Evaluation of NG-Test CARBA 5 version 2, Cepheid Xpert Carba-R, and carbapenem inactivation methods in comparison to whole-genome sequencing for the identification of carbapenemases in non-fermenting Gram-negative bacilli.

NG-Test CARBA 5 (NG-Biotech) is a rapid in vitro multiplex immunoassay for the phenotypic detection and differentiation of the "big five" carbapenemase families (KPC, OXA-48-like, VIM, IMP, and NDM). Version 2 of this assay was evaluated alongside the Xpert Carba-R assay (Cepheid, Inc.), the modified carbapenem inactivation method (mCIM), and the CIMTris assay, with a collection of carbapenem-resistant non-fermenting Gram-negative bacilli comprising 138 Pseudomonas aeruginosa and 97 Acinetobacter baumannii isolates. Whole-genome sequencing (WGS) was used as the reference standard. For P. aeruginosa, NG-Test CARBA 5 produced an overall percentage agreement (OPA) with WGS of 97.1%, compared with 92.8% forXpert Carba-R and 90.6% for mCIM. For A. baumannii, as OXA-type carbapenemases (non-OXA-48) are not included, both the NG-Test CARBA 5 and Xpert Carba-R only had an OPA of 6.2%, while the CIMTris performed well with an OPA of 99.0%. The majority of A. baumannii isolates (95.9%) tested falsely positive for IMP on NG-Test CARBA 5; no IMP genes were found on WGS. No clear cause was found for this phenomenon; a cross-reacting protein antigen unique to A. baumannii is a possible culprit. NG-Test CARBA 5 performed well for carbapenemase detection in P. aeruginosa. However, results from A. baumannii isolates should be interpreted with caution.

Treatment outcomes with benzylpenicillin and non-benzylpenicillin antibiotics, and the performance of the penicillin zone-edge test versus molecular detection of blaZ in penicillin-susceptible Staphylococcus aureus (PSSA) bacteraemia.

OBJECTIVES: The blaZ gene encodes penicillinase, which inactivates penicillin. As there were reports on suboptimal sensitivity for the penicillin zone-edge test, a phenotypic method for blaZ detection, we investigated treatment outcomes in patients with penicillin-susceptible Staphylococcus aureus (PSSA) bacteraemia (phenotypically negative for penicillinase), subjecting isolates to molecular testing for blaZ retrospectively. PATIENTS AND METHODS: A retrospective cohort study was conducted on 121 patients with a first episode of PSSA bacteraemia from 1 January 2012 to 31 October 2015 at Tan Tock Seng Hospital (TTSH), Singapore. Patients were grouped into IV benzylpenicillin and non-benzylpenicillin groups. The primary outcome was overall treatment failure, defined as either 30 day all-cause mortality and/or 90 day relapse. The penicillin (P10) zone-edge test was repeated on archived PSSA isolates, concurrently with penicillin MIC determination via gradient diffusion and PCR for blaZ. RESULTS: Among 121 patients, 57 patients (47.1%) received IV benzylpenicillin as the predominant antibiotic. There was no significant difference in overall treatment failure between treatment with the benzylpenicillin [7/57 (12.3%)] versus non-benzylpenicillin groups [12/64 (18.8%)] (P = 0.33) or cloxacillin/cefazolin [6/37 (16.2%)] (P = 0.59). For 112 PSSA isolates available for testing, repeat penicillin zone-edge testing was negative for penicillinase production, corroborating previous results. A single PSSA isolate with a negative penicillin zone-edge test was found to be positive for blaZ. CONCLUSIONS: We found no differences in overall treatment failure between patients with PSSA bacteraemia treated with benzylpenicillin, anti-staphylococcal ß-lactams cefazolin/cloxacillin and other antimicrobials, when using the penicillin zone-edge test as the phenotypic method for blaZ screening.

Designer broad-spectrum polyimidazolium antibiotics.

For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive Staphylococcus aureus we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a site-directed menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen Pseudomonas aeruginosa, PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections.

Accuracy of a Rapid Multiplex Polymerase Chain Reaction Plus a Chromogenic Phenotypic Test Algorithm for Detection of Extended-Spectrum ß-Lactamase and Carbapenemase-Producing Gram-Negative Bacilli in Positive Blood Culture Bottles.

