Routine Culture Research Articles

Practices to prevent central line-associated bloodstream infection: A 2021 survey of infection preventionists in US hospitals.

To determine prevalence of technical and behavioral interventions aimed at preventing central line-associated bloodstream infection (CLABSI) following the COVID19 pandemic. Cross-sectional survey. US acute care hospitals. Infection preventionists at participating hospitals. Surveys were sent to infection preventionists from a national random sample of 881 US acute care hospitals. Questions covered use of technical interventions to prevent CLABSI (eg, alcohol-containing chlorhexidine gluconate [CHG] for skin antisepsis, use of coated catheters), socio-adaptive interventions (eg, feedback of CLABSI rates, use of appropriateness criteria), and leadership support for CLABSI prevention. Survey response rate was 47% (415/881). Technical interventions such as maximal sterile barriers (99%) or CHG-impregnated dressings (92%) were highly prevalent, but routine use of CHG bathing was less common (68% indicated regular use in intensive care unit [ICU] vs 18% in non-ICU settings). Although 97% of respondents indicated use of systems to monitor CLABSI, feedback to providers on CLABSI events was reported by 89%. Only 53% of respondents indicated regular use of tools to determine appropriateness of central venous catheters (CVC). Three-quarters of respondents indicated their hospital assessed CVC necessity daily, but only 23% reported strategies to reduce routine blood cultures. CLABSI prevention was extremely important to hospital leadership at 82% of responding hospitals. Most US hospitals continue to use evidence-based methods to prevent CLABSI as recommended by leading organizations. Opportunities to focus on socio-adaptive interventions such as feedback of infection rates, use of appropriateness criteria for CVC placement, and improving the "culture of pan-culturing" remain.

Intact cell mass spectrometry coupled with machine learning reveals minute changes induced by single gene silencing

Intact (whole) cell MALDI TOF mass spectrometry is a commonly used tool in clinical microbiology for several decades. Recently it was introduced to analysis of eukaryotic cells, including cancer and stem cells. Besides targeted metabolomic and proteomic applications, the intact cell MALDI TOF mass spectrometry provides a sufficient sensitivity and specificity to discriminate cell types, isogenous cell lines or even the metabolic states. This makes the intact cell MALDI TOF mass spectrometry a promising tool for quality control in advanced cell cultures with a potential to reveal batch-to-batch variation, aberrant clones, or unwanted shifts in cell phenotype. However, cellular alterations induced by change in expression of a single gene has not been addressed by intact cell mass spectrometry yet. In this work we used a well-characterized human ovarian cancer cell line SKOV3 with silenced expression of a tumor suppressor candidate 3 gene (TUSC3). TUSC3 is involved in co-translational N-glycosylation of proteins with well-known global impact on cell phenotype. Altogether, this experimental design represents a highly suitable model for optimization of intact cell mass spectrometry and analysis of spectral data. Here we investigated five machine learning algorithms (k-nearest neighbors, decision tree, random forest, partial least squares discrimination, and artificial neural network) and optimized their performance either in pure populations or in two-component mixtures composed of cells with normal or silenced expression of TUSC3. All five algorithms reached accuracy over 90 % and were able to reveal even subtle changes in mass spectra corresponding to alterations of TUSC3 expression. In summary, we demonstrate that spectral fingerprints generated by intact cell MALDI-TOF mass spectrometry coupled to a machine learning classifier can reveal minute changes induced by alteration of a single gene, and therefore contribute to the portfolio of quality control applications in routine cell and tissue cultures.

Post-COVID reactivation of latent Bartonella henselae infection: a case report and literature review

Cat scratch disease (CSD) is caused by Bartonella henselae (B. henselae) and presents as lymphadenopathy following close contact with cats. However, in context of the global COVID-19 pandemic, clinical manifestations of CSD may vary, posing new challenges for healthcare professionals. Here we describe a case of a 54-year-old male with painful left upper arm mass, which gradually resolved until he was infected with COVID-19. The mass then rapidly progressed before admission. Meanwhile, pulmonary symptoms including pleural effusion emerged simultaneously. The cause was undetermined with routine blood culture and pathological test until the next generation sequencing (NGS) confirmed the presence of B. henselae. We believe this case is the first to report localized aggravation of CSD after COVID-19 infection and hopefully, offers treatment experience for clinicians worldwide.

