 strains of Enterohemorrhagic Escherichia coli (EHEC) O26 isolated from diarrhea patients in six prefectural institutes of public health in Japan during June 1996 and December 1997 and tried to establish an isolation medium for EHEC O26. None of the 101 EHEC O26 strains fermented rhamnose; Whereas all of the other EHEC including O157 and non-EHEC (166 strains) fermented rhamnose except 1 strain of non-EHEC. All of the randomly selected EHEC O26 (14 strains of O26:H11.2 strains of O26:H-) showed a very high resistance to potassium tellurite (Minimal Inhibitory Concentration (MIC) > or = 50 micrograms/ml), whereas all of the randomly selected non-EHEC (26 strains) but 1 showed a high sensitivity (MIC < or = 6.25 micrograms/ml) to this compound. On the basis of these results, we developed a Rhamnose MacConkey (RMAC) medium in which lactose in the MacConkey medium was replaced by rhamnose, and Cefixime-Potassium Tellurite-RMAC (CT-RMAC) medium in which Cefixime (0.05mg/l) and Potassium Tellurite (25mg/l) was added to RMAC for the isolation of EHEC O26 strains. We then evaluated the specifcity of these selective media by growing a selected number of O26 (24 strains) and 9 selected strains of bacteria. All of the EHEC O26 strains generated rhamnose non-fermented colonies (white color) on both media. In contrast to the EHEC O26, the vast majority of E. coli strains (166/167 = 99.4%) other than EHEC O26 were theoretically assumed to generate red colonies on the RMAC medium because of their rhamnose fermenting character and most of them were assumed not to grow on CT-RMAC medium because of their sensitivity to potassium tellurite. These findings and results indicate that EHEC O26 can be easily distinguished from other strains of E. coli including O157. Although EHEC O26 strains showed somewhat poor growth on CT-RMAC medium compared with that on RMAC medium, these O26 showed almost the same degree of growth on CT-RMAC as they showed on DHL media. The results of the present study demonstrated that the use of RMAC and CT-MRAC media for the isolation of EHEC O26 is very reliable and efficient with RMAC having good sensitivity and CT-RMAC having a better specificity for the isolation of this strain of EHEC.

[Usefulness of genotypic analysis for investigation of an epidemic of Shigella sonnei infections in Ooami-shirasato Town, Chiba Prefecture].

In March 1996, an epidemic of Shigella sonnei infection occurred in Ooamishira-sato Town, Chiba Prefecture. Colicine typing, antibiotic resistance patterns, plasmid profiles, pulsed-field gel electrophoresis (PFGE) and random amplified poly-morphic DNA (RAPD) were used for the investigation of the epidemic. Ninety-four isolates from patients exhibited three different colicine types and five different antibiotic resistance patterns. But the patterns of plasmid profile, PFGE and RAPD were uniform among the isolates with different colicine type and antibiotic resistance pattern. It is possible that these isolates belonged to a single bacterial clone and circulated through human to human.

[Prospective randomized trial comparing 1/2 FAM (5-fluorouracil (5-FU) + adriamycin + mitomycin C) versus palliative therapy for the treatment of unresectable pancreatic and biliary tract carcinomas (the 2nd trial in non-resectable patients). Japanese Study Group of Surgical Adjuvant Therapy for Carcinomas of the Pancreas and Biliary Tract].

The efficacy of 1/2 FAM, which consists of 5-fluorouracil (5-FU), adriamycin (ADM) and mitomycin C (MMC), was compared with that of palliative treatment in patients with unresectable pancreatic and biliary tract carcinomas in a multicenter randomized trial. The patients assigned to 1/2 FAM group were treated with 5-FU 200 mg/m2/day IV, ADM 15 mg/m2/day IV and MMC 5 mg/m2/day IV. These 3 drugs were given concurrently as the initial dose within a week after palliative operation, and this regimen was repeated for at least 2 whole courses, at 4-week intervals before the next course of therapy. Those randomized to the control group were subjected to palliative treatment alone. Completely eligible for analysis were 42 cases of the 1/2 FAM group and 41 of the control group. There was no significant difference between the groups with respect to the overall and differentiated survival times according to the tumor sites and the clinical efficacy. As for the duration of 50% inhibition of tumor progression, a significantly better outcome was obtained in 1/2 FAM group. Tumor progression was most significantly inhibited in patients with gallbladder carcinoma. In 1/2 FAM group, tumor reduction was achieved in 1 CR and 2 PR patients. The most frequent adverse reaction was gastrointestinal manifestations, along with diarrhea and alopecia. 1/2 FAM did not contribute to the life prolongation, but inhibited the tumor progression for a significantly longer duration and, to a lesser extent, reduced the tumor size in unresectable pancreatic and biliary tract carcinomas. This regimen is suggested to be useful particularly in the treatment of the latter carcinoma.

