In vitro activities of posaconazole, ravuconazole, terbinafine, itraconazole and fluconazole against dermatophyte, yeast and non-dermatophyte species.

The in vitro activities of two new triazole antifungal agents with broad-spectrum antifungal activity, posaconazole and ravuconazole, were compared with those of three well-established antifungal agents, terbinafine, itraconazole and fluconazole, against 184 clinical isolates. These included 129 dermatophyte isolates (twelve species), 25 yeast isolates (five species) and 28 non-dermatophyte isolates (nine species). In vitro testing was conducted using microdilution plates with RPMI 1640 and National Committee for Clinical Laboratory Standards (NCCLS) guidelines (M27-38P) were followed, except for the preparation of the dermatophyte inoculum. Both posaconazole and ravuconazole showed similar broad-spectrum activity against dermatophyte, yeast and non-dermatophyte species. Mean inhibitory concentrations (MIC) at which 90% [MIC90] of the isolates were inhibited by posaconazole and ravuconazole were 0.25 and 0.5 microg/ml for dermatophytes, 0.5 and 0.25 microg/ml for yeasts, and >4 and 8 microg/ml for non-dermatophytes. The MIC ranges against Trichophyton (six species), Microsporum (five species) and Epidermophyton flocossum were: posaconazole (0.007-1.0/0.007-0.25/0.007-1.0 microg/ml), ravuconazole (0.015-8.0/0.015-1.0/0.015-1.0 microg/ml), itraconazole (0.015- >8.0/0.015-0.5/ 0.015-8.0 microg/ml), fluconazole (0.125- >64.0/4.0 >64.0/0.5-64.0 microg/ml) and terbinafine (0.003 >2.0/0.007-2.0/0.007 >2.0 microg/ml). Overall ranking of the antifungal activity of the five antifungal agents was: terbinafine > posaconazole > ravuconazole > itraconazole > fluconazole, for dermatophytes; ravuconazole > posaconazole > itraconazole > fluconazole > terbinafine, against yeasts; and posaconazole > ravuconazole > terbinafine > itraconazole > fluconazole, for non-dermatophytes.

In vitro susceptibility testing of ciclopirox, terbinafine, ketoconazole and itraconazole against dermatophytes and nondermatophytes, and in vitro evaluation of combination antifungal activity.

