Development and operation of the defence COVID-19 lab as a SARS-CoV-2 diagnostic screening capability for UK military personnel.

BACKGROUND: In the face of the COVID-19 pandemic, the Defence Science and Technology Laboratory (Dstl) and Defence Pathology combined to form the Defence Clinical Lab (DCL), an accredited (ISO/IEC 17025:2017) high-throughput SARS-CoV-2 PCR screening capability for military personnel. LABORATORY STRUCTURE AND RESOURCE: The DCL was modular in organisation, with laboratory modules and supporting functions combining to provide the accredited SARS-CoV-2 (envelope (E)-gene) PCR assay. The DCL was resourced by Dstl scientists and military clinicians and biomedical scientists. LABORATORY RESULTS: Over 12 months of operation, the DCL was open on 289 days and tested over 72 000 samples. Six hundred military SARS-CoV-2-positive results were reported with a median E-gene quantitation cycle (Cq) value of 30.44. The lowest Cq value for a positive result observed was 11.20. Only 64 samples (0.09%) were voided due to assay inhibition after processing started. CONCLUSIONS: Through a sustained effort and despite various operational issues, the collaboration between Dstl scientific expertise and Defence Pathology clinical expertise provided the UK military with an accredited high-throughput SARS-CoV-2 PCR test capability at the height of the COVID-19 pandemic. The DCL helped facilitate military training and operational deployments contributing to the maintenance of UK military capability. In offering a bespoke capability, including features such as testing samples in unit batches and oversight by military consultant microbiologists, the DCL provided additional benefits to the UK Ministry of Defence that were potentially not available from other SARS-CoV-2 PCR laboratories. The links between Dstl and Defence Pathology have also been strengthened, benefitting future research activities and operational responses.

Clinical response correlates with 4-week postinjection ustekinumab concentrations in patients with moderate-to-severe psoriasis.

BACKGROUND: Cost-effective use of biologicals is important. As drug concentrations have been linked to clinical outcomes, monitoring drug concentrations is a valuable tool to guide clinical decision-making. A concentration-response relationship for ustekinumab at trough is uncertain owing to the contradictory results reported. OBJECTIVES: To investigate the relationship between 4-week postinjection ustekinumab concentrations and clinical response in patients with psoriasis. METHODS: Forty-nine patients with moderate-to-severe psoriasis treated with 45 mg or 90 mg ustekinumab every 12 weeks for ≥ 16 weeks were included. Ustekinumab serum concentrations and anti-ustekinumab antibodies were measured at week 4 after injection and disease severity was assessed by Psoriasis Area and Severity Index (PASI). RESULTS: At week 4 after injection, a significantly negative correlation was observed between ustekinumab concentrations and absolute PASI score up to 5·9 µg mL-1 (ρ = -0·357, P = 0·032). Ustekinumab concentrations were higher in optimal responders (PASI ≤ 2) than in suboptimal responders (PASI > 2) (4·0 vs 2·8 µg mL-1 , P = 0·036). The ustekinumab concentration threshold associated with optimal response was determined to be 3·6 µg mL-1 (area under the curve 0·71, sensitivity 86%, specificity 63%). Only one patient (2%) had anti-ustekinumab antibodies. Psoriatic arthritis was identified as an independent predictor of higher PASI scores and higher ustekinumab concentrations (P = 0·003 and P = 0·048, respectively). CONCLUSIONS: A concentration-response relationship at week 4 after injection was observed for patients with psoriasis treated with ustekinumab. Monitoring 4-week postinjection ustekinumab concentrations could timely identify underexposed patients who might benefit from treatment optimization. What's already known about this topic? Monitoring drug concentrations is a valuable tool that can guide clinical decision-making when drug concentrations are linked to clinical outcomes. The presence of a concentration-response relationship for ustekinumab at trough is still debated owing to the contradictory results reported. What does this study add? A concentration-response relationship at week 4 after injection for ustekinumab-treated patients with psoriasis was demonstrated. Monitoring 4-week postinjection ustekinumab concentrations could timely identify underexposed patients who might benefit from treatment optimization. Based on the findings of this study, a treatment algorithm for patients with a suboptimal response is proposed.

Cold storage to overcome dormancy affects the carbohydrate status and photosynthetic capacity of Rhododendron simsii.