The Headache of Post-Transurethral Prostate Surgery Pyuria: Pursuit for Evidence.

Objective: To prospectively assess early post-transurethral prostate surgery (TUPS) urinalysis changes and bacteriuria with its clinical relevance. Methods: Patients with benign prostate obstruction enrolled for TUPS were prospectively assessed. Patients were assessed at 2, 4, 8, 12, and 24 weeks postoperatively by the dysuria-visual-analogue-scale (DVAS), international prostate symptom scores (IPSS)-quality of life, uroflow, and postvoid residual. Routine urinalysis was performed before discharge and at all visits. Midstream urine culture (MSUC) was performed before discharge, and 4 and 12 weeks postoperatively. Results: At final analysis, 152 patients were evaluable. Significant pyuria was reported in 52%, 96.1%, 94.1%, 71.7%, 78.9%, and 52.5% in, before discharge, 2-, 4-, 8-, 12-, and 24-week urinalysis postoperative, respectively. The mean time to nonsignificant pyuria (95% confidence interval [CI]) was 19.1 (17.5-20.7), 20.1 (17.3-22.9), 15.8 (12.8-18.8), and 14 (10.3-17.8) weeks after prostate resection, vaporization, enucleation, and incision, respectively (p = 0.03). Regardless the TUPS technique, half of patients had significant pyuria at 24 weeks postoperative. MSUC was positive in 37/152 (24.3%), 3/152 (2%), 23/152 (15.1%), and 5/152 (3.3%) preoperatively, before discharge, and 4 and 12 weeks postoperative, respectively. Only positive preoperative urine leukocyte esterase independently predicted positive 4-week MSUC (odds ratio 3.8, 95% CI 1.3-11.1, p = 0.013). No significant correlation was found between IPSS or DVAS and positive MSUC, nor between IPSS and postoperative pyuria at different follow-up points (p > 0.05). However, the degree of postoperative dysuria was significantly correlated with postoperative pyuria count by urinalysis at 2 weeks (r = 0.69, p = 0.03), 8 weeks (r = 0.26, p = 0.001), and 12 weeks (r = 0.23, p = 0.004). Conclusion: There is a persistent but gradually declining pyuria and microhematuria following TUPS up to 6 months postoperative. An earlier resolution was noted following prostate incision and enucleation. While routine urine analysis screening in these months would be of no clear clinical value, a routine urine culture would be of a reasonable significance at 1 month postoperatively.

Evaluation of Biofire FilmArray Pneumonia Panel in Diagnosis of Pneumonia in Pediatric Patients in Intensive Care Unit in comparison to VITEK 2 Compact system and Routine Culture Methods

Evaluation of Biofire FilmArray Pneumonia Panel in Diagnosis of Pneumonia in Pediatric Patients in Intensive Care Unit in comparison to VITEK 2 Compact system and Routine Culture Methods

The significance of monitoring respiratory sample cultures and polymerase chain reaction tests for detecting bacterial pathogens in severely and critically ill patients with COVID-19

