Bioinspired Electro-RAFT Polymerization for Electrochemical Sensing of Nucleic Acids.

Sensing of ultralow-abundance nucleic acids (NAs) is integral to medical diagnostics and pathogen screening. We present herein an electrochemical method for the highly selective and amplified sensing of NAs, using a peptide nucleic acid (PNA) recognition probe and a bioinspired electro-RAFT polymerization (BERP)-based amplification strategy. The presented method is based on the recognition of target NAs by end-tethered PNA probes, the labeling of thiocarbonylthio reversible addition-fragmentation chain transfer (RAFT) agents, and the BERP-assisted growth of ferrocenyl polymers. The dynamic growth of polymers is electrochemically regulated by the reduction of 1-methylnicotinamide (MNA) organic cations, the redox center of nicotinamide adenine dinucleotide (NAD+, coenzyme I). Specifically, electroreduction of the MNA cations causes the fragmentation of thiocarbonylthio RAFT agents into radical species, triggering the polymerization of ferrocenyl monomers, thereby recruiting plenty of ferrocene electroactive tags for amplified sensing. It is obvious that the BERP-based strategy is inexpensive and simple in operation. Benefiting from the high specificity of the PNA recognition probe and the amplified signal by the BERP-based strategy, this method is highly selective and the detection limit is as low as 0.58 fM (S/N = 3). Besides, it is applicable to the sensing of NAs in serum samples, thus showing great promise in the selective and amplified sensing of NAs.

Highly sensitive detection of driver mutations from cytological samples and cfDNA in lung cancer.

BACKGROUND: Bronchoscopy is a minimally invasive procedure for establishing the diagnosis of lung cancer. It sometimes fails to obtain tissue samples but readily collects cytological samples. METHODS: We developed PNA-LNA dual-PCR (PLDP), which amplified mutant sequences by a high-fidelity DNA polymerase in the presence of a peptide nucleic acid (PNA) oligomer having a wild-type sequence. Mutations are detected either by locked nucleic acid (LNA) probes for quick detection of a limited number of mutations, which are EGFR, KRAS, and BRAF mutations in the current study, or by direct sequencing for a comprehensive screening. In a total of 233 lung cancer samples, the results for cytological samples by PLDP were compared with those for tissue samples by cobas® EGFR mutation test (cobas) or by the PNA-LNA PCR clamp method (P-LPC). Moreover, the performance of PLDP using cell-free DNA (cfDNA) was investigated. RESULTS: Peptide nucleic acid-LNA dual-PCR was able to detect each synthesized mutant sequence with high sensitivity. PLDP detected EGFR mutations in 80 out of 149 clinical samples, while the cobas or the P-LPC detected in 66 matched. The correctness of PLDP was confirmed both by clinical response and by the results of sequencing using a next-generation sequencer. PLDP detected mutations from cfDNA in approximately 70% of patients who harbors mutations in the tumor. CONCLUSIONS: Peptide nucleic acid-LNA dual-PCR exhibited an excellent performance, even using cytological samples. PLDP is applicable for the investigation of cfDNA. The combination of bronchoscopy and PLDP is attractive and will expand the utility of bronchoscopy in clinical practice.

A Robust Method for Preparing Optically Pure MiniPEG-Containing Gamma PNA Monomers.

We report the syntheses of chemical building blocks of a particular class of chiral PNAs, called miniPEG-containing (R)-gamma PNAs (or (R)-MPγPNAs). The strategy involves the application of 9-(4-bromophenyl)-9-fluorenyl as a temporary, safety-catch protecting group for the suppression of racemization in the alkylation and reductive amination steps. The methodology is general and robust, ideally suited for large-scale monomer productions with most synthetic steps providing excellent chemical yields without the need for purification other than a simple workup and precipitation.

Reactive Quantum Dot-Based FRET Systems for Target-Catalyzed Detection of RNA.

