1.
Abu Al-Soud, Waleed, et al.
(författare)
Assessment of PCR-DGGE for the identification of diverse Helicobacter species, and application to faecal samples from zoo animals to determine Helicobacter prevalence.
2003
Ingår i: Journal of Medical Microbiology. - : Microbiology Society. - 0022-2615 .- 1473-5644. ; 52:9, s. 765-771
Tidskriftsartikel (refereegranskat) abstract
Helicobacter species are fastidious bacterial pathogens that are difficult to culture by standard methods. A PCR-denaturing gradient gel electrophoresis (PCR-DGGE) technique for detection and identification of different Helicobacter species was developed and evaluated. The method involves PCR detection of Helicobacter DNA by genus-specific primers that target 16S rDNA and subsequent differentiation of Helicobacter PCR products by use of DGGE. Strains are identified by comparing mobilities of unknown samples to those determined for reference strains; sequence analysis can also be performed on purified amplicons. Sixteen DGGE profiles were derived from 44 type and reference strains of 20 Helicobacter species, indicating the potential of this approach for resolving infection of a single host by multiple Helicobacter species. Some more highly related species were not differentiated whereas in highly heterogeneous species, sequence divergence was observed and more than one PCR-DGGE profile was obtained. Application of the PCR-DGGE method to DNA extracted from faeces of zoo animals revealed the presence of Helicobacter DNA in 13 of 16 samples; a correlation was seen between the mobility of PCR products in DGGE analysis and DNA sequencing. In combination, this indicated that zoo animals are colonized by a wide range of different Helicobacter species; seven animals appeared to be colonized by multiple Helicobacter species. By this approach, presumptive identifications were made of Helicobacter bilis and Helicobacter hepaticus in a Nile crocodile, Helicobacter cinaedi in a baboon and a red panda, and Helicobacter felis in a wolf and a Taiwan beauty snake. All of these PCR products (400 bp) showed 100 % sequence similarity to 16S rDNA sequences of the mentioned species. These results demonstrate the potential of PCR-DGGE-based analysis for identification of Helicobacter species in complex ecosystems, such as the gastrointestinal tract, and could contribute to a better understanding of the ecology of helicobacters and other pathogens with a complex aetiology.
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6.
Asrat, D, et al.
(författare)
Prevalence of Helicobacter pylori infection among adult dyspeptic patients in Ethiopia
2004
Ingår i: Annals of Tropical Medicine and Parasitology. - : Informa UK Limited. - 1364-8594 .- 0003-4983. ; 98:2, s. 181-189
Tidskriftsartikel (refereegranskat) abstract
In developing countries such as Ethiopia, where chronic gastritis and peptic-ulcer disease are the most common endoscopic findings, it is important to study the association between Helicobacter pylori infection and gastroduodenal diseases. Both invasive and non-invasive diagnostic methods were therefore used to investigate 300, consecutive, adult patients with dyspepsia, from the gastrointestinal clinic of Tikur Anbassa University Hospital, Addis Ababa. The apparent overall prevalence of H. pylori infection varied according to the detection method employed. Culture revealed H. pylori in only 69%, of the patients but this pathogen appeared more common when rapid urease tests (71%), PCR-denaturating gradient gel electrophoresis (91%), histopathology (81%), silver staining (75%) or stool-antigen tests (81%) were employed. Antibodies to H. pylori were detected, both by enzyme immuno-assay (EIA) and immunoblotting, in approximately 80%, of the patients, whether the antigens used were of a reference strain or from a local isolate of H. pylon. When some of the EIA-positive and EIA-negative sera were cross-absorbed with antigens of Campylobacter jejuni and re-tested by EIA, the H. pylori-positive sera remained positive and the negative sera remained negative. Dyspeptic patients in Ethiopia, like most of those previously observed elsewhere in Africa, are often infected with H. pylon. It is important that the management of these patients should not be hampered by the misinterpretation of the African epidemiology of this pathogen.
7.
Asrat, Daniel, et al.
(författare)
Prevalence of Helicobacter pylori vacA and cagA genotypes in Ethiopian dyspeptic patients
2004
Ingår i: Journal of Clinical Microbiology. - 1098-660X. ; 42:6, s. 2682-2684
Tidskriftsartikel (refereegranskat) abstract
A total of 300 gastric biopsy samples and 50 Helicobacter pylori isolates were collected from Ethiopian adult dyspeptic patients. The vacA and cagA genes were detected in 90 and 79% of biopsy specimens, respectively, and in 100 and 87% of clinical isolates, respectively. Both genes were detected in 84% of the gastric biopsy samples and in 87% of the clinical isolates. Among vacA genotypes, the s1/m1 genotype was the most common in gastric biopsy samples (48%). The vacA and cagA positive H. pylori strains were detected to a higher degree in patients with chronic active gastritis (71%) than patients with other histopathological findings (29%) (P < 0.05).
