Prior Isolation Research Articles

Resistance to Ceftazidime/Avibactam in Klebsiella pneumoniae KPC-Producing Isolates: A Real-Life Observational Study.

Background: Ceftazidime/avibactam (CAZ-AVI) resistance amongst Enterobacterales is worryingly increasing worldwide. Objectives: The aim of this study was to collect and describe real-life data on CAZ-AVI-resistant Klebsiella pneumoniae (KP) isolates in our University Hospital, with the ultimate goal of evaluating possible risk factors related to the acquisition of resistance. Methods: This is a retrospective observational study, including unique Klebsiella pneumoniae (KP) isolates resistant to CAZ-AVI (CAZ-AVI-R) and producing only KPC, collected from July 2019 to August 2021 at Policlinico Tor Vergata, Rome, Italy. The pathogen's list was obtained from the microbiology laboratory; clinical charts of the corresponding patients were reviewed to collect demographic and clinical data. Subjects treated as outpatients or hospitalized for <48 h were excluded. Patients were then divided into two groups: S group, if they had a prior isolate of CAZ-AVI-susceptible KP-KPC, and R group, if the first documented isolate of KP-KPC was resistant to CAZ-AVI. Results: Forty-six unique isolates corresponding to 46 patients were included in the study. The majority of patients (60.9%) were hospitalized in an intensive care unit, 32.6% in internal medicine wards and 6.5% in surgical wards. A total of 15 (32.6%) isolates were collected from rectal swabs, representing a colonization. Amongst clinically relevant infections, pneumonia and urinary tract infections were the most commonly found (5/46, 10.9% each). Half of the patients received CAZ-AVI prior to isolation of the KP-KPC CAZ-AVI-R (23/46). This percentage was significantly higher in patients in the S group compared to patients in the R group (69.3% S group vs. 25% R group, p = 0.003). No differences between the two groups were documented in the use of renal replacement therapy or in the infection site. The clinically relevant CAZ-AVI-R KP infections (22/46, 47.8%) were all treated with a combination therapy, 65% including colistin and 55% including CAZ-AVI, with an overall clinical success of 38.1%. Conclusions: Prior use of CAZ-AVI was associated with the emergence of drug resistance.

Ladder-shaped microfluidic system for rapid antibiotic susceptibility testing

Rapid identification of antibiotic-resistant bacteria will play a key role in solving the global antibiotic crisis by providing a route to targeted antibiotic administration. However, current bacterial infection diagnoses take up to 3 days which can lead to antibiotic treatment that is less effective. Here we report a microfluidic system with a ladder shaped design allowing us to generate a twofold serial dilution of antibiotics comparable to current national and international standards. Our consolidated design, with minimal handling steps cuts down the testing time for antibiotic susceptibility from 16–20 h to 4–5 h. Our feasibility testing results are consistent with the commercial antibiotic susceptibility testing (AST) results, showing a 91.75% rate of agreement for Gram-negative and Gram-positive bacterial isolated from canine urinary tract infections (UTI) and may be used without prior isolation or enrichment. This platform provides an adaptable and efficient diagnostic tool for antibiotic susceptibility testing.

NMR Discrimination of d- and l-α-Amino Acids at Submicromolar Concentration via Parahydrogen-Induced Hyperpolarization

Differentiation of enantiomers represents an important research area for pharmaceutical, chemical, and food industries. However, enantiomer separation is a laborious task that demands complex analytical techniques, specialized equipment, and expert personnel. In this respect, discrimination and quantification of d- and l-α-amino acids is no exception, generally requiring extensive sample manipulation, including isolation, functionalization, and chiral separation. This complex sample treatment results in high time costs and potential biases in the quantitative determination. Here, we present an approach based on the combination of non-hydrogenative parahydrogen-induced hyperpolarization and nuclear magnetic resonance that allows detection, discrimination, and quantification of d- and l-α-amino acids in complex mixtures such as biofluids and food extracts down to submicromolar concentrations. Importantly, this method can be directly applied to the system under investigation without any prior isolation, fractionation, or functionalization step.