Oligonucleotide-templated reactions (OTRs) between two reactive hybridization probes allow for the detection of a DNA or RNA of interest by exploiting the target molecule as a catalyst of chemical reactions. The product of such a reaction commonly exhibits distinct fluorescence properties and can be detected by the means of fluorescence spectroscopy. The vast majority of OTR systems utilize organic dyes as fluorescent reporters. However, the use of brighter emitters, such as semiconductor quantum dots (QDs), has potential to improve the sensitivity of detection by providing brighter signals and permitting the use of probes at very low concentrations. Here we report an RNA-templated reaction between two fluorescently labeled peptide nucleic acid (PNA)-based probes, which proceeds on the surface of a QD. The QD-bound PNA probe bears a cysteine functionality, while the other PNA is functionalized with an organic dye as a thioester. OTR between these probes proceeds through a transfer of the organic dye to the QD and can be conveniently monitored via fluorescence resonance energy transfer (FRET) from the QD to the Cy5. The reaction was performed in a conventional fluorescence microplate reader and permits the detection of RNA in the picomolar range.

Nanopore-Assisted, Sequence-Specific Detection, and Single-Molecule Hybridization Analysis of Short, Single-Stranded DNAs.

We report here on the ability of the α-hemolysin (α-HL) nanopore to achieve label-free, selective, and real-time detection of 15 nt long ssDNA fragments in solution, by exploiting their hybridization with freely added, polycationic peptides-functionalized PNAs. At the core of our work lies the paradigm that when PNAs and ssDNA are mixed together, the bulk concentration of free PNA decreases, depending upon the (mis)match degree between complementary strands and their relative concentrations. We demonstrate that the ssDNA sensing principle and throughput of the method are determined by the rate at which nonhybridized, polycationic peptides-functionalized PNA molecules arrive at the α-HL's vestibule entrance and thread into the nanopore. We found that with the application of a 30-fold salt gradient across the nanopore, the method enhances single-molecule detection sensitivity in the nanomolar range of ssDNA concentrations. This study demonstrates that the transmembrane potential-dependent unzip of single PNA-DNA duplexes at the α-HL's ß-barrel entry permits discrimination between sequences that differ by one base pair.

Accelerated bacterial detection in blood culture by enhanced acoustic flow cytometry (AFC) following peptide nucleic acid fluorescence in situ hybridization (PNA-FISH).

Bacteraemia is a risk factor for subsequent clinical deterioration and death. Current reliance on culture-based methods for detection of bacteraemia delays identification and assessment of this risk until after the optimal period for positively impacting treatment decisions has passed. Therefore, a method for rapid detection and identification of bacterial infection in the peripheral bloodstream in acutely ill patients is crucial for improved patient survival through earlier targeted antibiotic treatment. The turnaround time for current clinical laboratory methods ranges from 12 to 48 hours, emphasizing the need for a faster diagnostic test. Here we describe a novel assay for accelerated generic detection of bacteria in blood culture (BC) using peptide nucleic acid fluorescence in situ hybridization enhanced acoustic flow cytometry (PNA-FISH-AFC). For assay development, we used simulated blood cultures (BCs) spiked with one of three bacterial species at a low starting concentration of 10 CFU/mL: Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa. Under current clinical settings, it takes a minimum of 12 hours incubation to reach positivity on the BacTEC system, corresponding to a bacterial concentration of 107-109 CFU/mL optimal for further analyses. In contrast, our PNA-FISH-AFC assay detected 103-104 CFU/mL bacteria in BC following a much shorter culture incubation of 5 to 10 hours. Using either PCR-based FilmArray assay or MALDI-TOF for bacterial detection, it took 7-10 and 12-24 hours of incubation, respectively, to reach the positive result. These findings indicate a potential time advantage of PNA-FISH-AFC assay for rapid bacterial detection in BC with significantly improved turnaround time over currently used laboratory techniques.

Peptide nucleic acid and amino acid modified peptide nucleic acid analysis by capillary zone electrophoresis.

A rapid, high resolution, and low sample consumption CZE method is developed for peptide nucleic acid (PNA) analysis for the first time. 30% v/v acetonitrile in PNA sample and 20% v/v acetonitrile in 50 mM borax-boric acid (pH 8.7) as BGE were employed after optimization. The calibration curves were linear for PNA concentration ranging from 1 to 50 µmol/L. LOD and LOQ of PNA were 0.2 and 1.0 µmol/L, respectively. Since the commercially available reagent gives rise to huge PNA peak and an apparent impurity peak, the purity of PNA was evaluated to be about 81.4% by CZE method, obviously lower than the supplier's purity value of 99.9% evaluated by RP-HPLC, and also lower than 94.8% determined with RP-HPLC by our research group. The CZE method takes only 5 min, needs only 90 nL PNA, much less than 20 min and 20 µL PNA in RP-HPLC method. Moreover, the CZE method is applicable for the analysis of glutamic acid modified and lysine modified PNAs, they show different migration time with their corresponding complementary PNAs. Our results show CZE provides a new choice for PNA and modified PNA analysis, also their purity or quality evaluation.

