PubMed Health⌕ Search

Biomedical subjects

Udo Reischl

Publications and source records attributed to Udo Reischl.

32 records · Page 2Linked to original sources
PubMed2003

Characterization of Mycobacterium tuberculosis complex DNAs from Egyptian mummies by spoligotyping.

Bone and soft tissue samples from 85 ancient Egyptian mummies were analyzed for the presence of ancient Mycobacterium tuberculosis complex DNA (aDNA) and further characterized by spoligotyping. The specimens were obtained from individuals from different tomb complexes in Thebes West, Upper Egypt, which were used for upper social class burials between the Middle Kingdom (since ca. 2050 BC) and the Late Period (until ca. 500 BC). A total of 25 samples provided a specific positive signal for the amplification of a 123-bp fragment of the repetitive element IS6110, indicating the presence of M. tuberculosis DNA. Further PCR-based tests for the identification of subspecies failed due to lack of specific amplification products in the historic tissue samples. Of these 25 positive specimens, 12 could be successfully characterized by spoligotyping. The spoligotyping signatures were compared to those in an international database. They all show either an M. tuberculosis or an M. africanum pattern, but none revealed an M. bovis-specific pattern. The results from a Middle Kingdom tomb (used exclusively between ca. 2050 and 1650 BC) suggest that these samples bear an M. africanum-type specific spoligotyping signature. The samples from later periods provided patterns typical for M. tuberculosis. This study clearly demonstrates that spoligotyping can be applied to historic tissue samples. In addition, our results do not support the theory that M. tuberculosis originated from the M. bovis type but, rather, suggest that human M. tuberculosis may have originated from a precursor complex probably related to M. africanum.

DNA, Bacterial↗
PubMed2003

Detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacterial isolates by real-time PCR.

Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. Despite advances in molecular diagnostics, the ability to rapidly diagnose M. tuberculosis infections by PCR is still inadequate, largely because of the possibility of false-negative reactions. We designed and validated a real-time PCR for mycobacteria by using the LightCycler system with 18 reference strains and 168 clinical mycobacterial isolates. All clinically significant mycobacteria were detected; the mean melting temperatures (with 99.9% confidence intervals [99.9% CI] in parentheses) for the different mycobacteria were as follows: M. tuberculosis, 64.35 degrees C (63.27 to 65.42 degrees C); M. kansasii, 59.20 degrees C (58.07 to 60.33 degrees C); M. avium, 57.82 degrees C (57.05 to 58.60 degrees C); M. intracellulare, 54.46 degrees C (53.69 to 55.23 degrees C); M. marinum, 58.91 degrees C (58.28 to 59.55 degrees C); rapidly growing mycobacteria, 53.09 degrees C (50.97 to 55.20 degrees C) or 43.19 degrees C (42.19 to 44.49 degrees C). This real-time PCR assay with melting curve analysis consistently accurately detected and differentiated M. tuberculosis from NTM. Detection of an NTM helps ensure that the negative result for M. tuberculosis is a true negative. The specific melting temperature also provides a suggestion of the identity of the NTM present, when the most commonly encountered mycobacterial species are considered. In a parallel comparison, both the LightCycler assay and the COBAS Amplicor M. tuberculosis assay correctly categorized 48 of 50 specimens that were proven by culture to contain M. tuberculosis, and the LightCycler assay correctly characterized 3 of 3 specimens that contained NTM.

Base Sequence↗
PubMed2003

Detection of Legionella pneumophila by real-time PCR for the mip gene.

A real-time PCR assay for the mip gene of Legionella pneumophila was tested with 27 isolates of L. pneumophila, 20 isolates of 14 other Legionella species, and 103 non-Legionella bacteria. Eight culture-positive and 40 culture-negative clinical specimens were tested. This assay was 100% sensitive and 100% specific for L. pneumophila.

Bacterial Proteins↗
PubMed2002

Helicobacter pylori and the risk of benign and malignant biliary tract disease.

BACKGROUND: The etiology of tumors arising in the biliary tract remains unclear. Several previous studies have detected Helicobacter pylori organisms in bile from patients with gallstones or cholecystitis. The objective of this study was to determine whether there is an association between H. pylori in bile and biliary tract carcinoma. METHODS: The authors used polymerase chain reaction (PCR) assays to detect the presence of H. pylori in the stomach and bile from 89 patients: Sixty-three disease free patients had biliary calculi, 15 patients had carcinoma of the biliary tract, and 11 patients had neither gallstones nor carcinoma. Bile was considered to contain H. pylori only if the results of PCR determinations were positive in two or more samples assayed independently in two separate laboratories. RESULTS: There was a strong association between the presence of H. pylori in the stomach and in the bile (P < or = 0.01). Biliary H. pylori was associated with age but not with gender, and it was associated strongly with the clinical diagnosis. Patients with gallstones were 3.5 times as likely to have H. pylori in the bile compared with patients in a control group (95% confidence interval [95%CI], 0.8-15.8; P = 0.100), and H. pylori was 9.9 times more frequent in patients with biliary tract carcinoma compared with patients in the control group (95%CI, 1.4-70.5; P = 0.022). CONCLUSIONS: There is a strong association between biliary tract carcinoma and H. pylori in bile. If these results are confirmed by prospective studies, H. pylori may be responsible for a significant proportion of malignant biliary tract disease.

