A multisite validation of a two hours antibiotic susceptibility flow cytometry assay directly from positive blood cultures.

BACKGROUND: Rapid antimicrobial susceptibility testing (AST) is urgently needed to provide safer treatment to counteract antimicrobial resistance. This is critical in septic patients, because resistance increases empiric therapy uncertainty and the risk of a poor outcome. We validate a novel 2h flow cytometry AST assay directly from positive blood cultures (PBC) by using a room temperature stable FASTgramneg and FASTgrampos kits (FASTinov® Porto, Portugal) in three sites: FASTinov (site-1), Hospital Ramon y Cajal, Madrid, Spain (site-2) and Centro Hospitalar S. João, Porto, Portugal (site-3). A total of 670 PBC were included: 333 spiked (site-1) and 337 clinical PBC (151 site-2 and 186 site-3): 367 gram-negative and 303 gram-positive. Manufacturer instructions were followed for sample preparation, panel inoculation, incubation (1h/37ºC) and flow cytometry analysis using CytoFlex (Site-1 and -2) or DxFlex (site-3) both instruments from Beckman-Coulter, USA. RESULTS: A proprietary software (bioFAST) was used to immediately generate a susceptibility report in less than 2 h. In parallel, samples were processed according to reference AST methods (disk diffusion and/or microdilution) and interpreted with EUCAST and CLSI criteria. Additionally, ten samples were spiked in all sites for inter-laboratory reproducibility. Sensitivity and specificity were >95% for all antimicrobials. Reproducibility was 96.8%/95.0% for FASTgramneg and 95.1%/95.1% for FASTgrampos regarding EUCAST/CLSI criteria, respectively. CONCLUSION: FASTinov® kits consistently provide ultra-rapid AST in 2h with high accuracy and reproducibility on both Gram-negative and Gram-positive bacteria. This technology creates a new paradigm in bacterial infection management and holds the potential to significantly impact septic patient outcomes and antimicrobial stewardship.

Ischaemic stroke in HIV-infected patients: a case-control study.

OBJECTIVES: The aim of the study was to provide insights into the contributions of HIV infection stage, antiretroviral therapy (ART) and vascular risk factors to the occurrence of ischaemic stroke in HIV-infected patients. METHODS: We performed a case-control study of HIV-infected patients followed in our clinic. We compared patients hospitalized between January 2006 and June 2014 with an ischaemic stroke or transient ischaemic attack to age- and gender-matched controls without stroke. RESULTS: Of 2146 patients followed in our clinic, we included 23 cases (20 men and three women; mean age 51.3 years) and 23 controls. Eighty-three per cent of cases had had a stroke and 17% a transient ischaemic attack. According to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification, small-vessel occlusion was the most frequent aetiology, followed by large-artery atherosclerosis and cardioembolism. Compared with controls, stroke was statistically significantly associated with diabetes, smoking and low concentrations of high-density lipoprotein (HDL) cholesterol. Illegal drug use, a low CD4 count and a high viral load were also associated with ischaemic cerebral events. There were no statistically significant differences between cases and controls in Centers for Disease Control and Prevention (CDC) HIV stage, CD4 count nadir and HIV infection time-to-event. No statistically significant differences were found concerning ART or treatment compliance. CONCLUSIONS: In our single centre study, we found associations of illegal drug use, HIV replication and some traditional vascular risk factors with the occurrence of ischaemic cerebral events. The paradigm of the care of HIV-infected patients is changing. Concomitant diseases in the ageing patient with HIV infection, including cerebrovascular disease, must also be addressed in view of their impacts on morbidity and mortality. Apart from controlling the HIV infection and immunosuppression with ART, vascular risk factors must also be addressed.

Assessment of gas compression and lung volume during air stacking maneuver.