Nanoscale Nucleic Acid Recognition at the Solid-Liquid Interface Using Xeno Nucleic Acid Probes.

Challenges in reliable nucleic acid detection are manifold. The major ones are related to false positive or negative signals due to a lack of target specificity in detection and to low sensitivity, especially when a plethora of background sequences are present that can mask the specific recognition signal. Utilizing designed synthetic nucleic acids that are commonly called xeno nucleic acids could offer potential routes to meeting such challenges. In this article, we present the general framework of nucleic acid detection, especially for nanoscale applications, and discuss how and why the xeno nucleic acids could be truly an alternative to the DNA probes. Two specific cases, locked nucleic acid (LNA) and peptide nucleic acid (PNA), which are nuclease-resistant and can form thermally stable duplexes with DNA, are addressed. It is shown that the relative ease of the conformationally rigid LNA probe to be oriented upright on the substrate surface and of the nonionic PNA probe to result into high probe density assists in their use in nanoscale nucleic acid recognition. It is anticipated that success with these probes may lead to important developments such as PCR-independent approaches where the major aim is to detect a small number of target sequences present in the analyte medium.

Peptide Nucleic Acid Probe-Based Analysis as a New Detection Method for Clarithromycin Resistance in Helicobacter pylori.

Background/Aims: Helicobacter pylori eradication rates are decreasing because of increases in clarithromycin resistance. Thus, finding an easy and accurate method of detecting clarithromycin resistance is important. Methods: We evaluated 70 H. pylori isolates from Korean patients. Dual-labeled peptide nucleic acid (PNA) probes were designed to detect resistance associated with point mutations in 23S ribosomal ribonucleic acid gene domain V (A2142G, A2143G, and T2182C). Data were analyzed by probe-based fluorescence melting curve analysis based on probe-target dissociation temperatures and compared with Sanger sequencing. Results: Among 70 H. pylori isolates, 0, 16, and 58 isolates contained A2142G, A2143G, and T2182C mutations, respectively. PNA probe-based analysis exhibited 100.0% positive predictive values for A2142G and A2143G and a 98.3% positive predictive value for T2182C. PNA probe-based analysis results correlated with 98.6% of Sanger sequencing results (κ-value=0.990; standard error, 0.010). Conclusions: H. pylori clarithromycin resistance can be easily and accurately assessed by dual-labeled PNA probe-based melting curve analysis if probes are used based on the appropriate resistance-related mutations. This method is fast, simple, accurate, and adaptable for clinical samples. It may help clinicians choose a precise eradication regimen.

Single-Molecule, Real-Time Dissecting of Peptide Nucleic Acid-DNA Duplexes with a Protein Nanopore Tweezer.

Peptide nucleic acids (PNAs) are artificial, oligonucleotides analogues, where the sugar-phosphate backbone has been substituted with a peptide-like N-(2-aminoethyl)glycine backbone. Because of their inherent benefits, such as increased stability and enhanced binding affinity toward DNA or RNA substrates, PNAs are intensively studied and considered beneficial for the fields of materials and nanotechnology science. Herein, we designed cationic polypeptide-functionalized, 10-mer PNAs, and demonstrated the feasible detection of hybridization with short, complementary DNA substrates, following analytes interaction with the vestibule entry of an α-hemolysin (α-HL) nanopore. The opposite charged state at the polypeptide-functionalized PNA-DNA duplex extremities, facilitated unzipping of a captured duplex at the lumen entry of a voltage-biased nanopore, followed by monomers threading. These processes were resolvable and identifiable in real-time, from the temporal profile of the ionic current through a nanopore accompanying conformational changes of a single PNA-DNA duplex inside the α-HL nanopore. By employing a kinetic description within the discrete Markov chains theory, we proposed a minimalist kinetic model to successfully describe the electric force-induced strand separation in the duplex. The distinct interactions of the duplex at either end of the nanopore present powerful opportunities for introducing new generations of force-spectroscopy nanopore-based platforms, enabling from the same experiment duplex detection and assessment of interstrand base pairing energy.