Adult↗
PubMed2002

Molecular analysis of ancient microbial infections.

The detection of ancient microbial DNA offers a new approach for the study of infectious diseases, their occurrence, frequency and host-pathogen interaction in historic times and populations. Moreover, data obtained from skeletal and mummified tissue may represent an important completion of contemporary phylogenetic analyses of pathogens. In the last few years, a variety of bacterial, protozoal and viral infections have been detected in ancient tissue samples by amplification and characterization of specific DNA fragments. This holds particularly true for the identification of the Mycobacterium tuberculosis complex, which seems to be more robust than other microbes due to its waxy, hydrophobic and lipid-rich cell wall. These observations provided useful information about the occurrence, but also the frequency of tuberculosis in former populations. Moreover, these studies suggest new evolutionary models and indicate the route of transmission between human and animals. Until now, other pathogens, such as Mycobacterium leprae, Yersinia pestis, Plasmodium falciparum and others, have occasionally been identified - mostly in single case studies or small sample sizes - as well, although much less information is available on these pathogens in ancient settings. The main reason therefore seems to be the degradation and modification of ancient DNA by progressive oxidative damage. Furthermore, the constant risk of contamination by recent DNA forces to take time and cost effective measures and renders the analysis of ancient microbes difficult. Nevertheless, the study of microbial ancient DNA significantly contributes to the understanding of transmission and spread of infectious diseases, and potentially to the evolution and phylogenetic pathways of pathogens.

Animals↗
PubMed2002

First evidence of an endogenous Spiroplasma sp. infection in humans manifesting as unilateral cataract associated with anterior uveitis in a premature baby.

PURPOSE: To elucidate a previously unknown aetiology of rapidly progressive unilateral cataract in a premature baby associated with severe anterior uveitis. METHODS: The lens and vitreous material were saved as part of a special protocol in a 4-month-old premature baby at the time of pars plana lensectomy with anterior vitrectomy. We performed (1) microbiological cultures to detect viable bacterial and fungal organisms; (2) PCR reaction to viral, bacterial and fungal agents; (3) transmission electron microscopy (TEM). In addition, serological examinations were performed for HSV-1 and -2, CMV, VZV and Mycoplasmainfection. RESULTS: PCR detected Spiroplasma sp.; TEM confirmed the presence of Spiroplasma within the lens fibres. Serological testing and microbiological cultures of the vitreous and lens were negative. CONCLUSION: Endogenous Spiroplasma infection in a premature baby may manifest as rapidly progressive acquired unilateral cataract with anterior uveitis. Beyond this, Spiroplasma infection has never been reported to occur naturally in vertebrates, although experimentally Spiroplasma mirum produces panophthalmitis associated with cataract in a wide range of rodents and in chicks. In acquired infantile cataract with inflammatory signs, PCR and TEM should be performed in the lensectomy/vitrectomy material to detect infectious agents not evident on routine laboratory and microbiological examinations.

Base Sequence↗
PubMed2002

Corneal haze after photorefractive keratectomy for myopia: role of collagen IV mRNA typing as a predictor of haze.

PURPOSE: To develop a test based on the individual expression of collagen type IV synthesis in corneal epithelial cells to identify patients who have the potential for significant corneal haze after myopic photorefractive keratectomy (PRK). SETTING: Department of Ophthalmology and the Institute of Microbiology, University of Regensburg, Germany. METHODS: The individual synthesis of collagen type IV alpha3 mRNA was quantitatively measured in corneal epithelial cells of 34 eye (34 patients) with myopia ranging from -1.5 to -10.0 diopters (D) by a polymerase chain reaction (PCR) test. The corneal epithelial cells were collected before the PRK procedure. Collagen type IV alpha3 mRNA levels were correlated to postoperative haze and regression at 12 months. RESULTS: In all samples, collagen type IV alpha3 mRNA was detected; the mean was 1.47 (range 0.11 to 6.42). There was a correlation between haze and the amount of collagen type IV alpha3 mRNA; that is, eyes with haze had more collagen IV expression. In contrast, no correlation was observed between regression and the amount of collagen type IV alpha3 mRNA. CONCLUSIONS: The results show that collagen type IV alpha3 is an important factor in the development of corneal haze after PRK. Based on a quantitative PCR test, the individual collagen IV mRNA concentration in corneal epithelial cells could be measured. Further development could establish a screening test by which eyes with pronounced synthesis of collagen IV could be identified as being at high risk for haze after PRK.

Adult↗
PubMed2002

Detection of Cryptococcus neoformans DNA in tissue samples by nested and real-time PCR assays.

