Detection of Helicobacter pylori DNA in human feces by PCR: DNA stability and removal of inhibitors.

In this study, the stability of Helicobacter pylori DNA in human feces and the effect of a diet lacking in plant material, the suspected source of PCR inhibitors in human feces, were investigated. In addition, a method to remove these inhibitors was developed. Stools inoculated with H. pylori were used as a model. For this purpose, a H. pylori suspension (10(8) CFU/ml) was used to spike stool samples obtained from four healthy adults known to be H. pylori negative. The evaluation of the stability of H. pylori DNA in feces showed that DNA was degraded after 3 days of contact with fecal material at 37 degrees C. A 2-day diet completely free of plant material was sufficient to eliminate PCR inhibitors from human feces. However, inhibitors were detected 48 h after a normal diet was resumed. A new technique consisting of agarose blocks containing embedded DNA as a template for PCR amplification was used for removal of inhibitors, following DNA extraction by a modified QIAamp tissue method (Qiagen, Hilden, Germany). When this method was applied to inhibiting stool samples known to have an inhibitory effect and spiked with H. pylori (5.10(8) CFU/g), a positive PCR was obtained showing that inhibitors present in the original DNA samples were completely removed. The agarose embedded DNA block method is highly efficient and provides clean, high quality template DNA for PCR purposes avoiding long and fastidious conventional extraction methods. In conclusion, this study confirms that H. pylori DNA degrades with time in stools. A diet free of plant material or a special DNA preparation can be used to remove inhibitors and to allow the detection of H. pylori.

Cryptolepis sanguinolenta activity against diarrhoeal bacteria.

Cryptolepine is the main alkaloid of Cryptolepis sanguinolenta (Lindl.) Schlechter, a plant used in traditional medicine in West Africa. The minimal inhibitory concentrations (MICs) of cryptolepine, ethanol and aqueous extracts of Cryptolepis sanguinolenta root were determined for 65 strains of Campylobacter jejuni, 41 strains of Campylobacter coli isolated from sporadic cases of gastroenteritis in Portugal and 86 strains of Vibrio cholerae isolated from patients with enteric infections in Angola, Brazil and Portugal. The ethanol extract activity against Campylobacter strains (MIC90% = 25 micrograms/ml) is higher than that of co-trimoxazole and sulfamethoxazole and Campylobacter strains susceptibility for cryptolepine (MIC90% = 12.5 micrograms/ml) is equal for ampicillin. The ethanol extract and cryptolepine show some activity against the Vibrio cholerae strains, although their activities are lower than that of tetracycline. The results suggest that these roots could be a therapeutic alternative for bacterial etiologic diarrhoea in West Africa.

Antimicrobial activity of Guinea-Bissau traditional remedies.

The ethanolic extracts of twelve plants selected through ethnomedical survey in Guinea-Bissau were investigated for their in vitro antimicrobial properties over ten bacteria and Candida albicans, using agar diffusion and dilution methods. All the tested extracts showed some activity against at least one of the bacteria. Most of the extracts (79%) showed activity against Staphylococcus aureus and only one (Cryptolepis sanguinolenta) against Escherichia coli. Cryptolepis sanguinolenta and Terminalia macroptera root extracts showed some activity against Candida albicans as well as showing an interesting profile of activity against most of the enteropathogen microorganisms. Inhibition zones against Staphylococcus aureus were localised on extract chromatograms by bioautographic techniques.

Antimicrobial activity of Terminalia macroptera root.

Terminalia macroptera Guill et Perr. (Combretaceae) is a medicinal plant used in Guinea-Bissau and other West African countries to treat infectious diseases. The ethanol extract from T. macroptera decorticated root and their liquid-liquid partition fractions, were screened for antimicrobial activity, by the twofold serial microdilution assay against seven reference bacterial strains and against Candida albicans. The extract and fractions showed some activity against at least one of the test microorganisms. The best results were obtained against Shigella dysenteriae and Vibrio cholerae. The minimum inhibitory concentrations (MIC) of T. macroptera ethanol extract were also determined for about 100 clinical strains of Campylobacter sp., Escherichia coli, Salmonella sp., Shigella sp. and Vibrio cholerae. The ethanol extract activity against Campylobacter strains is similar to co-trimoxazole, higher than sulfamethoxazole but lower than tetracycline, erythromycin, ampicillin and streptomycin. Ellagitannins are the major compounds in the extract and active fractions. The obtained results suggest a potential importance of this medicinal plant in the treatment of enteric diseases, particularly in Campylobacter infections.