Real-time wash-free detection of unlabeled PNA-DNA hybridization using discrete FET sensor.

We demonstrate an electrochemical sensor for detection of unlabeled single-stranded DNA using peptide nucleic acid (PNA) probes coupled to the field-effect transistor (FET) gate. The label-free detection relies on the intrinsic charge of the DNA backbone. Similar detection schemes have mainly concentrated on sensitivity improvement with an emphasis on new sensor structures. Our approach focuses on using an extended-gate that separates the FET and the sensing electrode yielding a simple and mass fabricable device. We used PNA probes for efficient hybridization in low salt conditions that is required to avoid the counter ion screening. As a result, significant part of the target DNA lies within the screening length of the sensor. With this, we achieved a wash-free detection where  typical gate potential shifts are more than 70 mV with 1 µM target DNA. We routinely obtained a real-time, label- and wash-free specific detection of target DNA in nanomolar concentration with low-cost electronics and the responses were achieved within minutes after introducing targets to the solution. Furthermore, the results suggest that the sensor performance is limited by specificity rather than by sensitivity and using low-cost electronics does not limit the sensor performance in the presented sensor configuration.

Pharmacokinetic Profiling of Conjugated Therapeutic Oligonucleotides: A High-Throughput Method Based Upon Serial Blood Microsampling Coupled to Peptide Nucleic Acid Hybridization Assay.

Therapeutic oligonucleotides, such as small interfering RNAs (siRNAs), hold great promise for the treatment of incurable genetically defined disorders by targeting cognate toxic gene products for degradation. To achieve meaningful tissue distribution and efficacy in vivo, siRNAs must be conjugated or formulated. Clear understanding of the pharmacokinetic (PK)/pharmacodynamic behavior of these compounds is necessary to optimize and characterize the performance of therapeutic oligonucleotides in vivo. In this study, we describe a simple and reproducible methodology for the evaluation of in vivo blood/plasma PK profiles and tissue distribution of oligonucleotides. The method is based on serial blood microsampling from the saphenous vein, coupled to peptide nucleic acid hybridization assay for quantification of guide strands. Performed with minimal number of animals, this method allowed unequivocal detection and sensitive quantification without the need for amplification, or further modification of the oligonucleotides. Using this methodology, we compared plasma clearances and tissue distribution profiles of two different hydrophobically modified siRNAs (hsiRNAs). Notably, cholesterol-hsiRNA presented slow plasma clearances and mainly accumulated in the liver, whereas, phosphocholine-docosahexaenoic acid-hsiRNA was rapidly cleared from the plasma and preferably accumulated in the kidney. These data suggest that the PK/biodistribution profiles of modified hsiRNAs are determined by the chemical nature of the conjugate. Importantly, the method described in this study constitutes a simple platform to conduct pilot assessments of the basic clearance and tissue distribution profiles, which can be broadly applied for evaluation of new chemical variants of siRNAs and micro-RNAs.

Predicting outcomes of EGFR-targeted therapy in non-small cell lung cancer patients using pleural effusions samples and peptide nucleic acid probe assay.

BACKGROUND: Mutation of epidermal growth factor receptor (EGFR) is a prediction marker of the response to tyrosine kinase inhibitor (TKI) drugs in non-small cell lung cancer (NSCLC) patients. As late stage lung cancer patients rarely undergo surgery, samples for EGFR mutation identification usually come from computed tomography (CT)-guided or endoscopic biopsies, which is invasive and costly. Pleural effusion may serve as a less invasive sample for EGFR mutation detection. METHODS: We designed a fluorophore-labeled peptide nucleic acid (PNA) probe assay for three types of EGFR mutations, including exon 19 deletions, L858R point mutations and T790M point mutations. The assay was applied in 39 pleural effusion samples from NSCLC patients. The correlation between detected EGFR status and clinical outcome were analyzed. RESULTS: In 15 paired samples, PNA probe assay in pleural effusion samples could detect all the mutations that were identified by conventional PCR plus Sanger sequencing in tissue biopsies. In addition, PNA probe assay detected three more T790M mutations. In all 39 pleural effusions, the PNA probe assay detected 27 having at least one of the three EGFR mutations. Among the patients before TKI treatment, those with a sensitizing mutation (either exon 19 deletion or L858R) but without T790M, had 94.1% response rate and longer progression-free survival (mean 10.8 months) than patients without detected mutation (mean 4.2 months) and patients with T790M (mean 1.7 months). CONCLUSIONS: Mutations detected in pleural effusions using PNA probe assay are highly associated with clinical outcome. This method appears to be a reliable way for the prediction of the efficacy of EGFR-targeted therapy.