Selection of probiotic candidate of lactic acid bacteria from Hermetia illucens larvae fed with different feeding substrates

Abstract. Siregar DJS, Julianti E, Tafsin M, Suryanto D. 2022. Selection of probiotic candidate of lactic acid bacteria from Hermetia illucens larvae fed with different feeding substrates. Biodiversitas 23: 6320-6326. Exploration of probiotics candidate have been accelerated from unique and novel source, one of which is from black soldier fly (Hermetia illucens). The black soldier flies are known as omnivores which are commonly reared, harvested and utilized to convert organic wastes into biomass yet reducing the amount of abundant waste in nature. Lactic acid bacteria (LAB) is one of beneficial gut symbionts which can be found across animal taxa, including insect, however, limited information is currently reported from H. illucens as a potential harboring insect. This study aimed to determine the probiotics candidate from the gut of H. illucens fed with different feeding substrates to improve the gut microbiota. The larvae were nourished with chicken manure, tofu solid wastes and vegetable wastes prior isolation of LAB. The LAB isolates were tested for their antagonistic activity against enteric pathogens, pH and bile salt tolerance, and adherence to chicken intestine (ileum). Based on the growth performance under physiological stresses and multivariate analysis using principal component analysis (PCA), four LAB isolates were designated as putative probiotics. The four isolates were Lactiplantibacillus. pentosus 5P2i1, Lacticaseibacillus. paracasei 5P2i5, Lactiplantibacillus. pentosus 5P2i9, and Lactiplantibacillus. plantarum 6P1i1 was then declared as novel strains that could be developed further as poultry probiotics.

Risk Factors Associated With Antimicrobial Resistance and Adverse Short-Term Health Outcomes Among Adult and Adolescent Female Outpatients With Uncomplicated Urinary Tract Infection.

Increasing antimicrobial resistance makes treating uncomplicated urinary tract infections (uUTIs) difficult. We compared whether adverse short-term outcomes among US female patients were more common when initial antimicrobial therapy did not cover the causative uropathogen. This retrospective cohort study used data from female outpatients aged ≥12 years, with a positive urine culture and dispensing of an oral antibiotic ±1 day from index culture. Isolate susceptibility to the antimicrobial initially dispensed, patient age, and history of antimicrobial exposure, resistance, and all-cause hospitalization within 12 months of index culture were evaluated for associations with adverse outcomes during 28-day follow up. Outcomes assessed were new antimicrobial dispensing, all-cause hospitalization, and all-cause outpatient emergency department/clinic visits. Of 2366 uUTIs, 1908 (80.6%) were caused by isolates susceptible and 458 (19.4%) by isolates not susceptible (intermediate/resistant) to initial antimicrobial treatment. Within 28 days, patients with episodes caused by not susceptible isolates were 60% more likely to receive a new antimicrobial versus episodes with susceptible isolates (29.0% vs 18.1%; 95% confidence interval, 1.3-2.1; P < .0001). Other variables associated with new antibiotic dispenses within 28 days were older age, prior antimicrobial exposure, or prior nitrofurantoin-not-susceptible uropathogens (P < .05). Older age, prior antimicrobial-resistant urine isolates, and prior hospitalization were associated with all-cause hospitalization (P < .05). Prior fluoroquinolone-not-susceptible isolates or oral antibiotic dispensing within 12 months of index culture were associated with subsequent all-cause outpatient visits (P < .05). New antimicrobial dispensing within the 28-day follow-up period was associated with uUTIs where the uropathogen was not susceptible to initial antimicrobial treatment. Older age and prior antimicrobial exposure, resistance, and hospitalization also identified patients at risk of adverse outcomes.

Development of multiplex PCR based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological samples using Oxford Nanopore platform

West Nile virus (WNV) can affect humans, birds, horses and another mammals, causing asymptomatic infection, mild febrile disease, neurological and systematic disease and death. In order to gain insight into the prevalence of WNV, a monitoring program has been established in the Republic of Serbia. Whole genome sequencing is essential for the molecular epizootiological analysis of virus entry and transmission routes, especially in high-risk regions. This paper describes the development of a multiplex PCR based NGS protocol for whole genome sequencing of WNV lineage 2 directly from biological samples using Oxford Nanopore (ONT) platform. The results obtained using this platform, confirmed by Sanger sequencing, indicate that this protocol can be applied to obtain whole sequences of the WNV genome, even when the virus concentration in the sample is medium, Ct value is approximately 30. The use of this protocol does not require prior virus isolation on cell culture nor the depletion of host nucleic acids.

