Next Generation Sequencing approaches for pathogen identification in clinical diagnostic routine lack FDA-cleared test systems so far. But technological improvements made in the last years are promising. One hurdle is the vast excess of human DNA compared to microbial DNA. Learn more about the problem and possible solutions in this blog post.
Cultures of clinical specimens often are negative due to prior antibiotic treatment, fastidious growth requirements or viable but non-culturable pathogens. Stavnsbjerg et al. compared two commercial broad-range 16S rDNA PCR assays, MicroSeq® ID and UMD-SelectNA™, regarding their capacity of identifying bacteria in culture-negative samples.
Salmonella enterica causes high morbidity and mortality worldwide. Culture detection is limited and affects timely antibiotic treatment. Masek et al. differentiated 9 serovars by 16S rDNA PCR and melt analysis. This and the high analytic sensitivity of the method (<10 cfu/ml) directly in blood promise to enhance current diagnosis of S. enterica.
The diagnostic value of rDNA PCR and sequencing is discussed referring to clinical evaluations of infective endocarditis. The overall positivity of PCR/sequencing was notably higher than culture, in particular of heart valves. Common and rare aetiologies were similar with both systems, whereas mixed infections were more diverse by PCR/sequencing.
Rapid pathogen detection is essential for timely patient treatment. In contrast to culture, molecular methods provide results within hours. Molzym’s highly sensitive approach combines a unique DNA extraction technique with 16S and 18S rRNA gene PCR and amplicon sequencing for the identification of bacterial and fungal infections. See how it works.
Direct molecular diagnosis of pathogens is a helpful adjunct to culturing. An important factor influencing assay performance is the isolation of microbial DNA. Notable differences in Real-Time PCR assay sensitivity were observed with DNAs extracted from mock blood samples of bacteria and yeast using 7 manual and 3 automated DNA isolation kits.
Structures of microbial communities can be distorted by reagent contaminations, especially in samples with low microbial loads as the placenta. Leon et al. used 16S targeted amplicon sequencing to examine the impact of potential contaminations on the microbiome analysis of spontaneous preterm, non-spontaneous preterm and term delivered placenta.
Pyomyositis is a bacterial infection of skeletal muscles, and antibiotic treatment leads to delayed or negative culture results. Gabas et al.  identified bacteria in all 10 patients by 16S rDNA PCR and sequencing, while culture was positive with 4 patients only. Antibiotic treatment was changed in all cases resulting in 90% recovery of patients.
In pathogen diagnosis, establishment of DNA extraction systems needs extensive validation processes. Usually, the eluate contains a mixture of microbial and human DNA, whereby the latter negatively influences downstream analysis, especially at low load samples. Molzym’s MolYsis™ Basic kits deplete human DNA in samples prior to standard DNA extraction.
Metagenomic sequencing is used to recover information regarding clinically relevant infections. In their report, Ruppé & Schrenzel summarise last year’s 2nd International Conference on Clinical Metagenomics (ICCMg2). This post focusses on the current status, experiences, diagnostic use and clinical impact of sample treatment.
Identification of aetiologies of infective endocarditis is crucial for initiation of targeted antibiotic treatment and patient outcome, but culture often fails. Marsch and his colleagues analysed 46 heart valves with negative or inconsistent culture results by 16S/18S rDNA PCR. In 7 patients, PCR results led to the change of the antibiotic regime.
The 70th AACC Annual Scientific Meeting & Clinical Lab Expo is the largest global scientific conference in laboratory medicine. Molzym will be exhibiting their innovative products for rapid culture-independent molecular diagnosis of microbes focussing on unique solutions for microbial DNA enrichment from clinical specimens and DNA-free PCR assays.
Rapid diagnosis of sepsis pathogens is crucial for an early targeted antibiotic treatment and improved outcome of patients. Blood culture needs days until pathogen identification and often stays negative. Tkadlec et al. discuss the pros and cons of broad-range 16S rDNA PCR and sequencing on the basis of blood sample results of 330 patients.
Invasive fungal disease is a major cause of morbidity and mortality in immuno-compromised patients. In a FP7 project of the European Commission, Molzym partners with 5 institutions from 4 countries to develop and evaluate in multi-centre clincial studies fast DNA, enzymatic and protein-based assays.
SelectNA™plus is the ‘Selective extraction of microbial Nucleic Acids’ from mixtures including host and microbial cells providing a ‘plus’ benefit by walk-away automated processing of clinical samples. This highly enriched microbial DNA guarantees highest analytical performance in research and routine diagnosis.