Discriminating typical and atypical cystic fibrosis-related bacteria by multiplex PNA-FISH.

This study aims to report the development of peptide nucleic acid (PNA) probes to specifically detect the cystic fibrosis (CF)-associated traditional and atypical species Pseudomonas aeruginosa and Inquilinus limosus, respectively. PNA probes were designed in silico, developed and tested in smears prepared in phosphate-buffer saline (PBS), and in artificial sputum medium (ASM). A multiplex fluorescent in situ hybridization (FISH) approach using the designed probes was further validated in artificially contaminated clinical sputum samples and also applied in polymicrobial 24 h-old biofilms involving P. aeruginosa, I. limosus, and other CF-related bacteria. Both probes showed high predictive and experimental specificities and sensitivities. The multiplex PNA-FISH assay, associated with non-specific staining, was successfully adapted in the clinical samples and in biofilms of CF-related bacteria, allowing differentiating the community members and inferring about microbial-microbial interactions within the consortia. This study revealed the great potential of PNA-FISH as a diagnostic tool to discriminate between classical and less common CF-associated bacteria, being suitable to further describe species-dependent prevention strategies and deliver more effective target control therapeutics. Biotechnol. Bioeng. 2017;114: 355-367. © 2016 Wiley Periodicals, Inc.

Oligonucleotide-based label-free detection with optical microresonators: strategies and challenges.

This review targets diversified oligonucleotide-based biodetection techniques, focusing on the use of microresonators of whispering gallery mode (WGM) type as optical biosensors mostly integrated with lab-on-a-chip systems. On-chip and microfluidics combined devices along with optical microresonators provide rapid, robust, reproducible and multiplexed biodetection abilities in considerably small volumes. We present a detailed overview of the studies conducted so far, including biodetection of various oligonucleotide biomarkers as well as deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs) and proteins. We particularly advert to chemical surface modifications for specific and selective biosensing.

Responsive Fluorescent PNA Analogue as a Tool for Detecting G-quadruplex Motifs of Oncogenes and Activity of Toxic Ribosome-Inactivating Proteins.

Fluorescent oligomers that are resistant to enzymatic degradation and report their binding to target oligonucleotides (ONs) by changes in fluorescence properties are highly useful in developing nucleic-acid-based diagnostic tools and therapeutic strategies. Here, we describe the synthesis and photophysical characterization of fluorescent peptide nucleic acid (PNA) building blocks made of microenvironment-sensitive 5-(benzofuran-2-yl)- and 5-(benzothiophen-2-yl)-uracil cores. The emissive monomers, when incorporated into PNA oligomers and hybridized to complementary ONs, are minimally perturbing and are highly sensitive to their neighboring base environment. In particular, benzothiophene-modified PNA reports the hybridization process with significant enhancement in fluorescence intensity, even when placed in the vicinity of guanine residues, which often quench fluorescence. This feature was used in the turn-on detection of G-quadruplex-forming promoter DNA sequences of human proto-oncogenes (c-myc and c-kit). Furthermore, the ability of benzothiophene-modified PNA oligomer to report the presence of an abasic site in RNA enabled us to develop a simple fluorescence hybridization assay to detect and estimate the depurination activity of ribosome-inactivating protein toxins. Our results demonstrate that this approach with responsive PNA probes will provide new opportunities to develop robust tools to study nucleic acids.

Prediction of melting temperatures in fluorescence in situ hybridization (FISH) procedures using thermodynamic models.

