Shrimp MultiPath™ multiplexed PCR white spot syndrome virus detection in penaeid shrimp.

White spot syndrome virus (WSSV), which causes white spot disease, is one of the notoriously feared infectious agents in the shrimp industry, inflicting estimated production losses world-wide of up to US$1 billion annually. Cost-effective accessible surveillance testing and targeted diagnosis are key to alerting shrimp industries and authorities worldwide early about WSSV carrier status in targeted shrimp populations. Here we present key validation pathway metrics for the Shrimp MultiPathTM (SMP) WSSV assay as part of the multi-pathogen detection platform. With superior throughput, fast turn-around time, and extremely low cost per test, the SMP WSSV assay achieves a high level of analytical sensitivity (~2.9 copies), perfect analytical specificity (~100%), and good intra- and inter-run repeatability (coefficient of variation <5%). The diagnostic metrics were estimated using Bayesian latent class analysis on data from 3 experimental shrimp populations from Latin America with distinct WSSV prevalence and yielded a diagnostic sensitivity of 95% and diagnostic specificity of 99% for SMP WSSV, which was higher than these parameters for the TaqMan quantitative PCR (qPCR) assays currently recommended by the World Organisation for Animal Health and the Commonwealth Scientific and Industrial Research Organisation. This paper additionally presents compelling data for the use of synthetic double-stranded DNA analyte spiked into pathogen-naïve shrimp tissue homogenate as a means to substitute clinical samples for assay validation pathways targeting rare pathogens. SMP WSSV shows analytical and diagnostic metrics comparable to qPCR-based assays and demonstrates fit-for-purpose performance for detection of WSSV in clinically diseased and apparently healthy animals.

Detection and genetic diversity of porcine rotavirus A, B and C in eastern Australian piggeries.

Rotaviruses (RV) have a high prevalence in piggeries worldwide and are one of the major pathogens causing severe diarrhoea in young pigs. RV species A, B, and C have been linked to piglet diarrhoea in Australian pig herds, but their genetic diversity has not been studied in detail. Based on sequencing of the structural viral protein 7 (VP7) RVA G genotypes G3, G4 and G5, and RVC types G1, G3, G5, and G6 have been identified in Australian piggeries in previous studies. Although occurrence of RVB was reported in Australia in 1988, no further genetic analysis has been conducted. To improve health management decisions in Australian pig herds, more information on RV prevalence and genetic diversity is needed. Here, 243 enteric samples collected from 20 pig farms within Eastern Australia were analysed for the presence of RV in different age groups using a novel PCR-based multiplex assay (Pork MultiPath™ enteric panel). RVA, RVB, and RVC were detected in 10, 14, and 14 farms, respectively. Further sequencing of VP7 in selected RV-positive samples revealed G genotypes G2, G5, G9 (RVA), G6, G8, G14, G16, G20 (RVB), and G1, G3, G5, G6 (RVC) present. RVA was only detected in young (<10 weeks old) pigs whereas RVB and RVC were also detected in older animals (>11 weeks old). Interestingly, RVB and RVC G-type occurrence differed between age groups. In conclusion, this study provides new insights on the prevalence and diversity of different RV species in pig herds of Eastern Australia whilst demonstrating the ability of the Pork MultiPath™ technology to accurately differentiate between these RV species.

Enterocytozoon hepatopenaei real-time and Shrimp MultiPathTM PCR assay validation for South-East Asian and Latin American strains of Penaeid shrimp.

