Real-Time Fluorescence PCR Assays for Detection and Characterization of Heat-Labile I and Heat-Stable I Enterotoxin Genes from Enterotoxigenic \u3ci\u3eEscherichia coli\u3c/i\u3e

To facilitate the diagnosis of enterotoxigenic Escherichia coli (ETEC) infections in humans, we developed and evaluated real-time fluorescence PCR assays for the Roche LightCycler (LC) against the enterotoxin genes commonly present in strains associated with human illness. Separate LC-PCR assays with identical cycling conditions were designed for the type I heat-labile enterotoxin (LT I) and the type I heat-stable enterotoxin (ST I) genes, using the LC hybridization probe format. A duplex assay for ST I with two sets of amplification primers and three hybridization probes was required to detect the major nucleotide sequence variants of ST I, ST Ia and ST Ib. LC-PCR findings from the testing of 161 E. coli isolates of human origin (138 ETEC and 23 non-ETEC) were compared with those obtained by block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the LT I and ST I genes. The LC-PCR and block cycler PCR assays were also compared for their abilities to detect LT I and ST I genes in spiked stool specimens with different methods of sample preparation. Findings from these experiments revealed that the limits of detection for the LC-PCR assays were the same or substantially lower than those observed for the block cycler PCR assay. Melting curve analysis of the amplified LT I and ST I genes revealed sequence variation within each gene, which for the ST I genes correlated with the presence of ST Ia and ST Ib. The rapidity, sensitivity, and specificity of the LC-PCR assays make them attractive alternatives to block cycler PCR assays for the detection and characterization of ETEC

Pulmonary vasculitis due to infection with Mycobacterium goodii : A case report

A 57-year-old Caucasian woman suffered from dyspnea on exertion. One year following a supposed pulmonary embolism event, a chronic thromboembolic vasculopathy was diagnosed and a pulmonary thromboendarterectomy was performed. However, a granulomatous pulmonary arterial vasculitis was identified upon examination. DNA of Mycobacterium goodii was detected as the most likely causative agent. Anti-inflammatory and anti-mycobacterial therapy was initiated for more than 12 months. Regular PET-CT scans revealed improvement under therapy. The last PET-CT did not show any tracer uptake following 10 months of therapy

Clinical impact of a commercially available multiplex PCR system for rapid detection of pathogens in patients with presumed sepsis

<p>Abstract</p> <p>Background</p> <p>Timely identification of pathogens is crucial to minimize mortality in patients with severe infections. Detection of bacterial and fungal pathogens in blood by nucleic acid amplification promises to yield results faster than blood cultures (BC). We analyzed the clinical impact of a commercially available multiplex PCR system in patients with suspected sepsis.</p> <p>Methods</p> <p>Blood samples from patients with presumed sepsis were cultured with the Bactec 9240™ system (Becton Dickinson, Heidelberg, Germany) and aliquots subjected to analysis with the LightCycler^®SeptiFast^®(SF) Test (Roche Diagnostics, Mannheim, Germany) at a tertiary care centre. For samples with PCR-detected pathogens, the actual impact on clinical management was determined by chart review. Furthermore a comparison between the time to a positive blood culture result and the SF result, based on a fictive assumption that it was done either on a once or twice daily basis, was made.</p> <p>Results</p> <p>Of 101 blood samples from 77 patients, 63 (62%) yielded concordant negative results, 14 (13%) concordant positive and 9 (9%) were BC positive only. In 14 (13%) samples pathogens were detected by SF only, resulting in adjustment of antibiotic therapy in 5 patients (7,7% of patients). In 3 samples a treatment adjustment would have been made earlier resulting in a total of 8 adjustments in all 101 samples (8%).</p> <p>Conclusion</p> <p>The addition of multiplex PCR to conventional blood cultures had a relevant impact on clinical management for a subset of patients with presumed sepsis.</p

Mechanisms behind variation in the Clostridium difficile 16S–23S rRNA intergenic spacer region

Clostridium difficile infection is an increasing problem in hospitals worldwide, mainly due to the recent emergence of a hypervirulent C. difficile strain. C. difficile PCR ribotyping, based on size variation of the 16S–23S rRNA intergenic spacer region (16S–23S ISR), is widely used in Europe for molecular epidemiological investigation. The mechanism underlying the 16S–23S ISR size variations in the genome of C. difficile is currently not completely understood. To elucidate this mechanism, isolates of six different PCR ribotypes were analysed by cloning and sequencing the 16S–23S ISR. A direct repeat, IB, of 9 bp was detected up to five times in the 16S–23S ISR in all 47 clones investigated. Thirty-five clones displayed differences either by ribotype or by nucleotide sequence. The sequences of the 16S–23S ISR of C. difficile showed a uniformly organized structure, composed of a tRNAAla gene and spacers of 33 and 53 bp separated by the 9 bp direct repeat IB. The results of the study support the hypothesis that this composition is responsible for the length variations seen in the 16S–23S ISR, and indicate that these length variations result from slipped-strand mispairing and intra- and possibly interchromosomal homologous recombination