We studied the performance of an algorithm combining multiplex polymerase chain reaction with phenotypic detection of extended-spectrum ß-lactamases and carbapenemases directly from positive blood culture bottles in patients with gram-negative bacteremia and found good concordance with routine cultures. Such an algorithm may be a tool to improve time to optimal therapy in patients with gram-negative bacteremia.

Epidemiological and Clinical Predictors of COVID-19.

BACKGROUND: Rapid identification of COVID-19 cases, which is crucial to outbreak containment efforts, is challenging due to the lack of pathognomonic symptoms and in settings with limited capacity for specialized nucleic acid-based reverse transcription polymerase chain reaction (PCR) testing. METHODS: This retrospective case-control study involves subjects (7-98 years) presenting at the designated national outbreak screening center and tertiary care hospital in Singapore for SARS-CoV-2 testing from 26 January to 16 February 2020. COVID-19 status was confirmed by PCR testing of sputum, nasopharyngeal swabs, or throat swabs. Demographic, clinical, laboratory, and exposure-risk variables ascertainable at presentation were analyzed to develop an algorithm for estimating the risk of COVID-19. Model development used Akaike's information criterion in a stepwise fashion to build logistic regression models, which were then translated into prediction scores. Performance was measured using receiver operating characteristic curves, adjusting for overconfidence using leave-one-out cross-validation. RESULTS: The study population included 788 subjects, of whom 54 (6.9%) were SARS-CoV-2 positive and 734 (93.1%) were SARS-CoV-2 negative. The median age was 34 years, and 407 (51.7%) were female. Using leave-one-out cross-validation, all the models incorporating clinical tests (models 1, 2, and 3) performed well with areas under the receiver operating characteristic curve (AUCs) of 0.91, 0.88, and 0.88, respectively. In comparison, model 4 had an AUC of 0.65. CONCLUSIONS: Rapidly ascertainable clinical and laboratory data could identify individuals at high risk of COVID-19 and enable prioritization of PCR testing and containment efforts. Basic laboratory test results were crucial to prediction models.

A Glycosylated Cationic Block Poly(ß-peptide) Reverses Intrinsic Antibiotic Resistance in All ESKAPE Gram-Negative Bacteria.

Carbapenem-resistant Gram-negative bacteria (GNB) are heading the list of pathogens for which antibiotics are the most critically needed. Many antibiotics are either unable to penetrate the outer-membrane or are excluded by efflux mechanisms. Here, we report a cationic block ß-peptide (PAS8-b-PDM12) that reverses intrinsic antibiotic resistance in GNB by two distinct mechanisms of action. PAS8-b-PDM12 does not only compromise the integrity of the bacterial outer-membrane, it also deactivates efflux pump systems by dissipating the transmembrane electrochemical potential. As a result, PAS8-b-PDM12 sensitizes carbapenem- and colistin-resistant GNB to multiple antibiotics in vitro and in vivo. The ß-peptide allows the perfect alternation of cationic versus hydrophobic side chains, representing a significant improvement over previous antimicrobial α-peptides sensitizing agents. Together, our results indicate that it is technically possible for a single adjuvant to reverse innate antibiotic resistance in all pathogenic GNB of the ESKAPE group, including those resistant to last resort antibiotics.

Antecedent Carbapenem Exposure as a Risk Factor for Non-Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae and Carbapenemase-Producing Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae (CRE) can be mechanistically classified into carbapenemase-producing Enterobacteriaceae (CPE) and non-carbapenemase-producing carbapenem nonsusceptible Enterobacteriaceae (NCPCRE). We sought to investigate the effect of antecedent carbapenem exposure as a risk factor for NCPCRE versus CPE. Among all patients with CRE colonization and infection, we conducted a case-control study comparing patients with NCPCRE (cases) and patients with CPE (controls). The presence of carbapenemases was investigated with phenotypic tests followed by PCR for predominant carbapenemase genes. We included 843 unique patients with first-episode CRE, including 387 (45.9%) NCPCRE and 456 (54.1%) CPE. The resistance genes detected in CPEs were blaNDM (42.8%), blaKPC (38.4%), and blaOXA-48-like (12.1%). After adjusting for confounders and clustering at the institutional level, the odds of prior 30-day carbapenem exposure was three times higher among NCPCRE than CPE patients (adjusted odds ratio [aOR], 3.48; 95% confidence interval [CI], 2.39 to 5.09; P < 0.001). The odds of prior carbapenem exposure and NCPCRE detection persisted in stratified analyses by Enterobacteriaceae species (Klebsiella pneumoniae and Escherichia coli) and carbapenemase gene (blaNDM and blaKPC). CPE was associated with male gender (aOR, 1.45; 95% CI, 1.07 to 1.97; P = 0.02), intensive care unit stay (aOR, 1.84; 95% CI, 1.24 to 2.74; P = 0.003), and hospitalization in the preceding 1 year (aOR, 1.42; 95% CI, 1.01 to 2.02; P = 0.05). In a large nationwide study, antecedent carbapenem exposure was a significant risk factor for NCPCRE versus CPE, suggesting a differential effect of antibiotic selection pressure.