Background. Bacterial superinfection is one of the most common and potentially lethal complications in severely and critically ill patients with COVID-19. Objectives. To determine the colonisation time frame and the spectrum of potential bacterial pathogens in respiratory samples from patients with severe and critical COVID-19, using routine culture and polymerase chain reaction (PCR) tests. Methods. A prospective observational study was conducted on patients aged ≥18 years with confirmed severe and critical COVID-19 who were admitted to or transferred to the intensive care unit (ICU). Respiratory samples were collected for microbial culture and PCR testing within the first 2 days after ICU admission/transfer, between days 3 and 6, and after 7 days of ICU stay. Results. A total of 82 patients, with a median (interquartile range) age of 74.5 (67.3 - 81.0) years and a median Charlson comorbidity index of 4 (3 - 5), were enrolled in the study. Colonisation with any pathogen was observed in 67% of patients, after a median of 4 (2 - 6) days in the ICU. On days 0 - 2 of the ICU stay, micro-organisms were detected in 18% of patients, with Klebsiella pneumoniae (without acquired antibiotic resistance) and methicillin-susceptible Staphylococcus aureus being most frequently identified. Later, Acinetobacter baumannii and carbapenem-resistant K. pneumoniae became the predominant micro-organisms, identified in nearly half of the patients. In 74% of the samples, the results of microbial culture and PCR tests were identical. In 17%, PCR revealed bacterial pathogens not identified by culture. Conclusion. Our study confirms that colonisation of the respiratory tract occurs early in the course of ICU stay. Superinfections are predominantly caused by multidrug-resistant Gram-negative bacteria.

Des fenêtres culturelles à l’hôpital : susciter de nouvelles perspectives cliniques dans les établissements de santé

ContextThis article is based on doctoral research interested in the effects of “médiation culturelle” – the bi-directional interplay and effects of embedding a cultural experience or artistic process – on institutional behavior, environment, daily routine, and corporate culture. The specific focus here is an original institutional experience, entitled “Windows Open to the World,” that examines the effect of embedding an artist and artistic process into the everyday life of a regional rehabilitation center for patients with acquired neurologic disease or traumatic injury. This study was completed in partnership with the Biennale d’Art Contemporain of Lyon, via the Veduta platform and the artist/photographer Karim Kal, as part of the national Culture and Health program. MethodThis is exploratory research. In an institutional clinical setting, we present clinical observations and propose lines of reflection and analysis. ObjectivesThis study seeks to analyze the function of art on healthcare institutions (daily routine, corporate culture, environment) and professional practice. This study further explores the modulatory effect of embedding artistic and cultural experiences (“médiation culturelle”) on clinical staff, including the potential to improve providers’ feelings of well-being, renew professional interest and commitment, spark individual and team creativity, improve team cohesion, and reduce burn-out. ResultsAs an interventional device, the “médiation culturelle” process created new opportunities at the institutional level: in a dynamic interplay, embedding the artistic process created new common space and spontaneous gatherings; and time spent in discussion promoted collaboration between professionals, allowing new projects to come to life despite the current constraints of the institutional environment. The preparatory work required for any project, often undocumented and hidden from view, is shown by these results to be essential.These interdisciplinary projects are conducive to group work, prompting a review of and a strengthening of the team's approach to collaborative clinical care. Moreover, through the interactive creative nature of the artistic cultural intervention, these projects serve to generate enduring psychological interactions and emotional experiences between patients, artists, and professionals, ultimately forging more vibrant relationships through these shared experiences. These cultural interventions make it possible to reset and re-ignite the imagination in the institution, in support and advocacy of artistic creativity. Similarly, they rejuvenate the professional staff, stimulating new ideas, fostering creative exchange, and prompting collaboration with natural extensions to their clinical practice. These cultural projects can also serve as a kind of libidinal recharge for professionals, supporting their ability to generate and maintain patient motivation over time. InterpretationsIn the current context of deteriorating working conditions for health care professionals, psychologists within multidisciplinary teams are increasingly led to find creative means to support their colleagues’ work. Incorporating a system of artistic and cultural processes into institutional healthcare systems is a novel and innovative approach that humanizes the hospital environment, enriches the patient experience, and re-invigorates the professionals who work there.

