Evaluation of Alternative Doxycycline Antibiotic Regimes in an Inhalational Murine Model of Q Fever.

The timing of the initiation of antibiotic treatment has been shown to impact the clinical outcome of many bacterial infections, including Q fever. Delayed, suboptimal or incorrect antibiotic treatment has been shown to result in poor prognosis, resulting in the progression of acute disease to long-term chronic sequalae. Therefore, there is a requirement to identify an optimal, effective therapeutic regimen to treat acute Q fever. In the study, the efficacies of different doxycycline monohydrate regimens (pre-exposure prophylaxis, post-exposure prophylaxis or treatment at symptom onset or resolution) were evaluated in an inhalational murine model of Q fever. Different treatment lengths (7 or 14 days) were also evaluated. Clinical signs and weight loss were monitored during infection and mice were euthanized at different time points to characterize bacterial colonization in the lungs and the dissemination of bacteria to other tissues including the spleen, brain, testes, bone marrow and adipose. Post-exposure prophylaxis or doxycycline treatment starting at symptoms onset reduced clinical signs, and also delayed the systemic clearance of viable bacteria from key tissues. Effective clearance was dependent on the development of an adaptive immune response, but also driven by sufficient bacterial activity to maintain an active immune response. Pre-exposure prophylaxis or post-exposure treatment at the resolution of clinical signs did not improve outcomes. These are the first studies to experimentally evaluate different doxycycline treatment regimens for Q fever and illustrate the need to explore the efficacy of other novel antibiotics.

Complications with hot, cold and altitude environments in disaster management.

INTRODUCTION: Disaster management is the process of preparing, responding and recovering to an emergency whether that be natural or man-made. It is a time-consuming, resource-heavy process with the aim of reducing the risk of certain events and, where not possible, reducing the impact of said disaster, ensuring that the risks have been identified and appropriate rescue and recovery plan is in place. METHODS: We carried out a thorough literature search on the complications of hot, cold and altitude environments in disaster management and distilled the learnings into this article. RESULTS: The incidence of disasters of natural, man-made and complex origin is likely to continue increasing as global temperatures continue to rise. CONCLUSION: Disaster management in the extreme environments of hot, cold and high altitude is fraught with unique challenges, especially around the physiological response of rescuers, resource constraints and logistics. Recognising these challenges is an important aspect of planning and preparation for disaster management in these environments.

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.

Finafloxacin, a Novel Fluoroquinolone, Reduces the Clinical Signs of Infection and Pathology in a Mouse Model of Q Fever.

Finafloxacin is a novel fluoroquinolone with optimal antibacterial activity in low pH environments, therefore offering a therapeutic advantage over some traditional antibiotics, in treating bacterial infections associated with acidic foci. Coxiella burnetii, the causative agent of Q fever, is a bacterium which resides and replicates in acidic intracellular parasitic vacuoles. The efficacy of finafloxacin was evaluated in vivo using the A/J mouse model of inhalational Q fever and was compared to doxycycline, the standard treatment for this infection and ciprofloxacin, a comparator fluoroquinolone. Finafloxacin reduced the severity of the clinical signs of infection and weight loss associated with Q fever, but did not reduce the level of bacterial colonization in tissues compared to doxycycline or ciprofloxacin. However, histopathological analysis suggested that treatment with finafloxacin reduced tissue damage associated with C. burnetii infection. In addition, we report for the first time, the use of viable counts on axenic media to evaluate antibiotic efficacy in vivo.

Evaluation of the Efficacy of Doxycycline, Ciprofloxacin, Levofloxacin, and Co-trimoxazole Using In Vitro and In Vivo Models of Q Fever.

Q fever, caused by the intracellular pathogen Coxiella burnetii, is traditionally treated using tetracycline antibiotics, such as doxycycline. Doxycycline is often poorly tolerated, and antibiotic-resistant strains have been isolated. In this study, we have evaluated a panel of antibiotics (doxycycline, ciprofloxacin, levofloxacin, and co-trimoxazole) against C. burnetii using in vitro methods (determination of MIC using liquid and solid media; efficacy assessment in a THP cell infection model) and in vivo methods (wax moth larvae and mouse models of infection). In addition, the schedule for antibiotic treatment has been evaluated, with therapy initiated at 24 h pre- or postchallenge. Both doxycycline and levofloxacin limited overt clinical signs during treatment in the AJ mouse model of aerosol infection, but further studies are required to investigate the possibility of disease relapse or incomplete bacterial clearance after the antibiotics are stopped. Levofloxacin was well tolerated and therefore warrants further investigation as an alternative to the current recommended treatment with doxycycline.