Pharmacokinetic/Pharmacodynamic Determinants of Vancomycin Efficacy in Enterococcal Bacteremia.

While pharmacokinetic-pharmacodynamic targets for vancomycin therapy are recognized for invasive methicillin-resistant Staphylococcus aureus infections, scant data are available to guide therapy for other Gram-positive infections. A retrospective single-center cohort of patients with Enterococcus bacteremia hospitalized between 1 January 2009 and 31 May 2015 were studied. The average vancomycin AUC0-24 was computed using a Bayesian approach. The MIC was determined by gradient diffusion (Etest; bioMérieux), and the average AUC0-24/MIC value over the initial 72 h of therapy was calculated. We assessed 30-day all-cause mortality as the primary outcome. Classification and regression tree analysis (CART) was used to identify the vancomycin AUC0-24/MIC value associated with 30-day mortality. Fifty-seven patients with enterococcal bacteremia (32 E. faecium, 21 E. faecalis, and 4 other Enterococcus spp.) were studied. The median vancomycin MIC was 0.75 mg/liter (range, 0.38 to 3 mg/liter). All-cause 30-day mortality occurred in 10 of 57 patients (17.5%). A CART-derived vancomycin AUC/MICEtest value of ≥389 was associated with reduced mortality (P = 0.017); failure to achieve this independently predicted 30-day mortality (odds ratio, 6.83 [95% confidence interval = 1.51 to 30.84]; P = 0.01). We found that a vancomycin AUC/MICEtest value of ≥389 achieved within 72 h was associated with reduced mortality. Larger, prospective studies are warranted to verify the vancomycin pharmacodynamic targets associated with maximal clinical outcomes and acceptable safety.

Clinical and Molecular Correlates of Escherichia coli Bloodstream Infection from Two Geographically Diverse Centers in Rochester, Minnesota, and Singapore.

Escherichia coli bacteremia is caused mainly by sequence type complex 131 (STc131) and two clades within its fluoroquinolone-resistance-associated H30 subclone, H30R1 and H30Rx. We examined clinical and molecular correlates of E. coli bacteremia in two geographically distinct centers. We retrospectively studied 251 unique E. coli bloodstream isolates from 246 patients (48 from the Mayo Clinic, Rochester, MN [MN], and 198 from Tan Tock Seng Hospital, Singapore [SG]), from October 2013 through March 2014. Isolates underwent PCR for phylogroup, STc, blaCTX-M type, and virulence gene profiles, and medical records were reviewed. Although STc131 accounted for 25 to 27% of all E. coli bacteremia isolates at each site, its extended-spectrum-ß-lactamase (ESBL)-associated H30Rx clade was more prominent in SG than in MN (15% versus 4%; P = 0.04). In SG only, patients with STc131 (versus other E. coli STc isolates) were more likely to receive inactive initial antibiotics (odds ratio, 2.8; P = 0.005); this was true specifically for patients with H30Rx (odds ratio, 7.0; P = 0.005). H30Rx comprised 16% of community-onset bacteremia episodes in SG but none in MN. In SG, virulence scores were higher for H30Rx than for H30R1, non-H30 STc131, and non-STc131 isolates (P < 0.02 for all comparisons). At neither site did mortality differ by clonal status. The ESBL-associated H30Rx clade was more prevalent and more often of community onset in SG, where it predicted inactive empirical treatment. The clonal distribution varies geographically and has potentially important clinical implications. Rapid susceptibility testing and clonal diagnostics for H30/H30Rx might facilitate earlier prescribing of active therapy.