Enterocytozoon hepatopenaei (EHP) infections are a global challenge for the Penaeid shrimp industry with a sharp rise in prevalence over the last 10 yr. EHP is known to cause sub-optimal growth, large size variation and reduced survival of shrimp. Molecular methods development has mainly focussed on 18S rRNA or spore wall protein 1 (SWP1). Due to the specificity and sensitivity issues with previously designed assays for both targets, new molecular assays are needed by the global shrimp industry and regulators to help manage the risks posed by EHP. This paper describes new real-time PCR (qPCR) methods developed for the novel EHP gene targets polar tube protein 2 (PTP2) and spore wall protein 26 (SWP26), whilst also presenting performance metrics of the new Shrimp MultiPathTM technology EHP assay. qPCR assays PTP2G and SWP26G show high amplification efficiency, a limit of detection (LOD) of between 1 and 4 copies, low assay variation and high diagnostic sensitivity (DSe) and specificity (DSp) compared to imperfect reference assays. Similar performance is seen with Shrimp MultiPathTM EHP showing an LOD of 8 copies, low assay variation and high DSe and DSp. These novel molecular targets for EHP and Shrimp MultiPathTM EHP strengthen global efforts to monitor and mitigate risks of EHP infections and outbreaks. Moreover, this study presents novel data on distribution of EHP in shrimp populations from South-East Asia and Latin America, and how sequence variations need to be considered when monitoring EHP in different geographies.

Contrast-enhanced ultrasound (CEUS) nephrostogram: utility and accuracy as an alternative to fluoroscopic imaging of the urinary tract.

AIM: To establish the feasibility and accuracy of contrast-enhanced ultrasound (CEUS) nephrostogram in comparison to the reference standard, fluoroscopic nephrostogram, in providing alternative imaging of the urinary tract post-nephrostomy insertion. MATERIALS AND METHODS: This prospective study was approved by the institutional and national ethics committee. All patients for whom a fluoroscopic nephrostogram was requested were included. Fluoroscopic and CEUS nephrostograms were performed within 24 hours. Image analysis (nephrostomy position, opacification of pelvicalyceal system, ureter, and bladder) was performed by two reviewers, and the diagnostic accuracy of the CEUS nephrostograms was compared to fluoroscopic nephrostograms. RESULTS: Sixty-two nephrostograms were performed in 48 patients from June 2011 to April 2016, (male: 25/48, 52.1%; mean age 65 years, range 28-90 years). Indications for nephrostomy were: malignancy (29/62; 46.8%), benign ureteric stricture (14/62; 22.6%), urinary diversion (8/62; 12.9%), renal calculus (5/62; 8.1%), haematoma (3/62; 4.8%) or pelvi-ureteric junction obstruction (3/62; 4.8%). Two nephrostomies were identified as displaced by both techniques. The pelvicalyceal system was visualised in 60/60 (100%) examinations in both fluoroscopic and CEUS nephrostograms. The entire ureter was visualised in 30/60 (50%) with CEUS compared to 32/60 (53.3%) fluoroscopically. The distal ureter was the least well-visualised segment for both techniques with no significant difference (p=0.815). Both CEUS and fluoroscopy could be used to correctly identify complications including entero-ureteric fistula or urine leak. Fluoroscopic nephrostogram demonstrated drainage into the bladder in 33/60 (55%), CEUS confirmed drainage in 34/60 (56.7%) cases (p=0.317). CONCLUSIONS: CEUS nephrostogram can determine the correct positioning of a nephrostomy and assess drainage into the bladder with statistically comparable results to fluoroscopy.

Endocavitary contrast enhanced ultrasound (CEUS): a novel problem solving technique.

Contrast-enhanced ultrasound (CEUS) is a technique that has developed as an adjunct to conventional ultrasound. CEUS offers a number of benefits over conventional axial imaging with computerised tomography and magnetic resonance imaging, primarily as a "beside" test, without ionising radiation or the safety concerns associated with iodinated/gadolinium-based contrast agents. Intravascular use of ultrasound contrast agents (UCAs) is widespread with extensive evidence for effective use. Despite this, the potential utility of UCAs in physiological and non-physiological cavities has not been fully explored. The possibilities for endocavitary uses of CEUS are described in this review based on a single-centre experience including CEUS technique and utility in confirming drain placement, as well as within the biliary system, urinary system, gastrointestinal tract and intravascular catheters. TEACHING POINTS: • CEUS offers an excellent safety profile, spatial resolution and is radiation free. • Endocavitary CEUS provides real-time imaging similar to fluoroscopy in a portable setting. • Endocavitary CEUS can define internal architecture of physiological cavities. • Endocavitary CEUS can confirm drain position in physiological and non-physiological cavities.