PURPOSE: We reasoned that the application of positive pressure through air stacking (AS) technique could cause gas compression and the absolute lung volumes could be estimated. The aim of this study was to estimate the amount of gas compression (ΔV comp) during AS in healthy subjects positioned at 45° trunk inclination and verify if the simultaneous measurements of chest wall volume changes (ΔV CW), by optoelectronic plethysmography, and changes in lung volume (ΔV ao), by pneumotachograph, combined with pressure variation at the airways opening (ΔP ao) during AS are able to provide reliable data on absolute lung volumes. METHODS: Twenty healthy subjects (mean age 23.5 ± 3.8 years) were studied during a protocol that included slow vital capacity and AS maneuvers. V comp was calculated by subtracting ΔV ao and ΔV CW occurring during AS and total lung capacity (TLC) was estimated by applying Boyle-Mariote's law using V comp and ΔP ao. RESULTS: During AS, 0.140 ± 0.050 L of gas was compressed with an average ΔP ao of 21.78 ± 6.18 cmH2O. No significant differences between the estimated TLC (-0.03 ± 3.0% difference, p = 0.6020), estimated FRC (-2.0 ± 12.4% difference, p = 0.5172), measured IC (1.2 ± 11.2% difference, p = 0.7627) and predicted values were found. CONCLUSION: During AS, a significant gas compression occurs and absolute lung volumes can be estimated by simultaneous measurements of ΔV CW, ΔV ao and ΔP ao.

Pain reduction and improvement of function following ultrasound-guided intra-articular injections of triamcinolone hexacetonide and hyaluronic acid in hip osteoarthritis.

The scientific literature has shown positive results regarding intra-articular injections of hyaluronic acid in osteoarthritic joints. When injecting in the hip joint, the guidance of ultrasound can provide higher injection accuracy and repeatability. However, due to the methodological limitations in the current available literature, its recommendation in the current practice is still controversial. This study shows that ultrasound-guided intra-articular injections of triamcinolone hexacetonide and hyaluronic acid can improve pain, function and quality of life in patients with symptomatic and radiographic hip osteoarthritis. In addition, the administration of triamcinolone hexacetonide and hyaluronic acid to the hip joint in these patients can delay the need for interventional surgery.

IL-4 and IL-6 levels and adipose tissue distribution in HIV-1 patients under antiretroviral therapy.

PURPOSE: Combined antiretroviral therapy (cART) for the treatment of HIV-1 infection has been associated with complications, including lipodystrophy. Several interleukins have been implicated in the pathology and physiology of lipodystrophy. The present study aimed to compare the levels of IL-4 and IL-6 in HIV-1 patients under cART with and without, clinically and fat mass ratio defined, lipodystrophy and in four different groups of fat distribution: (1) no lipodystrophy; (2) isolated central fat accumulation; (3) isolated lipoatrophy and (4) mixed forms of lipodystrophy. METHODS: In the present cross-sectional study we evaluated IL-4 and IL-6 levels, insulin resistance and insulin sensitivity indexes in 86 HIV-infected adults under cART. RESULTS: No significant differences in IL-4 and IL-6 levels between the four groups of body composition were observed. Patients with HOMA-IR >4 presented higher levels of IL-6 and lower levels of IL-4, although without statistical significance. No correlation between IL-6, or IL-4, HOMA-IR and quantitative body fat mass distribution was found. CONCLUSION: Although there was a tendency for patients with isolated lipoatrophy and isolated fat accumulation to present higher IL-6 levels, these differences were not statistically significant. No differences were found relating IL-4 levels.

Lung cancer: the immune system and radiation.

Lung cancer has a known relationship with smoking and is one of the leading causes of cancer-related death worldwide. Although the number of studies discussing lung cancer is vast, treatment efficacy is still suboptimal due to the wide range of factors that affect patient outcome. This review aims to collect information on lung cancer treatment, specially focused on radiation therapy. It also compiles information regarding the influence of radiotherapy on the immune system and its response to tumour cells. It evaluates how immune cells react after radiation exposure and the influence of their cytokines in the tumour microenvironment. The literature analysis points out that the immune system is a very promising field of investigation regarding prognosis, mostly because the stromal microenvironment in the tumour can provide some information about what can succeed in the future concerning treatment choices and perspectives. T cells (CD4+ and CD8+), interleukin-8, vascular endothelial growth factor and transforming growth factor-ß seem to have a key role in the immune response after radiation exposure. The lack of large scale studies means there is no common consensus in the scientific community about the role of the immune system in lung cancer patients treated with radiotherapy. Clarification of the mechanism behind the immune response after radiation can lead to better treatments and better quality life for patients.

