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Article ; Online: Novel approach for differentiating Shigella species and Escherichia coli by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Khot, Prasanna D / Fisher, Mark A

2013 Volume 51, Issue 11, Page(s) 3711–3716

Abstract: Shigella 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 ... ...

Abstract	Shigella 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. Sixty-six 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.
MeSH term(s)	Bacteriological Techniques/methods ; Biomarkers/analysis ; Escherichia coli/chemistry ; Escherichia coli/classification ; Humans ; Shigella/chemistry ; Shigella/classification ; Software ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
Chemical Substances	Biomarkers
Language	English
Publishing date	2013-08-28
Publishing country	United States
Document type	Evaluation Study ; Journal Article
ZDB-ID	390499-4
ISSN	1098-660X ; 0095-1137
ISSN (online)	1098-660X
ISSN	0095-1137
DOI	10.1128/JCM.01526-13
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Article ; Online: Identification of Nocardia Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Khot, Prasanna D / Bird, Brian A / Durrant, Robert J / Fisher, Mark A

Journal of clinical microbiology

2015 Volume 53, Issue 10, Page(s) 3366–3369

Abstract: Matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of Nocardia species remains challenging. By identifying 83.1% (64 of 77) and 80% (8 of 10) to the species and complex levels, respectively, and 94.3% (82 of ... ...

Abstract	Matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of Nocardia species remains challenging. By identifying 83.1% (64 of 77) and 80% (8 of 10) to the species and complex levels, respectively, and 94.3% (82 of 87) to the genus level, we show that an approach using routine sample preparation, an up-to-date commercial database minimally augmented with custom spectra, and testing at an early stage of growth is promising.
MeSH term(s)	Bacteriological Techniques/methods ; Humans ; Nocardia/chemistry ; Nocardia/classification ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
Language	English
Publishing date	2015-10
Publishing country	United States
Document type	Evaluation Studies ; Journal Article
ZDB-ID	390499-4
ISSN	1098-660X ; 0095-1137
ISSN (online)	1098-660X
ISSN	0095-1137
DOI	10.1128/JCM.00780-15
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Article ; Online: PCR-based diagnosis of human fungal infections.

Khot, Prasanna D / Fredricks, David N

Expert review of anti-infective therapy

2009 Volume 7, Issue 10, Page(s) 1201–1221

Abstract: 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- ... ...

Abstract	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.
MeSH term(s)	Aspergillosis/diagnosis ; Candidiasis/diagnosis ; DNA, Fungal/chemistry ; False Negative Reactions ; False Positive Reactions ; Humans ; Mycoses/diagnosis ; Pneumonia, Pneumocystis/diagnosis ; Polymerase Chain Reaction/methods ; Sensitivity and Specificity
Chemical Substances	DNA, Fungal
Language	English
Publishing date	2009-12-08
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Review
ZDB-ID	2181279-2
ISSN	1744-8336 ; 1478-7210
ISSN (online)	1744-8336
ISSN	1478-7210
DOI	10.1586/eri.09.104
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Article ; Online: Rapid microbial identification and colistin resistance detection via MALDI-TOF MS using a novel on-target extraction of membrane lipids.

Sorensen, Matthew / Chandler, Courtney E / Gardner, Francesca M / Ramadan, Salma / Khot, Prasanna D / Leung, Lisa M / Farrance, Christine E / Goodlett, David R / Ernst, Robert K / Nilsson, Erik

Scientific reports

2020 Volume 10, Issue 1, Page(s) 21536

Abstract: Rapid infection diagnosis is critical to improving patient treatment and outcome. Recent studies have shown microbial lipids to be sensitive and selective biomarkers for identifying bacterial and fungal species and antimicrobial resistance. Practical ... ...