Profiling non-tuberculous mycobacteria in an Asian setting: characteristics and clinical outcomes of hospitalized patients in Singapore.

BACKGROUND: Non-tuberculous mycobacteria (NTM) infection is an increasing problem worldwide. The epidemiology of NTM in most Asian countries is unknown. This study investigated the epidemiology, and clinical profile of inpatients in whom NTM was isolated from various anatomical sites in a Singaporean population attending a major tertiary referral centre. METHODS: Demographic profile, clinical data, and characteristics of patients hospitalized with NTM isolates at a major tertiary hospital over two-year period were prospectively assessed (2011-2012). Data collected included patient demographics, ethnicity, smoking status, co-morbidities, NTM species, intensive care unit (ICU) treatment, and mortality. RESULTS: A total of 485 patients (62.1% male) with 560 hospital admissions were analysed. The median patient age was 70 years. Thirteen different NTM species were isolated from this cohort. Mycobacterium abscessus (M. abscessus) (38.4%) was most frequently isolated followed by Mycobacterium fortuitum (M. fortuitum) (16.6%), Mycobacterium avium complex (MAC) (16.3%), Mycobacterium kansasii (M. kansasii) (15.4%), and Mycobacterium gordonae (M. gordonae) (6.8%). Most (91%) NTM was isolated from the respiratory tract. The three most common non-pulmonary sites were; blood (2.7%), skin wounds and abscesses (2.1%), and gastric aspirates (1.1%). A third (34.4%) of the study population had prior pulmonary tuberculosis (PTB). There was a significant association between isolated NTM species, and patient age (p = 0.0002). Eleven (2.2%) patients received intensive care unit (ICU) treatment during the study period and all cause mortality within 1 year of the study was 16.9% (n = 82). Of these, 72 (87.8%) patients died of pulmonary causes. CONCLUSIONS: The profile of NTM species in Singapore is unique. M. abscessus is the commonest NTM isolated, with a higher prevalence in males, and in the elderly. High NTM prevalence is associated with high rates of prior PTB in our cohort.

Clinical and Molecular Epidemiology of Carbapenem-Resistant Enterobacteriaceae Among Adult Inpatients in Singapore.

BACKGROUND: Since 2010, the incidence of carbapenem-resistant Enterobacteriaceae (CRE) has been increasing in Singapore. We analyzed the clinical and molecular epidemiology of CRE among adult inpatients in Singapore. METHODS: Quarterly incidence of unique subjects (per 100000 patient-days) with positive clinical and surveillance cultures for CRE were estimated based on mandatory data submitted to the National Public Health Laboratory by public hospitals between 2010 and 2015. CRE-positive adult inpatients were prospectively recruited from 6 public sector hospitals between December 2013 and April 2015. Subjects answered a standardized epidemiologic questionnaire and provided samples for this study. Further clinical information was extracted from subjects' electronic medical records. Whole-genome sequencing was performed on study isolates to determine transmission clusters. RESULTS: Incidence of CRE clinical cultures among adult inpatients plateaued from 2013 (range: 7.73 to 10.32 per 100000 patient-days) following an initial increase between 2010 and end-2012. We prospectively recruited 249 subjects. Their median age was 65 years, 108 (43%) were female, and 161 (64.7%) had carbapenemase-producing Enterobacteriaceae (CPE). On multivariate analysis, prior carbapenem exposure (OR: 3.23; 95% CI: 1.67-6.25) and hematological malignancies (OR: 2.85; 95% CI: 1.10-7.41) were associated with non-carbapenemase-producing CRE (NCPE) (n = 88) compared with CPE (n = 161) subjects. Among 430 CRE isolates from the 249 subjects, 307(71.3%) were CPE, of which 154(50.2%) were blaKPC-positive, 97(31.6%) blaNDM-positive, and 42 (13.7%) blaOXA-positive. Klebsiella pneumoniae (n = 180, 41.9%), Escherichia coli (n = 129, 30.0%) and Enterobacter cloacae (n = 62, 14.4%) were the main Enterobacteriaceae species. WGS (n = 206) revealed diverse bacterial strain type (STs). The predominant blaKPC-positive plasmid was pHS102707 (n = 62, 55.4%) and the predominant blaNDM-positive plasmid was pNDM-ECS01 (n = 46, 48.9%). Five transmission clusters involving 13 subjects were detected. CONCLUSIONS: Clinical CRE trend among adult inpatients showed stabilization following a rapid rise since introduction in 2010 potentially due to infection prevention measures and antimicrobial stewardship. More work is needed on understanding CPE transmission dynamics.

