Preclinical Evaluation of Sodium Butyrate's Potential to Reduce Alcohol Consumption: A Dose-Escalation Study in C57BL/6J Mice in Antibiotic-Enhanced Binge-Like Drinking Model.

INTRODUCTION: In our earlier efforts to establish gut-brain axis during alcohol use disorder (AUD), we have demonstrated that supplementation of C57BL/6J male mice with 8 mg/mL sodium butyrate, a major short-chain fatty acid, in drinking water reduced ethanol intake and neuroinflammatory response in antibiotic (ABX)-enhanced voluntary binge-like alcohol consumption model, drinking in the dark (DID). METHODS: To further evaluate the preclinical potential of SB, we have set a dose-escalation study in C57BL/6J male mice to test effects of ad libitum 20 mg/mL SB and 50 mg/mL SB and their combinations with ABX in the DID procedure for 4 weeks. Effects of these SB concentrations on ethanol consumption and bodily parameters were determined for the duration of the treatments. At the end of study, blood, liver, and intestinal tissues were collected to study any potential adverse effects ad to measure blood ethanol concentrations. RESULTS: Increasing SB concentrations in the drinking water caused a loss in the protective effect against ethanol consumption and produced adverse effects on body and liver weights, reduced overall liquid intake. The hypothesis that these effects were due to aversion to SB smell/taste at these high concentrations were further tested in a follow up proof-of-concept study with intragastric gavage administration of SB. The higher gavage dose (320 mg/kg) caused reduction in ethanol consumption without any adverse effects. CONCLUSION: Overall, these findings added more support for the therapeutic potential of SB in management of AUD, given a proper form of administration.

Optimizing Workflow in Combined Petrosectomy Approaches: Surgical Technique and Case Series.

BACKGROUND AND OBJECTIVE: The combined petrosectomy is one of the workhorse skull base approaches to the petroclival region. Traditionally, this approach starts with a temporosuboccipital craniotomy, followed by the mastoidectomy/anterior petrosectomy, and completed with the dural opening/tumor resection. This sequence of events (neurosurgery-neuro-otology-neurosurgery) involves at least 2 handoffs and change of surgical teams and instrumentation. This report describes a resequencing of events and a modification of the technique used to craft the temporosuboccipital craniotomy, with aims to reducing handoffs between surgical teams and improving operating room workflow. METHODS: Adhering to PROCESS guidelines, a case series is provided in addition to the surgical technique and surgical images. RESULTS: The technique for performing a combined petrosectomy is described with illustrations. This description shows that the temporal bone drilling may be performed before the craniotomy to allow for direct visualization of the dura and sinuses before completing the craniotomy. In doing so, only 1 transition between the otolaryngologist and neurosurgeon is necessary, thereby improving operating room workflow and time management. A series of 10 patients is presented, showing the feasibility of this procedure and providing operative details that were previously absent in the peer-reviewed literature. CONCLUSIONS: Combined petrosectomy, although often performed in a 3-step manner with the neurosurgeon starting the craniotomy, can be performed as described here in a 2-step manner, with similar outcomes and reasonable operating time.

Whole genome sequencing and molecular epidemiology of paediatric Staphylococcus aureus bacteraemia.

OBJECTIVES: The role Staphylococcus aureus antimicrobial resistance genes and toxins play in disease severity, management and outcome in childhood is an emerging field requiring further exploration. METHODS: A prospective multisite study of Australian and New Zealand children hospitalised with S. aureus bacteraemia (SAB) occurred over 24 months (2017-2018). Whole genome sequencing (WGS) data were paired with clinical information from the ISAIAH cohort. RESULTS: 353 SAB isolates were sequenced; 85% methicillin-susceptible S. aureus ([MSSA], 301/353) and 15% methicillin-resistant S. aureus ([MRSA], 52/353). There were 92 sequence types (STs), most commonly ST5 (18%) and ST30 (8%), grouped into 23 clonal complexes (CCs), most frequently CC5 (21%) and CC30 (12%). MSSA comprised the majority of healthcare-associated SAB (87%, 109/125), with principal clones CC15 (48%, 11/21) and CC8 (33%, 7/21). Panton-Valentine leukocidin (PVL)-positive SAB occurred in 22% (76/353); predominantly MSSA (59%, 45/76), community-onset (92%, 70/76) infections. For community-onset SAB, the only microbiological independent predictor of poor outcomes was PVL positivity (aOR 2.6 [CI 1.0-6.2]). CONCLUSION: From this WGS paediatric SAB data, we demonstrate the previously under-recognized role MSSA has in harbouring genetic virulence and causing healthcare-associated infections. PVL positivity was the only molecular independent predictor of poor outcomes in children. These findings underscore the need for further research to define the potential implications PVL-producing strains may have on approaches to S. aureus clinical management.