An unusual case of delayed-type hypersensitivity to ceftriaxone and meropenem.

Recent studies have demonstrated a low cross-reactivity between ß-lactam antibiotics and carbapenems in IgE-mediated reactions. There are no studies on cross-reactivity of meropenem in patients with non-immediate hypersensitivity to cephalosporins. We describe a case of a 13-year-old male, admitted in Neurosurgery with a severe extradural empyema complicating frontal sinusitis, submitted to an emergent bifrontal craniotomy. A generalized maculopapular exanthema, fever and malaise, appeared by the 7th day of meningeal doses of ceftriaxone, clindamycin and vancomycin. Those were replaced by meropenem, with posterior worsening of the reaction and mucosal involvement. A new scheme with amikacin, metronidazole and linezolid was done with improvement. Skin prick, intradermal and patch tests to penicillins, ceftriaxone and meropenem were negative. Lymphocyte transformation test was positive to ceftriaxone and negative to meropenem.Non-immediate T cell mechanism seems to be involved. Diagnosis work-up couldn't exclude cross-reactivity between ceftriaxone and meropenem.

Infliximab therapy increases the frequency of circulating CD16(+) monocytes and modifies macrophage cytokine response to bacterial infection.

Crohn's disease (CD) has been correlated with altered macrophage response to microorganisms. Considering the efficacy of infliximab treatment on CD remission, we investigated infliximab effects on circulating monocyte subsets and on macrophage cytokine response to bacteria. Human peripheral blood monocyte-derived macrophages were obtained from CD patients, treated or not with infliximab. Macrophages were infected with Escherichia coli, Enterococcus faecalis, Mycobacterium avium subsp. paratuberculosis (MAP) or M. avium subsp avium, and cytokine levels [tumour necrosis factor (TNF) and interleukin (IL)-10] were evaluated at different time-points. To evaluate infliximab-dependent effects on monocyte subsets, we studied CD14 and CD16 expression by peripheral blood monocytes before and after different infliximab administrations. We also investigated TNF secretion by macrophages obtained from CD16(+) and CD16(-) monocytes and the frequency of TNF(+) cells among CD16(+) and CD16(-) monocyte-derived macrophages from CD patients. Infliximab treatment resulted in elevated TNF and IL-10 macrophage response to bacteria. An infliximab-dependent increase in the frequency of circulating CD16(+) monocytes (particularly the CD14(++) CD16(+) subset) was also observed (before infliximab: 4·65 ± 0·58%; after three administrations: 10·68 ± 2·23%). In response to MAP infection, macrophages obtained from CD16(+) monocytes were higher TNF producers and CD16(+) macrophages from infliximab-treated CD patients showed increased frequency of TNF(+) cells. In conclusion, infliximab treatment increased the TNF production of CD macrophages in response to bacteria, which seemed to depend upon enrichment of CD16(+) circulating monocytes, particularly of the CD14(++) CD16(+) subset. Infliximab treatment of CD patients also resulted in increased macrophage IL-10 production in response to bacteria, suggesting an infliximab-induced shift to M2 macrophages.

Acute acalculous cholecystitis in malaria: a review of seven cases from an adult cohort.

PURPOSE AND METHODS: Acute acalculous cholecystitis (AAC) is an uncommon condition related to serious clinical conditions, such as surgery, trauma, burn injuries and sepsis. The diagnosis of AAC remains challenging to make, since it generally occurs as a secondary event in acutely ill patients with another disease. Imaging evaluation is crucial, and well-known criteria are accepted for the diagnosis. To our knowledge, only case reports of AAC related to 12 malaria adult patients have been published. In this series, seven cases of AAC from a cohort of 42 adult patients with severe imported falciparum malaria [according to the World Health Organization (WHO) criteria] are presented. The aim is to report the cases and look for malaria conditions that may affect the incidence of this unusual malaria complication. RESULTS: Ultrasonography revealed gallbladder with wall thickening in all patients, plus other(s) major criteria. Each patient presented five to nine WHO severe malaria criteria: all had hyperparasitaemia and hyperbilirubinaemia. All patients developed renal failure, six pulmonary oedema/acute respiratory distress syndrome (ARDS) (five were mechanically ventilated) and five shock. Treatment was non-operative in five patients, cutaneous cholecystostomy was done in two and the outcome was favourable in all. CONCLUSIONS: Patients with AAC have significantly more commonly five or more criteria of severe malaria: renal insufficiency, pulmonary oedema/ARDS, parasitaemia higher than 30 %, nosocomial infection and a prolonged intensive care unit (ICU) stay. Increased awareness for this unusual and potentially severe complication of malaria is needed.