Ultranarrow Optical Inhomogeneous Linewidth in a Stoichiometric Rare-Earth Crystal.

We obtain a low optical inhomogeneous linewidth of 25 MHz in the stoichiometric rare-earth crystal EuCl_{3}·6H_{2}O by isotopically purifying the crystal in ^{35}Cl. With this linewidth, an important limit for stoichiometric rare-earth crystals is surpassed: the hyperfine structure of ^{153}Eu is spectrally resolved, allowing the whole population of ^{153}Eu^{3+} ions to be prepared in the same hyperfine state using hole-burning techniques. This material also has a very high optical density, and can have long coherence times when deuterated. This combination of properties offers new prospects for quantum information applications. We consider two of these: quantum memories and quantum many-body studies. We detail the improvements in the performance of current memory protocols possible in these high optical depth crystals, and describe how certain memory protocols, such as off-resonant Raman memories, can be implemented for the first time in a solid-state system. We explain how the strong excitation-induced interactions observed in this material resemble those seen in Rydberg systems, and describe how these interactions can lead to quantum many-body states that could be observed using standard optical spectroscopy techniques.

Testicular Microlithiasis: Is Sonographic Surveillance Necessary? Single Centre 14 Year Experience in 442 Patients with Testicular Microlithiasis.

PURPOSE: Increased prevalence of germ cell tumour (GCT) is seen with testicular microlithiasis (TM) suggesting TM is a premalignant condition with US surveillance advocated. We present a cohort of patients with TM followed up in a single centre and deliberate on the value of US surveillance. MATERIALS AND METHODS: A retrospective analysis of subjects with underlying US diagnosis of TM between 1998 and 2012. One-yearly US follow-up was offered to all patients with TM and a database maintained. Any co-existing tumour at presentation with TM was recorded. TM was divided into limited (< 5 microliths/field), classical (≥ 5 microliths/field) and florid ('snowstorm' appearance). Patient demographics, follow-up details and the development of any scrotal abnormalities were recorded. The radiological and histological findings were documented when a testicular lesion occurred during the follow-up period. RESULTS: 20 224 patients were examined: 867/20 224 (4.3 %) had TM. 21/867 (2.4 %) patients had histology proven malignant tumours at presentation. All TM patients consented to follow-up with 442/867 (51.0 %) achieving this and entering into a follow-up program (mean duration 28 months, range 8 - 165 months). Two patients developed primary GCT during the follow up period. One patient (limited TM) had undergone a previous orchiectomy for contralateral GCT and developed a palpable mass at follow up month 21. The other (limited TM) had an atrophic testis; a tumour was found on US at follow up month 62. CONCLUSION: Two patients of 442 (0.5 %) followed up for all forms of TM in a single centre developed a GCT over a mean duration of 28 months, both had independent risk factors for the development of GCT. These findings suggest that US surveillance is not required when TM is the only abnormality in the absence of any clinical risk factors for the development of GCT.

Contrast enhanced ultrasound (CEUS) characterization of grey-scale sonographic indeterminate focal liver lesions in pediatric practice.