Global warming leads to increasing irregular and unexpected warm spells during autumn, and therefore natural chilling requirements to break dormancy are at risk. Controlled cold treatment can provide an answer to this problem. Nevertheless, artificial cold treatment will have consequences for carbon reserves and photosynthesis. In this paper, the effect of dark cold storage at 7 °C to break flower bud dormancy in the evergreen Rhododendron simsii was quantified. Carbohydrate and starch content in leaves and flower buds of an early ('Nordlicht'), semi-early ('M. Marie') and late ('Mw. G. Kint') flowering cultivar showed that carbon loss due to respiration was lowest in 'M. Marie', while 'Mw. G. Kint' was completely depleted of starch reserves at the end of cold treatment. Gene isolation resulted in a candidate gene for sucrose synthase (SUS) RsSus, which appears to be homologous to AtSus3 and had a clear increase in expression in leaves during cold treatment. Photosynthesis measurements on 'Nordlicht' and the late-flowering cultivar 'Thesla' showed that during cold treatment, dark respiration decreased 58% and 63%, respectively. Immediately after cold treatment, dark respiration increased and stabilised after 3 days. The light compensation point followed the same trend as dark respiration. Quantum efficiency showed no significant changes during the first days after cold treatment, but was significantly higher than in plants with dormant flower buds at the start of cold treatment. In conclusion, photosynthesis stabilised 3 days after cold treatment and was improved compared to the level before cold treatment.

Tuberculin skin test versus interferon-gamma release assays for the diagnosis of tuberculosis infection.

OBJECTIVE: Accurate detection of latent tuberculosis infection (LTBI) is becoming increasingly important due to the increasing use of immunosuppressive medications and the human immunodeficiency epidemic, which have increased the risk for reactivation to active tuberculosis (TB) infection. LTBI is detected by tuberculin skin test (TST) and interferon-gamma release assays (IGRAs). The latter include T-SPOT(®).TB (Oxford Immunotec) and QuantiFERON(®)-TB Gold In-Tube (QFT-GIT; Cellestis). We examined the value of TST versus IGRAs in the diagnosis of TB infection by meta-analysis based on data derived from a systematic literature review. METHODS: PubMed was searched for articles in English published between January 2010 and July 2012 in which TST and IGRA were performed simultaneously in individuals with and without active TB infection. A random effect model meta-analysis was performed to determine pooled sensitivity and specificity values for TST, T-SPOT.TB, and QFT-GIT. Owing to the absence of a gold standard for the diagnosis of LTBI, active TB infection was used as a surrogate for LTBI. RESULTS: Nineteen studies were included. T-SPOT.TB was significantly more sensitive [90% (95% confidence interval: 85-95) versus 64% (46-81)] than TST. The specificity of T-SPOT.TB was higher than the specificity of TST, but there was overlap between confidence intervals [77% (68-85) versus 57% (41-72)]. QFT-GIT seemed to be more sensitive than TST [75% (61-86) versus 64% (48-78)] but similarly specific [71% (62-86) versus 70% (57-81)]. CONCLUSIONS: IGRAs, especially T-SPOT.TB, are more effective at detecting TB infection than TST. Despite their higher cost, they have added value and can be requested in addition to TST.

Endometriosis: translation of molecular insights to management.

Endometriosis is a debilitating gynecologic disorder causing pelvic pain and infertility and characterized by the implantation of endometrial tissue to extrauterine locations. Though aspects of the condition remain enigmatic, the molecular pathophysiology of endometriosis appears to be clarifying. Estrogen dependence of the disease is a sentinel endocrine feature and reduction of estrogen bioavailability is the therapeutic principle upon which traditional treatment and prevention approaches have been based. Endometriosis is a chronic inflammatory condition associated with lesional neoangiogenesis and attenuated progesterone action at the level of the endometrium. The elucidation of the molecular pathways mediating these observations has revealed new targets for directed medical and surgical treatment. This paper will review current approaches to the management of endometriosis in the context of the molecular pathophysiology.

Cold storage to overcome dormancy affects the carbon balance of azalea.

Flower bud dormancy in azalea (Rhododendron simsii) is broken by artificial cold treatment and this will have its consequences on carbon reserves and photosynthesis. The effect of cold storage at 7 °C on carbohydrate and starch content in leaves and flower buds of an early ('Nordlicht') and semi-early ('M. Marie) flowering cultivar was quantified. Carbon loss due to respiration was lowest for 'M. Marie'. Photosynthetic measurements on 'Nordlicht' showed that photosynthesis 3 days after cold treatment (plants ready to flower) was improved compared to before cold treatment (plants with dormant flower buds).