Acute hepatitis E complicated by Guillain-Barre syndrome in Portugal, December 2012--a case report.

Autochthonous hepatitis E virus (HEV) infection has been increasingly reported in Europe and the United States, mostly arising from genotype 3 and less frequently genotype 4. We report here on a patient with HEV genotype 3a infection complicated by Guillain-Barré syndrome in Portugal in December 2012. We draw attention to the diagnosis of autochthonous HEV infection and to its rare, but important, neurological complications.

Population mobility and the changing epidemics of HIV-2 in Portugal.

INTRODUCTION: Portugal is the European country with the highest frequency of HIV-2 infection, which is mainly concentrated in West Africa. The cumulative number of notified HIV-2 infections in Portugal was 1813 by the end of December 2008. To better characterize the dynamics of HIV-2 infection in the country and to obtain data that may be of use in the prevention of the spread of HIV-2, we evaluated a large pooled sample of patients. PATIENTS AND METHODS: Five Portuguese hospitals provided data on HIV-2-infected patients from 1984 to the end of 2007. Data concerning demographic characteristics and clinical variables were extracted. Patients were stratified according to date of diagnosis in approximately 5-year categories. RESULTS: The sample included 442 patients, accounting for 37% of all HIV-2 infections notified in Portugal during that period. HIV-2-infected patients showed clearly different characteristics according to the period of diagnosis. Until 2000, the majority of HIV-2-infected patients were Portuguese-born males living in the north of the country. From 2000 to 2007, most of the patients diagnosed with HIV-2 infection had a West African origin, were predominantly female and were living in the capital, Lisbon. The average age at diagnosis and loss to follow-up significantly increased over time. CONCLUSION: HIV-2 infection has been documented in Portugal since the early 1980s and its epidemiology appears to reflect changes in population movement. These include the movements of soldiers and repatriates from African territories during the independence wars and, later, migration and mobility from high-endemicity areas. The findings of this study stress the importance of promoting migrant-sensitive health care.

Prevalence of obesity and its relationship to clinical lipodystrophy in HIV-infected adults on anti-retroviral therapy.

BACKGROUND: Combination antiretroviral therapy (cART) is associated with lipodystrophy (lipoatrophy and lipoaccumulation) and several metabolic abnormalities that together can contribute to an increased cardiovascular risk. The aim of this study was to evaluate the prevalence of obesity in patients on cART according to the presence of clinical lipodystrophy (CL) and to analyze factors associated with obesity. METHODS: We evaluated 368 HIV-infected adults on cART. RESULTS: CL was present in 59.0%. Independently of gender, patients with CL were more frequently underweight [5.7% (21/368)] and of normal weight [47.3% (174/368)], and less frequently overweight [33.2% (122/368)] or obese [13.9% (51/368)]. Mean body mass index was higher in patients with abdominal prominence regardless of the presence of clinical lipoatrophy. Patients with CL had lower waist circumference, higher waist/hip and fat mass ratio and lower total and subcutaneous fat, without significant difference in visceral fat but with a higher visceral/subcutaneous fat ratio, as evaluated by CT at abdominal level. CL was significantly less frequent in overweight [odds ratio (OR)=0.21, 95% confidence interval (CI): 0.05-0.92] and obese (OR=0.05, 95%CI: 0.01-0.26) patients, when compared to underweight ones, independent of age, gender, duration of infection, cART regimen, virological suppression, and HIV-infection risk factor. CONCLUSIONS: Being overweight or obese is highly prevalent in HIV-infected patients on cART. Patients with CL were more frequently under- or normal weight, and less frequently overweight or obese. Obesity is a condition that should be considered in HIV patients on cART.

