Direct 16S/18S rRNA Gene PCR Followed by Sanger Sequencing as a Clinical Diagnostic Tool for Detection of Bacterial and Fungal Infections: a Systematic Review and Meta-Analysis.

rRNA gene Sanger sequencing is being used for the identification of cultured pathogens. A new diagnostic approach is sequencing of uncultured samples by using the commercial DNA extraction and sequencing platform SepsiTest (ST). The goal was to analyze the clinical performance of ST with a focus on nongrowing pathogens and the impact on antibiotic therapy.

Fungal-Grade Reagents and Materials for Molecular Analysis.

Fungal DNA is present at very low loads in clinical specimens. Molecular detection by amplification assays generally is a challenge because of a potentially multiple input of contaminating DNA from exogenous sources. Besides airborne, handling and cross-contamination, materials and reagents used in the molecular laboratory can contain microbial DNA which is a long underestimated potential source of false positive results.

Higher blood volumes improve the sensitivity of direct PCR diagnosis of blood stream tuberculosis among HIV-positive patients: an observation study.

Background: Blood stream tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB) is common among HIV-positive patients, turning rapidly fatal unless detected and treated promptly. Blood culture is currently the standard test for the detection of MTB in whole blood but results take weeks; patients deteriorate markedly and often die before a diagnosis of blood stream TB is made. Rapid molecular tests on whole blood, with potential for same day diagnosis of blood stream TB usually show low sensitivity due to the problem of insufficient MTB DNA template when extraction is performed directly on low blood volumes.

Bacterial and fungal DNA extraction from blood samples: automated protocols.

Automation in DNA isolation is a necessity for routine practice employing molecular diagnosis of infectious agents. To this end, the development of automated systems for the molecular diagnosis of microorganisms directly in blood samples is at its beginning. Important characteristics of systems demanded for routine use include high recovery of microbial DNA, DNA-free containment for the reduction of DNA contamination from exogenous sources, DNA-free reagents and consumables, ideally a walkaway system, and economical pricing of the equipment and consumables.

Bacterial and fungal DNA extraction from blood samples: manual protocols.

A critical point of molecular diagnosis of systemic infections is the method employed for the extraction of microbial DNA from blood. A DNA isolation method has to be able to fulfill several fundamental requirements for optimal performance of diagnostic assays. First of all, low- and high-molecular-weight substances of the blood inhibitory to downstream analytical reactions like PCR amplification have to be removed.

Monitoring of patients supported by extracorporeal membrane oxygenation for systemic infections by broad-range rRNA gene PCR amplification and sequence analysis.

The rRNA gene PCR and sequencing test, SepsiTest, was compared with blood culture (BC) regarding the diagnosis of pathogens in 160 blood samples drawn from 28 patients during extracorporeal membrane oxygenation. With 45% of positive samples, SepsiTest was 13 to 75 h faster than BC. SepsiTest indicated bacteremias in 25% of patients who were BC negative.

The potential for intraspecific horizontal gene exchange by natural genetic transformation: sexual isolation among genomovars of Pseudomonas stutzeri.

The potential for natural genetic transformation among the seven genomovars (gvs) of Pseudomonas stutzeri was investigated. Of the 12 strains originating from a variety of environments, six strains (50%) from five gvs were competent for DNA uptake (Rif(R) marker). The transformation frequencies varied over more than three orders of magnitude.

Natural genetic transformation of Pseudomonas stutzeri in a non-sterile soil.

Natural transformation of the soil bacterium Pseudomonas stutzeri JM300 in a non-sterile brown earth microcosm was studied. For this purpose, the microcosm was loaded with purified DNA (plasmid or chromosomal DNA, both containing a high-frequency-transformation marker, his+, of the P. stutzeri genome), the non-adsorbed DNA was washed out with soil extract and then the soil was charged with competent cells (his-1).