Two EST-derived marker systems for cultivar identification in tree peony.

Tree peony (Paeonia suffruticosa Andrews), a woody deciduous shrub, belongs to the section Moutan DC. in the genus of Paeonia of the Paeoniaceae family. To increase the efficiency of breeding, two EST-derived marker systems were developed based on a tree peony expressed sequence tag (EST) database. Using target region amplification polymorphism (TRAP), 19 of 39 primer pairs showed good amplification for 56 accessions with amplicons ranging from 120 to 3,000 bp long, among which 99.3% were polymorphic. In contrast, 7 of 21 primer pairs demonstrated adequate amplification with clear bands for simple sequence repeats (SSRs) developed from ESTs, and a total of 33 alleles were found in 56 accessions. The similarity matrices generated by TRAP and EST-SSR markers were compared, and the Mantel test (r = 0.57778, P = 0.0020) showed a moderate correlation between the two types of molecular markers. TRAP markers were suitable for DNA fingerprinting and EST-SSR markers were more appropriate for discriminating synonyms (the same cultivars with different names due to limited information exchanged among different geographic areas). The two sets of EST-derived markers will be used further for genetic linkage map construction and quantitative trait locus detection in tree peony.

The use of relative quantitative RT-PCR for expression analysis in azalea flower color sports.

The fastest way to create new azalea (Rhododendron simsii hybrids) cultivars is by making use of flower colour sports, which appear spontaneously on azalea plants. Unfortunately, there is still very little known on how bud sport induction occurs. Therefore, genes coding for two key enzymes of the azalea flavonoid biosynthesis pathway, chalcon synthase (chs) and dihydroflavonol 4-reductase (dfr) that were reported before to be apt for modification by the action of bud sporting, were isolated and characterized. The expression of these two flower colour genes in the petals of azalea flowers will be compared between all 'Hellmut Vogel' flower colour sports. To measure the expression levels of both genes, relative quantitative RT-PCR analysis will be worked out on a real-time PCR machine. The expression of housekeeping genes, which is expected to be the same for all sports, will be used to calculate the relative expression level of the two genes of interest. The optimisation of this technique will be discussed.

Characterization of carotenoid biosynthesis in Rhododendron luteum.

In the color chart of Rhododendron simsii hybrids (Belgian pot azalea), yellow is not yet present, although this color is wanted by growers and consumers. Carotenoid pigments, which are absent in azalea, are responsible for this yellow color of the flower petals. The first objective of this project is characterizing the pathway of carotenoid biosynthesis in the yellow flowers of Rhododendron luteum.

A simplified simulator for the training and evaluation of laparoscopic skills.

BACKGROUND: Laparoscopic skills can be measured objectively in a video-laparoscopic cart simulator system. These scores have been shown to be sufficiently sensitive to distinguish differences in performance between residents at different levels of training. The purpose of this study was to compare a simplified mirrored-box simulator to the video-laparoscopic cart system. METHODS: A total of 22 surgical residents performed seven structured tasks in both simulators in random order. Scores reflected precision and speed. The tasks were transferring, cutting, clip + divide, looping, mesh placement + fixation, and suturing with intracorporeal and extracorporeal knots. RESULTS: There were no significant differences in mean raw scores between the simulators for six of the seven tasks. Resident total scores correlated well between simulators (r = 0.68, p = 0.001). Resident ranking also correlated well (r = 0.69, p < 0. 001). CONCLUSIONS: A mirrored-box simulator was shown to provide a reasonable reflection of relative performance of laparoscopic skills. Practical, effective laparoscopic skills training and evaluation can be accomplished without the need for cumbersome equipment.

Short-term outcomes in open vs. laparoscopic herniorrhaphy: confounding impact of worker's compensation on convalescence.

Over a 28-month period, 123 patients with a unilateral inguinal hernia were recruited into a randomized controlled trial comparing open herniorrhaphy (OH) to laparoscopic inguinal herniorrhaphy (LH). The primary end point was duration of convalescence. Sixty-five patients underwent OH and 58 underwent LH. Both groups were well matched for all baseline parameters, although LH patients anticipated a shorter convalescence than OH patients (14.3 +/- 9.4 days vs. 18.5 +/- 10.8 days; P = 0.021). The median duration of hospital stay was one day in both groups. No difference was observed in the duration of convalescence (LH 9.8 +/- 7.4 days; OH 11.6 +/- 7. 7 days) across groups. However, when the data were analyzed after removing patients receiving disability ("worker's") compensation (21 patients), patients undergoing LH recovered on average 3 days faster (LH 7.8 +/- 5.6 days; OH 10.9 +/- 7.5 days; P = 0.02). Patients not receiving worker's compensation appear to have a shorter convalescence after LH compared to OH. Disability compensation is a major confounding variable in determining convalescence and needs to be controlled for in any future trial design.

