PCR Followed by Electrospray Ionization Mass Spectrometry for Broad-Range Identification of Fungal Pathogens (original) (raw)
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Application of Mass Spectrometry Technology to Early Diagnosis of Invasive Fungal Infections
Journal of Clinical Microbiology
We recently developed a mass spectrometry (MS) procedure based on the detection of a serum disaccharide (MS-DS) in patients with invasive candidiasis (IC). Here, we compare the performance of MS-DS for the diagnosis of IC, invasive aspergillosis (IA), and mucormycosis (MM) with those of commercially available antigen detection tests. This retrospective study included 48 patients (23 IC patients [74 serum samples], 15 IA patients [40 serum samples], and 10 MM patients [15 serum samples]) and 49 appropriate controls (102 serum samples). MS-DS, mannan (Mnn), galactomannan (GM), and (1,3)-β- d -glucan (BDG) were detected by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS, Platelia, and Fungitell assays, respectively. For IC, the sensitivity and specificity of the MS-DS index, BDG detection, and Mnn detection were 62% and 84%, 82% and 60%, and 33% and 94% per serum sample and 83% and 69%, 96% and 31%, and 39% and 86% per patient, respectively. For IA, the corres...
MGM Journal of Medical Sciences, 2018
Background: Rapid identification of fungi and molds reduce turnaround time and cost for diagnosis of infections with these organisms in a clinical microbiology laboratory. We report here the clinical evaluation of the VITEK mass spectrometry system for rapid fungal identification in comparison to the internal transcribed spacer (ITS) DNA polymorphism method. Methods: Total 136 archived isolates comprising 126 yeast and 10 molds were analyzed by mass spectrometry (VITEK system) and ITS sequencing for identification of fungi. Results: Majority of the yeast isolates belonged to genus Candida (N = 123), followed by one isolate each of Trichosporon, Cryptococcus, and Rhodotorula. Amongst molds, Aspergillus (N = 4), Trichophyton (N = 3), Fusarium (N = 2) and Rhizopus (N = 1) were identified. Overall, correct species-level identification was obtained in 135/136 (99.26%) isolates with a single isolate of Candida auris misidentified as Candida haemulonii by VITEK MS. Conclusion: The VITEK MS system, a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) system, is a reliable and rapid method for the identification of most of the fungi. Further expansion of the database of the VITEK MS for emerging pathogens is needed to enhance its performance.
Journal of Fungi
A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the Candida Panel for the identification of Candida species, and the Filamentous Fungi Panel for the identification of Aspergillus species and Rhizopusarrhizus. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical sampl...
The new microbiologica, 2007
The increasing incidence of severe fungal infections highlights the need for rapid and precise identification methods in clinical mycology. The aim of this study was to develop and validate a culture-indipendent molecular approach that could allow the detection of fungal pathogens in clinical samples, with particular attention to the identification of drug-resistant Candida and Aspergillus species. A real-time multiplex PCR assay was developed using TaqMan probes specific for highly discriminating ITS sequences. In its multiplex format the assay showed a high specificity, clearly discriminating among different species, as well as a high sensitivity (20 CFU/1 mL sample), making it a potentially useful starting point for the development of a more complete molecular diagnostic assay.
Difficulties with molecular diagnostic tests for mold and yeast infections: where do we stand?
Clinical Microbiology and Infection, 2014
PCR assays have not reached the same level of acceptance for the detection of human fungal pathogens as for other microorganisms , mainly because the low number of microorganisms challenges the detection limits of PCR. Therefore, whereas meta-analyses focusing on clinical validation suggest interest in adding PCR results to the diagnostic workup for invasive fungal disease (IFD) along with clinical evaluation, CT scans, classical mycology and antigen detection, no consensual PCR method has emerged. Compared with the end-point format of the 1990s, real-time quantitative PCR is a major breakthrough. This format prevents contamination with previously amplified products, provides the yield of amplification, allows for developing consensus procedures and should therefore be the only format used. An internal control is now mandatory to avoid false-negative results. Primer design strongly impacts on the objectives: pan-fungal primers can provide false-positive results due to environmental fungal DNA contamination; conversely, species-specific primers miss infections caused by untargeted fungi. Unresolved issues include the best specimens to be used; serum is currently preferred to blood because of the ease of the DNA extraction step. Work is in progress to establish standards at least for Aspergillus PCR, and the implementation of quality controls should help centres to improve assays. Eventually, the classical analysis of biomarker performance does not consider the evolving risk factors and changing treatments during IFD, which can lead to variable conclusions. New statistical methods such as event history analysis should be considered.
