Intracellular peptides in SARS-CoV-2-infected patients.

Intracellular peptides (InPeps) generated by the orchestrated action of the proteasome and intracellular peptidases have biological and pharmacological significance. Here, human plasma relative concentration of specific InPeps was compared between 175 patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and 45 SARS-CoV-2 non-infected patients; 2,466 unique peptides were identified, of which 67% were InPeps. The results revealed differences of a specific group of peptides in human plasma comparing non-infected individuals to patients infected by SARS-CoV-2, following the results of the semi-quantitative analyses by isotope-labeled electrospray mass spectrometry. The protein-protein interactions networks enriched pathways, drawn by genes encoding the proteins from which the peptides originated, revealed the presence of the coronavirus disease/COVID-19 network solely in the group of patients fatally infected by SARS-CoV-2. Thus, modulation of the relative plasma levels of specific InPeps could be employed as a predictive tool for disease outcome.

Severe COVID-19 patients show a dysregulation of the NLRP3 inflammasome in circulating neutrophils.

SARS-CoV-2 triggers inflammasome-dependent release of pro-inflammatory cytokine IL-1ß and pyroptosis, therefore, contributes to the huge inflammatory response observed in severe COVID-19 patients. Less is known about the engagement of inflammasome in neutrophils, main players in tissue injury and severe infection. We studied the activation of the inflammasome in neutrophils from severe COVID-19 patients and assessed its consequence in term of cells contribution to disease pathogenesis. We demonstrated that NLRP3 inflammasome is dramatically activated in neutrophils from severe COVID-19 patients and that the specific inhibition of NLRP3 reverts neutrophils' activation. Next, the stimulation of severe patients' neutrophils with common NLRP3 stimuli was not able to further activate the inflammasome, possibly due to exhaustion or increased percentage of circulating immature neutrophils. Collectively, our results demonstrate that the NLRP3 inflammasome is hyperactivated in severe COVID-19 neutrophils and its exhaustion may be responsible for the increased susceptibility to subsequent (and possibly lethal) infections. Our findings thus include a novel piece in the complex puzzle of COVID-19 pathogenesis.

A common variant close to the "tripwire" linker region of NLRP1 contributes to severe COVID-19.

OBJECTIVE AND DESIGN: The heterogeneity of response to SARS-CoV-2 infection is directly linked to the individual genetic background. Genetic variants of inflammasome-related genes have been pointed as risk factors for several inflammatory sterile and infectious disease. In the group of inflammasome receptors, NLRP1 stands out as a good novel candidate as severity factor for COVID-19 disease. METHODS: To address this question, we performed an association study of NLRP1, DPP9, CARD8, IL1B, and IL18 single nucleotide variants (SNVs) in a cohort of 945 COVID-19 patients. RESULTS: The NLRP1 p.Leu155His in the linker region, target of viral protease, was significantly associated to COVID-19 severity, which could contribute to the excessive cytokine release reported in severe cases. CONCLUSION: Inflammasome genetic background contributes to individual response to SARS-CoV-2.

Immunogenicity decay and case incidence six months post Sinovac-CoronaVac vaccine in autoimmune rheumatic diseases patients.

The determination of durability and vaccine-associated protection is essential for booster doses strategies, however data on the stability of SARS-CoV-2 immunity are scarce. Here we assess anti-SARS-CoV-2 immunogenicity decay and incident cases six months after the 2nd dose of Sinovac-CoronaVac inactivated vaccine (D210) in 828 autoimmune rheumatic diseases patients compared with 207 age/sex-balanced control individuals. The primary outcome is the presence of anti-S1/S2 SARS-CoV-2 IgG at 6 months compared to 6 weeks after 2nd vaccine dose for decay evaluation. Secondary outcomes are presence of neutralizing antibodies, percent inhibition by neutralizing, geometric mean titers and cumulative incident cases at 6 months after 2nd dose. Anti-S1/S2 IgG positivity and titers reduce to 23.8% and 38% in patients (p < 0.001) during the six-month follow up and 20% and 51% in controls (p < 0.001), respectively. Neutralizing antibodies positivity and percent inhibition declines 41% and 54% in patients (p < 0.001) and 39.7% and 47% in controls (p < 0.001). Multivariate logistic regression analysis show males (OR = 0.56;95% CI0.40-0.79), prednisone (OR = 0.56; 95% CI0.41-0.76), anti-TNF (OR = 0.66;95% CI0.45-0.96), abatacept (OR = 0.29; 95% CI0.15-0.56) and rituximab (OR = 0.32;95% CI0.11-0.90) associate with a substantial reduction in IgG response at day 210 in patients. Although cellular immunity was not assessed, a decrease of COVID-19 cases (from 27.5 to 8.1/100 person-years; p < 0.001) is observed despite the concomitant emergence and spread of the Delta variant. Altogether we show a reduction in immunity 6-months of Sinovac-CoronaVac 2nd dose, particularly in males and those under immunosuppressives therapies, without a concomitant rise in COVID-19 cases. (CoronavRheum clinicaltrials.gov:NCT04754698).