Diagnosis of external ventricular drainage related infections with real-time 16S PCR and third-generation 16S sequencing

Objective Investigate the performance of real-time 16S PCR and third-generation 16S sequencing in the diagnosis of external ventricular drain related infections (EVDRI). Methods Subjects with suspected EVDRI were prospectively included at Uppsala University Hospital. Subjects were included into three groups: subjects with negative CSF culture with and without antibiotic treatment and subjects with positive CSF culture, respectively. CSF was analysed with real-time 16S PCR and third-generation 16S sequencing. Real-time 16S PCR positivity/negativity and number of 16S sequence reads were compared between groups. For culture positive subjects, species identification in third-generation sequencing and routine culture was compared. Results 84 subjects were included. There were 18, 44 and 22 subjects in the three groups. Real-time PCR was positive in 17 of 22 subjects in the culture positive group and negative in 61 of the 62 subjects in the two culture negative groups. The sensitivity and specificity for real-time 16S PCR compared to culture was estimated to 77% and 98%, respectively. Species identification in 16S sequencing and culture was concordant in 20 of 22 subjects. The number of 16S sequence reads were significantly higher in the culture positive group than in both culture negative groups (p < 0.001). There was no significant difference in number of 16S sequences between the two culture negative groups. Conclusions Real-time 16S PCR predict culture results with sufficient reliability. Third-generation 16S sequencing could enhance sensitivity and species identification in diagnostics of EVD-related infections. False negative culture results appear to be uncommon in patients with suspected EVDRI.

A LAMP point-of-care test to guide antimicrobial choice for treatment of Staphylococcus pseudintermedius pyoderma in dogs

Staphylococcus pseudintermedius is the most common cause of pyoderma in dogs. We validated a point-of-care (PoC) test based on colorimetric loop-mediated isothermal amplification (LAMP) for rapid S. pseudintermedius identification and susceptibility testing for first line antimicrobials for systemic treatment of canine pyoderma, i.e., lincosamides, first generation cephalosporins and amoxicillin clavulanate. Newly designed LAMP primers targeting clinically relevant resistance genes were combined with a previously validated set of primers targeting spsL for species identification. After laboratory validation on 110 clinical isolates, we assessed the performance of the test on 101 clinical specimens using routine culture and susceptibility testing as a reference standard. The average hands-on and turnaround times for the PoC test were 30 and 90 min, respectively. The assay showed sensitivity and specificity near 100% for both species identification and susceptibility testing when performed on bacterial cultures or clinical specimens in the laboratory. However, the PoC test yielded less accurate results when performed on-site by clinical staff (92% sensitivity and 64% specificity for species identification, 67% sensitivity and 96% specificity for β-lactam susceptibility, and 83% sensitivity and 71% specificity for lincosamide susceptibility). These results indicate that the PoC test should be adapted to a user-friendly technology to facilitate performance and interpretation of results by clinical staff. If properly developed, the test would allow veterinarians to gain rapid information on antimicrobial choice, limiting the risk of treatment failure and facilitating adherence to antimicrobial use guidelines in small animal veterinary dermatology.

PI3K/Akt/Erk signaling pathway mediates neuroprotection of CaMKⅡγ and CaMKⅡδ against ischemic reperfusion injury in mice

To observe neuroprotective effects of Ca2+/calmodulin-dependent kinase Ⅱ (CaMK Ⅱ)γ and CaMkII δ against acute neuronal ischemic reperfusion injury in mice and explore the underlying mechanism. Primary cultures of brain neurons isolated from fetal mice (gestational age of 18 days) were transfected with two specific siRNAs (si-CAMK2G and si-CAMK2D) or a control sequence (si-NT). After the transfection, the cells were exposed to oxygen-glucose deprivation/reperfusion (OGD/R) conditions for 1 h followed by routine culture. The expressions of phosphatidylinositol-3-kinase/extracellular signal-regulated kinase (PI3K/Akt/Erk) signaling pathway components in the neurons were detected using immunoblotting. The expressions of the PI3K/Akt/Erk signaling pathway proteins were also detected in the brain tissues of mice receiving middle cerebral artery occlusion (MCAO) or sham operation. The neuronal cells transfected with siCAMK2G showed significantly lower survival rates than those with si-NT transfection at 12, 24, 48, and 72 h after OGD/R (P < 0.01), and si-CAMK2G transfection inhibited OGD/R-induced upregulation of CaMKⅡγ expression. Compared to si-NT, transfection with si-CAMK2G and si-CAMK2D both significantly inhibited the expressions of PI3K/Akt/Erk signaling pathway components (P < 0.01). In the mouse models of MCAO, the expressions of CaMKⅡδ and CaMKⅡγ were significantly increased in the brain, where activation of the PI3K/Akt/Erk signaling pathway was detected. The expression levels of CaMKⅡδ, CaMKⅡγ, Erk, phosphorylated Erk, Akt, and phosphorylated Akt were all significantly higher in MCAO mice than in the sham-operated mice at 24, 48, 72, and 96 h after reperfusion (P < 0.05). The neuroprotective effects of CaMKⅡδ and CaMKⅡγ against acute neuronal ischemic reperfusion injury are mediated probably by the PI3K/Akt/Erk pathway.