The thermodynamics and kinetics of DNA hybridization, i.e. the process of self-assembly of one, two or more complementary nucleic acid strands, has been studied for many years. The appearance of the nearest-neighbor model led to several theoretical and experimental papers on DNA thermodynamics that provide reasonably accurate thermodynamic information on nucleic acid duplexes and allow estimation of the melting temperature. Because there are no thermodynamic models specifically developed to predict the hybridization temperature of a probe used in a fluorescence in situ hybridization (FISH) procedure, the melting temperature is used as a reference, together with corrections for certain compounds that are used during FISH. However, the quantitative relation between melting and experimental FISH temperatures is poorly described. In this review, various models used to predict the melting temperature for rRNA targets, for DNA oligonucleotides and for nucleic acid mimics (chemically modified oligonucleotides), will be addressed in detail, together with a critical assessment of how this information should be used in FISH.

A new method for ABO genotyping using fluorescence melting curve analysis based on peptide nucleic acid probes.

ABO genotyping has been routinely used to identify suspects or unknown remains in crime investigations. Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection and is based on melting temperature shifts due to thermal denaturation. In the present study, we developed a new method for ABO genotyping using peptide nucleic acid (PNA) probe-based FMCA. This method allowed for the simultaneous detection of three single nucleotide polymorphism (SNP) sites in the ABO gene (nucleotide positions 261, 526, and 803) and the determination of 14 ABO genotypes (A/A, A/O01 or A/O02, A/O03, B/B, B/O01 or B/O02, B/O03, O01/O01 or O01/O02 or O02/O02, O01/O03 or O02/O03, O03/O03, A/B, cis-AB01/A, cis-AB01/B, cis-AB01/O01 or cis-AB01/O02, and cis-AB01/cis-AB01). Using this method, we analyzed 80 samples and successfully identified ABO genotypes (A/A [n=5], A/O01 or A/O02 [n=23], B/B [n=3], B/O01 or B/O02 [n=18], A/B [n=9], O01/O01 or O01/O02 or O02/O02 [n=20], cis-AB01/A [n=1], and cis-AB01/O01 or cis-AB01/O02 [n=1]). In addition, all steps in the method, including polymerase chain reaction, PNA probe hybridization, and FMCA, could be performed in one single closed tube in less than 3h. Since no processing or separation steps were required during analysis, this method was more convenient and rapid than traditional methods and reduced the risk of contamination. Thus, this method may be an effective and helpful tool in forensic investigations.

PNA-based fluorescence in situ hybridization for identification of bacteria in clinical samples.

Fluorescence in situ hybridization with PNA probes (PNA-FISH) that target specific bacterial ribosomal RNA sequences is a powerful and rapid tool for identification of bacteria in clinical samples. PNA can diffuse readily through the bacterial cell wall due to its uncharged backbone, and PNA-FISH can be performed with high specificity due to the extraordinary thermal stability of RNA-PNA hybrid complexes. We describe a PNA-FISH procedure and provide examples of the application of PNA-FISH for the identification of bacteria in chronic wounds, cystic fibrosis lungs, and soft tissue fillers. In all these cases, bacteria can be identified in biofilm aggregates, which may explain their recalcitrance to antibiotic treatment.

Clinical consequences of using PNA-FISH in Staphylococcal bacteraemia.

To optimize patient treatment and rational use of antimicrobials, it is important to provide fast information on findings in blood-cultures (BCs). The purpose of this study was to evaluate the impact of using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) on positive BCs containing Gram-positive cocci in clusters to differentiate between Staphylococcus aureus (SA) and coagulase negative staphylococci (CoNS) on the prescribed antimicrobial therapy and on the number of contacts between microbiologist and clinician. All cases of positive BCs in our laboratory with SA or CoNS in the year 2011 were identified and the charts were reviewed retrospectively. The group of patients with BCs tested with PNA-FISH was compared to the group of patients with untested BCs. A total of 200 patients with SA and 725 patients with CoNS were included. The mean number of contacts was 0.82 when PNA-FISH showed CoNS and 1.39 when PNA-FISH was not done (p < 0.0001). More patients were recommended appropriate antimicrobial therapy for SA bacteraemia in the PNA-FISH group (98.0%) than in the non-PNA-FISH group (89.4 %) (p = 0.025). The percentage treated with dicloxacillin was 29.6 in the PNA-FISH group, and 8.2 in the non-PNA-FISH group (p = 0.0003). The use of PNA-FISH on BCs in this study was associated with more appropriate and narrow spectrum antimicrobial therapy for patients with SA in an area with low prevalence of methicillin-resistant SA, and a lower number of contacts between clinical microbiologist and clinician about BCs with CoNS as contaminants.