BACKGROUND: With the development of newer antifungal agents with activity against both yeasts and filamentous fungi, there is an increased need to develop and standardize in vitro assays that will evaluate the activity of antimycotics against filamentous fungi. In vitro analysis of antifungal activity of these agents would also allow for the comparison between different antimycotics, which in turn may clarify the reasons for lack of clinical response or serve as an effective therapy for patients with chronic infection. OBJECTIVES: To determine the in vitro susceptibility of fungal organisms to ciclopirox, terbinafine, ketoconazole and itraconazole and to evaluate the in vitro activity and mode of interaction of ciclopirox in combination with either terbinafine or itraconazole. MATERIALS AND METHODS: In the minimum inhibitory concentration (MIC) study 133 strains were evaluated, including dermatophytes (110 strains; 98 from Trichophyton spp.), Candida spp. (14 strains) and nondermatophyte moulds (nine strains). In vitro susceptibility testing was conducted in microbroth dilutions based on the National Committee for Clinical Laboratory Standards (NCCLS) M27-A proposed standard. The testing MIC ranges were 0.003-2 microg mL-1 for ciclopirox and terbinafine, and 0.06-32 microg mL-1 for itraconazole and ketoconazole. For inoculum preparation, dermatophytes were grown on Heinz oatmeal cereal agar slants. Inoculum suspensions of dermatophytes were diluted in RPMI 1640 (Sigma-Aldrich) with the desired final concentration being 2-5 x 103 c.f.u. mL-1. Once inoculated, the microdilution plates were set up according to the NCCLS M27-A method, incubated at 35 degrees C, and read visually following 7 days of incubation. For azole agents, the MIC was the lowest concentration showing 80% growth inhibition; for terbinafine and ciclopirox, the MIC was the lowest concentration showing 100% growth inhibition. In the synergy studies, 29 strains from nondermatophyte species were evaluated using a checkerboard microdilution method. The concentrations tested were: 0 and 0.06-32 microg mL-1 for itraconazole, and 0 and 0.003-4 microg mL-1 for both terbinafine and ciclopirox. Modes of interaction between drugs were classified as synergism, additivism, antagonism or indifference based on fractional inhibitory concentration index values (FIC index). Synergism was defined as an FIC index of < or = 0.50, additivity as an FIC index of < or = 1.0, and antagonism as an FIC index of > or = 2.0. The drug combination was interpreted as indifferent if neither of the drugs had any visible effect on the presence of the other drug. RESULTS: In the MIC study, the dermatophyte MIC values (microg mL-1) (mean +/- SEM) were: ciclopirox (0.04 +/- 0.02), terbinafine (0.04 +/- 0.23), itraconazole (2.28 +/- 7.42) and ketoconazole (0.83 +/- 1.99). The yeast MIC values (microg mL-1) (mean +/- SEM) were: ciclopirox (0.05 +/- 0.02), terbinafine (1.77 +/- 0.58), itraconazole (0.18 +/- 0.27) and ketoconazole (0.56 +/- 0.60). The non-dermatophyte fungi MIC values (microg mL-1) (mean +/- SEM) were: ciclopirox (1.04 +/- 2.62), terbinafine (1.04 +/- 0.95), itraconazole (17.87 +/- 16.75) and ketoconazole (10.69 +/- 13.09). In the synergy study, with ciclopirox in combination with terbinafine, mainly a synergistic or additive reaction was observed; there were no cases of antagonism. For ciclopirox in combination with itraconazole, there were some instances of additivism or synergism, with indifference in the majority of instances; there were no cases of antagonism. CONCLUSIONS: In vitro susceptibility testing indicates that ciclopirox may have a broad antimicrobial profile including dermatophytes, yeasts and other nondermatophytes. Terbinafine is extremely potent against dermatophytes. In vitro evaluation of activity of ciclopirox and terbinafine suggests many instances of synergy or additivism; for ciclopirox and itraconazole there may be indifference, synergy or additivism.

Variation in restriction fragment length polymorphisms among serial isolates from patients with Trichophyton rubrum infection.

Molecular genotyping of strains of Trichophyton rubrum and T. mentagrophytes from patients with onychomycosis of the toes was performed to ascertain whether the fungal genotype changes over the course of time as sequential samples were obtained from patients receiving antifungal therapy and during follow-up. Sixty-six serial strains of T. rubrum and 11 strains of T. mentagrophytes were obtained from 20 patients (16 patients with T. rubrum, 4 with T. mentagrophytes) who were treated with oral antifungal therapy and observed over periods of up to 36 months. These strains were screened for genetic variation by hybridization of EcoRI-digested genomic DNAs with a probe amplified from the small-subunit (18S) ribosomal DNA and adjacent internal transcribed spacer regions. A total of five restriction fragment length polymorphism (RFLP) types were observed among 66 strains of T. rubrum. Two major RFLP types, differentiated by one band shift, represented 68% of the samples. None of the patients had a unique genotype. More than one RFLP type was often observed from a single patient (same nail) over a period of 1, 2, or 3 years, even in cases that did not appear cured at any time. Samples taken from different nails of the same patient had either the same or a different genotype. The genotypic variation did not correspond to any detectable phenotypic variation. Furthermore, no correlation was observed between the efficacy of the treatment administered and the genotype observed. While the DNA region studied distinguished among T. rubrum, T. mentagrophytes, and T. tonsurans, intraspecific RFLP variation was observed for T. rubrum and T. mentagrophytes strains. While independent multiple infection and coinhabitation of multiple strains may explain the presence of different genotypes in a nail, microevolutionary events such as rapid substrain shuffling, as seen in studies of repetitive regions in Candida species, may also produce the same result. The recovery of multiple strains during the course of sequential sampling of uncured patients further suggests that the typing system is not able to distinguish between relapse or reinfection, ongoing infection, and de novo infection.