Pediatric Staphylococcus aureus Bacteremia: Clinical Spectrum and Predictors of Poor Outcome.

BACKGROUND: Staphylococcus aureus is a common cause of bacteremia, yet the epidemiology and predictors of poor outcome remain inadequately defined in childhood. METHODS: ISAIAH (Invasive Staphylococcus aureus Infections and Hospitalizations in children) is a prospective, cross-sectional study of S. aureus bacteremia (SAB) in children hospitalized in Australia and New Zealand over 24 months (2017-2018). RESULTS: Overall, 552 SABs were identified (incidence 4.4/100 000/year). Indigenous children, those from lower socioeconomic areas and neonates were overrepresented. Although 90-day mortality was infrequent, one-third experienced the composite of: length of stay >30 days (26%), intensive care unit admission (20%), relapse (4%), or death (3%). Predictors of mortality included prematurity (adjusted odds ratio [aOR],16.8; 95% confidence interval [CI], 1.6-296.9), multifocal infection (aOR, 22.6; CI, 1.4-498.5), necrotizing pneumonia (aOR, 38.9; CI, 1.7-1754.6), multiorgan dysfunction (aOR, 26.5; CI, 4.1-268.8), and empiric vancomycin (aOR, 15.7; CI, 1.6-434.4); while infectious diseases (ID) consultation (aOR, 0.07; CI .004-.9) was protective. Neither MRSA nor vancomycin trough targets impacted survival; however, empiric vancomycin was associated with nephrotoxicity (OR, 3.1; 95% CI 1.3-8.1). CONCLUSIONS: High SAB incidence was demonstrated and for the first time in a pediatric setting, necrotizing pneumonia and multifocal infection were predictors of mortality, while ID consultation was protective. The need to reevaluate pediatric vancomycin trough targets and limit unnecessary empiric vancomycin exposure to reduce poor outcomes and nephrotoxicity is highlighted. One in 3 children experienced considerable SAB morbidity; therefore, pediatric inclusion in future SAB comparator trials is paramount to improve outcomes.

Medical management of atraumatic Mycobacterium abscessus cutaneous infection: A case report.

Treatment for cutaneous infection from Mycobacterium abscessus is fraught with poorly established evidence. Given its antibiotic multi-resistance, surgical intervention is often recommended. We report a case of cutaneous M. abscessus infection that was successfully managed with medical therapy alone. A 55-year-old immunocompetent woman from the Bellarine peninsula in Victoria, Australia presented to our hospital with a 2-week history of a non-healing ulcer on her left forearm. The patient had no history of trauma or procedures to the skin. On presentation, the patient had a punch biopsy, which was culture positive for M. abscessus. The isolate was susceptible to clarithromycin and amikacin, had intermediate susceptibility to ciprofloxacin, cefoxitin and linezolid and was resistant to doxycycline, imipenem, cotrimoxazole and moxifloxacin. The tigecycline MIC was 0.25 µg/ml. The patient received a total of 12 weeks of oral clarithromycin 500 mg twice daily, 4 weeks of intravenous amikacin 500 mg daily, 6 weeks of intravenous tigecycline 100 mg over 24 hours via Baxter pump, and 4 weeks of oral clofazimine 100 mg daily. The patient made a good clinical recovery and had her medical therapy ceased after 12 weeks. M. abscessus cutaneous infection in an immunocompetent individual without antecedent trauma or surgery is rare. Our case illustrates the successful treatment of a deep M. abscessus cutaneous ulcer with relatively short duration macrolide-based antibiotic therapy without any surgical intervention.