Revista española de quimioterapia : publicación oficial de la Sociedad Española de Quimioterapia, 2013
Recently, bacterial identification by MALDI-TOF MS has acquired a high relevance in terms of speed and reliability. Conventional mycological identification has some disadvantages: it is frequently slow, reliability is sometimes low, and an extensive experience is required. The risk population for fungal infections, and therefore their clinical significance has progressively increased in recent years. 153 yeast and mould clinical isolates were analyzed by MALDI-TOF MS and conventional identification. When both methods were discrepant to the genus or species level, ITS-2 sequencing was performed. Results. The correlation in yeasts identification between conventional identification methods and MALDI-TOF MS was extremely high (99.2% to the species level and 100% to the genus level). The only discrepancy was checked by ITS-2 sequencing and confirmed the MALDI-TOF identification. The correlation in moulds identification was more heterogeneous. 68.7% of the isolates showed correlation at l...
Utility of PCR in Diagnosis of Invasive Fungal Infections: Real-Life Data from a Multicenter Study
Journal of Clinical Microbiology, 2012
Prospective studies addressing the clinical value of broad-range PCR using the internal transcribed spacer region (ITS) for diagnosis of microscopy-negative fungal infections in nonselected patient populations are lacking. We first assessed the diagnostic performance of ITS rRNA gene PCR compared with that of routine microscopic immunofluorescence examination. Second, we addressed prospectively the impact and clinical value of broad-range PCR for the diagnosis of infections using samples that tested negative by routine microscopy; the corresponding patients' data were evaluated by detailed medical record reviews. Results from 371 specimens showed a high concordance of >80% for broad-range PCR and routine conventional methods, indicating that the diagnostic performance of PCR for fungal infections is comparable to that of microscopy, which is currently considered part of the “gold standard.” In this prospective study, 206 specimens with a negative result on routine microscopy ...
Nucleic Acid Tools for Invasive Fungal Disease Diagnosis
Current Fungal Infection Reports, 2020
Purpose of Review This review has incorporated the knowledge and experience of the leads of each of the laboratory working parties of the fungal PCR initiative in order to provide up-to-date information on the performance and developments of PCR methods for the detection of fungi that commonly cause invasive fungal disease (IFD). Recent Findings Molecular diagnosis of IFD enhances the current repertoire of mycological investigations. Providing superior sensitivity and turnaround time over classical approaches, yet maintaining the benefits of classical tests (e.g. species level identification and identifying resistance). Standardization for Aspergillus PCR is almost complete; the recent release of commercial PCR assays for a wide range fungi (Aspergillus, Candida, Pneumocystis, Mucorales and Pan-fungal) and availability of external quality control schemes (e.g. Quality Control of Molecular Diagnostics for Aspergillus, Candida, Pneumocystis) means that fungal PCR testing is robust and ready for use, globally. Summary Further work is needed to ascertain the utility of PCR in routine practice and to determine whether combining it with other biomarkers is an optimal strategy. PCR for detecting Mucorales sp. and on tissue, together with direct antifungal resistance detection in body fluids, may increase its diagnostic value across the board. This and the ability to diagnose Pneumocystis pneumonia and invasive candidiasis would go a long way towards attaining the long-held ambition of medical mycology to provide a comprehensive range of tests that can be relied upon to diagnose, at least, the common IFD. In short, PCR has a clear future and is close to achieving its full potential in our laboratories. Keywords Aspergillus. Candida. Pneumocystis. Mucorales. Pan-fungal. Tissue. PCR This article is part of the Topical Collection on Advances in Diagnosis of Invasive Fungal Infections
Clinical Microbiology and Infection, 2006
This report describes the development of a real-time LightCycler assay for the detection and identification of Candida and Aspergillus spp., using the MagNa Pure LC Instrument for automated extraction of fungal DNA. The assay takes 5-6 h to perform. The oligonucleotide primers and probes used for species identification were derived from the DNA sequences of the 18S rRNA genes of various fungal pathogens. All samples were screened for Aspergillus and Candida to the genus level in the real-time PCR assay. If a sample was Candida-positive, typing to species level was performed using five species-specific probes. The assay detected and identified most of the clinically relevant Aspergillus and Candida spp. with a sensitivity of 2 CFU ⁄ mL blood. Amplification was 100% specific for all Aspergillus and Candida spp. tested. To assess clinical applicability, 1650 consecutive samples (1330 blood samples, 295 samples from other body fluids and 25 biopsy samples) from patients with suspected invasive fungal infections were analysed. In total, 114 (6.9%) samples were PCR-positive, 5.3% for Candida and 1.7% for Aspergillus spp. In patients with positive PCR results for Candida and Aspergillus, verification with conventional methods was possible in 83% and 50% of cases, respectively. In conclusion, the real-time PCR assay allows sensitive and specific detection and identification of fungal pathogens in vitro and in vivo.