Endoscopic Botulinum toxin as a treatment for delayed gastric emptying following oesophagogastrectomy.

INTRODUCTION: The incidence of delayed gastric emptying (DGE) following oesophagogastrectomy with gastric conduit reconstruction is reported to be between 1.7% and 50%. This variation is due to differing practices of intraoperative pylorus drainage procedures, which increase the risk of postoperative biliary reflux and dumping syndrome, resulting in significant morbidity. The aim of our study was to establish rates of DGE in people undergoing oesophagogastrectomy without routine intraoperative drainage procedures, and to evaluate outcomes of postoperative endoscopically administered Botulinum toxin into the pylorus (EBP) for people with DGE resistant to systemic pharmacological treatment. METHODS: All patients undergoing oesophagogastrectomy between 1 January 2016 and 31 March 2018 at our unit were included. No intraoperative pyloric drainage procedures were performed, and DGE resistant to systemic pharmacotherapy was managed with EBP. RESULTS: Ninety-seven patients were included. Postoperatively, 29 patients (30%) were diagnosed with DGE resistant to pharmacotherapy. Of these, 16 (16.5%) were diagnosed within 30 days of surgery. The median pre-procedure nasogastric tube aspirate was 780ml; following EBP, this fell to 125ml (p<0.001). Median delay from surgery to EBP in this cohort was 13 days (IQR 7-16 days). Six patients required a second course of EBP, with 100% successful resolution of DGE before discharge. There were no procedural complications. CONCLUSIONS: This is the largest series of patients without routine intraoperative drainage procedures. Only 30% of patients developed DGE resistant to pharmacotherapy, which was managed safely with EBP in the postoperative period, thus minimising the risk of biliary reflux in people who would otherwise be at risk following prophylactic pylorus drainage procedures.

Comparison of PCR and Viable Count as a Method for Enumeration of Bacteria in an A/J Mouse Aerosol Model of Q Fever.

Historically, disease progression in animal models of Q fever has been carried out using PCR to monitor the presence of Coxiella burnetii DNA in the host. However, the colonization and dissemination of other bacterial infections in animal models are tracked using viable counts, enabling an accurate assessment of viable bacterial load within tissues. Following recent advances in the culture methods, it has become possible to do the same with C. burnetii. Here we compare and contrast the different information gained by using PCR or viable counts to study this disease. Viable bacteria were cleared from organs much faster than previously reported when assessed by bacterial DNA, but weight loss and clinical signs improved while animals were still heavily infected.

Crystallisation in basaltic magmas revealed via in situ 4D synchrotron X-ray microtomography.

Magma crystallisation is a fundamental process driving eruptions and controlling the style of volcanic activity. Crystal nucleation delay, heterogeneous and homogeneous nucleation and crystal growth are all time-dependent processes, however, there is a paucity of real-time experimental data on crystal nucleation and growth kinetics, particularly at the beginning of crystallisation when conditions are far from equilibrium. Here, we reveal the first in situ 3D time-dependent observations of crystal nucleation and growth kinetics in a natural magma, reproducing the crystallisation occurring in real-time during a lava flow, by combining a bespoke high-temperature environmental cell with fast synchrotron X-ray microtomography. We find that both crystal nucleation and growth occur in pulses, with the first crystallisation wave producing a relatively low volume fraction of crystals and hence negligible influence on magma viscosity. This result explains why some lava flows cover kilometres in a few hours from eruption inception, highlighting the hazard posed by fast-moving lava flows. We use our observations to quantify disequilibrium crystallisation in basaltic magmas using an empirical model. Our results demonstrate the potential of in situ 3D time-dependent experiments and have fundamental implications for the rheological evolution of basaltic lava flows, aiding flow modelling, eruption forecasting and hazard management.

