Optimizing DNA Extraction Methods for Nanopore Sequencing of Neisseria gonorrhoeae Directly from Urine Samples.

Empirical gonorrhea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance. We investigated if Nanopore sequencing can detect sufficient Neisseria gonorrhoeae DNA to reconstruct whole genomes directly from urine samples. We used N. gonorrhoeae-spiked urine samples and samples from gonorrhea infections to determine optimal DNA extraction methods that maximize the amount of N. gonorrhoeae DNA sequenced while minimizing contaminating host DNA. In simulated infections, the Qiagen UCP pathogen mini kit provided the highest ratio of N. gonorrhoeae to human DNA and the most consistent results. Depletion of human DNA with saponin increased N. gonorrhoeae yields in simulated infections but decreased yields in clinical samples. In 10 urine samples from men with symptomatic urethral gonorrhea, ≥92.8% coverage of an N. gonorrhoeae reference genome was achieved in all samples, with ≥93.8% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections, if ≥104 CFU/ml of N. gonorrhoeae was present, sequencing of the large majority of the genome was frequently achieved. N. gonorrhoeae could also be detected from urine in cobas PCR medium tubes and from urethral swabs and in the presence of simulated Chlamydia coinfection. Using Nanopore sequencing of urine samples from men with urethral gonorrhea, sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.

A Candida auris Outbreak and Its Control in an Intensive Care Setting.

BACKGROUND: Candida auris is an emerging and multidrug-resistant pathogen. Here we report the epidemiology of a hospital outbreak of C. auris colonization and infection. METHODS: After identification of a cluster of C. auris infections in the neurosciences intensive care unit (ICU) of the Oxford University Hospitals, United Kingdom, we instituted an intensive patient and environmental screening program and package of interventions. Multivariable logistic regression was used to identify predictors of C. auris colonization and infection. Isolates from patients and from the environment were analyzed by whole-genome sequencing. RESULTS: A total of 70 patients were identified as being colonized or infected with C. auris between February 2, 2015, and August 31, 2017; of these patients, 66 (94%) had been admitted to the neurosciences ICU before diagnosis. Invasive C. auris infections developed in 7 patients. When length of stay in the neurosciences ICU and patient vital signs and laboratory results were controlled for, the predictors of C. auris colonization or infection included the use of reusable skin-surface axillary temperature probes (multivariable odds ratio, 6.80; 95% confidence interval [CI], 2.96 to 15.63; P<0.001) and systemic fluconazole exposure (multivariable odds ratio, 10.34; 95% CI, 1.64 to 65.18; P=0.01). C. auris was rarely detected in the general environment. However, it was detected in isolates from reusable equipment, including multiple axillary skin-surface temperature probes. Despite a bundle of infection-control interventions, the incidence of new cases was reduced only after removal of the temperature probes. All outbreak sequences formed a single genetic cluster within the C. auris South African clade. The sequenced isolates from reusable equipment were genetically related to isolates from the patients. CONCLUSIONS: The transmission of C. auris in this hospital outbreak was found to be linked to reusable axillary temperature probes, indicating that this emerging pathogen can persist in the environment and be transmitted in health care settings. (Funded by the National Institute for Health Research Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at Oxford University and others.).

Sonication versus Tissue Sampling for Diagnosis of Prosthetic Joint and Other Orthopedic Device-Related Infections.

Current guidelines recommend collection of multiple tissue samples for diagnosis of prosthetic joint infections (PJI). Sonication of explanted devices has been proposed as a potentially simpler alternative; however, reported microbiological yield varies. We evaluated sonication for diagnosis of PJI and other orthopedic device-related infections (DRI) at the Oxford Bone Infection Unit between October 2012 and August 2016. We compared the performance of paired tissue and sonication cultures against a "gold standard" of published clinical and composite clinical and microbiological definitions of infection. We analyzed explanted devices and a median of five tissue specimens from 505 procedures. Among clinically infected cases the sensitivity of tissue and sonication culture was 69% (95% confidence interval, 63 to 75) and 57% (50 to 63), respectively (P < 0.0001). Tissue culture was more sensitive than sonication for both PJI and other DRI, irrespective of the infection definition used. Tissue culture yield was higher for all subgroups except less virulent infections, among which tissue and sonication culture yield were similar. The combined sensitivity of tissue and sonication culture was 76% (70 to 81) and increased with the number of tissue specimens obtained. Tissue culture specificity was 97% (94 to 99), compared with 94% (90 to 97) for sonication (P = 0.052) and 93% (89 to 96) for the two methods combined. Tissue culture is more sensitive and may be more specific than sonication for diagnosis of orthopedic DRI in our setting. Variable methodology and case mix may explain reported differences between centers in the relative yield of tissue and sonication culture. Culture yield was highest for both methods combined.

