Validation of a multiplex-tandem RT-PCR for the detection of bovine respiratory disease complex using Scottish bovine lung samples.

The welfare and economic impact of bovine respiratory disease complex (BRDC), and its associated antibiotic usage, are major challenges to cattle rearing and beef cattle finishing industries. Accurate pathogen diagnosis is important to undertake appropriate treatment and long-term management strategies, such as vaccine selection. Conventional diagnostic approaches have several limitations including high costs, long turnaround times and difficulty in test interpretation, which could delay treatment decisions and lead to unnecessary animal losses. We describe the validation of a multiplex-tandem (MT) reverse transcription-polymerase chain reaction (RT-PCR) for the detection of seven common pathogens associated with BRDC. This test has the potential to advance pathogen identification and to overcome many of the limitations of current testing methods. It requires a single sample and results are obtained quickly and not influenced by prior antimicrobial therapy or overgrowth of contaminating organisms. We demonstrated a test specificity of 100% and sensitivity ranging from 93.5% to 100% for these seven common pathogens. This test will be a useful addition to advance BRDC investigation and diagnosis.

Molecular typing of enteroviruses: comparing 5'UTR, VP1 and whole genome sequencing methods.

Enteroviruses (EV) commonly cause hand, foot and mouth disease (HFMD), and can also cause potentially fatal neurological and systemic complications. In our laboratory, sequencing 5' untranslated region (UTR) of the viral genome has been the routine method of genotyping EVs. During a recent localised outbreak of aseptic meningitis, sequencing the 5'UTR identified the causative virus as EV-A71, which did not fit with the clinical syndrome or illness severity. When genotyped using a different target gene, VP1, the result was different. This led us to evaluate the accuracy of the two different target genome regions and compare them against whole genome sequencing (WGS). We aimed to optimise the algorithm for detection and characterisation of EVs in the diagnostic laboratory. We hypothesised that VP1 and WGS genotyping would provide different results than 5'UTR in a subset of samples. Clinical samples from around New South Wales which were positive for EV by commercial polymerase chain reaction (PCR) assays were genotyped by targeting three different viral genome regions: the 5'UTR, VP1 and WGS. Sequencing was performed by Sanger and next generation sequencing. The subtyping results were compared. Of the 74/118 (63%) samples that were successfully typed using both the 5'UTR and the VP1 method, the EV typing result was identical for 46/74 (62%) samples compared to WGS as the gold standard. The same EV group but different EV types were found in 22/74 (30%) samples, and 6/74 (8%) samples belonged to different EV groups depending on typing method used. Genotyping with WGS and VP1 is more accurate than 5'UTR. Genotyping by the 5'UTR method is very sensitive, but less specific.

Adjuvant immunosuppression for paradoxical deterioration in tuberculous meningitis including one case responsive to cyclosporine. A tertiary referral hospital experience.

BACKGROUND: Tuberculous meningitis (TBM) accounts for 1-4% of all tuberculosis (TB) presentations. Paradoxical deterioration in non-HIV patients is a common manifestation of anti-tuberculosis therapy, characterised by clinico-radiological deterioration. We report a case series of TBM admissions to our institution including one case with paradoxical deterioration refractory to corticosteroids who responded to adjuvant cyclosporine. METHODS: Retrospective review of 12 HIV-negative patients admitted to Liverpool Hospital, Sydney (2005-2016) with laboratory and/or radiologically confirmed TBM. RESULTS: Median patient age was 40 (range 22-81 years), M:F = 7:5. Eleven patients (92%) were of Asia-Pacific origin. Eleven initially presented with central nervous system manifestations and one had preceding miliary TB. Nine patients had extra-cranial TB involvement including eight with past or current pulmonary disease. Cerebrospinal fluid (CSF) TB PCR/culture was positive in 10 patients. Paradoxical deterioration developed in three patients despite concomitant corticosteroids in two. One patient with paradoxical deterioration was refractory to corticosteroids: A 22-year-old Vietnamese male with TBM developed worsening headaches and altered mentation after seven weeks concomitant anti-TB and corticosteroid treatment. Interval MRI brain demonstrated increased size and number of tuberculomas as well as hydrocephalus. Cyclosporine was added with gradual improvement and ultimately good outcome. CONCLUSION: Our case series highlights the seriousness of paradoxical deterioration in TBM and the potential role of adjuvant cyclosporine in patients refractory to corticosteroids.