[Endocarditis caused by Candida parapsilosis after total correction of tetralogy of Fallot. Medico-surgical treatment]. / Endocardite por Cândida parapsilosis após correcção total de tetralogia de Fallot. Tratamento médico-cirúrgico.

Three children with Candida Parapsilosis endocarditis after total surgical correction for Tetralogy of Fallot, are reported. Blood cultures became negative in one child with amphotericin-B and in the other two with Ketoconazole, before reoperation for septal patch replacement. Oral therapy with Ketoconazole continued for a total of 24 months, and during a mean follow-up of 36 months no side effects or reinfection occurred. Medical and surgical conjoined therapy, with surgical timing decided on clinical and laboratorial grounds, were important for good results and cure.

[Chromomycosis]. / Cromomicose.

A case of chromomycosis with an evolution of 25 years, in a caucasoid man, aged 57, living in Mozambique, is reported. Fonsecae pedrosoi, the most frequently responsible agent of chromomycosis in Mozambique's cases, was isolated. Prognosis seemed poor since good results from therapy were unexpected, considering the long course of disease, the extensive and fibrotic lesions, and the unsuccessful treatment with 5-fluorocytosine and ketoconazole. The patient was treated with associated amphotericin B-5-fluorocytosine, and topical heat. Clinical improvement and mycologic negative studies were obtained.

Self-report of symptom frequency and symptom distress in kidney transplant recipients.

OBJECTIVE: The aim of this study was to assess symptom frequency and symptom distress associated with adverse effects of immunosuppressive therapy in kidney transplant recipients and to correlate it with compliance and socio-demographic characteristics. METHODS: Symptom frequency and symptom distress were evaluated by the 'Adapted Transplant Symptom Frequency and Symptom Distress Scale'. Data were collected from 85 patients who had undergone a kidney transplant between 1995 and 1997. RESULTS: Women reported a significantly higher level of symptom frequency and symptom distress when compared with men. The most frequent symptoms reported by women were changed appearance and increased hair growth, whereas for men were increased appetite and 'moon face'. Nevertheless the most distressing symptoms for women were painful menstruation and insomnia and for men were impotence and decreased interest in sex. Concerning compliance the non-compliant group reported a higher level of symptom frequency and symptom distress when compared with the compliant group. These results suggest that complications caused by symptoms associated with the adverse effects of immunosuppressive therapy are perceived differently by men and women and may induce non-compliance.

Evaluation of performances of three DNA enzyme immunoassays for detection of Helicobacter pylori PCR products from biopsy specimens.

PCR is recognized as a promising method for the detection of Helicobacter pylori in gastric biopsy specimens. However, detection of PCR products by gel electrophoresis is difficult to implement in routine clinical laboratories. The aim of this study was to compare three new DNA enzyme immunoassays with the standard method in their ability to detect PCR products. The three assays were based on the amplification of a fragment of the ureC gene of H. pylori and a colorimetric hybridization assay. The first assay (GEN-ETI-K DNA enzyme immunoassay; Sorin, Sallugia, Italy) was based on the hybridization of amplified DNA with a probe bound in microtiter wells and detected with labelled anti-DNA antibody. The second assay (Pylori-prob; Biocode, Sclessin, Belgium) comprised a solid-phase sandwich hybridization system with a specific biotinylated probe being used for detection. Finally, the third assay (PCR enzyme-linked immunosorbent assay; Boehringer, Mannheim, Germany) was based on the hybridization of amplified DNA labelled with digoxigenin as a probe (used as a coating in microtiter wells) and detected with antidigoxigenin-peroxidase as conjugate. The sensitivity of the colorimetric assay was evaluated by using amplification products from PCR assays performed on several 10-fold dilutions of DNA from H. pylori CIP 101260, and the specificity was assessed with different urease-positive bacteria. Biopsy specimens from 199 patients were tested; 106 were classified as H. pylori positive, and 93 were classified as H. pylori negative by culture and/or histological examination as the "gold standard." The receiving operating characteristic curve was used to determine the best cutoff point for each assay. The detection of PCR products by colorimetric hybridization increases the sensitivity up to 100-fold compared to that with gel electrophoresis. The results are rapid (4 h) and easy to interpret and can be automated.