Quantitative culture of Malassezia species from different body sites of individuals with or without dermatoses.

Quantitative cultures were obtained using contact plates to determine whether the quantity and composition of Malassezia species at a given anatomic site in normal individuals differs from that of patients with various cutaneous dermatoses. The sample included 20 clinically healthy individuals (without any dermatosis) and 110 patients with dermatoses (including 31 with atopic dermatitis [AD], 28 with psoriasis [PS], 28 with seborrheic dermatitis [SD] and 23 with pityriasis versicolor [PV]). Contact plates filled with special culture medium were used to obtain a quantitative culture from five body sites (scalp, forehead, arm, trunk and leg) of every individual. The number of cfu were recorded for every plate that grew Malassezia yeasts, and 3-5 colonies were isolated for identification to species level using microscopic, physiological and molecular characteristics. The mean cfu counts observed among patients with AD, PS and SD was significantly lower than normal control subjects (P < 0.05). The mean cfu counts from PV patients was not different from that of healthy control subjects. Overall, for all conditions considered together, the mean cfu counts in lesional sites were significantly lower than in non-lesional sites (P <0.05). Furthermore, the mean cfu counts from lesional sites in patients with AD and PS were significantly lower than the corresponding value in patients with PV (P <0.05). Six Malassezia species were recovered from the different dermatoses. Malassezia sympodialis was the most common species associated with AD and PV patients and healthy control subjects, while M. globosa was most frequently isolated from PS and SD patients. More than one Malassezia species was recovered at any given anatomic site from both controls as well as individuals with dermatoses. M. globosa was equally likely to be recovered from scalp, forehead and trunk, but less likely to derive from arms and legs. M. restricta and M. slooffiae were recovered more frequently from the upper body (scalp and forehead) than from the lower body. Among normal individuals and for patients with AD and PV, M. sympodialis was significantly more likely to affect the forehead than the legs.

Epidemiology of Malassezia yeasts associated with pityriasis versicolor in Ontario, Canada.

The genus Malassezia was recently revised to include seven species, but the clinical significance of each of these species is not clearly understood. To obtain a better understanding of the contribution of individual Malassezia species to the epidemiology of pityriasis (tinea) versicolor, we used Leeming-Notman medium to culture patient skin specimens showing positive evidence of Malassezia infection in direct microscopy. Isolates were identified on the basis of recently published morphological and physiological tests for distinction of the new species. Identification using recently developed molecular criteria was also carried out for the ambiguous cases. Malassezia species were cultured from 111 cases of pityriasis versicolor in the Canadian province of Ontario. The most frequently isolated species were Malassezia sympodialis, M. globosa and M. furfur which respectively made up 59.4%, 25.2% and 10.8% of the isolated etiological agents. M. globosa was commonly isolated from a small number of pityriasis versicolor specimens obtained from investigators outside Canada. A large number of additional Ontario specimens with positive direct microscopy failed to yield a culture; however, it is suggested that this is consistent with the standard sampling practice of scraping the older portions of pityriasis lesions rather than the margins, where viable fungal cells are most likely to occur.

Helicobacter pylori infection induces hyperammonaemia in Mongolian gerbils with liver cirrhosis.

BACKGROUND AND AIMS: We previously reported the effect of Helicobacter pylori eradication on hyperammonaemia in patients with liver cirrhosis. However, the role of H pylori as a cause of hyperammonaemia is controversial. We developed an animal model with liver cirrhosis and investigated the effect of H pylori infection on hyperammonaemia. MATERIALS AND METHODS: Five week old male Mongolian gerbils were inoculated orally with broth culture of H pylori. Forty eight gerbils were divided into four groups. Gerbils not inoculated with H pylori were fed a commercial rodent diet (group A) or a choline deficient diet (group C). Gerbils inoculated with H pylori were fed the commercial rodent diet (group B) or the choline deficient diet (group D). Blood ammonia levels of the femoral vein and portal vein were measured 30 weeks later. RESULTS: All gerbils fed the choline deficient diet developed liver cirrhosis with fatty metamorphosis. The survival rate of group D was significantly lower than that of the other groups. Systemic and portal blood ammonia levels in group D were significantly higher than those in the other groups. CONCLUSIONS: H pylori infection induces hyperammonaemia in gerbils with liver cirrhosis.

