Peptide nucleic acid-dependent artifact can lead to false-positive triplex gene editing signals.

Triplex gene editing relies on binding a stable peptide nucleic acid (PNA) sequence to a chromosomal target, which alters the helical structure of DNA to stimulate site-specific recombination with a single-strand DNA (ssDNA) donor template and elicits gene correction. Here, we assessed whether the codelivery of PNA and donor template encapsulated in Poly Lactic-co-Glycolic Acid (PLGA)-based nanoparticles can correct sickle cell disease and x-linked severe combined immunodeficiency. However, through this process we have identified a false-positive PCR artifact due to the intrinsic capability of PNAs to aggregate with ssDNA donor templates. Here, we show that the combination of PNA and donor templates but not either agent alone results in different degrees of aggregation that result in varying but highly reproducible levels of false-positive signal. We have identified this phenomenon in vitro and confirmed that the PNA sequences producing the highest supposed correction in vitro are not active in vivo in both disease models, which highlights the importance of interrogating and eliminating carryover of ssDNA donor templates in assessing various gene editing technologies such as PNA-mediated gene editing.

[Identification of Mycobacterium species from BACTEC MGIT™ positive cultures with Oligo-FISH and PNA-FISH methods]. / BACTEC MGIT™ Pozitif Kültürlerden Mycobacterium türlerinin Oligo-FISH ve PNA-FISH Yöntemleriyle Tanimlanmasi.

Rapid and accurate diagnosis of mycobacteria is very important in the prevention and effective treatment of tuberculosis which is still a serious public health problem. Fluorescence in situ hybridization (FISH) method using rRNA targeted probes allows for precise and accurate identification of mixed microorganisms from cultures and directly from clinical samples within a few hours without the need for culture methods. In this study it was aimed to compare the diagnostic performance of two different FISH methods (Oligo-FISH and PNA-FISH) with the conventional culture methods for the identification of Mycobacterium spp. grown in BACTEC MGIT™ (Mycobacteria Growth Indicator Tube) system. A total of 60 MGIT (BD, USA) positive, 52 MGIT negative samples and 10 different reference strains were included in the study. 16S rRNA targeted oligonucleotide probes (Myc657: Mycobacterium subdivision, Eub338: Positive control, NonEub: Negative control) were used for oligo-FISH, and 16S rRNA targeted peptide nucleotide probes (MTC: Mycobacterium tuberculosis complex, NTM: Non-tuberculosis Mycobacterium, BacUni: Positive control) for PNA-FISH. Ehrlich-Ziehl-Neelsen staining (ARB) and Löwenstein-Jensen (LJ) culture methods were performed as conventional methods as well as MGIT 960 culture system. Of MGIT positive 60 samples (44 sputum, 4 tissue, 4 urine, 3 bronchoalveolar lavage, 3 CSF, 1 abscess, 1 peritoneal fluid), 29 (48.3%) were found positive for ARB and 44 (73.3%) with LJ culture methods giving a total of 59 positive results. Fifty-eight (96.6%) of those isolates were identified as MTC, and one (1.7%) as NTM by conventional methods. By using Oligo-FISH, 95% (57/60) of the isolates were identified as Mycobacterium spp., while three samples (5%) yielded negative result. By using PNA-FISH, 54 (91.5%) isolates were identified as mycobacteria, of them 53 (90%) were typed as MTC and 1 (1.7%) as NTM. Five isolates that were found positive with Oligo-FISH, but negative with PNA-FISH, yielded positive result with PNA-FISH method performed with minor modifications. It was determined that both FISH methods are more rapid (approximately 2-2.5 hours) and practical than the conventional culture methods and also PNA-FISH was more practical than Oligo-FISH. The sensitivity, specificity, positive and negative predictive values of the probes used for Oligo-FISH, were 96.6%, 100%, 100% and 96.4%, respectively. Those values for the probes used for PNA-FISH, were 91.5%, 100%, 100% and 91.4%, respectively (p< 0.0001). The compatibility of the methods was calculated with kappa statistical analysis, assigning perfect concordances between Oligo- and PNA-FISH methods, as well as between conventional and both of the FISH methods (κ: 0.964, 0.929, 0.964; p= 0.001). The coverage of oligonucleotide and PNA probes was also checked by using 16S rRNA gene sequence database retrieved from the SILVA 102. It was determined that the rates of coverage were 86.5% for Eub338, 41.7% for Myc657, 84.2% for BacUni, 76.3% for MTC (100% for only M.tuberculosis and M.bovis) and 25.8% for NTM probes. In conclusion, Oligo- and PNA-FISH methods seem to be successful for rapid and accurate identification of Mycobacterium spp. from MGIT positive cultures in routine mycobacteriology laboratories without the need for expensive methods.