Automated direct screening for resistance of Gram-negative blood cultures using the BD Kiestra WorkCell.

Early detection of resistance in sepsis due to Gram-negative organisms may lead to improved outcomes by reducing the time to effective antibiotic therapy. Traditional methods of resistance detection require incubation times of 18 to 48 h to detect resistance. We have utilised automated specimen processing, digital imaging and zone size measurements in conjunction with direct disc susceptibility testing to develop a method for the rapid screening of Gram-negative blood culture isolates for resistance. Positive clinical blood cultures with Gram-negative organisms were prospectively identified and additional resistant mock specimens were prepared. Broth was plated and antibiotic-impregnated discs (ampicillin, ceftriaxone, piperacillin-tazobactam, meropenem, ciprofloxacin, gentamicin) were added. Plates were incubated, digitally imaged and zone sizes were measured using the BD Kiestra WorkCell laboratory automation system. Minimum, clinically useful, incubation times and optimised zone size cut-offs for resistance detection were determined. We included 187 blood cultures in the study. At 5 h of incubation, > 90% of plates yielded interpretable results. Using optimised zone size cut-offs, the sensitivity for resistance detection ranged from 87 to 100%, while the specificity ranged from 84.7 to 100%. The sensitivity and specificity for piperacillin-tazobactam resistance detection was consistently worse than for the other agents. Automated direct disc susceptibility screening is a rapid and sensitive tool for resistance detection in Gram-negative isolates from blood cultures for most of the agents tested.

Establishment of an AUC0-24 Threshold for Nephrotoxicity Is a Step towards Individualized Vancomycin Dosing for Methicillin-Resistant Staphylococcus aureus Bacteremia.

Unlike vancomycin trough concentrations, data on the utility of vancomycin pharmacokinetic (PK) parameters, namely, the area under the concentration-time curve from 0 to 24 h (AUC0-24), in predicting acute kidney injury (AKI) are limited. Our aim was to investigate this relationship in patients receiving vancomycin therapy for methicillin-resistant Staphylococcus aureus bacteremia (MRSA-B). A single-center retrospective observational cohort study involving 127 consecutive MRSA-B patients was conducted to examine the incidence of AKI (defined as serum creatinine of ≥0.5 mg/liter and a 50% increase from baseline) and vancomycin exposure parameters associated with nephrotoxicity. Bayesian estimation was used to predict individual vancomycin AUC0-24 All patients received vancomycin monotherapy for a minimum of 14 days following the diagnosis of MRSA-B. AKI was observed in 15.7% of patients (20/127). Clinical characteristics were similar between patients with and without AKI. At steady state, higher vancomycin trough concentrations were associated with AKI (17.2 mg/liter versus 13.1 mg/liter; P = 0.003). A vancomycin AUC0-24 threshold for AKI of >563 mg · h/liter was detected by classification and regression tree (CART) analysis; patients with exposures above this threshold were significantly more likely to experience AKI than patients with lower vancomycin exposures (40% [8/20] versus 11.2% [12/107]; P = 0.002). This parameter remained an independent predictor of AKI on multivariate logistic regression (odds ratio [OR], 5.07; 95% confidence interval [CI], 1.57 to 16.29; P = 0.006) and was a better predictor of nephrotoxicity than vancomycin trough concentrations. Overall, AKI is associated with higher vancomycin exposure as measured by AUC0-24 These results suggest that individualized patient dosing may be possible with dose modifications directed toward established pharmacodynamic targets while balancing AKI risks.

Utility of 16S rRNA PCR performed on clinical specimens in patient management.