In vitro susceptibility of the seven Malassezia species to ketoconazole, voriconazole, itraconazole and terbinafine.

Fifty-five strains, either authentic or ex-type, of seven Malassezia species were investigated for in vitro susceptibility to various concentrations (0.03-64.0 microg/mL) of three azole drugs, ketoconazole, voriconazole and itraconazole, as well as the allylamine terbinafine, using the agar dilution method. All strains of the seven Malassezia species were susceptible to the three azole drugs at low concentrations. M. furfur, M. sympodialis, M. slooffiae, M. pachydermatis, M. globosa, M. obtusa and M. restricta were most sensitive to ketoconazole and itraconazole, with minimum inhibitory concentrations (MICs) ranging from < or = 0.03 to 0.125 microg/mL. The recently introduced antifungal, voriconazole, was also very effective, with MIC80 values < or = 0.03 microg/mL for 80% of strains. MICs of terbinafine against the seven Malassezia species ranged from

Molecular differentiation of seven Malassezia species.

A system based on PCR and restriction endonuclease analysis was developed to distinguish the seven currently recognized Malassezia species. Seventy-eight strains, including authentic culture collection strains and routine clinical isolates, were investigated for variation in the ribosomal DNA repeat units. Two genomic regions, namely, the large subunit of the ribosomal gene and the internal transcribed spacer (ITS) region, were amplified by PCR, and products were digested with restriction endonucleases. The patterns generated were useful in identification of five out of seven Malassezia species. M. sympodialis was readily distinguishable in that its ITS region yielded a 700-bp amplified fragment, whereas the other six species yielded an 800-bp fragment. M. globosa and M. restricta were very similar in the regions studied and could be distinguished only by performing a hot start-touchdown PCR on primers for the beta-tubulin gene. Primers based on the conserved areas of the Candida cylindracea lipase gene, which were used in an attempt to amplify Malassezia lipases, yielded an amplification product after annealing at 55 degrees C only with M. pachydermatis. This specific amplification may facilitate the rapid identification of this organism.

Helicobacter pylori infection occurs via close contact with infected individuals in early childhood.

BACKGROUND: The manner in which Helicobacter pylori is transmitted is of fundamental importance when considering strategies for its control, yet, to date, the exact mode of transmission remains uncertain. METHODS: The seroprevalence of H. pylori in a relatively isolated rural town in Japan (A-town) was examined to analyse the H. pylori infection route. The immunoglobulin G antibodies against H. pylori in 1684 subjects who had received public health examinations in A-town were determined with an enzyme-linked immunosorbent assay. The seroprevalence was compared in five areas according to the water source. The possibility and frequency of intrafamilial infection was analysed by comparing the seroprevalence among family members residing in the same home. RESULTS: The seroprevalence of H. pylori did not differ significantly between the five areas examined. Seropositivity was significantly more common in the children whose mothers were seropositive (45.0%, 27/60) than in the children whose mothers were seronegative (10.0%, 2/20; odds ratio (OR) = 7.36, P = 0.0036, 95% confidence interval (CI) = 1.57-34.59). Seropositivity was significantly more common in the children whose older siblings were seropositive (55.0%, 22/40) than in the children whose older siblings were seronegative (23.5%, 20/85; OR = 3.97, P = 0.00051, 95% CI = 1.79-8.84). There was no significant relationship in seroprevalence between children and fathers, grandchildren and grandfathers, grandchildren and grandmothers, or within couples. Seropositivity was significantly more common in the adolescents who had attended a nursery school (44.4%, 20/45) than in the adolescents who had not attended a nursery school (25.6%, 109/426) (OR = 2.33, P = 0.0070, 95% CI = 1.24-4.36). CONCLUSIONS: The acquisition of H. pylori infection occurs by close contact with infected individuals in early childhood, especially via contact with infected mothers and other infected children.