Dermatophytes in Portugal (1972-1981).

For the years 1972-1981, 7 333 isolates of dermatophytes belonging to 14 species were obtained from glabrous skin (32%), feet (28%), groin (19%), scalp (8%), toenails (7%), fingernails (3%) and beard (1%). T. rubrum represented 50% of all the isolates and was the most frequent species on glabrous skin, groin and nails. T. mentagrophytes (24%) was mainly obtained from the feet, E. floccosum (9%) from the groin and T. megninii (4%) from uncovered areas of the skin, fingernail and beard. These 4 species predominated in men. M. canis was the commonest agent on the scalp and in children up to 11 years. T. violaceum, previously the main cause of tinea capitis, and T. tonsurans have been decreasing for the period of this study, just as T. schoenleinii for the years 1962-71. The rising prevalence of T. rubrum was observed since 1962. In the whole it seems stable after 1969, but the analysis of the main sites involved shows that in the glabrous skin this species increased from 1962 to 1974; in the groin it was gone up from 30% during 1962-1965, to 64% in the years 1969-1971; in the feet the evolution was slower and only in 1980 T. rubrum became more frequent than T. mentagrophytes. The increase in certain species, whereas others become rare, lacks a satisfactory explanation.

Assessment of metronidazole susceptibility in Helicobacter pylori: statistical validation and error rate analysis of breakpoints determined by the disk diffusion test.

Metronidazole susceptibility of 100 Helicobacter pylori strains was assessed by determining the inhibition zone diameters by disk diffusion test and the MICs by agar dilution and PDM Epsilometer test (E test). Linear regression analysis was performed, allowing the definition of significant linear relations, and revealed correlations of disk diffusion results with both E-test and agar dilution results (r2 = 0.88 and 0.81, respectively). No significant differences (P = 0.84) were found between MICs defined by E test and those defined by agar dilution, taken as a standard. Reproducibility comparison between E-test and disk diffusion tests showed that they are equivalent and with good precision. Two interpretative susceptibility schemes (with or without an intermediate class) were compared by an interpretative error rate analysis method. The susceptibility classification scheme that included the intermediate category was retained, and breakpoints were assessed for diffusion assay with 5-microg metronidazole disks. Strains with inhibition zone diameters less than 16 mm were defined as resistant (MIC > 8 microg/ml), those with zone diameters equal to or greater than 16 mm but less than 21 mm were considered intermediate (4 microg/ml < MIC

Contribution to our knowledge of Trichophyton megninii.

In addition to its requirement for histidine, Trichophyton megninii can be readily differentiated from certain other dermatophytes, particularly Trichophyton rubrum, by its "+" mating type and a positive urease test on urea-indole broth.

Prevalence, biotypes, plasmid profile and antimicrobial resistance of Campylobacter isolated from wild and domestic animals from northeast Portugal.