Two PCR protocols targeting the 18S rRNA gene of Cryptococcus neoformans were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 x 10(1) to 2.9 x 10(4) CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of C. neoformans per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast Malassezia furfur, which are phylogenetically related to C. neoformans. Twenty-four strains, including species of human skin flora like M. furfur and Trichosporon spp., were PCR negative. Amplification was observed with Cryptococcus amylolentus, Filobasidiella depauperata, Cryptococcus laurentii, and five species unrelated to clinical specimens. LightCycler PCR products from F. depauperata and Trichosporon faecale could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections.

Animals↗
PubMed2002

Direct detection and differentiation of Legionella spp. and Legionella pneumophila in clinical specimens by dual-color real-time PCR and melting curve analysis.

A dual-color LightCycler PCR assay targeting the 16S rDNA gene of Legionella spp. was established. By using two pairs of hybridization probes, Legionella spp. and Legionella pneumophila could be detected and differentiated simultaneously. With 26 culture-positive and 42 culture-negative respiratory specimens from patients with atypical pneumonia, 100% sensitivity and specificity was observed for L. pneumophila.

Computer Systems↗
PubMed2002

Real-time LightCycler PCR for detection and discrimination of Bordetella pertussis and Bordetella parapertussis.

Real-time PCR assays based on the LightCycler technology were developed for individual (simplex PCR) and simultaneous (duplex PCR) detection and discrimination of Bordetella pertussis and Bordetella parapertussis in clinical samples. The assays were evaluated with 113 specimens from patients with and without symptoms of pertussis. Results were compared to those from conventional culture and TaqMan real-time PCR. The analytical sensitivity ranged from 0.1 to 10 CFU for B. pertussis and B. parapertussis, and intra- and interassay variations were less than 7%. Results were available within 2 h. With the simplex format, 21 of 100 samples from patients with clinical symptoms of pertussis were positive for B. pertussis and/or B. parapertussis. With the duplex format, 18 of 100 samples were positive. LightCycler PCR increased the diagnostic sensitivity over that of culture by 2.0-fold (duplex PCR) (P = 0.08) to 2.3-fold (simplex PCR) (P = 0.02). Our data suggest that duplex PCR in this format showed good analytical sensitivity but lost some sensitivity on clinical samples compared with the simplex format.

Bordetella↗
PubMed2002

Real-time fluorescence PCR assays for detection and characterization of Shiga toxin, intimin, and enterohemolysin genes from Shiga toxin-producing Escherichia coli.

PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx(1) and stx(2)) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx(1), eae, and E-hly genes and 96 and 100%, respectively, for the stx(2) gene. No stx(2) genes were detected from 10 stx(2f)-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains.

Adhesins, Bacterial↗
PubMed2002

Septicemia due to Acinetobacter junii.

Acinetobacter spp. are considered to be emerging nosocomial pathogens. Acinetobacter junii is a rare cause of disease in humans and was associated mainly with bacteremia in preterm infants and pediatric oncologic patients. In this report we describe a case of catheter-related infection by A. junii in an adult oncologic patient. Application of molecular methods for precise species identification of Acinetobacter spp. will help to further clarify their role as human pathogens.

Acinetobacter↗
PubMed2001

Molecular analysis of skeletal tuberculosis in an ancient Egyptian population.

A paleomicrobiological study was performed on 37 skeletal tissue specimens from cadavers in the necropolis of Thebes-West, Upper Egypt, (2120-500 BC) and four from the necropolis of Abydos (3000 BC). The subjects had typical macromorphological evidence of osseous tuberculosis (n = 3), morphological alterations that were not specific, but probably resulted from tuberculosis (n = 17), or were without morphological osseous changes (n = 21). DNA was extracted from these bone samples and amplified by PCR with a primer pair that recognised the Mycobacterium tuberculosis complex insertion sequence IS6110. To confirm specificity of the analysis, the amplification products of several samples were subjected to restriction enzyme digestion, or direct sequencing, or both. In 30 of the 41 cases analysed, ancient DNA was demonstrated by amplification by the presence of the human beta-actin or the amelogenin gene and nine of these cases were positive for M. tuberculosis DNA. The results were confirmed by restriction endonuclease digestion and sequencing. A positive result for M. tuberculosis DNA was seen in two of the three cases with typical morphological signs of tuberculosis and amplifiable DNA, in five of 13 non-specific, but probable cases (including two cases from c. 3000 BC), but also in two of 14 cases without pathological bone changes. These observations confirm that tuberculosis may be diagnosed unequivocally in skeletal material from ancient Egypt, even dating back to c. 3000 BC. As a positive molecular reaction was observed in most of the typical cases of skeletal tuberculosis, in about one-third of non-specific, but probable tuberculous osseous changes and, surprisingly, in about one-seventh of unremarkable samples, this suggests that infection with M. tuberculosis was relatively frequent in ancient Egypt.

Amelogenin↗