[Effect of 5'-deoxy-5-fluorouridine (5'-DFUR) on the activity of pyrimidine nucleoside phosphorylase (pyNPase) in normal and tumor tissues of human stomach].

PyNPase (pyrimidine nucleoside phosphorylase) activity and IL-1 alpha were measured in normal and tumor tissues of the specimens resected from gastric cancer patients who had been divided into two groups; one given preoperative 5'-DFUR (oral administration at a mean dose of 10.0 g) and one not given 5'-DFUR (control). PyNPase activities in both groups were higher in tumor than in normal tissues (p = 0.0001), but, less in tumor tissue of the preoperative administration group than the controls (p = 0.0376). IL-1 alpha levels in both groups were higher in tumor than in normal tissues (p < 0.01). Multiple regression analysis of the results showed that IL-1 alpha strongly influenced on PyNPase activity in tumor tissues (higher IL-1 alpha levels resulted in higher PyNPase activities in tumor tissues) (p = 0.0334).

High prevalence of thermostable direct hemolysin (TDH)-like toxin in Vibrio mimicus strains isolated from diarrhoeal patients.

A total of 17 isolates of Vibrio mimicus from patients, 29 from environment and 2 from food was examined for toxigenicity. Sixteen (94%) clinical isolates and one (50%) from food produced TDH-like toxin, whereas none of the environmental isolates did so. The food from which V. mimicus with TDH-like toxin production was isolated, was one which had caused food poisoning. Only one environmental strain produced CT-like toxin, whilst ST-like toxin was not detected from any strains tested.

Localized, aggregative, and diffuse adherence to HeLa cells, plastic, and human small intestines by Escherichia coli isolated from patients with diarrhea.

Adherence of diarrhea-associated Escherichia coli was studied by scanning electron microscopy. Enteropathogenic E. coli (EPEC) adherence factor-positive (EAF+) E. coli of EPEC serotypes (class I EPEC) adhered to plastic and human jejunal and ileal mucosa, similar to case and HeLa cells. Localized adherence, elongation of cell microvilli, and "locking" of the bacterial aggregates by the elongated microvilli were evident after incubation for 20 min. EAF+ E. coli adhered strikingly to mucus but rarely to M cells in Peyer's patch-associated epithelium. Most enteroaggregative E. coli (EAggEC) strains adhered to plastic, similar to HeLa cells. Some diffuse-adhering E. coli (DAEC) strains displayed no adherence to plastic but formed "dimples" on HeLa cells. Both EAggEC and DAEC adhered at lower levels to human small intestines (except M cells) than did EAF+ E. coli. In all cases of EAF+ E. coli, EAggEC, and DAEC, strains were found with atypical characteristics. The data demonstrate the unique adherence characteristics of EAF+ E. coli, EAggEC, and DAEC.

Production of hemagglutinins and pili by Vibrio mimicus and its adherence to human and rabbit small intestines in vitro.

Clinical isolate of Vibrio mimicus were examined for production of cell-associated hemagglutinin (HA) and pili and for adherence to formalin-fixed human intestinal mucosa. V. mimicus grown on CFA agar for 3 h at 37 degrees C possessed HA and adhered better to the mucus layer than to the epithelial cell surface. A significant correlation was found between the HA titers and adherence ability to the epithelial cell surface of villi (P less than 0.05); adherence to the ileal lymphoid follicle-associated epithelium occurred at higher levels. In contrast, V. mimicus grown on CFA agar for 20 h at 37 degrees C exhibited lower levels of HA and reduced adherence ability. The production of pili was more pronounced after 20 h of incubation than after 3 h of incubation. In comparison with V. cholerae 01 and V. cholerae non-01 cultured under similar conditions, V. mimicus showed inferior adherence, but with similar HA production or piliation.