Gonorrhoea treatment failure caused by a Neisseria gonorrhoeae strain with combined ceftriaxone and high-level azithromycin resistance, England, February 2018.

We describe a gonorrhoea case with combined high-level azithromycin resistance and ceftriaxone resistance. In February 2018, a heterosexual male was diagnosed with gonorrhoea in the United Kingdom following sexual intercourse with a locally resident female in Thailand and failed treatment with ceftriaxone plus doxycycline and subsequently spectinomycin. Resistance arose from two mechanisms combining for the first time in a genetic background similar to a commonly circulating strain. Urgent action is essential to prevent further spread.

Use of an automated blood culture system (BD BACTEC™) for diagnosis of prosthetic joint infections: easy and fast.

BACKGROUND: For the diagnosis of prosthetic joint infection (PJI) automated BACTEC™ blood culture bottle methods have comparable sensitivity, specificity and a shorter time to positivity than traditional cooked meat enrichment broth methods. We evaluate the culture incubation period required to maximise sensitivity and specificity of microbiological diagnosis, and the ability of BACTEC™ to detect slow growing Propionibacteria spp. METHODS: Multiple periprosthetic tissue samples taken by a standardised method from 332 patients undergoing prosthetic joint revision arthroplasty were cultured for 14 days, using a BD BACTEC™ instrumented blood culture system, in a prospective study from 1st January to 31st August 2012. The "gold standard" definition for PJI was the presence of at least one histological criterion, the presence of a sinus tract or purulence around the device. Cases where > =2 samples yielded indistinguishable isolates were considered culture-positive. 1000 BACTEC™ bottle cultures which were negative after 14 days incubation were sub-cultured for Propionibacteria spp. RESULTS: 79 patients fulfilled the definition for PJI, and 66 of these were culture-positive. All but 1 of these 66 culture-positive cases of PJI were detected within 3 days of incubation. Only one additional (clinically-insignificant) Propionibacterium spp. was identified on terminal subculture of 1000 bottles. CONCLUSIONS: Prolonged microbiological culture for 2 weeks is unnecessary when using BACTEC™ culture methods. The majority of clinically significant organisms grow within 3 days, and Propionibacteria spp. are identified without the need for terminal subculture. These findings should facilitate earlier decisions on final antimicrobial prescribing.

Miniature multimode monolithic flextensional transducers.

Traditional flextensional transducers classified in seven groups based on their designs have been used extensively in 1-100 kHz range for mine hunting, fish finding, oil explorations, and biomedical applications. In this study, a new family of small, low cost underwater, and biomedical transducers has been developed. After the fabrication of transducers, finite-elements analysis (FEA) was used extensively in order to optimize these miniature versions of high-power, low-frequency flextensional transducer designs to achieve broad bandwidth for both transmitting and receiving, engineered vibration modes, and optimized acoustic directivity patterns. Transducer topologies with various shapes, cross sections, and symmetries can be fabricated through high-volume, low-cost ceramic and metal extrusion processes. Miniaturized transducers posses resonance frequencies in the range of above 1 MHz to below 10 kHz. Symmetry and design of the transducer, polling patterns, driving and receiving electrode geometries, and driving conditions have a strong effect on the vibration modes, resonance frequencies, and radiation patterns. This paper is devoted to small, multimode flextensional transducers with active shells, which combine the advantages of small size and low-cost manufacturing with control of the shape of the acoustic radiation/receive pattern. The performance of the transducers is emphasized.

Short ultrasound exposure times for noninvasive insulin delivery in rats using the lightweight cymbal array.

The purpose of this study is to demonstrate the feasibility of using short ultrasound exposure times to noninvasively deliver insulin with a lightweight (<22 g), low-profile (37 x 37 x 7 mm3) cymbal array (f = 20 kHz). Using hyperglycemic rats, previous experiments using the array demonstrated that blood glucose would decrease approximately 250 mg/dl from 60 and 20 minutes of pulsed ultrasound exposure for transdermal insulin delivery. Using a similar intensity (Isptp = 100 mW/cm2, 20% duty cycle), the goal was to determine if the same effect can be achieved with only 5 minutes of ultrasound exposure. For these experiments, 20 Sprague Dawley rats were anesthetized and shaved, and a 1-mm watertight standoff reservoir that held the insulin or saline was placed between the rat's abdomen and the ultrasound array. At the beginning of the experiment and every 30 minutes, 0.3 ml of blood was collected from the jugular vein to determine the blood glucose level (milligrams per deciliter) for a total of 90 minutes. For comparison purposes between the rats, the change in the glucose level for each rat was normalized to a baseline (i.e., 0 mg/dl). The first control group used insulin in the reservoir without any ultrasound. The second control group had saline in the reservoir with ultrasound operating at Isptp = 100 mW/cm2 for 60 minutes. For the noncontrol experiments, the third group used insulin with ultrasound exposure for 10 minutes. The last group used insulin with ultrasound operating with a 5-minute exposure to examine the effects of using short ultrasound exposure times on delivery. For the 10- and 5-minute ultrasound exposure groups, the glucose level was found to decrease from the baseline to -174.6 +/- 67.2 and -200.4 +/- 43.4 mg/dl measured after 1 hour, respectively. These results indicated that ultrasound exposure times do not need to be long to deliver a clinically significant insulin dose to reduce a high blood glucose level.