Endoscopic [13C]-urea breath test for quantification of Helicobacter pylori infection.

BACKGROUND: We previously developed a new diagnostic method for Helicobacter pylori infection and called it the endoscopic [13C]-urea breath test (EUBT). Here we evaluate the relationship between the EUBT results and the histological findings. METHODS: The EUBT was performed on 137 patients with gastroduodenal diseases. After the collection of a baseline breath sample, gastroduodenal endoscopy was performed. Twenty milliliters of 0.05% phenol red solution containing 100 mg of [13C]-urea was sprayed over the entire gastric mucosa under endoscopic observation. A breath sample was collected 15 min after spraying. The content of 13CO2 in the breath samples was measured by ratio mass spectrometry. Two biopsy specimens each from the antrum and the middle corpus were obtained for culture and histology. Helicobacter pylori colonization, activity, inflammation, atrophy and intestinal metaplasia were classified on a four-point scale according to the Updated Sydney System. RESULTS: We found positive correlations between the EUBT values and the H. pylori colonization and activity score in the antrum and corpus, and negative correlations between the EUBT values and the atrophy and intestinal metaplasia scores in the corpus. CONCLUSIONS: The EUBT can be an indicator of the intragastric bacterial load and the histological findings for H. pylori.

Sequence analysis and clinical significance of the iceA gene from Helicobacter pylori strains in Japan.

The Helicobacter pylori iceA gene was recently identified as a genetic marker for the development of peptic ulcer in a Western population. To assess the significance of iceA subtypes of H. pylori in relation to peptic ulcer, 140 Japanese clinical isolates (88 from Fukui and 52 from Okinawa) were characterized. Sequence analysis of the iceA1 gene from 25 representative Japanese strains was also carried out to identify the differences in iceA between the ulcer group and the gastritis group. The iceA1 genotype was not correlated with the presence of peptic ulceration in either area. In addition, sequence analysis led to identification of five deletions and five point mutations (a nonsense mutation or a 1-bp insertion) within the iceA1 open reading frame corresponding to previously published sequences. These mutations were identified in both clinical groups (ulcer and gastritis groups) in each area. Local DNA sequence analysis revealed that the endpoints of all five deletions coincided with direct repeats. We also found four strains that carried longer iceA1 open reading frames compared with that for strain 60190. In conclusion, carriage of an iceA1 strain does not seem to be a risk factor for peptic ulcer in Japanese subjects. The critical mutations in the iceA1 gene in some isolates from patients with peptic ulcers suggested that IceA does not participate in the pathogenesis of peptic ulcer in Japan. We also found deletion hot spots that were associated with direct repeats in iceA1 and that favored a small-deletion model of slipped mispairing events during replication. We showed that iceA1 sequence variations may be useful tools for analysis of the population genetics of H. pylori.

The effect of helicobacter pylori eradication therapy on gastric antral myoelectrical activity and gastric emptying in patients with non-ulcer dyspepsia.

BACKGROUND: Dysmotility of the gastroduodenal region and delayed gastric emptying have been considered to play roles in non-ulcer dyspepsia. In addition, it has been reported that Helicobacter pylori induced inflammation of the gastric mucosa may affect gastric motility. AIM: To evaluate the effects of H. pylori eradication therapy on gastrointestinal motility and symptoms in non-ulcer dyspepsia patients. METHODS: A total of 46 non-ulcer dyspepsia patients were examined for gastric emptying, antral myoelectrical activity, H. pylori infection, and symptom scores. In H. pylori-positive non-ulcer dyspepsia patients, gastric emptying, antral myoelectrical activity, and symptom scores were also analysed 2 months after being cured of H. pylori infection. RESULTS: A total of 67.4% of the non-ulcer dyspepsia patients were H. pylori-positive. Both abnormal gastric emptying and antral myoelectrical activity were observed in non-ulcer dyspepsia patients. H. pylori-positive non-ulcer dyspepsia patients were divided into three groups according to their gastric emptying: the delayed gastric emptying group, the normal gastric emptying group, and the rapid gastric emptying group. In the delayed and rapid gastric emptying groups, the gastric emptying and symptom scores were improved significantly by the eradication therapy. However, there was no improvement in symptom scores in the normal gastric emptying non-ulcer dyspepsia group by the eradication therapy. CONCLUSIONS: Disturbed gastric emptying and antral myoelectrical activity play roles in non-ulcer dyspepsia. Helicobacter pylori infection, inducing disturbed gastric emptying, may cause some non-ulcer dyspepsia symptoms. Gastric emptying and symptom scores are improved by H. pylori eradication therapy in non-ulcer dyspepsia patients with disturbed gastric emptying. H. pylori eradication therapy is effective in H. pylori-positive non-ulcer dyspepsia patients with disturbed gastric emptying.