The incidence of Campylobacter jejuni and Campylobacter coli in wild and producing animals has been studied to evaluate their importance as potential reservoirs of campylobacter infection. These organisms were isolated from: 59 chicken (60.2%), 65 swine (59.1%), 31 black rats (57.4%), 61 sparrows (45.5%), 21 ducks (40.5%), 32 cows (19.5%) and 27 sheep (15.3%). Biotypes, plasmid and resistance profiles were studied in order to characterize the isolates. Biotypes I and II of C. jejuni were predominant in all reservoirs except swine, where C. coli I was more frequent. Plasmid prevalence was higher in strains isolated from swine (53.8%) and rats (45.5%). The size of the plasmids ranged from 1.3 to 82 MDa. A 2.3 MDa plasmid was the most frequent, detected in all the reservoirs except ducks. Antimicrobial susceptibility testing revealed that 5.5% of the strains were resistant to ampicillin, 5.5% to tetracycline, 12.6% to erythromycin and 23.5% to streptomycin. Resistance to erythromycin (26.2%) and to streptomycin (58.4%) was particularly high in isolates from swine. Tetracycline resistance was encoded by a 33 or a 41 MDa plasmid and transferred by conjugation.

Acyloxymethyl as a drug protecting group. Part 3. Tertiary O-amidomethyl esters of penicillin G: chemical hydrolysis and anti-bacterial activity.

PURPOSE: O-(N-alkylamido)methyl esters of penicillin G were studied as a new class of prodrugs. METHODS: The hydrolysis in aqueous buffers containing 20 % (v/v) of acetonitrile was investigated by HPLC. RESULTS: A U-shaped pH-rate profile was seen with a pH-independent process extending from pH ca. 2 to pH ca. 10. This pathway is characterised by kinetic data that are consistent with a unimolecular mechanism involving rate-limiting iminium ion formation and penicillinoate expulsion. Penicillin G and the corresponding amide are the ultimate products detected and isolated, indicating that beta-lactam ring opening is much slower than ester hydrolysis. The O-(N-alkylamido)methyl esters of penicillin G displayed similar in vitro antibacterial activity to penicillin G itself. CONCLUSIONS: Compared to the penicillin G derivatives, the much higher stability of the O-(N-methylbenzamido)methyl benzoate, acetate and valproate esters (which gave rise to a Bronsted Beta 1g value of ca. -1) suggests that tertiary N-acyloxymethylamides may be useful prodrugs for carboxylic acid drugs with pKa > 4.

Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model.

A model was developed to study inhibitors present in feces which prevent the use of PCR for the detection of Helicobacter pylori. A DNA fragment amplified with the same primers as H. pylori was used to spike samples before extraction by a modified QIAamp tissue method. Inhibitors, separated on an Ultrogel AcA44 column, were characterized. Inhibitors in feces are complex polysaccharides possibly originating from vegetable material in the diet.

Campylobacter enteritis in Portugal: epidemiological features and biological markers.

From 1984 to 1989, stool samples from 2811 gastroenteritis cases were examined for the presence of Campylobacter jejuni and C. Coli, Salmonella, Shigella and Yersinia species. Isolation rates were: Campylobacter jejuni and C. Coli, 5.3%, Salmonella spp., 14.8%, Shigella spp., 4.6% and Yersinia enterocolitica, 1.1%. Age group distribution analysis shows a higher Campylobacter isolation rate in children under one year of age. Seasonal distribution revealed a peak incidence in winter as in other Mediterranean countries. Predominant biotypes were C. jejuni I (51%), C. jejuni II (21.5%) and C. coli I (18.8%). Antimicrobial susceptibility testing did not reveal resistance to erythromycin. Thirty of the strains harboured plasmids with 7 different profiles.

Features and trends in Helicobacter pylori antibiotic resistance in Lisbon area, Portugal (1990-1999).

The features of Helicobacter pylori antibiotic resistance in Lisbon from 1990 to 1999 were studied. Overall resistance rates to amoxycillin, tetracycline, metronidazole, clarithromycin and ciprofloxacin were 0, 0, 30.6, 19.0 and 9.6%, respectively. The incidence of resistance to clarithromycin was much higher in isolates from children (44.8%) than adults (14.6%). For metronidazole, the contrary was observed (children: 19.0%, adults: 32.3%). Ciprofloxacin-resistant isolates were all from adult patients. Concerning the adult population, the resistance rate to metronidazole showed a slight increase during the decade, while for clarithromycin and ciprofloxacin a significant increase was observed (4.6 to 22.0% and 0 to 20.9%, respectively).