Use of axenic media to determine antibiotic efficacy against coxiella burnetii.

The traditional methods of measuring minimum inhibitory concentration (MIC) of antibiotics against Coxiella burnetii are time-consuming and technically difficult. The discovery of axenic media for C. burnetii culture provided an opportunity to determine the feasibility of using both broth dilution and an antimicrobial gradient method (Etest) as a convenient method of measuring MICs. The MICs for a range of antibiotics that have proven or potential use in the treatment of Q fever, namely doxycycline, ciprofloxacin, levofloxacin, moxifloxacin and co-trimoxazole, were measured. It was possible to measure MICs using both microdilution and Etest methods. MICs obtained were comparable to those from other methods. This study demonstrates the potential use of a relatively simple test to measure MIC in an organism that is difficult to culture.

Safety and effectiveness of antireflux surgery in obese patients.

Introduction The incidence of gastro-oesophageal reflux disease and obesity has increased significantly in recent years. The number of antireflux procedures being carried out on people with a higher body mass index (BMI) has been rising. Evidence is conflicting for outcomes of antireflux surgery in obese patients in terms of its safety and efficacy. Given the contradictory reports, this meta-analysis was undertaken to establish the outcomes of antireflux surgery (ARS) in obese patients and its associated safety. Methods A systematic electronic search was conducted using the PubMed, MEDLINE®, Ovid®, Cochrane Library and Google Scholar™ databases to identify studies that analysed the effect of BMI on the outcomes of ARS. A meta-analysis was performed using the random effects model. The intraoperative and postoperative outcomes that were examined included operative time, conversion to an open procedure, mean length of hospital stay, recurrence of acid reflux requiring reoperation and wrap migration. Results A total of 3,772 patients were included in 13 studies. There was no significant difference in procedure conversion rate, recurrence of reflux requiring reoperation or wrap migration between obese and non-obese patients. However, both the mean operative time and mean length of stay were longer for obese patients. Conclusions ARS in obese patients with gastro-oesophageal reflux disease is safe and outcomes are comparable with those in patients with a BMI in the normal range. A high BMI should therefore not be a deterrent to considering ARS for appropriate patients.

Imaging for Synovitis, Acne, Pustulosis, Hyperostosis, and Osteitis (SAPHO) Syndrome.

Multifocal osteomyelitis and synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome constitute a spectrum of disease that includes inflammatory bone lesions and dermatologic findings. Radiographic features resemble those of the spondyloarthropathies with anterior chest wall involvement. Early radiographic findings are osteodestructive with lytic lesions. Bone scintigraphy of the sternoclavicular region classically yields a 'bull's head' pattern of radionuclide uptake. Magnetic resonance imaging (MRI) can demonstrate corner lesions of vertebral bodies. Ultrasound often reveals peripheral enthesitis. Late radiographic features are usually osteoproliferative. PET/CT can identify chronic lesions. Differential diagnostic considerations include osteomyelitis and malignancy, which often prompt bone biopsy.

Multifocal Langerhans Cell Histiocytosis in an Adult.

Eosinophilic granuloma (EG) is the most common and benign form of the spectrum of disorders referred to as Langerhans cell histiocytosis (LCH). Langerhans cell histiocytosis is primarily regarded as a pediatric disease, with few adult cases of multifocal EG of bone reported. We report a case of multifocal EG in a 48-year-old woman, who presented with right knee pain. Radiographs showed a small lytic lesion in the medial femoral condyle. Diagnosis was confirmed by ultrasound-guided biopsy. She had had a previous EG lesion excised from her skull. Whole-body bone scan demonstrated a new skull lesion in the right diploic space, which was confirmed by magnetic resonance imaging. The patient underwent curettage, bone grafting, and prophylactic internal fixation of the right distal femur lesion. The skull lesion was treated with repeat craniectomy. Two years later, she developed a new lesion in the right distal femoral metaphysis, which was treated with intralesional corticosteroid injections. Now, more than 1 year later, the patient is pain-free with no evidence of new or recurrent disease. Because multifocal EG is a rare diagnosis in adults, appropriate clinical suspicion, in combination with radiographic findings and histologic examination, is essential for correct diagnosis and treatment.