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus (AAV) Capsid Variants.

Gene delivery vectors derived from Adeno-associated virus (AAV) are one of the most promising tools for the treatment of genetic diseases, evidenced by encouraging clinical data and the approval of several AAV gene therapies. Two major reasons for the success of AAV vectors are (i) the prior isolation of various naturally occurring viral serotypes with distinct properties, and (ii) the subsequent establishment of powerful technologies for their molecular engineering and repurposing in high throughput. Further boosting the potential of these techniques are recently implemented strategies for barcoding selected AAV capsids on the DNA and RNA level, permitting their comprehensive and parallel in vivo stratification in all major organs and cell types in a single animal. Here, we present a basic pipeline encompassing this set of complementary avenues, using AAV peptide display to represent the diverse arsenal of available capsid engineering technologies. Accordingly, we first describe the pivotal steps for the generation of an AAV peptide display library for the in vivo selection of candidates with desired properties, followed by a demonstration of how to barcode the most interesting capsid variants for secondary in vivo screening. Next, we exemplify the methodology for the creation of libraries for next-generation sequencing (NGS), including barcode amplification and adaptor ligation, before concluding with an overview of the most critical steps during NGS data analysis. As the protocols reported here are versatile and adaptable, researchers can easily harness them to enrich the optimal AAV capsid variants in their favorite disease model and for gene therapy applications.

Screening of antagonistic bacteria against the blue mold of citrus fruit from soil by a new parallel screening method without prior isolation of single strains

Antagonistic bacteria are effective in controlling fruit and vegetable diseases. However, screening for antagonistic bacteria is time-consuming. To simplify the procedure for screening antagonistic bacteria, we developed an ameliorated method for screening antagonistic bacteria against Penicillium italicum from the soil that requires no prior isolation of single colonies. The soil supernatant was directly spread on the P. italicum plate, then the antagonistic bacterial strains were isolated from the inhibition zones on the plate and further validated by dual culture assay. By using the novel method, three antagonistic bacterial strains were isolated from soil, which showed significant antagonistic effects on P. italicum. The results of 16S rDNA sequence analysis showed that the three antagonistic strains were classified as Paenibacillus sp., Bacillus sp., and Pseudomonas sp., respectively. Compared to the control, treatments with the three antagonistic trains decreased the disease incidence of blue mold by 32.5 %, 68.7 %, and 42.2 %, respectively. These results indicate that the ameliorated method is time-saving and effective for the screening of antagonistic bacteria.

Impact of Pseudomonas aeruginosa coverage on the prognosis of elderly patients with community-acquired pneumonia

BackgroundAlthough the 2019 American Thoracic Society and Infectious Diseases Society of America guidelines for community-acquired pneumonia (CAP) recommend the use of antibiotics with Pseudomonas aeruginosa coverage for patients with prior sputum isolation of P. aeruginosa, further research is needed to confirm its clinical outcomes. This study aimed to assess the impact of the use of antibiotics with P. aeruginosa coverage on mortality in elderly CAP patients with sputum isolation of P. aeruginosa. MethodsWe retrospectively included consecutive elderly patients who were hospitalized for CAP and P. aeruginosa-positive sputum culture. The association between the use of antibiotics with P. aeruginosa coverage and 28-day mortality was assessed based on propensity score to reduce selection bias. ResultsA total of 216 patients were included, and 68 (31%) of them were treated with antibiotics with P. aeruginosa coverage. The number of patients treated with antibiotics with P. aeruginosa coverage was significantly higher among nonsurvivors than among survivors. After adjustment using propensity score, the association between the use of antibiotics with P. aeruginosa coverage and the 28-day mortality was found to be statistically nonsignificant (odds ratio 2.182, 95% confidence interval 0.732–6.508, p = 0.162). ConclusionsThe use of antibiotics with P. aeruginosa coverage in elderly CAP patients with sputum isolation of P. aeruginosa did not improve their prognosis. A randomized control study is required to identify cases that should be treated with antibiotics covering P. aeruginosa.