Noninvasive ultrasonic transdermal insulin delivery in rabbits using the light-weight cymbal array.

OBJECTIVE: Recent studies have shown that ultrasound-mediated transdermal drug delivery offers a promising potential for noninvasive drug administration. The purpose of this study was to demonstrate ultrasonic transdermal delivery of insulin in vivo using rabbits with a novel, low-profile two-by-two ultrasound array based on the cymbal transducer. As a practical device, the cymbal array (f = 20 kHz) was 37 x 37 x 7 mm3 in size and weighed less than 22 g. Using the same array on hyperglycemic rats, our previous experiments demonstrated that blood glucose would decrease 233.3 +/- 22.2 mg/dL in 90 min from 5 min of pulsed ultrasound exposure. With a similar intensity (Isptp = 100 mW/cm2, 20% duty cycle), our goal was to determine if the same effect could be achieved with rabbits. METHODS: Experiments were performed in 16 New Zealand White rabbits (weighing 2.7-3.4 kg) divided into three groups: two controls and one ultrasound with insulin exposure. The rabbits were first anesthetized, and their thigh area was shaved for the exposure area. While the animal was lying in the lateral recumbent position, a 1-mm-thick, water-tight standoff (reservoir) that held insulin or saline was fastened between the thigh and the ultrasound array. The first control group (control 1: insulin-no ultrasound) had insulin placed in the reservoir with no ultrasound exposure, while the second control group (control 2: saline-ultrasound) had saline in the reservoir with ultrasound operating at Isptp = 100 mW/cm2 for 60 min. The third rabbit group (ultrasound-insulin) was subjected to insulin with ultrasound exposure for 60 min (Isptp = 100 mW/cm2). At the beginning of the experiment and every 15 min for 90 min, 0.3 mL of blood was collected from the ear vein to determine the blood glucose level (in mg/dL) using a glucose monitoring system. For comparison between individual rabbits, the change in the blood glucose level was normalized to a baseline value. The insulin reservoir was removed with the array after the ultrasound was turned off at 60 min of exposure. RESULTS: For both controls, insulin-no ultrasound and saline-ultrasound, the blood glucose level varied from the initial baseline by approximately +75 mg/dL. However, for the ultrasound-insulin group, the glucose level was found to decrease to -132.6 +/- 35.7 mg/dL from the initial baseline in 60 min. Even after the array and insulin reservoir were removed, the blood glucose level of ultrasound-insulin group continued to decrease to -208.1 +/- 29 mg/dL from the initial baseline. CONCLUSIONS: These results indicate the feasibility of using a low-cost, lightweight cymbal array for enhanced transdermal insulin delivery using ultrasound.

Transducer design for a portable ultrasound enhanced transdermal drug-delivery system.

For application in a portable transdermal drug-delivery system, novel transducers have been designed to enhance insulin transmission across skin using ultrasound. Previous research has shown transdermal delivery of insulin across skin using commercial sonicators operating at 20 kHz with intensities ranging from 12.5 to 225 mW/cm2. The goal of this research was to design and construct a small, lightweight transducer or array that could operate with a similar frequency and intensity range as a commercial sonicator used in previous transdermal ultrasound insulin experiments, but without the weight and mass of a sonicator probe. To obtain this intensity range, a cymbal transducer design was chosen because of its light, compact structure and low resonance frequency in water. To increase the spatial ultrasound field for drug delivery across skin, two arrays, each comprising of four cymbal transducers, were constructed. The first array, designated the standard array, used four cymbals transducer elements in parallel. A second array (named the stack array) used four cymbal transducers that used stacked piezoelectric discs to drive the titanium flextensional caps. Under similar driving conditions, the standard array produced intensities comparable to those achieved using a commercial sonicator.