Assessment of body fat composition disturbances by bioimpedance analysis in HIV-infected adults.

HIV-lipodystrophy syndrome is characterized by different patterns of body fat distribution (BFD) which are identified by clinical and body composition (BC) assessment, including bioimpedance analysis (BIA). Our aim was to compare BC in HIV-infected patients on combination antiretroviral therapy (cART) according to 4 distinct phenotypes of BFD (G1-no lipodystrophy, G2-isolated central fat accumulation, G3-lipoatrophy, G4-mixed forms of lipodystrophy) and assessed factors associated with them. Anthropometry and BIA were performed in 344 HIV-1 patients. G2 and G4 phenotype patients had significantly higher fat mass (FM) but no differences were observed in fat-free mass (FFM) and total body water among the 4 phenotypes. Significant negative associations were found between the presence of lipoatrophy and female gender, body mass index (BMI), waist (WC), hip (HC) and thigh circumferences, and total body FM estimated by BIA. After adjustment for gender, cART duration and BMI, G3 had significant lower WC [odds ratio (OR)=0.84; 0.78- 0.90] and HC (OR=0.88; 0.81-0.96) mean. Independently of gender, cART duration and BMI, G2 remained significantly associated with higher WC (OR=1.11; 1.05-1.18) and HC (OR=1.15; 1.07-1.23) mean, and with FM estimated by BIA [FM as %, OR=1.17 (1.09-1.26); and FM as kg, OR=1.15 (1.06- 1.25)]. There was a significant positive association between G4 and female gender (OR=1.66; 1.01-2.75), BMI (OR=1.10; 1.04-1.17) and WC (OR=1.15; 1.09-1.21). The similar FFM along the BFD spectrum describes the actual BC of these patients without sarcopenia. In a clinical setting, BIA is an easy and useful tool to evaluate fat mass and FFM and gives us a picture of BC that was not possible with anthropometry.

Diagnosis and treatment of acute humoral kidney allograft rejection.

Acute humoral rejection (AHR) is a severe form of rejection associated with poor graft survival. Prompt diagnosis and rapid institution of therapy are crucial to improve the prognosis. A therapeutic approach based on plasmapheresis, intravenous imunoglobulin, and rituximab seems to be effective in refractory cases. Herein we have described our experience with 11 patients with biopsy-proven AHR who were treated between January 2005 and June 2008. Seven of these patients had panel reactive antibodies titers more than 50%. The diagnosis was based on Banff 2001 criteria; treatment consisted of a combination of plasmapheresis and intravenous immunoglobulin. Four refractory cases were also treated with a single dose of rituximab. One graft was lost due to thrombosis. All other patients recovered graft function with an average creatinine level of 1.6 mg/dL at 8.6 +/- 2.7 months of follow-up.

Pancreas-kidney transplantation and the evolution of pancreatic autoantibodies.

The recurrence or persistence of pancreatic autoantibodies after pancreas-kidney transplantation (PKT) is an intriguing finding. We prospectively analyzed 77 PKTs, searching for risk factors for the expression of these autoimmune markers and their impact on pancreas graft function. Among the 77 PKTs, 24.7% had 0 HLA matches, 20.8% displayed delayed graft function, and 14.3% had acute rejection episodes. Immunosuppression included antithymocyte globulin (ATG), tacrolimus, mycophenolate mofetil (MMF), and steroids. Sixty-five patients had both grafts functioning as a follow-up of more than 6 months. In 11 patients anti-glutamic acid decarboxylase (GAD) positivity persists (n = 8) or has recurred (n = 3), 4 of whom show increasing titers. Two patients maintain positive islet cell antibodies (ICA) and anti-GAD antibodies. The 9 patients positive for ICA included 2 who were negative before PKT and 7 who remain positive. The "positive" group (22 patients with positive ICA and/or anti-GAD) did not differ from the global group of 65 functioning PKT in terms of acute rejection episodes, HLA match, and steroid withdrawal. Among the positive patients, there were 2 with borderline glucose levels; however, among the entire "positive" group, the mean fasting glucose, HbA1c, and C-peptide measurements were not significantly different, when compared with the other 65 PKTs. In conclusion, pancreatic autoantibodies may be persistently positive or recur after PKT, despite appropriate immunosuppression. Its impact on long-term pancreas graft survival is unknown. We could not identify risk factors for their expression. An extended follow-up with monitoring and search for other risk factors may be necessary to increase our knowledge in this field.

