Rapid molecular identification of fungal pathogens in corneal samples from suspected keratomycosis cases

Mancini, Nicasio; Perotti, Mario; Ossi, C.; Cavallero, Annalisa; Matuška, Stanislav; Paganoni, Giorgio; Burioni, Roberto; Rama, Paolo; Clementi, Massimo

doi:10.1099/jmm.0.46638-0

Cited by 18 publications

(15 citation statements)

References 21 publications

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“…In this context, among the most exploited molecular targets are internal transcribed spacer 1 (ITS1) and ITS2 ribosomal DNA sequences. The ITS regions are noncoding sequences interspaced among highly conserved bacterial rRNA genes and fungal ribosomal DNA, showing a high level of heterogeneity among different bacterial and fungal genera and species (37,117,138,160). These characteristics allow the use of a limited pool of slightly degenerated primers for the PCR and species-specific detection with probes targeting the ITS regions (37,101).…”

Section: Nat-based Assays For Detection and Identification Ofmentioning

confidence: 99%

The Era of Molecular and Other Non-Culture-Based Methods in Diagnosis of Sepsis

et al. 2010

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SUMMARY Sepsis, a leading cause of morbidity and mortality throughout the world, is a clinical syndrome with signs and symptoms relating to an infectious event and the consequent important inflammatory response. From a clinical point of view, sepsis is a continuous process ranging from systemic inflammatory response syndrome (SIRS) to multiple-organ-dysfunction syndrome (MODS). Blood cultures are the current “gold standard” for diagnosis, and they are based on the detection of viable microorganisms present in blood. However, on some occasions, blood cultures have intrinsic limitations in terms of sensitivity and rapidity, and it is not expected that these drawbacks will be overcome by significant improvements in the near future. For these principal reasons, other approaches are therefore needed in association with blood culture to improve the overall diagnostic yield for septic patients. These considerations have represented the rationale for the development of highly sensitive and fast laboratory methods. This review addresses non-culture-based techniques for the diagnosis of sepsis, including molecular and other non-culture-based methods. In particular, the potential clinical role for the sensitive and rapid detection of bacterial and fungal DNA in the development of new diagnostic algorithms is discussed.

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Section: Nat-based Assays For Detection and Identification Ofmentioning

confidence: 99%

The Era of Molecular and Other Non-Culture-Based Methods in Diagnosis of Sepsis

et al. 2010

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“…This technique is the most promising for routine use for the diagnosis of BSI in clinical microbiology laboratories because it is based on amplification of the internal transcribed spacer. This non-coding region of the ribosomal DNA is Page 24 of 63 A c c e p t e d M a n u s c r i p t localized among highly conserved genes, shows a high level of heterogeneity among bacterial and fungal genera and species [114][115][116] and allows a high level of identification using a limited pool of slightly degenerated primers [76].…”

Section: Multiplex Pcr Assaymentioning

confidence: 99%

Conventional and molecular techniques for the early diagnosis of bacteraemia

Paolucci

Landini

Sambri

2010

International Journal of Antimicrobial Agents

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Bloodstream infections account for 30-40% of all cases of severe sepsis and septic shock, and are major causes of morbidity and mortality. Diagnosis of bloodstream infections must be performed promptly so that adequate antimicrobial therapy can be started and patient outcome improved. An ideal diagnostic technology would identify the infecting organism(s) and their determinants of antibiotic resistance, in a timely manner, so that appropriate pathogen-driven therapy could begin promptly. Unfortunately, despite the essential information it provides, blood culture, the gold standard, largely fails in this purpose because time is lost waiting for bacterial or fungal growth. Several efforts have been made to optimize the performance of blood culture, such as the development of technologies to obtain rapid detection of microorganism(s) directly in blood samples or in a positive blood culture. The ideal molecular method would analyze a patient's blood sample and provide all the information needed to immediately direct optimal antimicrobial therapy for bacterial or fungal infections. Furthermore, it would provide data to assess the effectiveness of the therapy by measuring the clearance of microbial nucleic acids from the blood over time. None of the currently available molecular methods is sufficiently rapid, accurate or informative to achieve this. This review examines the principal advantages and limitations of some traditional and molecular methods commercially available to help the microbiologist and the clinician in the management of bloodstream infections.