The effect of Helicobacter pylori eradication therapy on gastric antral myoelectrical activity and gastric emptying in patients with non-ulcer dyspepsia.

BACKGROUND: Dysmotility of the gastroduodenal region and delayed gastric emptying have been considered to play roles in non-ulcer dyspepsia (NUD). Helicobacter pylori-induced inflammation of the gastric mucosa may affect gastric motility. AIM: To evaluate the effects of H. pylori eradication therapy on gastrointestinal motility and symptoms in NUD patients. METHODS: : Forty-six NUD patients were examined for gastric emptying, antral myoelectrical activity, H. pylori infection, and symptom scores. In H. pylori-positive NUD patients, gastric emptying, antral myoelectrical activity, and symptom scores were also analysed 2 months after cure of H. pylori infection. RESULTS: Sixty-seven per cent of NUD patients were H. pylori-positive. Both abnormal gastric emptying and antral myoelectrical activity were observed in NUD patients. H. pylori-positive NUD patients were divided into three groups according to their gastric emptying: the delayed group, the normal group, and the rapid group. In the delayed and rapid gastric emptying groups, the emptying and symptom scores were improved significantly by eradication. There was no improvement in symptom scores in the normal gastric emptying NUD group by the eradication therapy. CONCLUSIONS: Disturbed gastric emptying and antral myoelectrical activity play roles in NUD. H. pylori-induced disturbed gastric emptying may cause some NUD symptoms. Gastric emptying and symptom scores are improved by H. pylori eradication therapy in NUD patients with disturbed gastric emptying; H. pylori eradication therapy is effective in H. pylori-positive NUD patients with disturbed gastric emptying.

Helicobacter pylori-negative gastric and duodenal ulcers.

It is unclear whether Helicobacter pylori infection is essential to the development of peptic ulcers. In this study, we examined the rates of H. pylori-negativity among patients with peptic ulcers. We also attempted to clarify the characteristics of H. pylori-negative peptic ulcers to throw light on the pathogenesis of peptic ulcers. The study included 215 consecutive patients with gastric ulcers (GUs) and 120 consecutive patients with duodenal ulcers (DUs). After routine endoscopic examination and phenol red dye endoscopy, forceps biopsies were performed for culture, histology, and the rapid urease test. A patient was considered H. pylori-negative when the serum anti-H. pylori IgG and the three tests on biopsied specimens were all negative. H. pylori-negative rates were 3.2% in the patients with GUs and 1.7% in the patients with DUs. Lack of atrophy of the gastric mucosa was significantly more common in the H. pylori-negative patients with GUs. A history of ulcer disease was less common and antral ulcers were more common in H. pylori-negative GU patients, but not significantly so. As the urea breath test had not been performed, the possibility of a false-negative result cannot be completely ruled out, but we believe that the H. pylori-negative rate in our study is more reliable than these rates in previous reports, because we visualized H. pylori distribution by phenol red dye endoscopy to avoid false-negative results in biopsies, and we used both biopsy and serum anti-H. pylori IgG findings to establish an H. pylori-negative diagnosis. Since H. pylori-negative peptic ulcers certainly exist, H. pylori infection is thought not to be essential to the development of peptic ulcers. There were few differences between the characteristics of H. pylori-negative and H. pylori-positive peptic ulcers in our study. A large-scale study is required to clarify the characteristics of H. pylori-negative peptic ulcers.