BACKGROUND: Broad-range 16S rRNA PCR can be used for the detection and identification of bacteria from clinical specimens in patients for whom there is a high suspicion of infection and cultures are negative. The aims of this study were (1) to compare 16S rRNA PCR results with microbiological culture results, (2) to assess the utility of 16S rRNA PCR with regard to antimicrobial therapy, and (3) to compare the yield of 16S rRNA PCR for different types of clinical specimen and to perform a cost analysis of the test. METHODS: A retrospective study was performed on different clinical specimens which had 16S performed over 3 years (2012-2015). Standard microbiological cultures were performed on appropriate media, as per the laboratory protocol. Patient clinical and microbiological data were obtained from the electronic medical records and laboratory information system, respectively. 16S rRNA PCR was performed in a reference laboratory using a validated method for amplification and sequencing. The outcomes assessed were the performance of 16S rRNA PCR, change of antimicrobials (rationalization, cessation, or addition), and duration of therapy. Concordance of 16S rRNA PCR with bacterial cultures was also determined for tissue specimens. RESULTS: Thirty-two patients were included in the study, for whom an equal number of specimens (n=32) were sent for 16S rRNA PCR. 16S rRNA PCR could identify an organism in 10 of 32 cases (31.2%), of which seven were culture-positive and three were culture-negative. The sensitivity was 58% (confidence interval (CI) 28.59-83.5%) and specificity was 85% (CI 61.13-96%), with a positive predictive value of 70% (CI 35.3-91.9%) and negative predictive value of 77.2% (CI 54.17-91.3%). Antimicrobial therapy was rationalized after 16S rRNA PCR results in five patients (15.6%) and was ceased in four based on negative results (12.5%). Overall the 16S rRNA PCR result had an impact on antimicrobial therapy in 28% of patients (9/32). The highest concordance of 16S rRNA PCR with bacterial culture was found for heart valve tissue (80%), followed by joint fluid/tissue (50%). CONCLUSIONS: Despite the low diagnostic yield, results of 16S rRNA PCR can still have a significant impact on patient management due to rationalization or cessation of the antimicrobial therapy. The yield of 16S rRNA PCR was highest for heart valves.

Exposure to viral and bacterial pathogens among Soay sheep (Ovis aries) of the St Kilda archipelago.

We assessed evidence of exposure to viruses and bacteria in an unmanaged and long-isolated population of Soay sheep (Ovis aries) inhabiting Hirta, in the St Kilda archipelago, 65 km west of Benbecula in the Outer Hebrides of Scotland. The sheep harbour many metazoan and protozoan parasites but their exposure to viral and bacterial pathogens is unknown. We tested for herpes viral DNA in leucocytes and found that 21 of 42 tested sheep were infected with ovine herpesvirus 2 (OHV-2). We also tested 750 plasma samples collected between 1997 and 2010 for evidence of exposure to seven other viral and bacterial agents common in domestic Scottish sheep. We found evidence of exposure to Leptospira spp., with overall seroprevalence of 6·5%. However, serological evidence indicated that the population had not been exposed to border disease, parainfluenza, maedi-visna, or orf viruses, nor to Chlamydia abortus. Some sheep tested positive for antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) but, in the absence of retrospective faecal samples, the presence of this infection could not be confirmed. The roles of importation, the pathogen-host interaction, nematode co-infection and local transmission warrant future investigation, to elucidate the transmission ecology and fitness effects of the few viral and bacterial pathogens on Hirta.

Impact of source of infection and vancomycin AUC0-24/MICBMD targets on treatment failure in patients with methicillin-resistant Staphylococcus aureus bacteraemia.

Despite recent controversies about toxicity and reduced efficacy, vancomycin remains the current treatment of choice for methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia. The parameter associated with treatment success is the vancomycin 24-h area under concentration-time curve to MIC ratio (AUC0-24/MIC). We aimed to determine the utility of calculated AUCs and explore the optimal AUC0-24/MIC targets associated with treatment success. In this single-centre retrospective observational cohort study of 127 patients with MRSA bacteraemia, forty-five (35.4%) did not respond to vancomycin treatment. Patient characteristics were essentially the same between those who did not respond to vancomycin treatment and those with treatment success, with independent predictors of treatment failure being source of bacteraemia (odds ratio (OR), 4.29; 95% confidence interval (CI), 1.50-12.26; p 0.007) and not achieving an AUC0-24/MICBMD (using broth microdilution) target of ≥398 (OR, 11.4; 95% CI, 4.57-28.46; p< 0.001). Bacteraemic source-specific thresholds were observed with a higher AUC0-24/MICBMD target of 440 required for high-risk sources (e.g. infective endocarditis) compared with 330 for low-risk sources (line related bacteraemia). Overall treatment success in patients with MRSA bacteraemia was associated with a vancomycin AUC0-24/MICBMD target of ≥398, with source-specific targets observed. Future vancomycin practice guidelines will need to take into account MIC methodology, source of bacteraemia and patient populations prior to setting targets and monitoring recommendations.