Rapid detection of Enterococcus spp. direct from blood culture bottles using Enterococcus QuickFISH method: a multicenter investigation.

The performance of a diagnostic method for detection and identification of Enterococcus spp. directly from positive blood culture was evaluated in a clinical study. The method, Enterococcus QuickFISH BC, is a second-generation peptide nucleic acid (PNA) fluorescence in situ hybridization (FISH) test, which uses a simplified, faster assay procedure. The test uses fluorescently labeled PNA probes targeting 16S rRNA to differentiate Enterococcus faecalis from other Enterococcus spp. by the color of the cellular fluorescence. Three hundred fifty-six routine blood culture samples were tested; only 2 discordant results were recorded. The sensitivities for detection of Enterococcus faecalis and non-faecalis Enterococcus were 100% (106/106) and 97.0% (65/67), respectively, and the combined specificity of the assay was 100%. The combined positive and negative predictive values of the assay were 100% (171/171) and 98.9% (185/187), respectively.

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.

A novel rapid peptide nucleic acid fluorescence in situ hybridization (FISH) method, Staphylococcus QuickFISH, 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 QuickFISH 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.

Rapid detection of Streptococcus agalactiae from swabs by peptide nucleic acid fluorescence in situ hybridization.

The applicability of the PNA FISH (peptide nucleic acid fluorescence in situ hybridization) method for detection of Streptococcus agalactiae [group B streptococci (GBS)] from swab samples was evaluated. Three swab-sample-processing protocols with different time-to-result (TTR) values were compared: (i) direct smearing of fresh swabs onto microscope slides (n=153, TTR 2.5 h), (ii) further extraction and concentration of cells from these same swabs (n=153, TTR 2.7 h), and (iii) short-term LIM broth enrichment culture incubation (7 h, 37 degrees C) of fresh swabs (n=120, TTR 9.5 h). The sensitivity, specificity, positive predictive value and negative predictive value for GBS PNA FISH for sample processing procedures, with TTR values of 2.5, 2.7 and 9.5 h, were 68, 100, 100 and 95 %; 91, 100, 100 and 98 %; and 100, 100, 100 and 100 %; respectively. Improved test results were achieved by subjecting swabs to an extraction procedure or abbreviated LIM broth enrichment culture incubation prior to performing GBS PNA FISH.

Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.

The present study was undertaken to investigate the appearance and location of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung and in sputum. Samples include preserved tissues of CF patients who died due to chronic P. aeruginosa lung infection prior to the advent of intensive antibiotic therapy, explanted lungs from 3 intensively treated chronically P. aeruginosa infected CF patients and routine sputum from 77 chronically P. aeruginosa infected CF patients. All samples were investigated microscopically using hematoxylin-eosin (HE), Gram and alcian-blue stain, PNA FISH and immunofluorescence for alginate.Investigation of the preserved tissues revealed that prior to aggressive antibiotic therapy, P. aeruginosa infection and destruction of the CF lung correlated with the occurrence of mucoid (alginate) bacteria present in aggregating structures surrounded by pronounced polymorphonuclear-leukocyte (PMN) inflammation in the respiratory zone (9/9). Non-mucoid bacteria were not observed here, and rarely in the conductive zone (1/9). However, in the explanted lungs, the P. aeruginosa aggregates were also mucoid but in contrast to the autopsies, they were very rare in the respiratory zone but abundant in the sputum of the conductive zone (3/3), which also contained abundances of PMNs (3/3). Non-mucoid and planktonic P. aeruginosa were also observed here (3/3).In conclusion, the present intensive antibiotic therapy of chronic P. aeruginosa infections, at the Copenhagen CF Centre, seems to restrain but not eradicate the bacteria from the conductive zone, whereas the remaining healthy respiratory zone appears to be protected, for a long period, from massive biofilm infection. This strongly suggests that the conductive zone serves as a bacterial reservoir where the bacteria are organized in mucoid biofilms within the mucus, protected against antibiotics and host defenses.