Resveratrol Downmodulates Neutrophil Extracellular Trap (NET) Generation by Neutrophils in Patients with Severe COVID-19.

The formation of microthrombi in lung autopsies indicates the involvement of NETs in the immunopathogenesis of severe COVID-19. Therefore, supplements inhibiting NET formation, in association with drugs with fewer adverse effects, should be a relevant strategy to attenuate the disease. Resveratrol (RESV) is a natural polyphenol with an important antiviral and antioxidant role. To modulate neutrophils from patients infected with SARS-CoV-2, we evaluated the in vitro effect of RESV on NET formation. Herein, we investigated 190 patients hospitalized with moderate, severe, and critical symptoms at Hospital das Clínicas, Brazil. We observed that neutrophilia in patients with severe COVID-19 infection is composed of neutrophils with activated profile able to release NET spontaneously. Notably, RESV decreased the neutrophil-activated status and the release of free DNA, inhibiting NET formation even under the specific PMA stimulus. At present, there is no evidence of the role of RESV in neutrophils from patients with COVID-19 infection. These findings suggest that adjunctive therapies with RESV may help decrease the inflammation of viral or bacterial infection, improving patient outcomes.

SARS-CoV-2/COVID-19: Clinical Course Among Subjects HIV-1-Infected in Sao Paulo.

INTRODUCTION: People living with Human Immunodeficiency Virus (HIV) are under risk for co-infection with SARS-CoV-2. This population may be more prone to complications from COVID-19 due to persistent inflammation caused by HIV and higher incidence of metabolic syndromes, cardiovascular diseases, and malignancies, as well as being considered elderly at 50 years of age. The objective of this study was to report SARS-CoV-2 infection frequency, clinical evolution, and mortality in HIV-positive patients on antiretroviral therapy. METHODS: The period of inquiry ranged from January to September 2020. Due to the social distance and the suspension of in-person medical care during the time of the investigation, we sent electronic questions about demographic, epidemiological, and clinical data to 403 HIV-infected patients. RESULTS: Among 260 patients who answered the questionnaire, thirty-nine patients (15%) had suggestive symptoms and were tested for SARS-CoV-2 infection. Of this, 11 had positive results (32.4%) and no patient died of COVID-19 complications. Nine were male (3.4%), and the mean age of the patients with positive results was 43.2 years (± 9.6). 107 patients (41.1%) were over 50 years of age and their mean T-CD4+ cell count was 768. Eleven patients (4.2%) had a detectable HIV RNA viral load and 127 (48.8%) had comorbidities. These variables were not associated with an increased risk for infection. CONCLUSION: The frequency of SARS-COV2 infection among HIV-infected is similar to the general population, and the clinical course is associated with the presence of comorbidities and not due to the HIV infection. However, new studies should be done to assess if this vulnerable population could answer the vaccine anti-SARS-Cov2.

Genetic risk factors and COVID-19 severity in Brazil: results from BRACOVID study.

The coronavirus disease 2019 (COVID-19) pandemic has changed the paradigms for disease surveillance and rapid deployment of scientific-based evidence for understanding disease biology, susceptibility and treatment. We have organized a large-scale genome-wide association study (GWAS) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected individuals in Sao Paulo, Brazil, one of the most affected areas of the pandemic in the country, itself one of the most affected in the world. Here, we present the results of the initial analysis in the first 5233 participants of the BRACOVID study. We have conducted a GWAS for COVID-19 hospitalization enrolling 3533 cases (hospitalized COVID-19 participants) and 1700 controls (non-hospitalized COVID-19 participants). Models were adjusted by age, sex and the 4 first principal components. A meta-analysis was also conducted merging BRACOVID hospitalization data with the Human Genetic Initiative (HGI) Consortia results. BRACOVID results validated most loci previously identified in the HGI meta-analysis. In addition, no significant heterogeneity according to ancestral group within the Brazilian population was observed for the two most important COVID-19 severity associated loci: 3p21.31 and Chr21 near IFNAR2. Using only data provided by BRACOVID, a new genome-wide significant locus was identified on Chr1 near the genes DSTYK and RBBP5. The associated haplotype has also been previously associated with a number of blood cell related traits and might play a role in modulating the immune response in COVID-19 cases.

Antiviral Response Induced by Toll-Like Receptor (TLR) 7/TLR8 Activation Inhibits Human Immunodeficiency Virus Type 1 Infection in Cord Blood Macrophages.

Vertical transmission is the main mechanism of human immunodeficiency virus type 1 (HIV-1) infection in infants, who may develop high viremia and rapidly progress to AIDS. Innate immunity agonists can control HIV-1 replication in vitro, but the protective effect in the neonatal period remains unknown. Herein, we evaluated the immunomodulatory and antiviral effects of type I interferon (IFN-I) adjuvants on cord blood monocyte-derived macrophages upon HIV-1 infection. Despite the phenotypic and transcriptional similarities between cord blood and adult macrophages, cord blood cells were prone to viral replication when infected with HIV-1. However, treatment with CL097 efficiently promoted the antiviral and inflammatory responses and inhibited HIV-1 replication in cord blood cells in an NF-κB and autophagy activation-independent manner. Our data suggest that cord blood macrophages are able to establish antiviral responses induced by IFN-I adjuvants similar to those of their adult counterparts, revealing a potential adjuvant candidate to enhance the neonatal immune response.

