Microbial diagnosis of bloodstream infection: towards molecular diagnosis directly from blood.

When a bloodstream infection (BSI) is suspected, most of the laboratory results-biochemical and haematologic-are available within the first hours after hospital admission of the patient. This is not the case for diagnostic microbiology, which generally takes a longer time because blood culture, which is to date the reference standard for the documentation of the BSI microbial agents, relies on bacterial or fungal growth. The microbial diagnosis of BSI directly from blood has been proposed to speed the determination of the etiological agent but was limited by the very low number of circulating microbes during these paucibacterial infections. Thanks to recent advances in molecular biology, including the improvement of nucleic acid extraction and amplification, several PCR-based methods for the diagnosis of BSI directly from whole blood have emerged. In the present review, we discuss the advantages and limitations of these new molecular approaches, which at best complement the culture-based diagnosis of BSI.

Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation.

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

Diagnostic microbiologique de la pneumonie [Microbiological diagnosis of pneumonia].

Les pneumonies causent une mortalité et une morbidité significatives. De manière simplifiée, deux types de pneumonie sont décrits : la pneumonie communautaire et la pneumonie nosocomiale avec le pneumocoque et l'Haemophilus influenzae comme causes principales pour la première, le Pseudomonas et diverses entérobactéries pour la deuxième. La réalité est cependant plus complexe puisque l'on distingue aussi la pneumonie d'aspiration par exemple. La culture est très importante dans le cas des pneumonies nosocomiales car elle permet de déterminer la sensibilité aux antibiotiques de l'agent infectieux et d'adapter le traitement. Pour les patients immunosupprimés, le diagnostic différentiel est plus large et la recherche par tests moléculaires de certains virus, de champignons filamenteux et du Pneumocystis peut se révéler informative. Pneumonia is an importance cause of mortality and morbidity in adults. Two types of pneumonia are defined: community-acquired and nosocomial pneumonia with their corresponding etiology such as pneumococci or Haemophilus influenzae and Pseudomonas or enterobacteriaceae, respectively. However, the reality is more complex with aspiration pneumonia, pneumonia in immunocompromised patient, and pneumonia in ventilated patients. Culture in the case of nosocomial pneumonia is especially important to obtain the antibiotic susceptibility of the infectious agent and to adjust therapy. Moreover for immunocompromised patients, the differential diagnosis is much wider looking for viruses, filamentous fungi and Pneumocystis can be very informative, using new molecular assays.