Abstract	Rapid infection diagnosis is critical to improving patient treatment and outcome. Recent studies have shown microbial lipids to be sensitive and selective biomarkers for identifying bacterial and fungal species and antimicrobial resistance. Practical procedures for microbial lipid biomarker analysis will therefore improve patient outcomes and antimicrobial stewardship. However, current lipid extraction methods require significant hands-on time and are thus not suited for direct adoption as a clinical assay for microbial identification. Here, we have developed a method for lipid extraction directly on the surface of stainless-steel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) plates, termed fast lipid analysis technique or FLAT, which facilitates the identification of bacterial and fungal species using a sub-60-minute workflow. Additionally, our method detects lipid A modifications in Gram-negative bacteria that are associated with antimicrobial resistance, including to colistin.
MeSH term(s)	Biomarkers/analysis ; Colistin ; Drug Resistance, Bacterial ; Gram-Negative Bacteria/classification ; Gram-Negative Bacteria/metabolism ; Gram-Positive Bacteria/classification ; Gram-Positive Bacteria/metabolism ; Lipids/analysis ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Chemical Substances	Biomarkers ; Lipids ; Colistin (Z67X93HJG1)
Language	English
Publishing date	2020-12-09
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-020-78401-3
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Article ; Online: Rapid microbial identification and colistin resistance detection via MALDI-TOF MS using a novel on-target extraction of membrane lipids

Matthew Sorensen / Courtney E. Chandler / Francesca M. Gardner / Salma Ramadan / Prasanna D. Khot / Lisa M. Leung / Christine E. Farrance / David R. Goodlett / Robert K. Ernst / Erik Nilsson

Scientific Reports, Vol 10, Iss 1, Pp 1-

2020 Volume 9

Abstract: Abstract Rapid infection diagnosis is critical to improving patient treatment and outcome. Recent studies have shown microbial lipids to be sensitive and selective biomarkers for identifying bacterial and fungal species and antimicrobial resistance. ... ...

Abstract	Abstract Rapid infection diagnosis is critical to improving patient treatment and outcome. Recent studies have shown microbial lipids to be sensitive and selective biomarkers for identifying bacterial and fungal species and antimicrobial resistance. Practical procedures for microbial lipid biomarker analysis will therefore improve patient outcomes and antimicrobial stewardship. However, current lipid extraction methods require significant hands-on time and are thus not suited for direct adoption as a clinical assay for microbial identification. Here, we have developed a method for lipid extraction directly on the surface of stainless-steel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) plates, termed fast lipid analysis technique or FLAT, which facilitates the identification of bacterial and fungal species using a sub-60-minute workflow. Additionally, our method detects lipid A modifications in Gram-negative bacteria that are associated with antimicrobial resistance, including to colistin.
Keywords	Medicine ; R ; Science ; Q
Language	English
Publishing date	2020-12-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Enhanced fungal DNA-extraction from formalin-fixed, paraffin-embedded tissue specimens by application of thermal energy.

Rickerts, Volker / Khot, Prasanna D / Ko, Daisy L / Fredricks, David N

Medical mycology

2012 Volume 50, Issue 6, Page(s) 667–672

Abstract: Determining the etiology of invasive fungal infections (IFI) is critical for patient management as fungi vary in their susceptibility to antifungals. However, the etiology remains obscure in many cases due to negative culture results. The identification ... ...

Abstract	Determining the etiology of invasive fungal infections (IFI) is critical for patient management as fungi vary in their susceptibility to antifungals. However, the etiology remains obscure in many cases due to negative culture results. The identification of fungal DNA by PCR in pathology blocks and sequencing it is an alternative approach to determine the cause of IFI. Previous studies identified fungal DNA in only 50% of samples with positive histopathology results, probably due to DNA damage by tissue fixation. We used realtime PCR to quantify human and fungal DNA from formalin-fixed, paraffin-embedded tissue specimens in order to study the effect of thermal energy during extraction on the yield of amplifiable DNA and subsequent identification of fungal DNA. Tissue sections from eight patients with proven IFI were subjected to DNA extraction with varying exposure to thermal energy. Amplifiable DNA increased up to 76-fold by increasing the incubation temperature from 65°C to 90°C and an additional increase was documented by incubating samples for up to 6 hours at this temperature. The augmented amplification of fungal DNA was associated with improved species identification by the sequencing of the PCR amplicons. This may help illuminate the etiology of IFI and thereby improve patient management by guiding antifungal therapy.
MeSH term(s)	Aspergillosis/microbiology ; Aspergillus/genetics ; Aspergillus/isolation & purification ; Candida/genetics ; Candida/isolation & purification ; DNA, Fungal/isolation & purification ; Formaldehyde/chemistry ; Genome, Human ; Hot Temperature ; Humans ; Lung/microbiology ; Nucleic Acid Amplification Techniques/methods ; Paraffin Embedding ; Pathology, Molecular/methods ; Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Tissue Fixation/methods
Chemical Substances	DNA, Fungal ; Formaldehyde (1HG84L3525)
Language	English
Publishing date	2012-03-13
Publishing country	England
Document type	Journal Article
ZDB-ID	1421796-x
ISSN	1460-2709 ; 1369-3786
ISSN (online)	1460-2709
ISSN	1369-3786
DOI	10.3109/13693786.2012.665613
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Article ; Online: Sequencing and analysis of fungal rRNA operons for development of broad-range fungal PCR assays.

