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- Abu Al-Soud, Waleed, et al.
(author)
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Assessment of PCR-DGGE for the identification of diverse Helicobacter species, and application to faecal samples from zoo animals to determine Helicobacter prevalence.
- 2003
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In: Journal of Medical Microbiology. - : Microbiology Society. - 0022-2615 .- 1473-5644. ; 52:9, s. 765-771
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Journal article (peer-reviewed)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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- Abu Al-Soud, Waleed, et al.
(author)
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DNA of Helicobacter spp. and common gut bacteria in primary liver carcinoma.
- 2008
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In: Digestive and Liver Disease. - : Elsevier BV. - 1590-8658. ; 40:2, s. 126-131
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Journal article (peer-reviewed)abstract
- BACKGROUND AND AIM: Gastric and enteric Helicobacter species have been associated with the pathogenesis of some extragastric diseases. METHODS: We retrospectively investigated the presence of DNA of Helicobacter species in samples of the cancer and the surrounding tumour-free liver tissues of patients with hepatocellular carcinoma (HCC, n=12) and cholangiocarcinoma (CC, n=13). The patients were from an area with low liver cancer incidence and with low hepatitis B and C prevalence. Patients with a benign liver disease (n=24) were included as controls. Paraffin-embedded liver samples were examined by a Helicobacter genus-specific PCR assay as well as group-specific PCR assays for Enterobacteriaceae, Bacteroides, Lactobacillus and Enterococcus. PCR products of positive samples were characterised by denaturing gradient gel electrophoresis (DGGE) and DNA sequencing. RESULTS: PCR assay detected Helicobacter DNA in seven of 12 (58%) and eight of 13 (62%) normal liver tissue specimens from HCC and CC patients, respectively. Two cancer samples from HCC patients were Helicobacter-positive but none of the CC cancers. In the control group, three of 24 (12.5%) patients with a benign liver condition were positive for Helicobacter species (p<0.01 compared to results of tumour-free liver tissue from the cancer patients). DGGE and DNA sequence analysis showed that 90% of the detected PCR products were "H. pylori-like". DNA of some other enteric bacteria was detected in the liver of one cancer patient and one control (4% of all patients). CONCLUSION: The presence of DNA of Helicobacter species in liver specimens, but not of other common gut bacteria, was associated with human hepatic carcinogenesis.
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- Abu Al-Soud, Waleed
(author)
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Optimisation of diagnostic PCR a study of PCR inhibitors in blood and sample pretreatment
- 2000
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Doctoral thesis (other academic/artistic)abstract
- PCR is widely employed as a rapid, sensitive and specific molecular diagnostic technique in clinical diagnosis, environmental investigations and for monitoring biotechnical processes. However, the full potential of diagnostic PCR is limited, in part, by the presence of PCR-inhibitory substances in biological samples. Among the mechanisms by which these inhibitors may act are through interfering with the DNA polymerase, with target nucleic acids and with lysis of cells. Therefore characterisation of PCR inhibitors is an important step for the development of more efficient sample preparation methods, which are used to reduce the effect of inhibitors and/or to concentrate target DNA. The aim of this work was to identify PCR-inhibitory components in blood and to develop pre-PCR treatment methods to eliminate the effect of inhibitors. Three major inhibitors were identified in blood namely, immunoglobulin G in plasma, haemoglobin in erythrocytes and lactoferrin in leukocytes. In addition, the quantitative effects of inhibitors on DNA synthesis were also investigated. Pre-PCR treatment methods based on selection of DNA polymerases more resistant to PCR-inhibitory components and the addition of amplification facilitators were tested. This approach was very efficient in reducing inhibition of PCR by blood and other complex biological samples. For example, when rTth was used instead of Taq DNA polymerase it became possible to amplify DNA in the presence of 20% (vol/vol) blood without reduced amplification sensitivity, whereas Taq DNA polymerase was totally inhibited in the presence of 0.004% (vol/vol) blood in the PCR mixture. Furthermore, the addition of bovine serum albumin to rTth the reaction mixtures allowed DNA amplification in the presence of 20% instead of 2% (vol/vol) meat and 4% instead of 0.4% (vol/vol) faeces. Thus, the PCR-inhibitory effect of various components in biological samples can, to some extent, be eliminated by the use of an appropriate thermostable DNA polymerase and amplification facilitators. This approach will allow direct detection of pathogens in biological samples, or facilitate the development of simple and rapid sample preparation methods more suitable for automation. Blood culture is an important step for the detection of blood pathogens, due to the low number of pathogens in the blood (often less than one per ml) and the small sample volume of blood obtainable from neonates and young infants. Therefore, a pre-PCR treatment was developed to remove the effect of PCR inhibitors in blood cultures, and to concentrate the bacterial cells prior to PCR. This method was based on a combination of dilution, centrifugation, washing with NaOH, and the addition of bovine serum albumin. Using this pre-PCR treatment allowed detection of 1 CFU of bacteria per ml of blood culture medium.
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- Asrat, D, et al.
(author)
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Prevalence of Helicobacter pylori infection among adult dyspeptic patients in Ethiopia
- 2004
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In: Annals of Tropical Medicine and Parasitology. - : Informa UK Limited. - 1364-8594 .- 0003-4983. ; 98:2, s. 181-189
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Journal article (peer-reviewed)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.
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| 10. |
- Asrat, Daniel, et al.
(author)
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Prevalence of Helicobacter pylori vacA and cagA genotypes in Ethiopian dyspeptic patients
- 2004
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In: Journal of Clinical Microbiology. - 1098-660X. ; 42:6, s. 2682-2684
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Journal article (peer-reviewed)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).
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