Optimization of three FISH procedures for in situ detection of anaerobic ammonium oxidizing bacteria in biological wastewater treatment.

Fluorescence in situ hybridization (FISH) using fluorochrome-labeled DNA oligonucleotide probes has been successfully applied for in situ detection of anaerobic ammonium oxidizing (anammox) bacteria. However, application of the standard FISH protocols to visualize anammox bacteria in biofilms from a laboratory-scale wastewater reactor produced only weak signals. Increased signal intensity was achieved either by modifying the standard FISH protocol, using peptide nucleic acid probes (PNA FISH), or applying horse radish peroxidase- (HRP-) labeled probes and subsequent catalyzed reporter deposition (CARD-FISH). A comparative analysis using anammox biofilm samples and suspended anammox biomass from different laboratory wastewater bioreactors revealed that the modified standard FISH protocol and the PNA FISH probes produced equally strong fluorescence signals on suspended biomass, but only weak signals were obtained with the biofilm samples. The probe signal intensities in the biofilm samples could be enhanced by enzymatic pre-treatment of fixed cells, and by increasing the hybridization time of the PNA FISH protocol. CARD-FISH always produced up to four-fold stronger fluorescent signals but unspecific fluorescence signals, likely caused by endogenous peroxidases as reported in several previous studies, compromised the results. Interference of the development of fluorescence intensity with endogenous peroxidases was also observed in cells of aerobic ammonium oxidizers like Nitrosomonas europea, and sulfate-reducers like Desulfobacter postgatei. Interestingly, no interference was observed with other peroxidase-positive microorganisms, suggesting that CARD-FISH is not only compromised by the mere presence of peroxidases. Pre-treatment of cells to inactivate peroxidase with HCl or autoclavation/pasteurization failed to inactive peroxidases, but H(2)O(2) significantly reduced endogenous peroxidase activity. However, for optimal inactivation, different H(2)O(2) concentrations and incubation time may be needed, depending on nature of sample and should therefore always be individually determined for each study.

Candida bracarensis detected among isolates of Candida glabrata by peptide nucleic acid fluorescence in situ hybridization: susceptibility data and documentation of presumed infection.

Molecular taxonomic studies have revealed new Candida species among phenotypically delineated species, the best example being Candida dubliniensis. This study was designed to determine the occurrence of two new molecularly defined species, Candida bracarensis and Candida nivariensis, which are closely related to and identified as Candida glabrata by phenotypic assays. A total of 137 recent clinical isolates of C. glabrata identified by phenotypic characteristics was tested with C. bracarensis and C. nivariensis species-specific peptide nucleic acid fluorescence in situ hybridization probes. Three of 137 (2.2%) isolates were positive with the C. bracarensis probe, whereas the control strain, but none of the clinical isolates, was positive with the C. nivariensis probe. D1/D2 sequencing confirmed the identification of the three isolates as representing C. bracarensis. Clinically, one C. bracarensis isolate was recovered from a presumed infection, a polymicrobial pelvic abscess in a patient with perforated diverticulitis. The other two isolates were recovered from two adult oncology patients who were only colonized. C. bracarensis was white on CHROMagar Candida, had variable API-20C patterns that overlapped with C. nivariensis and some C. glabrata isolates, and had variable results with a rapid trehalose assay. Interestingly, an isolate from one of the colonized oncology patients was resistant to fluconazole, itraconazole, voriconazole, and posaconazole in vitro. In summary, C. bracarensis was detected among clinical isolates of C. glabrata, while C. nivariensis was not. One C. bracarensis isolate causing a presumed deep infection was recovered, and another isolate was azole resistant. Whether clinical laboratories should identify C. bracarensis will require more data.

Multicenter evaluation of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization method for simultaneous dual-color identification of C. albicans and C. glabrata directly from blood culture bottles.