Khot, Prasanna D / Ko, Daisy L / Fredricks, David N

Applied and environmental microbiology

2009 Volume 75, Issue 6, Page(s) 1559–1565

Abstract: rRNA genes are attractive targets for developing PCR assays targeting human fungal pathogens. Most studies have focused on the 18S rRNA gene, internal transcribed spacers, and the 5' end of the 28S rRNA gene. An approximately 2,900-bp region of the 28S ... ...

Abstract	rRNA genes are attractive targets for developing PCR assays targeting human fungal pathogens. Most studies have focused on the 18S rRNA gene, internal transcribed spacers, and the 5' end of the 28S rRNA gene. An approximately 2,900-bp region of the 28S rRNA gene remains largely unexplored because sequences of many medically relevant fungi are either unavailable or undefined in genomic databases. The internal transcribed spacers and 28S rRNA gene of nine medically and phylogenetically important fungi were sequenced. In addition, 42 sequences from this region were acquired from public databases, resulting in an alignment of 51 fungal sequences spanning 30 fungal genera. For the nearly 3,950-bp region from the 3' end of 18S rRNA gene to the 3' end of the 28S rRNA gene, 27 broad-range PCR primers were designed such that their sequence homology with the human rRNA gene was minimal. All 62 possible amplicons in the size range from 75 to 400 bp from 27 primers were screened using fungal genomic DNA from 26 species spanning 14 genera. Eleven of the 62 amplicons did not cross-react with 1 microg/PCR human DNA but simultaneously amplified 10 fg of fungal DNA. Phylogenetic distance matrices were calculated for regions covered by these 11 amplicons based on 51 fungi. Two of these 11 amplicons successfully amplified 30 fg of fungal DNA from 25 of 26 fungi and provided the most phylogenetic information for species identification based on the distance matrices. These PCR assays hold promise for detection and identification of fungal pathogens in human tissues.
MeSH term(s)	Animals ; DNA Primers/genetics ; DNA, Fungal/chemistry ; DNA, Fungal/genetics ; DNA, Ribosomal/chemistry ; DNA, Ribosomal/genetics ; DNA, Ribosomal Spacer/chemistry ; DNA, Ribosomal Spacer/genetics ; Fungi/genetics ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction/methods ; RNA, Ribosomal, 28S/genetics ; Sensitivity and Specificity ; Sequence Analysis, DNA ; rRNA Operon
Chemical Substances	DNA Primers ; DNA, Fungal ; DNA, Ribosomal ; DNA, Ribosomal Spacer ; RNA, Ribosomal, 28S
Language	English
Publishing date	2009-01-09
Publishing country	United States
Document type	Evaluation Study ; Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	223011-2
ISSN	1098-5336 ; 0099-2240
ISSN (online)	1098-5336
ISSN	0099-2240
DOI	10.1128/AEM.02383-08
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Article: Candida albicans viability after exposure to amphotericin B: assessment using metabolic assays and colony forming units.

Khot, Prasanna D / Suci, Peter A / Tyler, Bonnie J

Journal of microbiological methods

2008 Volume 72, Issue 3, Page(s) 268–274

Abstract: Metabolic assays are a preferred method for evaluation of Candida albicans viability after exposure to antimicrobial agents in cases in which the culture is a complex mixture of yeast and filamentous forms. There is a lack of published data indicating ... ...