IGH cytogenetic abnormalities can be detected in multiple myeloma by imaging flow cytometry

AimsCytogenetic abnormalities involving the IGH gene are seen in up to 55% of patients with multiple myeloma. Current testing is performed manually by fluorescence in situ hybridisation (FISH) on purified...

Domino Synthesis of Thiazolo-Fused Six- and Seven-Membered Nitrogen Heterocycles via Intramolecular Heteroannulation of In-Situ-Generated 2-(Het)aryl-4-amino-5-functionalized Thiazoles.

Synthesis of novel 2-(het)aryl-substituted thiazolo-fused six- and seven-membered heterocycles, such as thiazolo[4,5-b]pyridin-5(4H)-ones, thiazolo[4,5-c]isoquinolin-5(4H)-ones, thiazolo[4,5-b]quinolin-9(4H)-ones, 4H-benzo[e]thiazolo[4,5-b]azepine-5,10-diones, have been developed in a single-pot operation via intramolecular heteroannulation of in-situ-generated 2-(het)aryl-4-amino-5-functionalized thiazoles. These 4-amino-5-functionalized thiazoles were readily obtained in a one-pot process by treatment of a range of (het)aryldithioesters with cyanamide in the presence of NaH, followed by in situ S-alkylation-intramolecular condensations of the resulting thioimidate salts with appropriate activated methylene halides. On the other hand, the corresponding 4H-benzo[b]thiazolo[4,5-e][1,4]diazepin-10(9H)-ones were synthesized in a two-step process, requiring prior isolation of 5-carboethoxy-4-(2-nitrophenyl)aminothiazoles and their subsequent reductive cyclization. The activated methylene halides employed in these reactions for the synthesis of various thiazolo-fused heterocycles were methyl bromocrotonate, ethyl 2-(bromomethyl)benzoate, 2-fluorophenacyl bromides, ethyl 2-(2-bromoacetyl)benzoate, and ethyl bromoacetate. Several of these thiazolo-fused heterocycles display yellow green to green fluorescence, and their absorption and emission spectra have also been examined.

PCR Detection of Epstein-Barr Virus (EBV) DNA in Patients with Head and Neck Squamous Cell Carcinoma, in Patients with Chronic Tonsillitis, and in Healthy Individuals.

Epstein-Barr virus (EBV) is a common virus worldwide that is an etiologic agent in the development of many diseases, including cancer. Recent reports have shown the association of EBV with tumorigenesis in head and neck squamous cell carcinoma (HNSCC). Moreover, EBV has been reported to be present in tonsillar tissues, which suggests a close relationship between viral infections and tonsillar diseases, including chronic tonsillitis. The aim of the study was to analyze the prevalence of EBV DNA in 86 patients with HNSCC, in 70 patients with chronic tonsillitis, and in 144 healthy individuals (control group) and the associations between EBV infection and clinicopathological and demographic characteristics and the use of stimulants in all study groups. The objective of this study was also to analyze the prevalence of coinfection with human papillomavirus (HPV). After prior DNA isolation, EBV detection was performed using an EBV kit by real-time polymerase chain reaction. The prevalence of EBV infection in patients with HNSCC, patients with chronic tonsillitis, and the control group was 47.7%, 60%, and 24.3%, respectively. Compared to controls, a significantly higher prevalence of EBV in patients with chronic tonsillitis and HNSCC may suggest that EBV is a potential risk factor. No association was found between EBV infection and demographic or clinical data. Further studies are warranted due to inconclusive reports that were mainly related to geographic distribution, sample type, and detection technique. Considering the prevalence of the virus and the risk of serious diseases, attention should be paid to screening diagnosis and prevention of the infection.

