A large number of different protocols for the efficient isolation of highly purified DNA from eukaryotic and prokaryotic cells is extant. (1-4) These procedures usually include treatment with proteinase K in the presence of SDS, which efficiently lyses the cells and nuclei and liberates the DNA tightly bound in chromatin. (s) Proteins are then extracted with phenol and chloroform, and the nucleic acids are precipitated with ethanol. This procedure is tedious and time-consuming, and significant amounts of DNA may be lost, especially when working with small specimens (e.g., joint biopsies). Therefore, this approach is not appropriate for diagnostic tests. Direct amplification of digested samples without phenol/chloroform extraction and precipitation is not possible because SDS is inhibitory to

Broad-range PCR amplification of part of the 16S rRNA gene followed by single-strand sequencing was applied to samples of 18 resected heart valves from patients with infective endocarditis. The PCR results were compared with those of cultures of valves and with those of previous blood cultures. For two patients there was agreement with the cultures of the valves; for nine patients there was agreement with the previous blood cultures, which were positive, while the cultures of the valves were negative; a Streptococcus sp. and Tropheryma whippelii each were found in one patient with negative cultures (valve and blood); for two patients the cultures of the valves as well as the PCR results were negative but the blood cultures were positive; for one patient amplification was inhibited; and for two patients the PCR results were positive but the amplicons could not be sequenced. It is concluded that broad-range PCR is a promising tool for patients with culture-negative endocarditis and allows the detection of rare, noncultivable organisms.

Toxigenic Corynebacterium diphtheriae is an important and potentially fatal threat to patients and public health. During the current dramatic influx of refugees into Europe, our objective was to use whole genome sequencing for the characterization of a suspected outbreak of C. diphtheriae wound infections among refugees. After conventional culture, we identified C. diphtheriae using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and investigated toxigenicity by PCR. Whole genome sequencing was performed on a MiSeq Illumina with >70×coverage, 2×250 bp read length, and mapping against a reference genome. Twenty cases of cutaneous C. diphtheriae in refugees from East African countries and Syria identified between April and August 2015 were included. Patients presented with wound infections shortly after arrival in Switzerland and Germany. Toxin production was detected in 9/20 (45%) isolates. Whole genome sequencing-based typing revealed relatedness between isolates using neighbour-joining algorithms. We detected three separate clusters among epidemiologically related refugees. Although the isolates within a cluster showed strong relatedness, isolates differed by >50 nucleotide polymorphisms. Toxigenic C. diphtheriae associated wound infections are currently observed more frequently in Europe, due to refugees travelling under poor hygienic conditions. Close genetic relatedness of C. diphtheriae isolates from 20 refugees with wound infections indicates likely transmission between patients. However, the diversity within each cluster and phylogenetic time-tree analysis suggest that transmissions happened several months ago, most likely outside Europe. Whole genome sequencing offers the potential to describe outbreaks at very high resolution and is a helpful tool in infection tracking and identification of transmission routes.

Whipple's disease is a systemic illness caused by a specific agent. Despite recognition of bacteria in lesions, efforts to isolate the causative agent remained futile. A novel strategy was devised to culture Whipple bacilli in deactivated mononuclear phagocytes. Infected tissue was inoculated into human phagocytes deactivated with interleukin (IL)-4, IL-10, and dexamethasone. Within 8-10 days, diastase-resistant periodic acid-Schiff-positive inclusions appeared, corresponding to intact and degenerating bacteria shown to be Tropheryma whippelii by electron microscopy and molecular analyses. T. whippelii was passaged several times in deactivated monocytes and a monoblastic cell line. Time-kinetics growth studies and comparative polymerase chain reaction analysis documented multiplication of T. whippelii in deactivated macrophages. Complementary studies showed that IL-4 rendered phagocytes permissive for T. whippelii, a strong indication that host factors contribute to the pathogenesis of Whipple's disease. The propagation of T. whippelii will permit microbiologic, immunologic, seroepidemiologic, and therapeutic studies of this pathogen.

PCR-based detection of Mucorales species could improve diagnosis of suspected invasive fungal infection, leading to a better patient outcome. This study describes two independent probe-based real-time PCR tests for detection of clinically relevant Mucorales, targeting specific fragments of the 18S and the 28S rRNA genes. Both assays have a short turnaround time, allow fast, specific and very sensitive detection of clinically relevant Mucorales and have the potential to be used as quantitative tests. They were validated on various clinical samples (fresh and formalin-fixed paraffin-embedded specimens, mainly biopsies, n517). The assays should be used as add-on tools to complement standard techniques; a combined approach of both real-time PCR assays has 100 % sensitivity. Genus identification by subsequent sequencing is possible for amplicons of the 18S PCR assay. In conclusion, combination of the two independent Mucorales assays described in this study, 18S and 28S, detected all clinical samples associated with proven Mucorales infection (n510). Reliable and specific identification of Mucorales is a prerequisite for successful antifungal therapy as these fungi show intrinsic resistance to voriconazole and caspofungin.