PCR is a very appealing technology for the detection of human pathogens, but the detection of fungal pathogens is particularly challenging. Fungi have cell walls that impede the efficient lysis of organisms and liberation of DNA, which can lead to false-negative PCR results. Conversely, some human pathogens are also ubiquitous environmental saprophytes that can contaminate PCR reagents and cause false-positive results. We examine the quality of PCR-based studies for fungal diagnostics using 42 variables within the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines. This review focuses on taxon-directed PCR assays for the diagnosis of invasive aspergillosis, candidiasis and Pneumocystis pneumonia. Finally, we evaluate broad-range fungal PCR assays capable of detecting a wide spectrum of human pathogens. Keywordsaspergillosis; candidiasis; diagnosis; fungal infection; PCR; pneumocystis; review The PCR method for DNA amplification was developed by Kary Mullis and colleagues in 1984 and was rapidly adapted to detect a variety of infectious agents, particularly viruses. Despite this success, PCR has not been widely adopted to detect fungal pathogens in human infections and has been eclipsed by other technologies such as fungal antigen detection assays. However, the diagnosis of human fungal infections continues to be a challenge. Conventional diagnostic techniques such as radiological imaging, culture and histology fall short in terms of specificity, sensitivity and time to diagnosis. In addition, diagnostic tests based on galactomannan (GM) antigen and glucan do not detect all fungal pathogens and have problems with specificity.PCR assays offer several features that could overcome current shortcomings for the diagnosis of fungal infections. PCR assays can have detection limits of a few gene copies per reaction, providing the ability to detect a fraction of an organism when targeting genes present in multiple copies per fungal genome. Primers and probes can be designed such that the target can be refined to a specific phylogenetic/taxonomic level; for example, species or genus, or broadened to include most fungi using a consensus sequence PCR approach. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript quantification of fungal load in the clinical specimen, which may provide information about burden or progression of disease. In addition, real-time PCR formats can provide rapid pathogen detection and confirmation when using a taxon-targeted assay with a probe, or when using methods such as broad-range PCR with melt-curve analysis of the amplified product. Multiplexed assays enable the simultaneous detection of numerous pathogens at varying levels of phylogeny/taxonomy. Furthermore, sequence variation within the amplified product can enable accurate species-level identification.There is no lack of genomic information for developing fungal PCR assays, so why has adoption of PCR-based diagnostic methods been so glacial? The design of appropriate exper...

bShigella species are so closely related to Escherichia coli that routine matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) cannot reliably differentiate them. Biochemical and serological methods are typically used to distinguish these species; however, "inactive" isolates of E. coli are biochemically very similar to Shigella species and thus pose a greater diagnostic challenge. We used ClinProTools (Bruker Daltonics) software to discover MALDI-TOF MS biomarker peaks and to generate classification models based on the genetic algorithm to differentiate between Shigella species and E. coli. Sixtysix Shigella spp. and 72 E. coli isolates were used to generate and test classification models, and the optimal models contained 15 biomarker peaks for genus-level classification and 12 peaks for species-level classification. We were able to identify 90% of E. coli and Shigella clinical isolates correctly to the species level. Only 3% of tested isolates were misidentified. This novel MALDI-TOF MS approach allows laboratories to streamline the identification of E. coli and Shigella species.

The resistance of Candida albicans biofilms to a broad spectrum of antimicrobial agents has been well documented. Biofilms are known to be heterogeneous, consisting of microenvironments that may induce formation of resistant subpopulations. In this study we characterized one such subpopulation. C. albicans biofilms were cultured in a tubular flow cell (TF) for 36 h. The relatively large shear forces imposed by draining the TF removed most of the biofilm, which consisted of a tangled mass of filamentous forms with associated clusters of yeast forms. This portion of the biofilm exhibited the classic architecture and morphological heterogeneity of a C. albicans biofilm and was only slightly more resistant than either exponential-or stationary-phase planktonic cells. A submonolayer fraction of blastospores that remained on the substratum was resistant to 10 times the amphotericin B dose that eliminated the activity of the planktonic populations. A comparison between planktonic and biofilm populations of transcript abundance for genes coding for enzymes in the ergosterol (ERG1, -3, -5, -6, -9, -11, and -25) and ␤-1,6-glucan (SKN and KRE1, -5, -6, and -9) pathways was performed by quantitative RT-PCR. The results indicate a possible association between the high level of resistance exhibited by the blastospore subpopulation and differential regulation of ERG1, ERG25, SKN1, and KRE1. We hypothesize that the resistance originates from a synergistic effect involving changes in both the cell membrane and the cell wall.

Background: The diagnosis of invasive pulmonary aspergillosis (IPA) remains challenging. Culture and histopathological examination of bronchoalveolar lavage (BAL) fluid are useful but have suboptimal sensitivity and in the case of culture may require several days for fungal growth to be evident. Detection of Aspergillus DNA in BAL fluid by quantitative PCR (qPCR) offers the potential for earlier diagnosis and higher sensitivity. It is important to adopt quality control measures in PCR assays to address false positives and negatives which can hinder accurate evaluation of diagnostic performance.

b Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively new addition to the clinical microbiology laboratory. The performance of the MALDI Biotyper system (Bruker Daltonics) was compared to those of phenotypic and genotypic identification methods for 690 routine and referred clinical isolates representing 102 genera and 225 unique species. We systematically compared direct-smear and extraction methods on a taxonomically diverse collection of isolates. The optimal score thresholds for bacterial identification were determined, and an approach to address multiple divergent results above these thresholds was evaluated. Analysis of identification scores revealed optimal species-and genus-level identification thresholds of 1.9 and 1.7, with 91.9% and 97.0% of isolates correctly identified to species and genus levels, respectively. Not surprisingly, routinely encountered isolates showed higher concordance than did uncommon isolates. The extraction method yielded higher scores than the direct-smear method for 78.3% of isolates. Incorrect species were reported in the top 10 results for 19.4% of isolates, and although there was no obvious cutoff to eliminate all of these ambiguities, a 10% score differential between the top match and additional species may be useful to limit the need for additional testing to reach single-species-level identifications. Recent decades have seen advances in automation of traditional phenotypic and biochemical methods for microbial identification (ID), and advances in sequencing and the proliferation of genomic data hold great promise for further improvements. The development of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has brought microbial diagnostics to another cusp of rapid development. The speed and low cost of bacterial identification by MALDI-TOF MS make it an attractive technology in the clinical microbiology laboratory, and it has shown promise for identification of Gram-positive cocci (2, 6, 8), enteric and nonfermenting Gram-negative rods (11,21,24), HACEK organisms (10), anaerobes (14,17,19,20,31), and broad cohorts of clinically relevant bacteria (3,4,22,27,30).Commercial MALDI-TOF systems identify a broad range of microorganisms based on analysis of unique "fingerprints" of abundant proteins from whole cells or cellular extracts (15,23,26,28). These profiles are searched against databases of reference spectra, and similarity scores for the top database matches are used to determine the identification of unknown isolates. As observed previously, a systematic evaluation of scoring criteria on diverse isolates could improve results (2,10,25,27,29). Identification may be complicated when multiple species-or genus-level matches are among the top 10 results. Most current publications on the MALDI Biotyper system (Bruker Daltonics, Billerica, MA) do not address these complicated situations; however, one example where this problem is addressed is the use of the "10% rule," which stat...