We evaluated the performance of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method, a rapid two-color assay for detection of C. albicans and C. glabrata, in a multicenter study. The assay is designed for use directly from positive blood culture bottles in a FISH format. Intact, fixed cells are labeled fluorescent green (C. albicans) or fluorescent red (C. glabrata) by rRNA hybridization of fluorophore-labeled PNA probes. Results are available <3 h after cultures signal positive. An evaluation of 197 routine blood culture bottles newly positive for yeast by Gram staining was performed at five hospitals. The sensitivities of detection for C. albicans, and C. glabrata were 98.7% (78/79) and 100% (37/37), respectively, and the specificity for both components of the assay was 100% (82/82). The assay was also evaluated with 70 fungal reference strains and was challenged in the BacT/ALERT microbiological detection system with spiked blood culture bottles. These results support the use of the assay for rapid, simultaneous identification of C. albicans and C. glabrata in positive blood culture bottles. This rapid assay may aid in the selection of initial antifungal drugs, leading to improved patient outcomes.

Peptide nucleic acid fluorescence in situ hybridization for rapid detection of Klebsiella pneumoniae from positive blood cultures.

This study evaluated a novel peptide nucleic acid (PNA) probe targeting a region of the 23S rRNA gene of Klebsiella pneumoniae by fluorescence in situ hybridization (FISH). Analytical performance was determined using 39 reference strains and other well-characterized strains of Klebsiella spp. and Enterobacter aerogenes. The probe was found to be specific for the K. pneumoniae complex (K. pneumoniae including Klebsiella ozaenae and Klebsiella variicola). The diagnostic accuracy was evaluated with 264 blood cultures containing Gram-negative rods. Using conventional identification as the reference, performance specifications were as follows: sensitivity 98.8 %, specificity 99.5 %, positive predictive value 98.8 % and negative predictive value 99.5 %. Discrepancies were resolved by PNA FISH retest and phenotypic tests. In conclusion, the K. pneumoniae probe provided an accurate diagnosis within 3 h and may supplement other methods for direct identification of Gram-negative bacteria.

Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets.

Molecular beacons are sensitive fluorescent probes hybridizing selectively to designated DNA and RNA targets. They have recently become practical tools for quantitative real-time monitoring of single-stranded nucleic acids. Here, we comparatively study the performance of a variety of such probes, stemless and stem-containing DNA and PNA (peptide nucleic acid) beacons, in Tris-buffer solutions containing various concentrations of NaCl and MgCl(2). We demonstrate that different molecular beacons respond differently to the change of salt concentration, which could be attributed to the differences in their backbones and constructions. We have found that the stemless PNA beacon hybridizes rapidly to the complementary oligodeoxynucleotide and is less sensitive than the DNA beacons to the change of salt thus allowing effective detection of nucleic acid targets under various conditions. Though we found stemless DNA beacons improper for diagnostic purposes due to high background fluorescence, we believe that use of these DNA and similar RNA constructs in molecular-biophysical studies may be helpful for analysis of conformational flexibility of single-stranded nucleic acids. With the aid of PNA "openers", molecular beacons were employed for the detection of a chosen target sequence directly in double-stranded DNA (dsDNA). Conditions are found where the stemless PNA beacon strongly discriminates the complementary versus mismatched dsDNA targets. Together with the insensitivity of PNA beacons to the presence of salt and DNA-binding/processing proteins, the latter results demonstrate the potential of these probes as robust tools for recognition of specific sequences within dsDNA without denaturation and deproteinization of duplex DNA.

PNA for rapid microbiology.

The acceptance of rRNA sequence diversity as a criterion for phylogenetic discrimination heralds the transition from microbiological identification methods based on phenotypic markers to assays employing molecular techniques. Robust amplification assays and sensitive direct detection methods are rapidly becoming the standard protocols of microbiology laboratories. The emergence of peptide nucleic acid (PNA) from its status as an academic curiosity to that of a promising and powerful molecular tool, coincides with, and complements, the transition to rapid molecular tests. The unique properties of PNA enable the development of assay formats, which go above and beyond the possibilities of DNA probes. PNA probes targeting specific rRNA sequences of yeast and bacteria with clinical, environmental, and industrial value have recently been developed and applied to a variety of rapid assay formats. Some simply incorporate the sensitivity and specificity of PNA probes into traditional methods, such as membrane filtration and microscopic analysis; others involve recent techniques such as real-time and end-point analysis of amplification reactions.