Bile reflux due to disturbed gastric movement is a cause of spontaneous gastric ulcer in W/Wv mice.

c-Kit is a receptor tyrosine kinase, and it is encoded by the mouse W locus. Mutant W/Wv mice develop spontaneous gastric antral ulcers. The aim of the present study was to investigate the pathogenesis of these gastric ulcers and to examine the effects of two antiulcer drugs; a proton pump inhibitor (2{[4-(3-methoxypropoxy)-3-methylpyridine-2-yl]methyl-sulfinyl}-1H -benzimidazole sodium salt, rabeprazole) and a mucosal protective drug (geranylgeranylacetone, GGA), on the gastric ulcers. The inhibition of the gastric acid secretion by rabeprazole (30 mg/kg body weight, subcutaneous injection once a day for six weeks) significantly increased the gastric ulcer formation compared to the controls. In contrast, the GGA treatment (100 mg/kg body weight, oral administration for six weeks) significantly inhibited the ulcer formation. Bile reflux was seen in these mutant mice, and they showed no cyclic intense contractions in the gastric antrum. These results suggest that bile reflux due to the disturbance of gastric antral movement is a cause of the spontaneous gastric ulcers in W/Wv mice.

Evaluation of endoscopic 13C-urea breath test for assessment of Helicobacter pylori eradication.

To minimize the inaccuracies of the diagnostic methods for Helicobacter pylori infection, we developed a new diagnostic method and called it the endoscopic 13C-urea breath test (EUBT). We evaluated the accuracy of EUBT for detecting this infection and assessing its eradication. EUBT was conducted on 267 patients with gastroduodenal disease. After the collection of a baseline breath sample, gastroduodenal endoscopy was performed. A 20-ml aliquot of a solution containing 100 mg of 13C-urea was sprayed over the entire gastric mucosa via the endoscope. A breath sample was then collected 15 min after spraying. The content of 13CO2 was measured in the breath samples by ratio mass spectrometry. Two biopsy specimens each from the antrum and the middle corpus were obtained for culture and histology. The EUBT cutoff level was 1.4%. The sensitivity and specificity of EUBT for the diagnosis of H. pylori infection were both 98.2%, and for assessment of the eradication they were 100% and 98.9%, respectively. EUBT is an accurate method not only for the diagnosis of H. pylori infection but also for assessment of its eradication.

Full-length sequence analysis of the vacA gene from cytotoxic and noncytotoxic Helicobacter pylori.

Some clinical isolates of Helicobacter pylori fail to express vacuolating cytotoxin, despite possessing a copy of the vacA gene on the chromosome. To gain insight into the differences between vacA from cytotoxic and noncytotoxic strains, the vacA open-reading frames from 16 cytotoxic and 22 noncytotoxic strains were sequenced. Mutations that cause truncation of VacA in 11 of 22 noncytotoxic strains were identified, including internal duplication, large deletion, 1-bp insertion, and non-sense mutations. In contrast, none of the 16 cytotoxic strains had any truncation of VacA. Four cytotoxic strains had inserted sequences downstream of vacA. Three were mini-IS605, and the other was a putative rfaJ gene that encodes lipopolysaccharide glucosyltransferase. The rfaJ gene identified in this study had a poly(C) tract, resulting in premature termination of the gene product. The phylogenetic tree based on the vacA open-reading frame indicated that two different H. pylori lineages are circulating in Japan and the West.

[Non-real-time computed tomography-guided percutaneous ethanol injection therapy for hepatocellular carcinoma undetectable by ultrasonography].