The role of ultrasound in the management of patients with occult groin hernias.

INTRODUCTION: Groin ultrasound scanning is commonly used to examine patients with obscure groin pain or swelling. A recent study has shown ultrasound has a poor positive predictive value (PPV) in diagnosing groin hernias although earlier studies reported PPV values as high as 100%. Our aims were to calculate ultrasound's accuracy in diagnosing occult groin hernias in symptomatic patients and assess how management of these patients is affected by ultrasound result. METHODS: We retrospectively analysed 375 symptomatic adult patients, who between February 2008 and March 2010, had ultrasound to diagnose groin hernias when clinical examination was inconclusive. Patients were identified on a prospective radiology database and all groin ultrasounds were performed by either one consultant radiologist or one radiographer. RESULTS: Ultrasound was positive in 199 patients, of which 118 underwent surgery. Using operative findings as the gold standard, ultrasound's PPV for groin hernias was 70% (95% CI: 62-78%). Ultrasound was equivocal in 42 patients of which hernias were diagnosed in 7 of the 10 who had surgery. Ultrasound was negative in 151 patients of which none were later diagnosed with hernias during 3 years' median follow-up. CONCLUSION: Ultrasound is poor in diagnosing occult groin hernias with a PPV of 70% suggesting a 30% chance of negative groin exploration. The equivocal ultrasound group requires careful follow-up as a considerable number were later diagnosed with hernia. The absence of subsequent hernia diagnosis in the negative ultrasound group suggests it may be a useful rule-out test to exclude occult groin hernias in symptomatic patients.

Control of intracellular Francisella tularensis by different cell types and the role of nitric oxide.

Reactive nitrogen is critical for the clearance of Francisella tularensis infections. Here we assess the role of nitric oxide in control of intracellular infections in two murine macrophage cell lines of different provenance: the alveolar macrophage cell line, MH-S, and the widely used peritoneal macrophage cell line, J774A.1. Cells were infected with the highly virulent Schu S4 strain or with the avirulent live vaccine strain (LVS) with and without stimuli. Compared to MH-S cells, J774A.1 cells were unresponsive to stimulation and were able to control the intracellular replication of LVS bacteria, but not of Schu S4. In MH-S cells, Schu S4 demonstrated control over cellular NO production. Despite this, MH-S cells stimulated with LPS or LPS and IFN-γ were able to control intracellular Schu S4 numbers. However, only stimulation with LPS induced significant cellular NO production. Combined stimulation with LPS and IFN-γ produced a significant reduction in intracellular bacteria that occurred whether high levels of NO were produced or not, indicating that NO secretion is not the only defensive cellular mechanism operating in virulent Francisella infections. Understanding how F. tularensis interacts with host macrophages will help in the rational design of new and effective therapies.

Galleria mellonella as an alternative model of Coxiella burnetii infection.

Coxiella burnetii is a Gram-negative intracellular bacterium and is the causative agent of the zoonotic disease Q fever. Several rodent and non-human primate models of virulent phase I C. burnetii [Nine Mile (NM)I] have been developed, and have been used to determine the efficacy of antibiotics and vaccine candidates. However, there are several advantages to using insect models to study host-microbe interactions, such as reduced animal use, lowered cost and ease of manipulation in high containment. In addition, many laboratories use the avirulent phase II C. burnetii clone (NMII) to study cellular interactions and identify novel virulence determinants using genetic manipulation. We report that larvae of the greater wax moth, Galleria mellonella, were susceptible to infection with both C. burnetii NMI and NMII. Following subcutaneous infection, we report that intracellular bacteria were present within haemocytes and that larval death occurred in a dose-dependent manner. Additionally, we have used the model to characterize the role of the type 4 secretion system in C. burnetii NMII and to determine antibiotic efficacy in a non-mammalian model of disease.

The ability of CpG oligonucleotides to protect mice against Francisella tularensis live vaccine strain but not fully virulent F. tularensis subspecies holarctica is reflected in cell-based assays.