PURPOSE: To determine the usefulness of contrast-enhanced ultrasound (CEUS) in characterizing grey-scale sonographic indeterminate focal liver lesions (FLL) in pediatric practice. MATERIALS AND METHODS: Local Ethics Board approval waiver was attained. Consent for CEUS examinations was acquired from parents. Forty-four children referred for CEUS assessment of grey-scale sonographic indeterminate FLL over a 5-year period underwent standard multiphase CEUS performed by experienced operators. A phospholipid microbubble agent was used and low mechanical index ultrasound imaging techniques employed. Interpretation by consensus of the CEUS examination was compared to consensus interpretation of other imaging and to histology. Follow-up imaging was used to confirm stability of benign abnormalities. Any contrast reactions were recorded. RESULTS: The CEUS examination interpretation agreed with reference imaging in 29/34 (85.3 %) of cases. In discordant cases, reference imaging showed no abnormality (n = 5), with fatty change (n = 4) and regenerating nodules (n = 1) on CEUS and follow-up sonography. Where reference imaging was not performed (n = 10), histology (n = 7) or follow-up sonography (n = 3) confirmed the diagnosis. In one discordant case, all imaging modalities showed concordance identifying a malignant lesion; however histology demonstrated a benign hepatocellular adenoma. The specificity was 98.0 % (95 % CI; 86 - 100 %) and the negative predictive value was 100 %. No adverse effects to the contrast material were noted. CONCLUSION: These findings demonstrate the usefulness of CEUS in characterizing indeterminate grey-scale sonography FLL in pediatric patients with the potential to reduce exposure to ionizing radiation.

Storage and retrieval of collective excitations on a long-lived spin transition in a rare-earth ion-doped crystal.

Robust, long-lived optical quantum memories are important components of many quantum information and communication protocols. We demonstrate coherent generation, storage, and retrieval of excitations on a long-lived spin transition via spontaneous Raman scattering in a rare-earth ion-doped crystal. We further study the time dynamics of the optical correlations in this system. This is the first demonstration of its kind in a solid and an enabling step toward realizing a solid-state quantum repeater.

Precision measurement of electronic ion-ion interactions between neighboring Eu3+ optical centers.

We report measurements of discrete excitation-induced frequency shifts on the 7F0→5D0 transition of the Eu+ center in La:Lu:EuCl3·6D2O resulting from the optical excitation of neighboring Eu3+ ions. Shifts of up to 46.081±0.005 MHz were observed. The magnitude of the interaction between neighboring ions was found to be significantly larger than expected from the electric dipole-dipole mechanism often observed in rare earth systems. We show that a large network of interacting and individually addressable centers can be created by lightly doping crystals otherwise stoichiometric in the optically active rare earth ion, and that this network could be used to implement a quantum processor with more than ten qubits.

Ultrastructure of putative germ granules in the penaeid shrimp Marsupenaeus japonicus.

Knowledge about the specification of the germ line in penaeid shrimp would allow development of techniques to control germ cell formation and/or fate to produce reproductively sterile shrimp for genetic copyright purposes. Recent studies have traced the localization of an RNA-enriched intracellular body (ICB) in the putative germ line of four penaeid shrimp species. It is hypothesized that the ICB may serve as a putative germ granule and marker of germ line fate. In this study semi-thin and ultra-thin sections of Marsupenaeus japonicus embryos were prepared, and the dimensions and ultrastructure of the ICB was examined at different stages of embryogenesis. The ICB was an aggregation of electron dense granules, small vesicles and multi-vesicular bodies (MVBs), similar to germ granules from other species. Lamellar membranes and mitochondria were localized at the periphery of the ICB. Using fluorescence microscopy, microtubules were also observed between the centrosome and the ICB. The localization of the ICB in the D lineage and putative germ cell line, the enrichment of RNA in the ICB, and the ultrastructural similarities to other germ granules characterized in this study support the hypothesis that the ICB contains germ granules.

Scrotal calcification in a symptomatic paediatric population: Prevalence, location, and appearance in a cohort of 516 patients.