Short duration of lamivudine for the prevention of hepatitis B virus transmission in pregnancy: lack of potency and selection of resistance mutations.

This study sought to assess the antiviral efficacy of lamivudine (LMV) administered during third trimester to reduce maternal viraemia and to identify the emergence of LMV resistance. A prospective observational analysis was performed on 26 mothers with high viral load (>107 IU/mL). Twenty-one women received LMV (treated group) for an average of 53 days (range 22-88 days), and the remaining five formed the untreated control group. Serum samples from two time points were used to measure HBV DNA levels and antiviral drug resistance. The LMV-treated women achieved a median HBV DNA reduction of 2.6-log10 IU/mL. Although end-of-treatment (EOT) HBV DNA in four (18%) LMV-treated women remained at >10(7) IU/mL (± 0.5 log IU/mL), no mother-to-baby transmission was observed. In contrast, a baby from the untreated mother was HBsAg positive at 9 months postpartum. Four technologies were used for drug resistance testing. Only ultra-deep pyrosequencing (UDPS) was sufficiently sensitive to detect minor viral variants down to <1%. UDPS showed that LMV therapy resulted in increased viral quasispecies diversity and positive selection of HBV variants with reverse transcriptase amino acid substitutions at sites associated with primary LMV resistance (rtM204I/V and rtA181T) in four (19%) women. These viral variants were detected mostly at low frequencies (0.63-5.92%) at EOT, but one LMV-treated mother had an rtA181T variant that increased from 2.2% pretherapy to 25.59% at EOT. This mother was also infected with the vaccine escape variant (sG145R), which was inhibited by LMV treatment. LMV therapy during late pregnancy only reduced maternal viraemia moderately, and drug-resistant viral variants emerged.

Decontamination of ultrasound equipment used for peripheral ultrasound-guided regional anaesthesia.

Portable ultrasound machines are frequently used in operating theatres for peripheral single-shot nerve block procedures. This equipment must be decontaminated by reducing the microbial load to a sufficient level to reduce the risk of nosocomial infection. In our institution we use a simple three-step decontamination protocol utilising 70% isopropyl alcohol as chemical disinfectant. We performed a prospective, quality assurance study to assess the efficacy of this protocol, as it is unclear if this is suitable for disinfecting semi-critical equipment. The primary endpoint was presence of microbial contamination prior to re-use of equipment. Over a four-week period, 120 swabs were taken from multiple sites on our ultrasound machines and linear array transducers for microbial culture. Swabs were taken after decontamination and immediately prior to patient contact. Any pathogenic and environmental bacterial organisms were isolated and identified. No pathogenic organisms were grown from any of the collected swabs. In 85% (n=102) of cultures, no growth was detected. Of the remaining 15% (n=18), commensal organisms commonly found on skin, oral and environmental surfaces were isolated. Our results suggest that our decontamination protocol may be an effective, rapid and cost-effective method of cleaning ultrasound equipment used for peripheral invasive single-shot nerve blocks. Further guidance from national bodies is required to define appropriate cleaning protocols for these machines.

The first report of otarine herpesvirus-1-associated urogenital carcinoma in a South American fur seal (Arctocephalus australis).