CpG DNA is a potent activator of the innate immune system. Here the protective effects of CpG DNA are assessed against the facultative intracellular pathogen Francisella tularensis. Dosing of mice with CpG DNA provided protection against disease caused by F. tularensis subsp. holarctica live vaccine strain (LVS) but did not protect against the fully virulent F. tularensis subsp holarctica strain HN63. Similarly, in vitro studies in J774A murine macrophage-like cells demonstrated that stimulation with CpG DNA enables control of intracellular replication of LVS but not HN63. These data confirm findings that CpG DNA may have limited efficacy in providing protection against fully virulent strains of F. tularensis and also suggest that in vitro assays may be useful for the evaluation of novel treatments for virulent F. tularensis.

The therapeutic potential of immune cross-talk in leishmaniasis.

Veterans of infection, Leishmania parasites have been plaguing mammals for centuries, causing a morbidity toll second only to that of malaria as the most devastating protozoan parasitic disease in the world. Cutaneous leishmaniasis (CL) is, by far, the most prevalent form of the disease, with symptoms ranging from a single self-healing lesion to chronic metastatic leishmaniasis (ML). In an increasingly immunocompromised population, complicated CL is becoming a more likely outcome, characterized by severely inflamed, destructive lesions that are often refractory to current treatment. This is perhaps because our ageing arsenal of variably effective antileishmanial drugs may be directly or indirectly immunomodulatory and may thus have variable effects in each type and stage of CL. Indeed, widely differing immune biases are created by the various species of Leishmania, and these immunological watersheds are further shifted by extrinsic disturbances in immune homeostasis. For example, we recently showed that a naturally occurring RNA virus (Leishmania RNA virus (LRV)) within some Leishmania parasites creates hyperinflammatory cross-talk, which can predispose to ML: a case of immunological misfire that may require a different approach to immunotherapy, whereby treatments are tailored to underlying immune biases. Understanding the intersecting immune pathways of leishmaniasis and its co-infections will enable us to identify new drug targets, and thereby design therapeutic strategies that work by untangling the immunological cross-wires of pathogenic cross-talk.

Adherence to acceptability and repeatability criteria for spirometry in complex lung function laboratories.

BACKGROUND: Few published data exist for adherence rates to spirometry acceptability and repeatability criteria in clinical respiratory laboratories. This study quantified adherence levels in this setting and observed changes in adherence levels as a result of feedback and ongoing training. METHODS: Two tertiary hospital-based, lung function laboratories (L1 and L2) participated. Approximately 100 consecutive, FVC spirometry sessions were reviewed for each year from 2004 to 2008 at L1 and for years 2004 and 2008 at L2. Each spirometric effort and session was interrogated for adherence to the acceptability and repeatability criteria of international spirometry standards of the time. Feedback of audit results and refresher training were provided at L1 throughout the study; in addition, a quality rating scale was implemented in 2006. No formal feedback or follow-up training was provided at L2. RESULTS: We reviewed 707 test sessions over the 5 years. There was no difference in adherence rates to acceptability and repeatability criteria between sites in 2004 (L1 61%, L2 59%, P = .89). There was, however, a significant difference between sites in 2008 (L1 92%, L2 65%, P < .001). No difference was seen at L2 between 2004 and 2008 (P = .26), while L1 experienced a significant increase in adherence levels between 2004 and 2008 (61% to 92% P < .001). CONCLUSIONS: Clinical respiratory laboratories met published spirometry acceptability and repeatability criteria only 60% of the time in the first audit period. This improved with regular review, feedback, and implementation of a rating scale. Auditing of spirometry quality, feedback, and implementation of test rating scales need to be incorporated as an integral component of laboratory quality assurance programs to improve adherence to international acceptability and repeatability criteria.

Peripheral human γδ T cells control growth of both avirulent and highly virulent strains of Francisella tularensis in vitro.

In this paper we evaluate the role of human γδ T cells in control of Francisella tularensis infection. Using an in vitro model of infection, a reduction in bacterial numbers was detected in the presence of human γδ T cells for both attenuated LVS and virulent SCHU S4 strains of F. tularensis. Antibody neutralisation of IFN-γ caused an increase in survival of F. tularensis LVS suggesting that γδ T cell-mediated control of F. tularensis infection is partially mediated by IFN-γ.