Rate of methicillin-resistant Staphylococcus aureus in pediatric emergency departments in Spain

IntroductionStaphylococcus aureus is a common germ in bacterial infections in children. The rate of methicillin-resistant S. aureus (MRSA) is increasing lately. ObjectivesThe main aim is to know the rate of positive cultures to MRSA in Spanish pediatric emergency departments. The secondary aims are to analyse the risk factors for MRSA isolation (patient origin, history of hospitalization or surgery in the previous 90 days, antibiotherapy in the previous 60 days, presence of comorbidity, invasive devices, prior MRSA isolation) and to analyse the morbidity of these infections. MethodologyRetrospective multicenter study (07/01/2017–06/30/2018) with review of patient histories with isolation of S. aureus in samples of any origin obtained in 8 pediatric emergency departments of the Infectious Diseases Working Group of the Spanish Society of pediatric Emergencies. ResultsDuring this period, S. aureus was detected in 403 patients (average age 75.8 ± 59.2 months; 54.8% male): 28.8% hospital-related infections (HRI) and 71.2% community-related infections (CRI). Overall, MRSA rate was 16.6% (95% CI: 13−20.2%); 18.1% in HRI and 16.2% in CRI (p > 0.05). The highest rates of MRSA were obtained in skin abscesses (29.3%, CI 95%: 21.8−36.8%), patients not born in Spain (52%; CI 95%: 32−72%) or patients with a previous MRSA infection (90%; CI 95% 71.4−100%).167 (41%) patients were admitted, 12 (3%) had complications and 4 (1%) suffered sequels. There were no deaths. ConclusionsThe overall MRSA rate was one in 6 staphylococcal infections. Higher MRSA rates were detected in samples of suppurating skin injuries and in foreign children or in children with a history of previous MRSA infection. In suppurative skin lesions, early drainage is essential and the change to an antibiotic with MRSA coverage should be considered if the evolution is inadequate.

An imaging flow cytometry-based methodology for the analysis of single extracellular vesicles in unprocessed human plasma

Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. To rule out selection bias or introduction of artefacts caused by EV isolation techniques, we present a clinically feasible, imaging flow cytometry (IFCM)–based methodology to phenotype and determine the concentration of EVs with a diameter ≤400 nm in human platelet-poor plasma (PPP) without prior isolation of EVs. Instrument calibration (both size and fluorescence) were performed with commercial polystyrene beads. Detergent treatment of EVs was performed to discriminate true vesicular events from artefacts. Using a combination of markers (CFSE & Tetraspanins, or CD9 & CD31) we found that >90% of double-positive fluorescent events represented single EVs. Through this work, we provide a framework that will allow the application of IFCM for EV analysis in peripheral blood plasma in a plethora of experimental and potentially diagnostic settings. Additionally, this direct approach for EV analysis will enable researchers to explore corners of EVs as cellular messengers in healthy and pathological conditions.

Risk factor-based analysis of community-acquired pneumonia, healthcare-associated pneumonia and hospital-acquired pneumonia: Microbiological distribution, antibiotic resistance, and clinical outcomes.

BackgroundHealthcare-associated pneumonia (HCAP) lies in the intersection of community-acquired pneumonia (CAP) and hospital-acquired pneumonia (HAP). Although HCAP is excluded from the revised HAP guideline, reassessment for HCAP is needed considering its heterogeneous characteristics.MethodsThe microbiological distribution, antibiotic resistance, and clinical outcomes in CAP, HCAP, and HAP were studied retrospectively. The susceptibility to standard CAP regimens (β-lactams plus macrolide or fluoroquinolone monotherapy) and rates of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa) infections were evaluated in the CAP group and HCAP subgroups.ResultsIn total, 933 cases were included (CAP, n = 557; HCAP, n = 264; HAP, n = 112). In the CAP and HCAP cases, Streptococcus pneumoniae (7.4% vs. 5.7%) and P. aeruginosa (9.2% vs. 18.6%) were the most common gram-positive and gram-negative pathogens. Staphylococcus aureus (methicillin-resistant, 2.7%; methicillin-susceptible, 2.4%) and carbapenem-resistant Acinetobacter baumannii (20.5%) were the most common Gram-positive and Gram-negative pathogens in the HAP group, respectively. Higher susceptibility to levofloxacin was observed in CAP and HCAP isolates than that to β-lactam agents. However, levofloxacin non-susceptibility was significantly higher in long-term care facility (LTCF)-onset HCAP compared to community-onset HCAP (43.6% vs. 22.7%, P = 0.014).ConclusionHCAP showed higher rates of P. aeruginosa and MRSA infections than CAP. Empirical antipseudomonal therapy should be considered in the treatment of HCAP. Prior isolation of P. aeruginosa was the most important risk factor for P. aeruginosa infection.