Otarine herpesvirus (OtHV)-1-associated urogenital carcinoma has been well documented in the California sea lion (Zalophus californianus, CSL), but this is the first report of this tumour in a captive South American fur seal (Arctocephalus australis, SAFS). The gross and microscopical morphology of the tumour in the SAFS was identical to that described previously in CSLs and the tumour in the present case had metastasized within the urogenital tract and draining lymph nodes and to the lungs and one kidney. Immunohistochemistry revealed intra- and extracytoplasmic labelling of herpesvirus antigen in the cells of the tumour tissue and transitional epithelium of the urethra. OtHV-1 nucleic acids were detected within tumour tissue and from a urogenital swab by polymerase chain reaction. The ranges of these two species of pinniped do not overlap normally in the wild, suggesting that transmission of OtHV-1 probably occurred in captivity. This confirmed susceptibility of the SAFS to the development of OtHV-1-associated urogenital carcinoma suggests that all species of Otariidae should be screened for OtHV-1 infection prior to movement within and between zoological collections.

Demographic screening for MRSA may compromise the effectiveness of ring fencing on a joint replacement unit.

Ring fencing of joint replacement (JR) units has been reported to reduce infections and is recommended by health authorities in Australia and the UK. It has not been determined whether a demographic risk assessment is adequate to prevent the admission of patients colonized with meticillin-resistant Staphylococcus aureus (MRSA) to ring-fenced units. As such, 250 admissions to the JR unit of a suburban Sydney hospital were screened, and MRSA colonization was identified in 2.8% of patients complying with the demographic risk assessment. Demographic risk assessment is not an adequate substitute for physical MRSA screening, and undermines the effectiveness of ring-fencing procedures.

Systemic herpesvirus and morbillivirus co-infection in a striped dolphin (Stenella coeruleoalba).

During 2007 a dolphin morbillivirus epizootic affected the western Mediterranean and several striped dolphins (Stenella coeruleoalba) stranded on the Catalonian coasts. One of those animals had severe lymphoid depletion, necrosis and syncytial formation in lymph nodes and spleen, with large basophilic nuclear inclusions compatible with herpesvirus detected by immunohistochemical and ultrastructural examination. Non-suppurative encephalitis with associated morbillivirus antigen and morbillivirus antigen within alveolar macrophages were also observed. A pan-herpesvirus nested polymerase chain reaction amplified a sequence virtually identical to two cetacean herpesvirus sequences previously identified in systemic infections in an Atlantic Cuvier's beaked whale (Ziphius cavirostris) and in a Mediterranean striped dolphin. The herpesviral infection was probably secondary to the immunosuppression caused by the morbillivirus. To our knowledge, this is the first report of a cetacean co-infected by dolphin morbillivirus and herpesvirus with evidence of lesions attributable to both viruses.

Multi-centre evaluation of pre-transfusional routine tests using 8-column format gel cards (DG Gel®).

BACKGROUND: Advances in immunohaematology laboratory practice to improve performance, cost-effectiveness and patient safety are desirable. OBJECTIVES: To perform a multi-centre evaluation of the 8-column Grifols DG Gel(®) cards and reagent system to assess its performance, suitability and adaptability to the daily blood transfusion laboratory routine in the United Kingdom. METHODS/MATERIALS: A total of 4281 immunohematological analyses {1825 ABO/D grouping, 1921 antibody screening, 75 Rh phenotyping and K antigen determination, 361 antibody identification and 99 neonates [ABO/D and DAT (direct anti-globulin test)]} were performed on 2255 specimens. All cases were run in parallel with the reference method of each laboratory (DiaMed-ID(®) cards or conventional tube technique in some cases). RESULTS: Concordant results between Grifols DG Gel(®) system and the reference method were obtained in 97·7% of tests. For ABO grouping by the Grifols DG Gel(®) system, sensitivity was 99·95%, specificity was 99·96%, predictive positive value (PPV) was 99·89% and predictive negative value (PNV) was 99·98%. For D grouping, sensitivity was 99·78%, specificity was 100%, PPV was 100% and PNV was 99·78%. For antibody screening, sensitivity was 90·63%, specificity was 99·94%, PPV was 99·32% and PNV was 99·15%. Of the Rh subgroups and K types, results were 100% concordant. For antibody specificity detection, accuracy was 96·95% for Grifols DG Gel(®) system and 95·29% for DiaMed-ID(®) system. For the newborn tests, concordant results were obtained in 100% of ABO/D grouping and in 89·9% of DAT. CONCLUSION: The Grifols DG Gel(®) 8-column system is reliable and safe for routine tests performed in the immunohaematology laboratory.