Optimizing practices to prevent urinary tractinfection after cystoscopy and urodynamics in women: A quality improvement study.

The objective of this study was to reduce the incidence of urinary tract infection (UTI) in women undergoing outpatient cystoscopy and/or urodynamic studies (UDS) at our centre by identifying and then altering modifiable risk factors through an analysis of incidence variability among physicians. This was a quality improvement study involving adult women undergoing outpatient cystoscopy and/or UDS at an academic tertiary urogynecology practice. Prophylactic practices for cystoscopy/UDS were surveyed and division and physician-specific UTI rates following cystoscopy/UDS were established. In consultation with key stakeholders, this delineated change concepts based on associations between prophylactic practices and UTI incidence, which were then implemented while monitoring counterbalance measures. Two "Plan-Do-Study-Act-Cycles" were conducted whereby 212 and 210 women were recruited, respectively. Change concepts developed and implemented were: (1) to perform routine urine cultures at the time of these outpatient procedures, and (2) to withhold routine prophylactic antibiotics for outpatient cystoscopy/UDS, except in patients with signs of cystitis. There was no change in the incidence of early presenting UTI (9.0% vs. 9.2%, p = 0.680), but there were significantly fewer antibiotic-related adverse events reported (8.5% vs. 1.5%, p = 0.001). There was no significant change in the total incidence of UTI rates between cycles (7.8% vs. 5.6%, p = 0.649). No specific strategies to decrease the incidence of UTI following outpatient cystoscopy/UDS were identified, however, risk factor-specific antibiotic prophylaxis, as opposed to universal antibiotic prophylaxis, did not increase UTI incidence.

Clinical utility of multiplex PCR in the detection of pathogens from sterile body fluids.

The identification of pathogens in normally sterile body fluid (NSBF) is performed using routine culture, the current gold standard. Limitations of this method include sensitivity and increased turnaround times which could potentially delay vital patient care, especially antimicrobial therapy. Adaptations of NSBF in blood culture bottles prompted us to consider the utility of additional methods to bridge the gap in diagnostic challenges for these life-threatening infections. Multiplex molecular panels have been manufactured for use with multiple specimen types including blood, cerebral spinal fluid, stool, and respiratory. Therefore, the purpose of this study was to evaluate the off-label use of ePlex Blood Culture Identification Panels on positive body fluids grown in blood culture bottles for the detection of microorganisms for research purposes.