Rapid and cost‐effective detection of Fusarium euwallaceae from woody tissues

AbstractThe polyphagous shot hole borer (PSHB) Euwallacea fornicatus is an ambrosia beetle that poses a serious threat to hundreds of tree species in invaded countries. In its invaded range, it relies on a mutualistic fungus, Fusarium euwallaceae, for colony establishment and nutrition. Together, the beetle–fungus complex can cause Fusarium dieback disease that can lead to the death of highly susceptible hosts. The key to mitigation of the spread and control of PSHB is its rapid detection. Current identification relies on DNA extraction and sequencing of barcoding genes of the beetle and/or the fungus, which is expensive and time‐consuming. Often the beetle is not present in a sample, and isolation and purification of the fungus is needed before employing molecular procedures. Because PSHB has a strong association with its fungal symbiont, at least in its invaded range, and the fungus can infect trees without successful PSHB colony establishment, a molecular tool that can rapidly detect F. euwallaceae from infected wood would aid monitoring efforts. Here we developed a PCR amplification protocol that targets a species‐specific gene present in F. euwallaceae from within infected wood of various hosts without prior fungal isolation, DNA purification or sequencing. The technique can detect F. euwallaceae within minimal woody material (c.15 mg) and even from degraded samples of many hosts. Additionally, identification of F. euwallaceae using lysate prepared from mycelia provided a reliable secondary measure. This method drastically reduces the time and costs associated with the identification of F. euwallaceae from field samples.

Ascorbate peroxidase-mediated in situ labelling of proteins in secreted exosomes.

The extracellular vesicle exosome mediates intercellular communication by transporting macromolecules such as proteins and ribonucleic acids (RNAs). Determining cargo contents with high accuracy will help decipher the biological processes that exosomes mediate in various contexts. Existing methods for probing exosome cargo molecules rely on a prior exosome isolation procedure. Here we report an in situ labelling approach for exosome cargo identification, which bypasses the exosome isolation steps. In this methodology, a variant of the engineered ascorbate peroxidase APEX, fused to an exosome cargo protein such as CD63, is expressed specifically in exosome‐generating vesicles in live cells or in secreted exosomes in the conditioned medium, to induce biotinylation of the proteins in the vicinity of the APEX variant for a short period of time. Mass spectrometry analysis of the proteins biotinylated by this approach in exosomes secreted by kidney proximal tubule‐derived cells reveals that oxidative stress can cause ribosomal proteins to accumulate in an exosome subpopulation that contains the CD63‐fused APEX variant.

Multiplexed mRNA analysis of brain-derived extracellular vesicles upon experimental stroke in mice reveals increased mRNA content with potential relevance to inflammation and recovery processes

Extracellular vesicles (EVs) are lipid bilayer-enclosed structures that represent newly discovered means for cell-to-cell communication as well as promising disease biomarkers and therapeutic tools. Apart from proteins, lipids, and metabolites, EVs can deliver genetic information such as mRNA, eliciting a response in the recipient cells. In the present study, we have analyzed the mRNA content of brain-derived EVs (BDEVs) isolated 72 h after experimental stroke in mice and compared them to controls (shams) using nCounter® Nanostring panels, with or without prior RNA isolation. We found that both panels show similar results when comparing upregulated mRNAs in stroke. Notably, the highest upregulated mRNAs were related to processes of stress and immune system responses, but also to anatomical structure development, cell differentiation, and extracellular matrix organization, thus indicating that regenerative mechanisms already take place at this time-point. The five top overrepresented mRNAs in stroke mice were confirmed by RT-qPCR and, interestingly, found to be full-length. We could reveal that the majority of the mRNA cargo in BDEVs was of microglial origin and predominantly present in small BDEVs (≤ 200 nm in diameter). However, the EV population with the highest increase in the total BDEVs pool at 72 h after stroke was of oligodendrocytic origin. Our study shows that nCounter® panels are a good tool to study mRNA content in tissue-derived EVs as they can be carried out even without previous mRNA isolation, and that the mRNA cargo of BDEVs indicates a possible participation in inflammatory but also recovery processes after stroke.