The purpose of this study was to evaluate the feasibility of non-real-time CT-guided percutaneous ethanol injection therapy (PEIT) for hepatocellular carcinoma (HCC, 37 lesions) untreatable by ultrasonography-guided (US)-PEIT. The HCC lesion was localized on the lipiodol CT image with a graduated grid system. We advanced a 21 G or 22 G needle in a stepwise fashion with intermittent localization scans using a tandem method to position the tip of the needle in the lesion. Ethanol containing contrast medium was injected with monitoring scans obtained after incremental volumes of injection, until perfusion of the lesion was judged to be complete. A total of 44 CT-PEIT procedures were performed. The average number of needle passes from the skin to the liver in each CT-PEIT procedure was 2.3, the average amount of ethanol injected was 14.4 ml, and the average time required was 49.3 minutes. Complete perfusion of the lesion by ethanol on monitoring CT images was achieved in all lesions with only a single or double CT-PEIT procedure without severe complication. Local recurrence was detected only in 5 lesions. At present, it is more time-consuming to perform CT-PEIT than US-PEIT because conventional CT guidance is not real-time imaging. However, it is expected that this limitation of CT-PEIT will be overcome in the near future with the introduction of CT fluoroscopy. In conclusion, CT-PEIT should prove to be a feasible, acceptable treatment for challenging cases of HCC undetectable by US.

Random association among alleles in clonal populations of Sclerotinia sclerotiorum.

Multiple loci identified in DNA fingerprints were used to test for random association in two agricultural populations of S. sclerotiorum. In linkage disequilibrium tests among pairs of loci with frequencies between 0.1 and 0.9, 44.5 and 80.5% of pairs of loci were consistent with random association in the clone-corrected samples of the Canadian canola and the North Carolina cabbage populations, respectively. In estimates of corrected (Bonferroni) P value, 70.66 and 98.89% of pairs of loci were in random association. All four possible genotypes for each pair of loci were observed in the Canadian canola sample, consistent with random association among loci. In multilocus association tests across all loci, however, significant association was observed in both populations. In the Canadian canola population, 40 possible heterokaryons were identified. Our data suggest that populations of S. sclerotiorum are predominantly clonal and that occasional genetic exchange and recombination, and not mutation alone, may be a source of new genotypes.

The role of the HLA-DQA1 gene in resistance to atrophic gastritis and gastric adenocarcinoma induced by Helicobacter pylori infection.

BACKGROUND: It has been suggested that immunogenetic factors for susceptibility or resistance to disease caused by Helicobacter pylori exist in the host. To examine host genetic factors that increase the risk of gastric adenocarcinoma among H. pylori-infected persons, the HLA-DQA1 locus was examined in patients with gastric adenocarcinoma. METHODS: Eighty-two gastric adenocarcinoma patients and 167 unrelated controls were examined for H. pylori infection and HLA-DQA1 genotyping. In addition, serum pepsinogen A (PGA) and pepsinogen C (PGC) values and the PGA/PGC ratio, which have been characterized as markers of gastric mucosal atrophy, also were analyzed. RESULTS: Of the 167 controls, 121 were H. pylori positive (+) and 46 were H. pylori negative (-). All H. pylori (-) individuals had normal endoscopic and histologic findings. Among the 121 H. pylori (+) controls, 36 had superficial gastritis and 85 had atrophic gastritis. The allele frequency of DQA1*0102 was significantly lower in the H. pylori (+) atrophic gastritis group than in the H. pylori (+) superficial gastritis and H. pylori (-) normal control groups. In addition, the allele frequency of DQA1*0102 also was significantly lower in the H. pylori (+) intestinal type gastric adenocarcinoma group than in the H. pylori (-) normal control, H. pylori (+) superficial gastritis, and H. pylori (-) diffuse type gastric adenocarcinoma groups. Serum PGA and PGC values and the PGA/PGC ratio did not differ significantly among HLA-DQA1 genotypes; however, the PGA/PGC ratio was significantly lower in the H. pylori (+) atrophic gastritis and H. pylori (+) intestinal type gastric adenocarcinoma groups than in the H. pylori (-) normal control and H. pylori (+) superficial gastritis groups. CONCLUSIONS: The DQA1*0102 allele may contribute to resistance against H. pylori-associated gastric atrophy and its association with intestinal type gastric adenocarcinoma, whereas the absence of DQA1*0102 may be a host genetic risk factor for H. pylori-associated atrophic gastritis and intestinal type gastric adenocarcinoma.