Yenidoğanlarda Uzamış Sarılıkta İdrar Yolu Enfeksiyonunun Sıklığı ve Rolü

Objective: The study aims to investigate the frequency and role of urinary tract infection (UTI) in prolonged jaundice in preterm and term neonates.&#x0D; Materials and Methods: This retrospective study occurred at Zeynep Kamil Maternity and Children's Hospital in Istanbul between January 2014 and April 2018. The study involved 391 neonates who presented to our outpatient clinics with prolonged jaundice. UTI was defined as urine culture growth of at least 10.000 colony-forming units per millilitre (CFU/ml) in samples obtained via urethral catheterization. Birth weight, sex, gestational age, chronological age, laboratory results, hospital courses of patients, ultrasound findings and phototherapy history were recorded. &#x0D; Results: UTI frequency was found to be 2.8% in 391 neonates with prolonged jaundice. Nine of them were male (81.8%), and 2 were female (18.2%). Eight patients with UTI (72.7%) were term and 3 (29.3%) were preterm. History of phototherapy, presence of leukocyturia or nitrituria, white blood cell count, and thrombocyte counts were significantly higher in the UTI group.&#x0D; Conclusion: Prolonged jaundice may be the first sign of UTI in neonates. Routine urine culture in neonates with prolonged jaundice may be useful, especially in those with a history of phototherapy and high white blood cell and thrombocyte counts.

Oleuropein activates autophagy to circumvent anti-plasmodial defense

Antimalarial drug resistance and unavailability of effective vaccine warrant for newer drugs and drug targets. Hence, anti-inflammatory activity of phyto-compound (oleuropein; OLP) was determined in antigen (LPS)-stimulated human THP-1 macrophages (macrophage model of inflammation; MMI). Reduction in the inflammation was controlled by the PI3K-Akt1 signaling to establish the "immune-homeostasis." Also, OLP treatment influenced the cell death/autophagy axis leading to the modulated inflammation for extended cell survival. The findings with MII prompted us to detect the antimalarial activity of OLP in the wild type (3D7), D10-expressing GFP-Atg18 parasite, and chloroquine-resistant (Dd2) parasite. OLP did not show the parasite inhibition in the routine invitro culture of P.falciparum whereas OLP increased the antimalarial activity of artesunate. The molecular docking of autophagy-related proteins, investigations with MMI, and parasite inhibition assays indicated that the host activated the autophagy to survive OLP pressure. The challenge model of P.berghei infection showed to induce autophagy for circumventing anti-plasmodial defenses.

In planta transformation in wheat: an improved protocol to develop wheat transformants.

Lack of efficient transformation protocol continues to be a major bottleneck for successful genome editing or transgenic development in wheat. An in planta transformation method was developed in Indian bread wheat in earlier study (Vasil et al. in Nat Biotechnol 10:667-674, 1992) which was labour-intensive and time-consuming. In the present study, in planta transformation method was improved to make it simple, efficient, less labour-intensive and time-saving. PCR-based screening for generated transformants at T0 stage was introduced in this method. Shoot apical meristem of two days old wheat seedling was inoculated with the routine active culture of Agrobacterium tumefaciens harboring plasmid pCAMBIA1300-Ubi-GFP having gene GFP under the control of Zea mays ubiquitin promoter. PCR analysis at T0 stage confirmed 27 plants to be transgene positive. These 27 plants were only taken to the next generation (T1) and the rest were discarded. At T1 generation 6 plants were analyzed to be PCR positive. Out of them, 4 plants were confirmed to have stable integration of transgene (GFP). Fluorescent microscopy at T1 stage confirmed the 4 Southern hybridization positive plants to be expressing reporter gene GFP. Screening at T0 stage, reduced the load of plants to be taken to T1 generation and their screening thereof at T1 with no overall loss in transformation efficiency. We successfully transformed wheat genotype HD2894 with 3.33% transformation efficiency using a simple, effective method which was less labour-intensive and less time-consuming. This method may be utilized to develop wheat transgenic as well as genome edited lines for desirable traits.

Uncovering the spread of drug-resistant bacteria through next-generation sequencing based surveillance: transmission of extended-spectrum β-lactamase-producing Enterobacterales by a contaminated duodenoscope