Abstract	Metabolic assays are a preferred method for evaluation of Candida albicans viability after exposure to antimicrobial agents in cases in which the culture is a complex mixture of yeast and filamentous forms. There is a lack of published data indicating the strength of the correlation between metabolic assays and viable cell numbers determined by a standard assay such as colony forming units (CFU). We developed a kinetic metabolic assay (KMA) for quantifying viable cells which was tested on yeast cells in both exponential and stationary phase using alamarBlue and XTT as metabolic indicators. The KMA enabled quantification of the viable population over a range of 10(1) to 10(7) cells that linearly correlated (R(2)>0.98) with estimates made by enumeration of CFU regardless of the indicator or growth phase of the cells. Linear relationships were used to calibrate the KMA in terms of equivalent CFU. Viable cell populations were then determined after exposure to AmB. These results were compared with those obtained by direct enumeration of CFU. There were significant correlations between KMA-derived equivalent CFU and direct CFU estimates of viable cell populations for exponential-phase cells. However, the proportions of viable cells based on the KMA were consistently lower than those obtained directly by CFU. This trend was substantially more pronounced for stationary phase cells. These results show that even in the relatively simple case in which only the yeast form is present, the relationship between assessment by metabolic assays and CFU is perturbed by exposure to an antimicrobial and that, furthermore, growth phase alters the nature of the perturbation.
MeSH term(s)	Amphotericin B/pharmacology ; Antifungal Agents/pharmacology ; Candida albicans/drug effects ; Candida albicans/growth & development ; Candida albicans/metabolism ; Chromogenic Compounds/metabolism ; Microbial Viability ; Oxazines/metabolism ; Stem Cells ; Tetrazolium Salts/metabolism ; Xanthenes/metabolism
Chemical Substances	Antifungal Agents ; Chromogenic Compounds ; Oxazines ; Tetrazolium Salts ; Xanthenes ; 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide (117038-70-7) ; resazurin (1FN9YD6968) ; Amphotericin B (7XU7A7DROE)
Language	English
Publishing date	2008-03
Publishing country	Netherlands
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	604916-3
ISSN	1872-8359 ; 0167-7012
ISSN (online)	1872-8359
ISSN	0167-7012
DOI	10.1016/j.mimet.2007.12.005
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Article ; Online: Optimization of matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis for bacterial identification.

Khot, Prasanna D / Couturier, Marc R / Wilson, Andrew / Croft, Ann / Fisher, Mark A

Journal of clinical microbiology

2012 Volume 50, Issue 12, Page(s) 3845–3852

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Bacteria/chemistry ; Bacteria/classification ; Bacteria/isolation & purification ; Bacterial Infections/diagnosis ; Bacterial Infections/microbiology ; Bacterial Typing Techniques ; Bacteriological Techniques/methods ; Humans ; Molecular Typing ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
Language	English
Publishing date	2012-09-19
Publishing country	United States
Document type	Comparative Study ; Evaluation Study ; Journal Article
ZDB-ID	390499-4
ISSN	1098-660X ; 0095-1137
ISSN (online)	1098-660X
ISSN	0095-1137
DOI	10.1128/JCM.00626-12
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Article ; Online: Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in formalin fixed, paraffin-embedded tissue specimens.

Rickerts, Volker / Khot, Prasanna D / Myerson, David / Ko, Daisy L / Lambrecht, Evelyn / Fredricks, David N

BMC infectious diseases

2011 Volume 11, Page(s) 202

Abstract: Background: Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, ... ...

Abstract	Background: Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients. Methods: We designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broad-range fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach. Results: PCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background. Conclusions: While PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples.
MeSH term(s)	Adult ; Aged ; Aged, 80 and over ; Female ; Fixatives/pharmacology ; Formaldehyde/pharmacology ; Fungi/genetics ; Fungi/isolation & purification ; Humans ; In Situ Hybridization, Fluorescence/methods ; Male ; Middle Aged ; Mycology/methods ; Mycoses/diagnosis ; Paraffin Embedding ; Pathology, Molecular/methods ; RNA, Fungal/genetics ; RNA, Ribosomal, 28S/genetics ; Real-Time Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Sequence Analysis, DNA/methods
Chemical Substances	Fixatives ; RNA, Fungal ; RNA, Ribosomal, 28S ; Formaldehyde (1HG84L3525)
Language	English
Publishing date	2011-07-26
Publishing country	England
Document type	Comparative Study ; Evaluation Study ; Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1471-2334
ISSN (online)	1471-2334
DOI	10.1186/1471-2334-11-202
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