8.
Bielawski, Krzysztof Plotr, et al.
(författare)
Determination of lamivudine-resistant variants of hepatitis B virus by denaturing gradient gel electrophoresis: A novel approach to monitoring drug resistance
2008
Ingår i: Medical Science Monitor. - 1643-3750. ; 14:5, s. 281-285
Tidskriftsartikel (refereegranskat) abstract
Background: A DGGE-based assay for hepatitis B virus (HBV) drug-resistance monitoring was designed and checked for feasibility. It detects mutations within the YMDD motif related to lamivudine resistance. Material/Methods: The YMDD motif of HBV polymerase was amplified by the set of primers designed in this study. DGGE analysis of the amplicons was performed on 9% polyacrylamide gels containing a 20-40% gradient of urea plus formamide and electrophoresis was performed. DNA sequencing was performed using a standard protocol. Results: Based on the DGGE pattern of previously sequenced HBV variants, a reference ladder consisting of bands was constructed within and near the YMDD motif of HBV with excellent resolution. The genotypes of all the fragments included in the ladder were confirmed by sequencing after DGGE analysis. The flexibility of DGGE was demonstrated by the ability to add more bands to the migration ladder when new variants were discovered during the analysis of patient specimens. Clinical samples from HBV-infected patients were also used to demonstrate the performance of this approach. Conclusions: This preliminary feasibility study of HBV drug-resistance monitoring by means of DGGE shows the potential advantage of this approach for low-cost screening for viral drug resistance in clinical settings. The presented example can be extended to detect other mutations related to drug resistance in the HBV genome as well as other viruses.
9.
Garcia-Amado, M. Alexandra, et al.
(författare)
Non-pylori Helicobacteraceae in the upper digestive tract of asymptomatic venezuelan subjects: Detection of helicobacter cetorum-like and candidatus Wolinella africanus-like DNA
2007
Ingår i: Helicobacter. - : Wiley. - 1083-4389 .- 1523-5378. ; 12:5, s. 553-558
Tidskriftsartikel (refereegranskat) abstract
Background: The spectrum of human non-pylori Helicobacter infections is expanding, with species such as H. heilmannii and H. felis occasionally being associated with gastritis. However, the existence of non-pylori Helicobacter colonization in asymptomatic subjects has not been evaluated. The aim of this study was to investigate whether Helicobacter species other than pylori are present in the upper digestive tract of asymptomatic human subjects. Materials and methods: A Helicobacteraceae-specific semi-nested polymerase chain reaction (PCR) assay was used to detect Helicobacter-like organisms in the upper digestive tract of 91 Venezuelan volunteers (aged 18-68 years, 41 females, 50 males). Species were identified by denaturing gradient gel electrophoresis analysis and sequencing of the PCR products. Results: We detected DNA sharing 99-100% sequence identity in over 300-400 bp with the 16S rRNA genes of H. pylori, H. cetorum, and Candidatus Wolinella africanus in 76%, 16%, and 15% of the subjects, respectively. Multiple colonization was documented in 10% of the subjects: H. cetorum and Candidatus W. africanus (4%), H. pylori and Candidatus W. africanus (4%), and H. pylori and H. cetorum (2%). Conclusions: Our results suggest that non-pylori Helicobacteraceae colonization is relatively common in the Venezuelan asymptomatic population. This is the first report documenting the presence of H. cetorum DNA in the human upper digestive tract, and the second report of the recently discovered Candidatus W. africanus.
10.
Nakonieczna, Joanna, et al.
(författare)
Detection of Helicobacter rodentium-like DNA in the liver tissue of patients with chronic liver diseases by polymerase chain reaction-denaturing gradient gel electrophoresis and DNA sequence analysis
2010
Ingår i: Diagnostic Microbiology and Infectious Disease. - : Elsevier BV. - 1879-0070 .- 0732-8893. ; 68:3, s. 201-207
Tidskriftsartikel (refereegranskat) abstract
Many Helicobacter spp. were isolated from the stomach, intestinal tract, and liver of different animals and humans. The association between Helicobacter spp. and hepatobiliary diseases, including hepatocellular carcinoma, was thoroughly examined, indicating a potential role of the bacteria in the progression toward cancer. In our work, we screened 97 liver biopsies from patients with chronic liver diseases for the presence of Helicobacter spp. DNA. With the use of genus-specific polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing, we found that the majority of Helicobacter spp. DNA detected was similar to Helicobacter rodentium DNA (71%). The DNA of other detected Helicobacter spp. was similar to Helicobacter pylori DNA. This is the first indication of H. rodentium-like DNA presence in human liver tissue. We also conclude that PCR DGGE is a useful screening method for assigning species designation and heterogeneity. (C) 2010 Elsevier Inc. All rights reserved.