Contamination of duodenoscopes is a significant concern due to the transmission of multidrug-resistant organisms (MDROs) among patients who undergo endoscopic retrograde cholangiopancreatography (ERCP), resulting in outbreaks worldwide. In July 2020, it was determined that three different patients, all had undergone ERCP with the same duodenoscope, were infected. Two patients were infected with blaCTX-M-15 encoding Citrobacter freundii, one experiencing a bloodstream infection and the other a urinary tract infection, while another patient had a bloodstream infection caused by blaSHV-12 encoding Klebsiella pneumoniae. Molecular characterization of isolates was available as every ESBL-producing isolate undergoes Next-Generation Sequencing (NGS) for comprehensive genomic analysis in our center. After withdrawing the suspected duodenoscope, we initiated comprehensive epidemiological research, encompassing case investigations, along with a thorough duodenoscope investigation. Screening of patients who had undergone ERCP with the implicated duodenoscope, as well as a selection of hospitalized patients who had ERCP with a different duodenoscope during the outbreak period, led to the discovery of three additional cases of colonization in addition to the three infections initially detected. No microorganisms were detected in eight routine culture samples retrieved from the suspected duodenoscope. Only after destructive dismantling of the duodenoscope, the forceps elevator was found to be positive for blaSHV-12 encoding K. pneumoniae which was identical to the isolates detected in three patients. This study highlights the importance of using NGS to monitor the transmission of MDROs and demonstrates that standard cultures may fail to detect contaminated medical equipment such as duodenoscopes.

Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection

BackgroundThe timely and accurate diagnosis of bloodstream infection (BSI) is critical for patient management. With longstanding challenges for routine blood culture, metagenomics is a promising approach to rapidly provide sequence-based detection and characterisation of bloodborne bacteria. Long-read sequencing technologies have successfully supported the use of clinical metagenomics for syndromes such as respiratory illness, and modified approaches may address two requisite factors for metagenomics to be used as a BSI diagnostic: depletion of the high level of host DNA to then detect the low abundance of microbes in blood.MethodsBlood samples from healthy donors were spiked with different concentrations of four prevalent causative species of BSI. All samples were then subjected to a modified saponin-based host DNA depletion protocol and optimised DNA extraction, whole genome amplification and debranching steps in preparation for sequencing, followed by bioinformatical analyses. Two related variants of the protocol are presented: 1mL of blood processed without bacterial enrichment, and 5mL of blood processed following a rapid bacterial enrichment protocol—SepsiPURE.ResultsAfter first identifying that a large proportion of host mitochondrial DNA remained, the host depletion process was optimised by increasing saponin concentration to 3% and scaling the reaction to allow more sample volume. Compared to non-depleted controls, the 3% saponin-based depletion protocol reduced the presence of host chromosomal and mitochondrial DNA < 106 and < 103 fold respectively. When the modified depletion method was further combined with a rapid bacterial enrichment method (SepsiPURE; with 5mL blood samples) the depletion of mitochondrial DNA improved by a further > 10X while also increasing detectable bacteria by > 10X. Parameters during DNA extraction, whole genome amplification and long-read sequencing were also adjusted, and subsequently amplicons were detected for each input bacterial species at each of the spiked concentrations, ranging from 50–100 colony forming units (CFU)/mL to 1–5 CFU/mL.ConclusionIn this proof-of-concept study, four prevalent BSI causative species were detected in under 12 h to species level (with antimicrobial resistance determinants) at concentrations relevant to clinical blood samples. The use of a rapid and precise metagenomic protocols has the potential to advance the diagnosis of BSI.

Comparison of the Simplexa GBS Direct and ARIES GBS assays for the detection of S. agalactiae in broth-enriched swab specimens.

We conducted a comparative evaluation of the FDA-cleared Simplexa GBS Direct and ARIES GBS molecular assays for the detection of Streptococcus agalactiae (Group B Streptococcus, GBS) in 386 prospectively collected, broth-enriched vaginal/rectal swab specimens. The sensitivity of each test was 96.2% and specificity was ≥98.7% when compared to a combined direct and enriched culture method using chromogenic culture medium. A total of four specimens were called positive by both molecular assays but negative by culture, likely representing specimens with a low burden of GBS in these specimens. Two specimens were reported positive by culture but negative by both molecular assays. One of these specimens demonstrated atypically colored colonies on chromogenic agar; the other yielded typically colored colonies only observed after broth enrichment. Our data demonstrate equivalent performance of Simplexa and ARIES molecular assays for the detection of GBS in clinical specimens.IMPORTANCEClinical laboratories often face decisions regarding which of the multiple available molecular platforms would best fit their needs based on cost, workflow, menu, and diagnostic performance. Therefore, objective clinical comparisons of similar molecular tests are valuable resources to aid these decisions. We provide a clinical comparison of two FDA-cleared tests to routine culture and to each other that can be used by clinical laboratories when determining which of the available molecular platforms would best fit their laboratory in terms of workflow, cost, and performance.