Bilateral osteomyelitis and liver abscess caused by hypervirulent Klebsiella pneumoniae- a rare clinical manifestation (case report).

BACKGROUND: Hypervirulent strains of Klebsiella pneumoniae are a recognized cause of a distinct invasive syndrome that results in pyogenic liver abscesses and metastatic complications, particularly in the Asia Pacific region. Reports of hypervirulent K.pneumoniae in Europe, the Americas and Australia indicate worldwide spread. We present a case of multi-focal osteomyelitis, a rarely described complication of hypervirulent K.pneumoniae in the medical literature. The prevalence of this condition in countries outside Asia may be expected to rise with increasing travel. CASE PRESENTATION: A 20-year-old Chinese man residing in Australia for 2 years presented with a 2-week history of gradually worsening leg pain preceded by 2 weeks of constitutional symptoms. Imaging with computerized axial tomography (CT) and other modalities revealed bilateral tibial lesions described as lattice-like linear lucencies involving the cortices with scalloping of the outer involved cortex. Cultures of tissue from a left tibial bone biopsy were positive cultures for K.pneumoniae. Whole-genome sequencing identified the isolate as K1 serotype ST23, a well-recognized hyper virulent strain capable of causing invasive disease. An abdominal CT revealed a 27x22mm liver abscess. The patient had no other metastatic manifestations of the disease, and responded to 6 weeks of intravenous ceftriaxone followed by 3 months of oral Ciprofloxacin. CONCLUSIONS: Increased awareness of the manifestations and subsequent management of hyper virulent strains of K.pneumoniae by clinicians is important to assist early recognition and help minimize serious sequelae. Cases with overseas links, such as previous residence in the Asia Pacific area, are at higher risk for infection with the hyper virulent strain. This case highlights the need for clinicians to be able to recognize this important disease, especially in patients with the right epidemiological links, and to investigate and treat appropriately to prevent severe metastatic complications.

Increased Severity and Spread of Mycobacterium ulcerans, Southeastern Australia.

Reported cases of Mycobacterium ulcerans disease (Buruli ulcer) have been increasing in southeastern Australia and spreading into new geographic areas. We analyzed 426 cases of M. ulcerans disease during January 1998-May 2017 in the established disease-endemic region of the Bellarine Peninsula and the emerging endemic region of the Mornington Peninsula. A total of 20.4% of cases-patients had severe disease. Over time, there has been an increase in the number of cases managed per year and the proportion associated with severe disease. Risk factors associated with severe disease included age, time period (range of years of diagnosis), and location of lesions over a joint. We highlight the changing epidemiology and pathogenicity of M. ulcerans disease in Australia. Further research, including genomic studies of emergent strains with increased pathogenicity, are urgently needed to improve the understanding of disease to facilitate implementation of effective public health measures to halt its spread.

Endovascular Treatment of Large Unruptured Fusiform Fenestrated Vertebrobasilar Junction Aneurysm.

Fenestrated vertebrobasilar junction aneurysms are rare vascular lesions. Microsurgical intervention is extremely difficult due to the complex anatomy in the vicinity of these aneurysms. Endovascular neurosurgery appears to be safe and should be considered as the first modality of treatment. This case study details the treatment of an unruptured fusiform fenestrated vertebrobasilar junction aneurysm with endovascular occlusion with stent-assisted coiling. The optimal angiographic exposure and selective microcatheterization of the aneurysm were challenging due to the patient's body habitus, and the aneurysm was large with one dominant fenestrated limb.

Management of dengue in Australian travellers: a retrospective multicentre analysis.