Pentastarch versus albumin in cardiopulmonary bypass prime: impact on blood loss.

BACKGROUND: Albumin is commonly used as a volume expander in cardiopulmonary bypass (CPB) prime. Pentastarch, a low molecular weight hetastarch, may provide similar efficacy at decreased cost but is known to alter coagulation profiles. Infectious concerns forced the temporary withdrawal of albumin in our institution. Therefore we evaluated pentastarch as an alternative with regards to perioperative hemostasis and blood loss. METHODS: One hundred consecutive adult patients undergoing first-time aorto-coronary bypass were given 750 mL of 10% pentastarch (represented as P in calculations) diluted in 1000 mL of Ringer's solution added in their CPB prime. A similar control group of 100 consecutive patients had received 200 mL of 25% albumin (represented as A in calculations) diluted in 1500 mL of Ringer's solution. RESULTS: Postoperative prothrombin time (PT) was slightly higher with pentastarch (P: 14.9 +/- 1.5 seconds, A: 14.2 +/- 1.3 seconds, p = 0.003). Postoperative bleeding was also increased (P: 2337 +/- 1242 mL, A: 1981 +/- 1121 mL, p = 0.034), mostly because of recirculated shed mediastinal blood (P: 834 +/- 499 mL, A: 640 +/- 388, p = 0.002) rather than lost pleural tube blood (P: 1503 +/- 821 mL, A: 1341 +/- 824 mL, p = 0.16). Overall net blood loss (P: 2014 +/- 914 mL, A: 2061 +/- 1015, p = 0.73) was similar. Blood-product transfusion requirements and postoperative daily hematocrits did not differ. CONCLUSION: The diminished coagulability associated with this dose of pentastarch resulted in increased postoperative bleeding. However, with recirculation of shed mediastinal blood, there was no net increase in blood loss. In this setting, pentastarch may serve as a suitable alternative to albumin.

Mycoplasma pneumoniae pneumonia in a mouse model.

To investigate the pathogenesis of acute Mycoplasma pneumoniae infection, BALB/c mice were anesthetized with metofane, and M. pneumoniae was introduced intranasally on days 0, 1, and 2. Mice were sacrificed on days 0-15. A histopathologic scoring system defined inflammatory changes in the lungs on a scale of 0-26 (least to most severe). Broth cultures were positive for all nasal passage and bronchoalveolar lavage (BAL) specimens. Histopathologic scores ranged from 0 to 21. The mean log10 (cfu/mL) were 4.1-6.4 on days 1-10 and >/=1.7 on days 13-15 for nasal passage and BAL specimens. Serum polymerase chain reaction was negative. ELISA for serum IgM and immunoblots for M. pneumoniae antibody were positive in 21 (62%) of 34 and 33 (97%) of 34 infected animals, respectively, at days 8-15. ELISA for IgG antibody was negative. This mouse pneumonia model can be used to study the immunologic and therapeutic responses to acute M. pneumoniae infection.

Double closed loop obstruction and perforation in a previous Roux-en-Y gastric bypass.

A healthy 45-year-old woman with a previous Roux-en-Y gastric bypass presented with the signs, symptoms and blood analysis results consistent with acute pancreatitis. She was initially treated nonoperatively and subsequently went into circulatory shock. Computerized tomographic scan and exploratory laparotomy revealed a volvulus of the afferent jejunal limb with secondary obstruction, necrosis, and perforation of the bypassed stomach.

Chest wall infarction following bilateral internal mammary to coronary arterial bypass in a patient with a thoracoabdominal aneurysm.

A 69-year-old man with severe peripheral vascular disease and a known thoracoabdominal aortic aneurysm underwent bilateral internal mammary artery (BIMA) to coronary artery bypass grafting and aortic hemiarch replacement. He immediately thereafter developed massive chest wall ischemia and infarction with a severe metabolic acidosis, and subsequently died. Chest wall infarction following BIMA harvesting has not been previously described. Cautious use of internal mammary arterial grafting may be in order in the severe vasculopath with significant thrombo-occlusive thoracoabdominal aortic disease.