Analytical performance of the Roche Lightcycler® Mycobacterium Detection Kit for the diagnosis of clinically important mycobacterial species

BACKGROUND: The LightCyclerH Mycobacterium Detection Kit based on real-time PCR technology for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii was recently developed. This study evaluated its analytical sensitivity, specificity and reproducibility. METHODOLOGY/PRINCIPAL FINDINGS: Plasmid standards were prepared and used to determine the limit of detection. The assay was also performed against organisms other than mycobacteria, other mycobacterial strains and interfering substances to exclude cross-reactivity and interference. Reference standards were prepared and tested to assess the assay’s reproducibility. All PCR assays were performed using the LightCyclerH 2.0 Instrument. The detection limit for M. tuberculosis was 28 copies per microlitre. Neither cross-reactivity nor interference occurred with non-mycobacterial organisms and substances tested. Overall reproducibility for consecutive measurements, run-to-run, lot-to-lot, day-to-day and laboratory-to-laboratory achieved a coefficient of variance of less than two percent. SIGNIFICANCE: The LightCyclerH Mycobacterium Detection kit has shown to be a robust and accurate assay with the potential to be used as a rapid TB diagnostic test.http://www.plosone.or

Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes

BACKGROUND: Toxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damnage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid. METHODS: Two separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity. RESULTS: Comparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii. CONCLUSION: We have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis

Application of molecular biology-based methods to the diagnosis of infectious diseases. Front. Biosci

ABSTRACT The basis for effective treatment and cure of a patient is the rapid diagnosis of the disease and its causative agent, which is founded on the analysis of the clinical symptoms coupled with laboratory tests. As we approach the 21st century, clinicians are becoming increasingly able to diagnose and treat diseases at the molecular level. The rapid development of new methods and techniques in the area of molecular biology has gained new insights into the genetic and structural features of a considerable number of human pathogens. These results obtained by intensive basic research are currently leading to improved diagnostic procedures. Basically, there are four different possibilities for laboratory diagnosis of infections: 1. direct detection of the pathogens (e.g., microscopy and/or culture), 2. detection of protein components of the pathogens with the help of specific antibodies (e.g., antigen capture ELISA) 3. IgA-, IgM-and IgGspecific detection of antibodies directed against a given pathogen and changes in their corresponding titer, and as the most sensitive method, 4. specific detection of nucleic acids (e.g., PCR) of the pathogens. SERODIAGNOSIS OF VIRAL PATHOGENS IS IMPROVED BY THE AVAILABILITY OF RECOMBINANT ANTIGENS. Infection by the human immunodeficiency virus, for example, is routinely diagnosed by the identification of specific antibodies in the serum. ELISA (enzyme linked immunosorbent assay) is the most common test format for screening purposes (1). First generation HIV tests are based on purified viral antigen preparations derived from virus culture (2, 3). Therefore, original test systems embody an appreciable rate of false-positive results, mainly due to the presence of residual cellular antigens which were incorporated into the virus particle during maturation. With the advent of &quot;nucleic acid engineering&quot; and recombinant technology, a number of strategies have been developed to produce recombinant proteins for diagnostic purposes. Several plasmid vectors and derivatives have been established for the high-level expression of foreign genes like recombinant antigens and antigenic peptides in prokaryotic (e.g., Escherichia coli) and eukaryotic host microorganisms. Once the genetic information for the antigenic viral protein of interest is introduced into the host cell with the help of a suitable expression vector, in addition to the proteins of the host microorganism, this foreign protein is also produced. Moreover, the production of recombinant antigens in undemanding bacteria like E.coli is usually a rapid

Multicenter quality assessment of 16S ribosomal DNA-sequencing for microbiome analyses reveals high inter-center variability

The composition of human as well as animal microbiota has increasingly gained in interest since metabolites and structural components of endogenous microorganisms fundamentally influence all aspects of host physiology. Since many of the bacteria are still unculturable, molecular techniques such as high-throughput sequencing have dramatically increased our knowledge of microbial communities. The majority of microbiome studies published thus far are based on bacterial 16S ribosomal RNA (rRNA) gene sequencing, so that they can, at least in principle, be compared to determine the role of the microbiome composition for host metabolism and physiology, developmental processes, as well as different diseases. However, differences in DNA preparation and purification, 16S rDNA PCR amplification, sequencing procedures and platforms, as well as bioinformatic analysis and quality control measures may strongly affect the microbiome composition results obtained in different laboratories. To systematically evaluate the comparability of results and identify the most influential methodological factors affecting these differences, identical human stool sample replicates spiked with quantified marker bacteria, and their subsequent DNA sequences were analyzed by nine different centers in an external quality assessment (EQA). While high intra-center reproducibility was observed in repetitive tests, significant inter-center differences of reported microbiota composition were obtained. All steps of the complex analysis workflow significantly influenced microbiome profiles, but the magnitude of variation caused by PCR primers for 16S rDNA amplification was clearly the largest. In order to advance microbiome research to a more standardized and routine medical diagnostic procedure, it is essential to establish uniform standard operating procedures throughout laboratories and to initiate regular proficiency testing. 2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND licens