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“…The ITS regions (ITS 1–4) of the rDNA sequences in fungi are interspersed between the highly conserved regions and may have sufficient heterogeneity for species-specific or genus-specific identification [54,55]. In 24 cases of keratomycoses, PCR amplification of the ITS region followed by sequencing significantly reduced time to diagnosis (24 h vs 5–10 days), with 71% sensitivity [55]. Multiplexing with two probes labeled with two different fluorescent dyes or combining one pan-bacterial probe with one species-specific probe have been useful in the diagnosis of polymicrobial infections [56].…”

Section: Multiplex Pcr Assaysmentioning

confidence: 99%

Molecular microbiological methods in the diagnosis of neonatal sepsis

Venkatesh

Flores

Luna

et al. 2010

Expert Review of Anti-infective Therapy

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Neonatal sepsis is a major cause of neonatal mortality and morbidity. The current gold standard for diagnosis of sepsis, namely blood culture, suffers from low sensitivity and a reporting delay of approximately 48-72 h. Rapid detection of sepsis and institution of antimicrobial therapy may improve patient outcomes. Rapid and sensitive tests that can inform clinicians regarding the institution or optimization of antimicrobial therapy are urgently needed. The ideal diagnostic test should have adequate specificity and negative predictive value to reliably exclude sepsis and avoid unnecessary antibiotic therapy. We comprehensively searched for neonatal studies that evaluated molecular methods for diagnosis of sepsis. We identified 19 studies that were assessed with respect to assay methodology and diagnostic characteristics. In addition, we also reviewed newer molecular microbiological assays of relevance that have not been fully evaluated in neonates. Molecular methods offer distinct advantages over blood cultures, including increased sensitivity and rapid diagnosis. However, diagnostic accuracy and cost-effectiveness should be established before implementation in clinical practice. Keywords diagnosis; FISH; microarray; molecular; neonate; PCR; sepsis Neonatal sepsis is a frequent life-threatening problem in neonatal intensive care units, particularly in very-low-birthweight infants (VLBW; infants with birthweight <1500 g) [1][2][3]. Early-onset sepsis (EOS; sepsis in infants <72 h old) occurs in 1.5-1.9% of VLBW infants, and late-onset sepsis (LOS; onset after 72 h of life) in approximately 20% [1]. Coagulasenegative staphylococci (CONS), Staphylococcus aureus and fungi are responsible for most neonatal infections [1]. Neonatal mortality in LOS is approximately 18% overall and 36% in Gram-negative sepsis. Sepsis also increases neonatal morbidities including patent ductus arteriosus, need for intravascular access, need for parenteral nutrition, bronchopulmonary † Author for correspondence: Tel.: +1 832 824 3206, Fax: +1 832 825 3204, mohanv@bcm.edu.For reprint orders, please contact reprints@expert-reviews.com Financial & competing interests disclosureFunding for color printing of the figures was provided by AdvanDx (MA, USA). Mohan Pammi Venkatesh is funded by CHRCDA grant (NIH K12 HD41648). James Versalovic is supported by R01 AT004326-01A1 NIH/NCCAM, R01 DK065075-01 NIH/NIDDK and 1UH2HG083990-01 NIH/NHGRI. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. NIH Public Access Author ManuscriptExpert Rev Anti Infect Ther. Author manuscript; available in PMC 2011 July 1. . In a large cohort of 6093 extremely low birthweight infants (ELBW; birthweight ≤1000 g), infected infants had a significantly higher incidence of adverse developmental outc...

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Rapid molecular identification of fungal pathogens in corneal samples from suspected keratomycosis cases

Cited by 18 publications

References 21 publications

The Era of Molecular and Other Non-Culture-Based Methods in Diagnosis of Sepsis

The Era of Molecular and Other Non-Culture-Based Methods in Diagnosis of Sepsis

Conventional and molecular techniques for the early diagnosis of bacteraemia

Molecular microbiological methods in the diagnosis of neonatal sepsis

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