Human Immunodeficiency Virus (HIV) 2 Superinfection in a Patient Receiving Antiretroviral Therapy With Longstanding HIV-1 Viral Load Suppression.

Dual human immunodeficiency virus (HIV) 1 and HIV-2 superinfections are rare but challenging. A HIV-1-infected patient receiving effective antiretroviral therapy was investigated for a severe CD4+ cell count decline. HIV-2 superinfection was diagnosed and genotypic test revealed mutations conferring resistance to most drug class, limiting options for treatment.

Life-Threatening Everolimus-Associated Pneumonitis: A Case Report and a Review of the Literature.

INTRODUCTION: Noninfective pneumonitis is a class-related effect within mammalian target of rapamycin (mTOR) inhibitors, including everolimus, and can occasionally be severe. CASE REPORT: A 62-year-old man, medicated with everolimus due to a heart transplantation 17 years previously and with chronic kidney disease, was admitted to the intensive care unit (ICU) with acute respiratory failure, cardiovascular shock, and impaired renal function requiring dialysis. Computed tomography (CT) scan revealed right upper lobe consolidation. Extensive microbiological workup, autoimmune testing, and cytology were negative and echocardiography showed preserved heart function. Everolimus levels were normal (5.7-6.1 ng/mL) and the drug was suspended at day 9. The patient was difficult to ventilate and responded poorly to broad-spectrum antibiotic and antifungal therapy. On day 25, CT scan and bronchoscopy revealed left-sided alveolar hemorrhage, and corticosteroid pulses were performed. The patient gradually improved. After discharge and 6 months of follow-up, clinical recovery was complete and chest imaging substantially improved. DISCUSSION: Pneumonitis occurs in up to 4.3% of transplant recipients using everolimus for immunosuppression. Despite usually presenting as a mild and self-limited disease, severe cases have been described. Alveolar hemorrhage can occur and is associated with poor outcome. Everolimus levels do not seem to accurately predict toxicity. Corticosteroid therapy has been used with success in severe disease. We review the pathophysiological, clinical, and management-related aspects of this entity with emphasis on its potential severity. CONCLUSION: Our case was a rare occurrence of severe life-threatening pulmonary disease related to everolimus. Awareness of the potential severity of this entity is important for the management of patients using mTOR inhibitors.

The influence of HLA mismatches and immunosuppression on kidney graft survival: an analysis of more than 1300 patients.

New immunosuppressive drugs used in kidney transplantation decreased the incidence of acute rejection. It was hypothesized that, with their power, the importance of HLA matching was decreased. To evaluate the influence of HLA matching, immunosuppression, and other possible risk factors, we analyzed data of 1314 consecutive deceased donor kidney transplantation. We divided the patient population into 4 cohorts, according to the era of transplantation: era 1, before 1990, azathioprine (Aza) and cyclosporine (Csa) no microemulsion; era 2, between 1990 and 1995, Csa microemulsion; era 3, between 1996 and 2000, wide use of mycophenolate mofetil (MMF) and anti-thymocyte globulin (ATG); and era 4, after 2000, marked by sirolimus and tacrolimus (TAC) use. Multivariate analysis compared death-censored graft survival. Using as reference the results obtained with 0 HLA mismatches, we verified, during era 1 and era 2, an increased risk of graft loss for all of the subgroups with HLA mismatch >0. However, during era 3 and era 4, the number of HLA mismatches did not influence graft survival. Although acute rejection and delayed graft function, which decreased in the later periods, remained as prognostic factors for graft loss. Considering the immunosuppressive protocol with Csa+Aza+Pred as reference, protocols used after 1995 with Pred+Csa+ATG, with Pred+Csa+MMF, and with Pred+Tac+MMF presented better survival results. Results showed that the significance of HLA matching decreased while the results improved with the new immunosuppressant drugs. These observations support the hypothesis that the weakened importance of HLA matching may be a consequence of the increasing efficacy of the immunosuppression.