Identification of indicator microorganisms using a standardized PNA FISH method

Perry‐O'Keefe, Heather; Rigby, S. V.; Oliveira, Kenneth; Sørensen, Ditte; Stender, Henrik; Coull, James; Hyldig‐Nielsen, J. J.

doi:10.1016/s0167-7012(01)00303-7

Cited by 119 publications

(57 citation statements)

References 23 publications

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“…To overcome these limitations, peptide nucleic acid (PNA) probes are being used instead of the typical DNA molecules (28). PNA molecules are synthetic DNA mimics with a neutrally charged chemical backbone that confers higher affinity for DNA or RNA complementary sequences (30,31). PNA probes are usually smaller (approximately 13 to 18 bp) than DNA probes (Ͼ18 bp), increasing their ability to penetrate the bacterial cell wall, and are more resistant to nucleases and proteases (30).…”

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Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens

et al. 2013

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h Here, we evaluated a previously established peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method as a new diagnostic test for Helicobacter pylori clarithromycin resistance detection in paraffin-embedded gastric biopsy specimens. Both a retrospective study and a prospective cohort study were conducted to evaluate the specificity and sensitivity of a PNA-FISH method to determine H. pylori clarithromycin resistance. In the retrospective study (n ‫؍‬ 30 patients), full agreement between PNA-FISH and PCR-sequencing was observed. Compared to the reference method (culture followed by Etest), the specificity and sensitivity of PNA-FISH were 90.9% (95% confidence interval [CI], 57.1% to 99.5%) and 84.2% (95% CI, 59.5% to 95.8%), respectively. In the prospective cohort (n ‫؍‬ 93 patients), 21 cases were positive by culture. For the patients harboring clarithromycin-resistant H. pylori, the method showed sensitivity of 80.0% (95% CI, 29.9% to 98.9%) and specificity of 93.8% (95% CI, 67.7% to 99.7%). These values likely represent underestimations, as some of the discrepant results corresponded to patients infected by more than one strain. PNA-FISH appears to be a simple, quick, and accurate method for detecting H. pylori clarithromycin resistance in paraffin-embedded biopsy specimens. It is also the only one of the methods assessed here that allows direct and specific visualization of this microorganism within the biopsy specimens, a characteristic that allowed the observation that cells of different H. pylori strains can subsist in very close proximity in the stomach.

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Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens

et al. 2013

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“…The principal disadvantage of latex agglutination coagulase tests, traditional growth-dependent culture methods, or automated broth microdilution identification systems is the prolonged turnaround time (TAT) for results. In contrast, rapid molecular techniques, such as peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH), provide results in Ͻ2 h. Assays that generate identifications directly from positive blood cultures in real time are likely to have a significant clinical impact and become mainstream diagnostic tools of clinical microbiology laboratories.The PNA FISH method has been used in clinical microbiology labs for over 10 years for identification of a variety of organisms (12,13,18). In clinical evaluations, the PNA FISH method has demonstrated sensitivities and specificities ranging from 96 to 100% (6,15,16).…”

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confidence: 99%

“…The PNA FISH method has been used in clinical microbiology labs for over 10 years for identification of a variety of organisms (12,13,18). In clinical evaluations, the PNA FISH method has demonstrated sensitivities and specificities ranging from 96 to 100% (6,15,16).…”

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Multicenter Evaluation of the Staphylococcus QuickFISH Method for Simultaneous Identification of Staphylococcus aureus and Coagulase-Negative Staphylococci Directly from Blood Culture Bottles in Less than 30 Minutes

Deck¹,

Anderson²,

Buckner

et al. 2012

J Clin Microbiol

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A novel rapid peptide nucleic acid fluorescence in situ hybridization (FISH) method, Staphylococcus Quick FISH, for the direct detection of Staphylococcus species from positive blood culture bottles was evaluated in a multicenter clinical study. The method utilizes a microscope slide with predeposited positive- and negative-control organisms and a self-reporting 15-min hybridization step, which eliminates the need for a wash step. Five clinical laboratories tested 722 positive blood culture bottles containing Gram-positive cocci in clusters. The sensitivities for detection of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) were 99.5% (217/218) and 98.8% (487/493), respectively, and the combined specificity of the assay was 89.5% (17/19). The combined positive and negative predictive values of the assay were 99.7% (696/698) and 70.8% (17/24), respectively. Studies were also performed on spiked cultures to establish the specificity and performance sensitivity of the method. Staphylococcus Quick FISH has a turnaround time (TAT) of <30 min and a hands-on time (HOT) of <5 min. The ease and speed of the method have the potential to improve the accuracy of therapeutic intervention by providing S. aureus /CoNS identification simultaneously with Gram stain results.

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“…[32,33] Haase et al [33] and Perry-O'Keefe et al [34] have shown that mixtures of isolates in cultures can be detected by FISH method and it has been indicated that PNA-FISH is more sensitive than conventional methods in the detection of mixtures of isolates in blood cultures. [33,34] Similarly, the rate of multi-species candidemia with FISH is higher than PCR-RFLP and conventional methods in the present study. With FISH, the rate of multi-species candidemia was 3.4 times higher than that of PCR-RFLP, and about 10.2 times higher than that of conventional methods.…”

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confidence: 99%

Identification Of Candida Species From Blood Cultures With Fluorescent In Situ Hybridization (fish), Polymerase Chain Reaction-restriction Fragment Length Polymorphism (pcr-rflp) And Conventional Methods

Börekçi¹,

Ersöz²,

Otağ³

et al. 2009

TUTFD

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Objectives: Rapid and accurate identification of Candida species from blood cultures is crucial to ensure effective antifungal therapy and to reduce the morbidity and mortality associated with bloodstream fungal infections. In this study, we aimed to identify Candida spp. from blood culture samples with fluorescent in situ hybridization (FISH), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and conventional methods. Materials and Methods:A total of 50 yeast positive samples out of 325 blood culture positive samples and 50 blood culture negative samples were examined by FISH, PCR-RFLP and conventional methods to identify Candida spp.Results: All three methods generally were compatible for identification of single-species Candida spp. (p<0.001). But, FISH and PCR-RFLP for the identification of multi-species Candida spp. were found more compatible than conventional methods (p<0.001). Besides, FISH is cheaper and quicker than the other two methods in the identification of Candida spp. from blood culture positive samples. The rates of multi-species candidemia with FISH, PCR-RFLP and conventional methods were 20%, 6% and 4%, respectively. Conclusion:Both PCR-RFLP and FISH methods might be preferred for the rapid identification of Candida spp. from blood culture positive samples. However, FISH is a more suitable method for the detection of multi-species candidemia.

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Identification of indicator microorganisms using a standardized PNA FISH method

Cited by 119 publications

References 23 publications

Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens

Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens

Multicenter Evaluation of the Staphylococcus QuickFISH Method for Simultaneous Identification of Staphylococcus aureus and Coagulase-Negative Staphylococci Directly from Blood Culture Bottles in Less than 30 Minutes

Identification Of Candida Species From Blood Cultures With Fluorescent In Situ Hybridization (fish), Polymerase Chain Reaction-restriction Fragment Length Polymorphism (pcr-rflp) And Conventional Methods

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