AIM: To describe the prevalence of all forms of scrotal calcification within a symptomatic paediatric population and to compare this with previous reported data in paediatric and adult populations. MATERIALS AND METHODS: A retrospective analysis of testicular ultrasound examinations performed in a single institution over a 55 month period. All examinations were performed by experienced operators using high-frequency linear array transducers. Types of scrotal calcification and position were recorded with all available images analysed by experienced radiologists. RESULTS: A total 516 male patients under the age of 19 years (mean age 10.5 years) were included. The prevalence of testicular microlithiasis (TM) was 8.7% and the prevalence of non-TM macrocalcification was 0.4%. 2.3% of the patients had scrotal pearls and 0.2% had epididymal calcification recorded. No other form of calcification was identified. A single patient had a co-existing testicular tumour and TM at examination. CONCLUSION: The prevalence of TM in the symptomatic paediatric population is greater than that reported in the symptomatic adult population, whereas the prevalence of intra-testicular macrocalcification is lower. It is speculated that TM and macrocalcification represent different pathways for the possible risk of testicular tumour development.

Contrast-enhanced ultrasound in testicular trauma: role in directing exploration, debridement and organ salvage.

We describe the use of contrast-enhanced ultrasound as an additional imaging technique during an ultrasound examination of a traumatised testis, allowing for confident testicular preserving surgery to be performed.

Triploidy induction in the Pacific white shrimp Litopenaeus vannamei: an assessment of induction agents and parameters, embryo viability, and early larval survival.

In this study, we trialed 6-dimethylaminopurine (6-DMAP) chemical shocks to induce meiosis I or meiosis II Pacific White shrimp, Litopenaeus vannamei, triploids for the first time, and cold temperature shocks to induce meiosis II L. vannamei triploids as done previously. Inductions were performed on 37 spawnings in total with experiments being progressively designed in a factorial manner to allow optimization of induction parameters. Treatment with a 200-µm 6-DMAP final concentration at 1 min post-spawning detection for a 6 to 8 min duration resulted in the most consistent induction of chemically induced meiosis I triploids while treatment at 7 min 30 s post-spawning detection for a 10-min duration resulted in the most consistent induction of chemically induced meiosis II triploids. A cold temperature shock of 11.7°C to 13.25°C (final treatment temperature; spawning water temperature 28.5°C) applied at 8 min post-spawning detection for a 4 to 10 min duration resulted in the most consistent induction of cold-temperature-induced meiosis II triploids. 6-DMAP shocks resulted in meiosis I induction rates from 29% to 100% in unhatched embryos and 50% in nauplii, and meiosis II induction rates from 65% to 100% in unhatched embryos and 52% to 100% in nauplii. Cold shocks resulted in induction rates from 5% to 100% in unhatched embryos and nauplii. Confocal microscopy analysis of embryos revealed that there are major developmental abnormalities in a large proportion of later stage triploid L. vannamei embryos compared to their diploid sibling controls. Despite this, however, some triploid embryos did appear normal and both shock agents induced small numbers of viable triploid L. vannamei nauplii which were successfully reared to protozoeal stage 3 as confirmed by flow cytometry. Triploids beyond this life-history stage were not observed in the present study as confirmed by flow cytometry at mysis stages. This study adds to our knowledge base of triploid induction in L. vannamei and further highlights the inherent difficulties with triploid embryonic and larval viability in this species.

Tracheal diameter at birth in severe congenital diaphragmatic hernia treated by fetal endoscopic tracheal occlusion.

OBJECTIVE: To investigate tracheal dimensional differences seen at birth following fetal endoscopic tracheal occlusion (FETO) in cases of severe congenital diaphragmatic hernia (CDH) and to report on their clinical follow-up. PATIENTS AND METHODS: In chest X-rays, taken within 48 h after birth, we measured the tracheal diameter at the level of the tracheal entry into the chest, 1 cm above the level of the carina and at middistance between these sites in 37 fetuses with severe CDH treated by FETO. These measurements were compared with those in 74 preterm and term neonates with no congenital lung abnormalities. RESULTS: In the CDH group, compared to the controls, the tracheal diameter corrected for gestational age was significantly larger at all three levels of the trachea. Regression analysis showed that significant predictors of the tracheal diameter at the level of tracheal entry into the chest were the observed to expected (o/e) lung area to head circumference ratio (LHR) and the duration of tracheal occlusion. In the CDH group, postnatal follow-up until the age of 22 months (1-70) showed that 5 of 24 neonates had an effort-induced barking cough. CONCLUSION: A large number of infants with severe CDH surviving after FETO have a degree of tracheomegaly that is associated with the severity of CDH as assessed by pre-FETO LHR. This tracheomegaly does not constitute an obvious clinical problem.