Diagnosis of enteroviral meningitis by using PCR with a colorimetric microwell detection assay.

A 5-h, user-friendly PCR assay for the diagnosis of enteroviral meningitis was developed. Reverse transcription and amplification were performed in a one-step reaction using rTth polymerase. Carryover contamination was prevented with dUTP and uracil N-glycosylate. Detection was performed colorimetrically on a microwell titer plate. Sensitivity, specificity, positive predictive value, and negative predictive value were 94.7, 97.4, 94.7, and 97.4%, respectively.

Diagnosis of enteroviral central nervous system infection by polymerase chain reaction during a large community outbreak.

Enteroviruses are common causes of localized and systemic infection in patients of all ages and are the most frequent cause of epidemic aseptic meningitis in the United States. We have developed a polymerase chain reaction (PCR) assay of cerebrospinal fluid (CSF) for rapid diagnosis of enteroviral meningitis. This assay was applied to 257 CSF specimens during a large community outbreak of enterovirus disease; 109 (97%) of 112 enterovirus culture-positive CSF samples contained enterovirus RNA. In addition 35 (66%) of 53 samples from patients with suspected central nervous system disease with negative or no CSF viral cultures were positive by enterovirus PCR. The enterovirus PCR detected 13 different enterovirus serotypes. PCR results are available within 24 hours compared with a mean of 6.8 days for enterovirus culture. The clinical characteristics of 141 patients with enterovirus central nervous system disease are presented. This study demonstrates the usefulness of enterovirus PCR for the rapid diagnosis of enterovirus central nervous system disease and the potential for PCR tests to shorten hospitalization.

Accuracy and utility of differential white blood cell count in the neonatal intensive care unit.

The clinical utility of the complete blood cell count (including the differential white blood cell count) as a means to follow the course of infants in a neonatal intensive care unit was assessed. Utility was judged for three purposes: (1) predicting the onset of clinically unrecognized disease, (2) assessing the severity of current disease, and (3) following a trend during treatment. Neither conventional nor automated differential counts were useful for surveillance (predicting the onset of clinically unrecognized disease). The white blood cell count, the platelet count, and the absolute immature neutrophil count and immature/total neutrophil ratio were useful to assess the severity of current clinical events. The white blood cell count was superior to the differential count for following trends in patients' conditions. Information regarding nuclear immaturity derived from automated counts and the cost and slowness of the manual differential count are good indications for decreased use of conventional counts, increased use of certain features of the automated differential, or both, in neonatal intensive care units.

Laboratory evaluation of differential white blood cell count information from the Coulter S-plus IV and Technicon H-1 in patient populations requiring rapid "turnaround" time.

Automated differential counts produced by the Coulter S-Plus IV (S + 4) and the Technicon H-1 (H-1) were compared with routine and reference manual differentials with the use of samples from the adult emergency room (ER) and the neonatal intensive care unit (NICU), populations in which rapid reporting of laboratory results is considered important. Error rates for routine technologists were 9.3% in the ER and 15.3% in the NICU. Error rates for the two instruments were higher than those for technologists with samples from the ER but could be reduced to 4-5% if instrument flags and additional criteria were used to signal the need for a conventional differential. Instrument error rates were higher yet with NICU samples, and specificity was very low (10% for each device). There were small differences between the instruments in detection of immature neutrophils, but flags from the H-1 were more specific (except for detection of nucleated red blood cells in samples from the ER). If either instrument were used in an adult ER and flags and additional criteria were used to signal the need for conventional differentials, 64-75% fewer manual counts would be performed with no decrease in accuracy and a considerable improvement in turnaround time.

A clinical trial of three-part electronic differential white blood cell counts.

Three-part white blood cell differential counts (diffs), performed during electrical impedance counting of blood cells, can accurately classify lymphocytes, granulocytes, and mononuclear cells in 85% of specimens, with an error rate not exceeding that of conventional diffs. To assess the clinical utility of the new test, we tried it for three months on a medical unit. Conventional diffs were no better than three-part diffs or total white blood cell counts in signaling clinical changes, and the same decisions would have been made in 78% of instances if only three-part diffs were available. The three-part diff is as reliable a clinical index for monitoring most inpatients as the conventional diff or white blood cell count. Although its use could effect significant cost savings, acceptance of the new test seems unlikely unless enforced by administrative flat.