Materials science challenges for high-temperature superconducting wire.

Twenty years ago in a series of amazing discoveries it was found that a large family of ceramic cuprate materials exhibited superconductivity at temperatures above, and in some cases well above, that of liquid nitrogen. Imaginations were energized by the thought of applications for zero-resistance conductors cooled with an inexpensive and readily available cryogen. Early optimism, however, was soon tempered by the hard realities of these new materials: brittle ceramics are not easily formed into long flexible conductors; high current levels require near-perfect crystallinity; and--the downside of high transition temperature--performance drops rapidly in a magnetic field. Despite these formidable obstacles, thousands of kilometres of high-temperature superconducting wire have now been manufactured for demonstrations of transmission cables, motors and other electrical power components. The question is whether the advantages of superconducting wire, such as efficiency and compactness, can outweigh the disadvantage: cost. The remaining task for materials scientists is to return to the fundamentals and squeeze as much performance as possible from these wonderful and difficult materials.

Development of a real time RT-PCR to detect and type ovine pestiviruses.

A real time one-step RT-PCR was designed to detect and type border disease virus (BDV), bovine viral diarrhea virus (BVDV) type 1 and BVDV type 2 in ovine samples. The real time RT-PCR was shown to behave in a linear manner and had limits of detection of 100-1000 copies of viral RNA as judged by in vitro transcribed RNA. The real time RT-PCR was validated on 50 clinical samples from UK flocks and was more sensitive than a virus isolation and a classical nested RT-PCR (nRT-PCR). The results of real time RT-PCR virus typing agreed completely with sequencing. The majority of ovine isolates were BDV; a small proportion were BVDV type 1. BVDV type 2 was not detected in any sample. This test appears reliable and can be used for the typing of ovine pestiviruses in the UK.

Spectroscopy of magnetic excitations in magnetic superconductors using vortex motion.

In magnetic superconductors a moving vortex lattice is accompanied by an ac magnetic field which leads to the generation of spin waves. At resonance conditions the dynamics of vortices in magnetic superconductors changes drastically, resulting in strong peaks in the dc I-V characteristics at voltages at which the washboard frequency of the vortex lattice matches the spin wave frequency omegaS(g), where g are the reciprocal vortex lattice vectors. We show that if the washboard frequency lies above the magnetic gap, measurement of the I-V characteristics provides a new method to obtain information on the spectrum of magnetic excitations in borocarbides and cuprate layered magnetic superconductors.

A multiplex polymerase chain reaction to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus -species venerealis: use on UK isolates of C. fetus and other Campylobacter spp.

AIMS: Subspeciation of Campylobacter fetus subsp. fetus (CFF) and Campylobacter fetus subsp. venerealis (CFV) is important for international animal import regulations. Phenotyping can be unreliable, and genotyping by techniques like pulsed field gel electrophoresis is difficult in routine diagnostic laboratories. A PCR subspeciation technique has been reported [Aust Vet J (1997) 75, 827]; we aimed to develop this PCR and investigate its use on UK C. fetus isolates. METHODS AND RESULTS: We augmented the PCR with further primers, and tested 76 isolates of C. fetus and 16 isolates of other Campylobacter spp. PCR failed to correlate well with phenotyping, especially for CFV. We characterized the amplicon of the CFV-specific primers (reported as plasmid derived, but unavailable on the public databases); and predicted a parA gene sequence, anticipated to be plasmid-associated. However, although plasmid isolations from selected CFV isolates demonstrated the presence of several plasmids, there was no correlation between plasmid profile and PCR result. Further, the parA sequence was not detected by PCR in any of the plasmid bands. CONCLUSIONS: This PCR is not suitable for subspeciation of C. fetus in the UK. The results suggest that this is a reflection of the presence of an unusual clone of CFV currently present in cattle in this country. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR cannot substitute for phenotyping of C. fetus isolates in the UK. The reasons for failure of PCR genotyping may reflect local strains and/or plasmid profiles. Further study is required to better elucidate molecular sub-speciation of C. fetus.