Molzym turns 15 years this month. Over the years, Molzym won reputation as a specialist in the fast molecular diagnosis of microbial pathogens without the need of cultivation. Our expertise in unique DNA extraction solutions and the supply of DNA-free amplification reagents supports the standardisation of qPCR, gene clone library and NGS assaying.
Molzym partners EU Horizon 2020 project “Fast Assay for Pathogen Identification and Characterization” (FAPIC). Ten members from 7 countries joined to develop and clinically evaluate fast molecular assays for the identification of bacterial pathogens. Health system-relevant issues led to the prototyping of new automated diagnostic systems.
Remove your host DNA for increased sensitivity for detection of microbes in a great variety of specimens, e.g body fluids and tissues. Here, we provide a collection of applications of MolYsis™ sample pre-treatment in combination with various analytical methods, including NGS, PCR and Real-time PCR, DNA arrays and amplicon cloning.
Efficient cell destruction is a key problem of microbial DNA isolation. As a component of Molzym's DNA extraction kits, the BugLysis reagent ensures degradation of the very different cell walls among prokaryotes and fungi. The high efficiency of BugLysis becomes obvious when considering that 1,330 species have been identified in studies and assaying so far.
The Molzym-Team was delighted to participate and exhibit at the European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) that took place in Madrid, Spain, from 21st to 24th of April 2018. Here we want to share some impressions and highlights of this year’s ECCMID with you.
In a poster presentation at this year’s ECCMID congress, Edelmann et al.  compared 4 DNA extraction kits and the influence of human DNA removal for use in microbiome analysis. Additionally, two master mixes were tested for bacterial DNA contamination. An efficient workflow towards a standardised use of NGS in infection diagnosis was proposed.
Micro-Dx™ combines automated pathogen enrichment and DNA extraction with broad-range PCR and sequencing ID of species. A multi-centre analysis of 409 routine samples of tissues, fluids and swabs indicated acceptable diagnostic performance in comparison to other molecular tests and culture. Additional pathogens could be identified by Micro-Dx™.
In most clinical samples, human DNA outnumbers the amount of pathogen DNA. Thus, pathogen analysis with metagenomic whole genome sequencing (MWGS) is often limited by unspecific primer binding to human sequences. Read a comparison study of two commercial kits with respect to their enrichment of microbial DNA and effect on MWGS.
The European Congress on Clinical Microbiology and Infectious Diseases is an important event that attracts specialists in the fields globally. Molzym will be exhibiting their innovative products for rapid culture-independent molecular pathogen diagnosis focussing on unique automation of microbial DNA enrichment from diverse clinical specimens.
The SelectNA™plus robot with DNA-free Micro-Dx™ kit reagents (SNP) enriches pathogens and isolates microbial DNA from specimens automatically. In this post, a 16S/18S PCR and sequencing analysis of heart valves and other biopsies from 52 infectious endocarditis patients is discussed. SNP found substantially more relevant pathogens than culture.
SepsiTest™-UMD uses rDNA PCR and sequencing analysis to identify pathogenic bacteria and fungi from uncultured clinical material. Bacterial pathogens from 160 genera within 8 phyla have been identified in studies. SepsiTest™-UMD reflects an unbiased approach of culture diagnosis but is faster and more comprehensive by detecting non-growing pathogens.
Whole-metagenome sequencing generates vast non-target reads at a high host to bacterial DNA relation in some clinical samples. The removal of host DNA increases the reads and coverage of bacterial sequences. Read a summary of an exemplary publication showing the benefits of host DNA removal and how it can positively affect your NGS results.
Molzym introduces its latest development in automation. Micro-Dx™ comprises automated human DNA removal and pathogen DNA enrichment together with broad-range 16S/18S rDNA amplification and sequencing analysis. The system went through clinical studies showing its benefit for culture-independent pathogen diagnosis in daily routine.
In clinical specimens with low microbial load, host DNA at large excess negatively influences amplification of target pathogen DNA. This contribution guides through Molzym’s MolYsis™ technology of host DNA removal in front of microbial DNA extraction systems. The positive effect of host DNA removal on molecular analysis is discussed.
One of the major challenges in molecular microbe detection are false-positive results due to DNA contaminations of amplification reagents. Molzym’s new proprietary Hot MolTaq 16S/18S is a high quality, DNA-free Taq DNA polymerase ideally suited for the sensitive PCR analysis of microbial target DNA even at very low concentrations. Find out the benefits.