Wide antral circumferential re-ablation in case of recurrent atrial fibrillation despite of prior pulmonary vein isolation increases freedom from atrial tachyarrhythmias

Abstract Funding Acknowledgements Type of funding sources: None. Background Despite repeated pulmonary vein isolation (re-PVI) due to recurrent atrial fibrillation (AF) after PVI has been a standard procedure, the detailed ablation strategy in case of re-PVI remains disputable. Objective The aim of this study was to assess the efficacy of re-PVI using wide antral circumferential ablation (WACA) supported by high density mapping (HDM) for recurrent AF after PVI as compared to simple repeated PVI supported by circular mapping catheter. Methods Consecutive patients with recurrent AF after PVI were prospectively enrolled in this study, who underwent left atrial HDM and subsequently WACA antral (re-)isolation ("Re-WACA" group). The historical controls with re-PVI between 2016 and 2018 using circular mapping catheter, but without HDM were also enrolled ("control group"). These control patients underwent re-PVI with gap ablation at the pulmonary vein ostium. Primary endpoint was defined as any recurrence and ECG documentation of atrial tachyarrhythmias (AT) including AF or atrial tachycardias over 30 seconds. The patients were routinely followed up for 1 year with at least twice annual holter-monitoring. Results In total, 116 patients were enrolled in this study (Re-WACA group [N=56, 68±10 years], control group [N=58, 65±10 years]). There were no significant differences in clinical characteristics including the number of previous left atrial ablation procedures between two groups. In all 56 patients with Re-WACA, residual PV antral potentials were demonstrated (100%), whereas 7 patients (13%) showed no electrical potentials inside any PVs. During a mean follow-up period of 402±71 days, 6 out of 56 Re-WACA patients (11%) and 18 out of 58 controls (31%) experienced AT recurrences. Kaplan-Meier analysis demonstrated that the patients who underwent Re-WACA showed significantly lower AT recurrence after the index Re-PVI procedure as compared to the controls (log-rank, P = 0.010). Multivariate Cox regression showed that Re-WACA was an independent predictor of freedom from AT recurrence (hazard ratio = 0.39; 95% confidence-interval 0.16-0.93; P=0.034). The number of previous PVI procedures predicted AT recurrence during follow-up (hazard ratio = 2.35; 95% confidence-interval 1.20-4.46; P=0.010). Conclusions Residual pulmonary vein antral potential in patients with recurrent AF after previously performed PVI is a frequent finding. These antral potentials can be easily visualized by HDM. Repeated isolation of wide PV antrum (Re-WACA) is an effective strategy to reduce further AF recurrence as compared to conventional re-PVI without left atrial HDM.

One-Pot Syntheses of Substituted 2-Aminothiazoles and 2-Aminoselenazoles via Meerwein Arylation of Alkyl Vinyl Ketones

AbstractBoth one-pot and two-step procedures for the synthesis of substituted 2-aminothiazoles and 2-aminoselenazoles are described. Anilines are first converted into arenediazonium bromides, which are then reacted with methyl vinyl ketone or cyclopropyl vinyl ketone in the presence of copper(II) bromide to give 4-aryl-3-bromobutan-2-ones (40–71%) and 3-aryl-2-bromo-1-cyclopropylpropan-1-ones (41–79%), respectively. These products are reacted, without prior isolation, with thiourea or selenourea to prepare 4-methyl- and 4-cyclopropyl-5-(R-benzyl)thiazol-2-amines (14 examples) and their selenium analogs (14 examples). The yields of the one-pot procedure are higher (40–81%) than those of the two-step procedure (32–70%).