Evaluation of gallbladder and biliary duct disease using microbubble contrast-enhanced ultrasound.

Ultrasound examination of the gallbladder is accepted as the primary imaging modality in the assessment of gallbladder disease, with inherent superiority in comparison to other imaging modalities. Contrast-enhanced ultrasound is established as a reliable tool in the detection and characterisation of focal liver lesions. It is less well recognised in gallbladder and biliary disease but can be a valuable complement to baseline ultrasound examination. Contrast-enhanced ultrasound provides the advantages of real-time, repeatable, multiplanar imaging without compromising patient safety or exposing patients to radiation. It can provide specific information as pathology often becomes more conspicuous following the administration of contrast, allowing detailed assessment of benign and malignant conditions arising in the gallbladder and biliary tree. This review illustrates the application of contrast-enhanced ultrasound in the evaluation of a variety of gallbladder and biliary duct diseases. The examination allows clearer delineation of the disease process and more confident diagnosis.

Congenital segmental emphysema: an evolving lesion.

INTRODUCTION: Congenital segmental emphysema (CSE) is a newly-recognised sub-type of congenital parenchymal lung anomaly. It is characterised by antenatal detection and post-natal evolution from an initially solid segmental appearance to a hyperlucent and hyperinflated segment. METHODS: A retrospective review of a single-centre tertiary referral database between Jan 1994 and Dec 2007 was performed. MAIN RESULTS: 130 infants had antenatally detected lung anomalies, and of these 12 (9.2%) infants (initially labelled as congenital cystic adenomatoid malformation (CCAM)), showed features better defined as CSE. The lesions were described antenatally as non-progressive microcystic (n=6), hyperechogenic (n=2) or both (n=2). Early post-natal CT scans showed areas of solid segmental parenchyma, initial hyperlucency or microcysts. Subsequent CT imaging, however, showed evolution to segmental hyperlucency in areas previously solid and in 2 cases a central bronchocele was noted. Ten children underwent resectional surgery (segmentectomy n=4, lobectomy n=6) at a median age of 1 (range 0.4-5.2) year and the gross appearance of the resected specimen confirmed hyperinflated (not cystic) segments. Histological review showed localised abnormally dilated alveolar spaces in 7 cases. Adjacent areas consistent with type 2 CCAM were also seen (n=3). CONCLUSION: CSE lies within the spectrum of both CCAM and sequestration but there is a definite post-natal evolution and volume change which presage symptoms. This may be associated with segmental bronchial atresia and progressive air trapping via collateral airways such as the interalveolar pores of Kohn.

Observation and control of blinking nitrogen-vacancy centres in discrete nanodiamonds.

Nitrogen-vacancy colour centres in diamond can undergo strong, spin-sensitive optical transitions under ambient conditions, which makes them attractive for applications in quantum optics, nanoscale magnetometry and biolabelling. Although nitrogen-vacancy centres have been observed in aggregated detonation nanodiamonds and milled nanodiamonds, they have not been observed in very small isolated nanodiamonds. Here, we report the first direct observation of nitrogen-vacancy centres in discrete 5-nm nanodiamonds at room temperature, including evidence for intermittency in the luminescence (blinking) from the nanodiamonds. We also show that it is possible to control this blinking by modifying the surface of the nanodiamonds.