Potential to grow carp oedema virus (genogroup I) in monolayers of carp-derived primary cells with further implication in cell analysis.

Carp oedema virus (CEV) has distinct molecularly identified genogroups of viral mutations, denoted as I, IIa, and IIb. Failure to propagate CEV invitro limits studies towards understanding its interactions with host cells. Here, virus isolates belonging to genogroup I collected during natural outbreaks in the Czech Republic were employed for routine CEV cultivation in monolayers of carp-derived primary cells, common carp brain (CCB) cells, and epithelioma papulosum cyprinid (EPC) cells. Induction of cytopathic effects (CPEs) was observed and recorded in affected cells. Cell survival rate was evaluated under serial dilutions of the CEV inoculum. Virus cell entry was quantified and visualized by qPCR and transmission electron microscopy, respectively. Study findings indicate primary gills epithelia likely present the most suitable matrix for CEV growth invitro. Cells of the head kidney and spleen facilitate virus entry with microscopically confirmed CPEs and the presence of cytoplasmic pleomorphic virus particles. Cells of the trunk kidney and gonads are unlikely to permit virus cell entry and CPEs development. Although CEV cultivation in cell lines was inconclusive, EPC cells were CEV permissible. Monolayers of carp-derived primary cells show promise for CEV cultivation that could enable elaborate study of mechanisms underlying cellular binding and responses.

Effect of Piper Cubeba Extract on Biofim Producing Pseudomonas Aeruginosa Isolated from Clinical Samples

Background: The species of Pseudomonas aeruginosa is a nosocomial bacterium that increases the risk of multidrug-resistant infections, especially in vulnerable patients. The plant Piper cubeba, commonly referred to as cubeb or tailed pepper, it is specifically cultivated for its fruit and essential oil. The plant belongs to the genus Piper. The study aims at assessing the effectiveness of Piper cubeba extract against different P. aeruginosa strains that could produce biofilm and carry blaIMP gene. Method: Isolates from 85 samples (17 sputum, 40 urine and 28 diarrhea samples). Collected from Al Nasiriyah Teaching Hospital during the period of December 2021 to March 2022. Samples were cultivated using routine cultivation methods, catalase test, oxidase reaction, and the Api20E system were performed to identify P. aeruginosa. Results: Out of a total of 17 isolated samples of Pseudomonas aeruginosa, 3 (17.64%) were from sputum, 6(35.29%) from urine, and 8 (47.05%) diarrhea samples. Out of 17 isolates 6 were sensitive in stool isolates 4 isolates were moderate sensitive and 7 isolates were resistance. Conclusion at concentrations of 25, 50, 75, and 100 mg/mL for Piper cubeba, respectively, the greatest MBC of plant extracts was noted. Also, all the isolates were capable of producing a biofilm by using presterilized 96-well polystyrene microtiter plates. Out of 17 isolates, 9 had a potent biofilm ability and the remaining 7 were moderate biofilm producers. Additionally, 7 of the isolates (41.17%) carried the blaIMP gene. Conclusion: Piper cubeba extract has potential as an antimicrobial agent against both sensitive and moderately resistant strains of Pseudomonas aeruginosa. This antimicrobial effect becomes more pronounced with an increase in the concentration of the extract. Additionally, Pseudomonas aeruginosa that produce biofilms tend to consistently produce more Metallo-beta-lactamase (MBL), which can further contribute to antibiotic resistance.