Black gram (Vigna Mungo L.) foliage supplementation to crossbred cows: effects on feed intake, nutrient digestibility and milk production.

OBJECTIVE: An experiment was conducted to examine the effect of dietary supplementation of dried and ground foliage of black gram (Vigna mungo L.) on feed intake and utilization, and production performance of crossbred lactating cows. METHODS: Eighteen lactating crossbred (Bos taurus×Bos indicus) cows (body weight 330.93± 10.82 kg) at their second and mid lactation (milk yield 6.77±0.54 kg/d) were randomly divided into three groups of six each in a completely randomized block design. Three supplements were formulated by quantitatively replacing 0, 50, and 100 per cent of dietary wheat bran of concentrate mixture with dried and ground foliage of black gram. The designated supplement was fed to each group with basal diet of rice straw (ad libitum) to meet the requirements for maintenance and milk production. Daily feed intake and milk yield was recorded. A digestion trial was conducted to determine the total tract digestibility of various nutrients. RESULTS: The daily feed intake was increased (p<0.05) with the supplementation of black gram foliage. Although the digestibility of dry matter, organic matter, crude protein, and ether extract did not vary (p>0.05), the fibre digestibility was increased (p<0.05), which ultimately improved (p<0.05) the total digestible nutrients content of composite diet. Although, the average milk yield (kg/animal/d) and composition did not differ (p>0.05) among the groups, milk yield was increased by 10 per cent with total replacement of wheat bran in concentrate mixture with of black gram foliage. The economics of milk production calculated as feed cost per kg milk yield (INR 10.61 vs 7.98) was reduced by complete replacement of wheat bran with black gram foliage. CONCLUSION: Black gram foliage could be used as complete replacement for wheat bran in concentrate mixture of dairy cows in formulating least cost ration for economic milk production in small holders' animal production.

Group B Streptococcus Sequence Type 283 Disease Linked to Consumption of Raw Fish, Singapore.

An outbreak of invasive group B Streptococcus (GBS) disease occurred in Singapore in mid-2015. We conducted a case-control study of 22 adults with invasive GBS infections during June 21-November 21, 2015. Consumption of raw fish was strongly associated with invasive sequence type 283 infections, but not with non-sequence type 283 infections.

In Vitro Activities of Ceftazidime-Avibactam, Aztreonam-Avibactam, and a Panel of Older and Contemporary Antimicrobial Agents against Carbapenemase-Producing Gram-Negative Bacilli.

Among 177 carbapenemase-producing Gram-negative bacilli (108 KPC, 32 NDM, 11 IMP, 8 OXA-48, 4 OXA-181, 2 OXA-232, 5 IMI, 4 VIM, and 3 SME producers), aztreonam-avibactam was active against all isolates except two NDM producers with elevated MICs of 8/4 and 16/4 mg/liter; ceftazidime-avibactam was active against all KPC-, IMI-, SME-, and most OXA-48 group-producing isolates (93%) but not metallo-ß-lactamase producers. Among older and contemporary antimicrobials, the most active were colistin, tigecycline, and fosfomycin, with overall susceptibilities of 88%, 79%, and 78%, respectively.

Influence of Condensed Tannins from Ficus bengalensis Leaves on Feed Utilization, Milk Production and Antioxidant Status of Crossbred Cows.