OBJECTIVES: To describe the epidemiology, clinical and laboratory features and outcomes of dengue in returned Australian travellers, applying the revised WHO dengue classification (2009) to this population. DESIGN, SETTING AND PARTICIPANTS: Retrospective case series analysis of confirmed dengue cases hospitalised at one of four Australian tertiary hospitals, January 2012 - May 2015. MAIN OUTCOME MEASURES: Clinical features, laboratory findings and outcomes of patients with dengue; dengue classification according to 2009 WHO guidelines. RESULTS: 208 hospitalised patients (median age, 32 years; range, 4-76 years) were included in the study. Dengue was most frequently acquired in Indonesia (94 patients, 45%) and Thailand (40, 19%). The most common clinical features were fever (98% of patients) and headache (76%). 84 patients (40%) met the WHO criteria for dengue with warning signs, and one the criteria for severe dengue; the most common warning signs were mucosal bleeding (44 patients, 21%) and abdominal pain (43, 21%). Leukopenia (176 patients, 85%), thrombocytopenia (133, 64%), and elevated liver enzyme levels (154, 76%) were the most common laboratory findings. 46 patients (22%) had serological evidence of previous exposure to dengue virus. WHO guidelines were documented as a management benchmark in ten cases (5%); 46 patients (22%) received non-steroidal anti-inflammatory drugs (NSAIDs). CONCLUSIONS: A significant proportion of returning Australian travellers hospitalised for dengue have unrecognised warning signs of severe disease. Many received NSAIDs, which can increase the risk of haemorrhage in dengue. As travel to Asia from Australia continues to increase, it is vital for averting serious outcomes that clinicians can recognise and manage dengue.

Delayed Temporal Lobe Hemorrhage After Initiation of Acyclovir in an Immunocompetent Patient with Herpes Simplex Virus-2 Encephalitis: A Case Report.

Herpes simplex virus (HSV) is the most common cause of non-epidemic, sporadic, acute focal encephalitis in the United States. Inflammation of the vasculature makes them friable and susceptible to hemorrhage. Massive hemorrhage, though rare, can present in a delayed fashion after initiation of acyclovir and often requires surgical intervention. We report a unique case of delayed temporal lobe hemorrhage after initiation of acyclovir in an immunocompetent patient, specifically for its presentation, virology, and surgical management. A 40-year-old left-handed Caucasian female with chronic headaches, along with a 20-pack-year smoking history, presented to an outside facility with one week of diffuse, generalized headache, fever, nausea, and vomiting. Initial cranial imaging was negative for hemorrhage. Cerebrospinal fluid (CSF) studies showed a lymphocytic pleocytosis with elevated protein, along with polymerase chain reaction (PCR) positive staining for HSV, establishing the diagnosis of HSV-2 encephalitis, which is less common in adults. Acyclovir was initiated and one week later while still hospitalized, the patient developed acute altered mental status with cranial imaging showing a large right temporal lobe hemorrhage with significant midline shift. She was transferred to our facility for surgical intervention. Computed tomography angiography (CTA) was negative for any underlying vascular lesion. She was taken to the operating room for a decompressive unilateral (right) hemicraniectomy and temporal lobectomy. She had no postoperative complications and completed a three-week course of acyclovir. She was discharged to acute rehab with plans to return at a later date for cranioplasty. Intracerebral hemorrhage is an uncommon, although possible sequela, of herpes encephalitis. Despite initiation of early antiviral therapy, close monitoring is warranted, given the pathophysiology of the vasculature. Any decline in the neurological exam and/or increasing symptomatology of increased intracranial pressure mandates immediate cranial imaging to evaluate for possible hemorrhage. Emergent surgical intervention is warranted with large temporal lobe hemorrhages.

A review of the public health management of shigellosis in Australia in the era of culture-independent diagnostic testing.