This study was conducted to examine the effects of condensed tannins (CT) from Ficus bengalensis leaves on the feed utilization, milk production and health status of crossbred cows. Eighteen crossbred dairy cows at their second and mid lactation (avg. BW 351.6±10.6 kg) were randomly divided into two groups of nine each in a completely randomized block design and fed two iso-nitrogenous supplements formulated to contain 0% and 1.5% CT through dried and ground leaves of Ficus bengalensis. The diets were designated as CON and FBLM, respectively and fed to cows with a basal diet of rice straw to meet requirements for maintenance and milk production. The daily milk yield was significantly (p<0.05) increased due to supplementation of FBLM diet. The 4% fat corrected milk yield was also significantly (p<0.01) higher due to increased (p<0.05) milk fat in cows under diet FBLM as compared to CON. The inclusion of CT at 1.5% in the supplement did not interfere with the feed intake or digestibility of DM, OM, CP, EE, NDF, and ADF by lactating cows. Digestible crude protein (DCP) and total digestible nutrients (TDN) values of the composite diets were comparable between the groups. The blood biochemical parameters remained unaltered except significantly (p<0.05) lowered serum urea concentration in cows fed FBLM diet. There was a significant (p<0.05) increase intracellular reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) activity in cows supplemented with condensed tannins. The total thiol group (T-SH) was found to be higher with reduction in lipid peroxidation (LPO) in cows of FBLM group. The cost of feeding per kg milk production was also reduced due to supplementation of Ficus bengalensis leaves. Therefore, a perceptible positive impact was evident on milk production and antioxidant status in crossbred cows during mid-lactation given supplement containing 1.5% CT through Ficus bengalensis leaves.

Cognitive facet and paradigm of the caregivers of children with developmental problems in Eastern India.

Background: The role of caregivers in grooming the neuro-developmental outcome of high-risk newborns and developmental challenges in children needs to be explored. Objectives: To find the knowledge and perception among parents regarding the neuro-developmental outcome of high-risk newborns, methods adopted to address these problems, and to identify areas on which awareness generation needs to focus. Materials and Methods: A questionnaire-based awareness survey was conducted to understand the knowledge, attitude, and practices of families of children with developmental challenges. Results: The study revealed that more than 70 percent of families lack information about child development, developmental challenges, and means to deal with them. They are unaware of the available health care services and other resources. One in three families has misconceptions on developmental disabilities; consider them as curse or jinx and consequently neglected. Female children with developmental problems are further ostracized due to gender inequity in families. About 10 percent of families have shown great openness toward acquiring new skills and knowledge for handling their children with developmental delays. Conclusions: This study is based on the précis research findings of our grass-root level fieldwork conducted in remote rural Bengal areas. The observation will be of interest and learning materials for general primary care practitioners, family physicians, and stakeholders to initiate appropriate intervention strategies for properly rehabilitating children with developmental delay at grass-root levels of primary health care.

Predictors of mortality in culture-proven Burkholderia pseudomallei infections in a Singapore Tertiary Hospital.

Burkholderia pseudomallei is a pathogen expanding in geographic range. We performed a retrospective study analyzing the clinical, microbiologic features of culture-proven melioidosis, and predictors of mortality based on data from a Singapore tertiary hospital between 2006- 2016. We found ICU admission, bacteremia, and mechanical ventilation to be associated with mortality.

Rapid Screening of Urinary Tract Infection Using Microfluidic Inertial-Impedance Cytometry.

Urinary tract infection (UTI) diagnosis based on urine culture for bacteriuria analysis is time-consuming and often leads to wastage of hospital resources due to false-positive UTI cases. Direct cellular phenotyping (e.g., RBCs, neutrophils, epithelial cells) of urine samples remains a technical challenge as low cell concentrations, and urine characteristics (conductivities, pH, microbes) can affect the accuracy of cell measurements. In this work, we report a microfluidic inertial-impedance cytometry technique for label-free rapid (<5 min) neutrophil sorting and impedance profiling from urine directly. Based on size-based inertial focusing effects, neutrophils are isolated, concentrated, and resuspended in saline (buffer exchange) to improve consistency in impedance-based single-cell analysis. We first observed that both urine pH and the presence of bacteria can affect neutrophil high-frequency impedance measurements possibly due to changes in nucleus morphology as neutrophils undergo NETosis and phagocytosis, respectively. As a proof-of-concept for clinical testing, we report for the first time, rapid UTI testing based on multiparametric impedance profiling of putative neutrophils (electrical size, membrane properties, and distribution) in urine samples from non-UTI (n = 20) and UTI patients (n = 20). A significant increase in cell count was observed in UTI samples, and biophysical parameters were used to develop a UTI classifier with an area under the receiver operating characteristic curve of 0.84. Overall, the developed platform facilitates rapid culture-free urine screening which can be further developed to assess disease severity in UTI and other urologic diseases based on neutrophil electrical signatures.