OBJECTIVE: To review the national case definition for shigellosis following the introduction of culture independent diagnostic testing by clinical laboratories and provide evidence to reform jurisdictional public health practices for the management shigellosis., . METHODS: A review of all Australian jurisdictional public health guidelines for shigellosis was conducted. Victorian 2014 shigellosis data were analysed: demographics and risk factors for cases identified by conventional culture or culture-independent diagnostic methods were described. RESULTS: There was considerable variation in reporting of cases to the National Notifiable Disease Surveillance System (NNDSS) by the eight Australian jurisdictions, with an array of classifications based on diagnostic testing methodologies. Analysis of Victorian 2014 shigellosis data found that culture positive cases were more likely to have reported men who have sex with men (MSM) as a risk factor than PCR positive only cases (p<0.0001) and less likely to have reported overseas travel during their incubation period (p<0.0001). Over a 10-year period (2005 to 2014), only two of 86 cases who were employed in high-risk occupations had ongoing positive faecal cultures after appropriate treatment. CONCLUSIONS: The national surveillance case definition for shigellosis should be reviewed to facilitate standardised reporting across Australia. All jurisdictions must consider the public health significance of PCR positive only results in their surveillance risk assessments to inform management of shigellosis cases.

Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes.

Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.

Overcoming preexisting humoral immunity to AAV using capsid decoys.

Adeno-associated virus (AAV) vectors delivered through the systemic circulation successfully transduce various target tissues in animal models. However, similar attempts in humans have been hampered by the high prevalence of neutralizing antibodies to AAV, which completely block vector transduction. We show in both mouse and nonhuman primate models that addition of empty capsid to the final vector formulation can, in a dose-dependent manner, adsorb these antibodies, even at high titers, thus overcoming their inhibitory effect. To further enhance the safety of the approach, we mutated the receptor binding site of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a target cell. Our work suggests that optimizing the ratio of full/empty capsids in the final formulation of vector, based on a patient's anti-AAV titers, will maximize the efficacy of gene transfer after systemic vector delivery.

Pharmacological modulation of humoral immunity in a nonhuman primate model of AAV gene transfer for hemophilia B.

Liver gene transfer for hemophilia B has shown very promising results in recent clinical studies. A potential complication of gene-based treatments for hemophilia and other inherited disorders, however, is the development of neutralizing antibodies (NAb) against the therapeutic transgene. The risk of developing NAb to the coagulation factor IX (F.IX) transgene product following adeno-associated virus (AAV)-mediated hepatic gene transfer for hemophilia is small but not absent, as formation of inhibitory antibodies to F.IX is observed in experimental animals following liver gene transfer. Thus, strategies to modulate antitransgene NAb responses are needed. Here, we used the anti-B cell monoclonal antibody rituximab (rtx) in combination with cyclosporine A (CsA) to eradicate anti-human F.IX NAb in rhesus macaques previously injected intravenously with AAV8 vectors expressing human F.IX. A short course of immunosuppression (IS) resulted in eradication of anti-F.IX NAb with restoration of plasma F.IX transgene product detection. In one animal, following IS anti-AAV6 antibodies also dropped below detection, allowing for successful AAV vector readministration and resulting in high levels (60% or normal) of F.IX transgene product in plasma. Though the number of animals is small, this study supports for the safety and efficacy of B cell-targeting therapies to eradicate NAb developed following AAV-mediated gene transfer.

Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography.

Adeno-associated virus (AAV) empty capsids typically co-purify with genome containing AAV2 vectors purified by column chromatography. This study describes a method to remove empty capsids from genome containing vector particles by anion exchange chromatography. The separation is based on the slightly less anionic character of empty particles compared to vectors. Detailed methods to achieve AAV2 vector purification and particle separation using cation exchange resin POROS 50HS followed by anion exchange resin Q-Sepharose(xl) are described. Chromatographic separation of AAV2 particles was achieved using gradients based on sodium acetate and ammonium acetate, and was optimal at pH 8.5. Efficient removal of particle surface nucleic acid impurities was found to be important to achieve good particle separation. In a large scale experiment performed using partially purified vector containing a mixture of 1.56 x 10(14)vg and 2.52 x 10(15) empty capsids as a starting material, the optimized anion exchange chromatography method resulted in a vector peak of 1.15 x 10(14)vg containing 0.25 x 10(14) empty capsids, corresponding to 74% vector yield and 86-fold reduction in empty capsids in the vector product.