Immunogenicity and safety of two doses of the CoronaVac SARS-CoV-2 vaccine in SARS-CoV-2 seropositive and seronegative patients with autoimmune rheumatic diseases in Brazil: a subgroup analysis of a phase 4 prospective study.

BACKGROUND: We aimed to examine the immunogenicity pattern induced by the inactivated SARS-CoV-2 vaccine CoronaVac (Sinovac Life Sciences, Beijing, China) in SARS-CoV-2 seropositive patients with autoimmune rheumatic diseases compared with seropositive controls, seronegative patients with autoimmune rheumatic diseases, and seronegative controls. METHODS: CoronavRheum is an ongoing, prospective, controlled, phase 4 study, in which patients aged 18 years or older with autoimmune rheumatic diseases, and healthy controls were recruited from a single site (Rheumatology Division of Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo) in São Paulo, Brazil Participants were vaccinated with two doses of CoronaVac (intramuscular injection, 3 µg in 0·5 mL of ß-propiolactone inactivated SARS-CoV-2) on day 0 and on day 28. Blood samples were taken pre-vaccination on day 0, day 28, and also on day 69. For this subgroup analysis, participants were defined as being SARS-CoV-2 seropositive or seronegative prevaccination via anti-SARS-CoV-2 spike (S)1 or S2 IgG (cutoff of 15·0 arbitrary units [AU] per mL) or neutralising antibody titres (cutoff of ≥30%) and were matched for age and sex, via convenience sampling, in a 1:3:1:1 ratio (seropositive patients to seronegative patients to seropositive controls to seronegative controls). The primary outcomes were rates of anti-SARS-CoV-2 S1 and S2 IgG seropositivity and SARS-CoV-2 neutralising antibody positivity at day 28 and day 69 and immunogenicity dynamics assessed by geometric mean titres (GMTs) of IgG and median neutralising activity in seropositive patients with autoimmune rheumatic diseases compared with seronegative patients and seropositive and seronegative controls. We assessed safety in all participants randomly selected for this subgroup analysis. This study is registered with ClinicalTrials.gov, NCT04754698, and is ongoing for long-term immunogenicity evaluation. FINDINGS: Between Feb 4 and Feb 8, 2021, 1418 patients and 542 controls were recruited, of whom 1685 received two vaccinations (1193 patients and 492 controls). After random sampling, our immunogenicity analysis population comprised 942 participants, of whom 157 were SARS-CoV-2 seropositive patients with autoimmune rheumatic diseases, 157 were seropositive controls, 471 were seronegative patients, and 157 were seronegative controls; the median age was 48 years (IQR 38-56) and 594 (63%) were female and 348 (37%) were male. For seropositive patients and controls, an increase in anti-SARS-CoV-2 S1 and S2 IgG titres (seropositive patients GMT 52·3 [95% CI 42·9-63·9] at day 0 vs 128·9 [105·6-157·4] at day 28; seropositive controls 53·3 [45·4-62·5] at day 0 vs 202·0 [174·8-233·4] at day 28) and neutralising antibody activity (seropositive patients 59% [IQR 39-83] at day 0 vs 82% [54-96] at day 28; seropositive controls 58% [41-79] at day 0 vs 92% [79-96] at day 28), was observed from day 0 to day 28, without further increases from day 28 to day 69 (at day 69 seropositive patients' GMT was 137·1 [116·2-161·9] and neutralising antibody activity was 79% [57-94]); and seropositive controls' GMT was 188·6 [167·4-212·6] and neutralising antibody activity was 92% [75-96]). By contrast, for seronegative patients and controls, the second dose was required for maximum response at day 69, which was lower in seronegative patients than in seronegative controls. GMTs in seronegative patients were 2·3 (95% CI 2·2-2·3) at day 0, 5·7 (5·1-6·4) at day 28, and 29·6 (26·4-33·3) at day 69, and in seronegative controls were 2·3 (2·1-2·5) at day 0, 10·6 (8·7-13·1) at day 28, and 71·7 (63·5-81·0) at day 69; neutralising antibody activity in seronegative patients was 15% (IQR 15-15) on day 0, 15% (15-15) at day 28, and 39% (15-65) at day 69, and in seronegative controls was 15% (15-15) at day 0, 24% (15-37) at day 28, and 61% (37-79) at day 69. Neither seronegative patients nor seronegative controls reached the GMT or antibody activity levels of seropositive patients at day 69. INTERPRETATION: By contrast with seronegative patients with autoimmune rheumatic diseases, seropositive patients have a robust response after a single dose of CoronaVac. Our findings raise the possibility that the reduced immunogenicity observed in seronegative patients might not be the optimum response potential to SARS-CoV-2 vaccination, and therefore emphasise the importance of at least a single booster vaccination in these patients. FUNDING: Fundação de Amparo à Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and B3-Bolsa de Valores do Brasil. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Supplement-based nutritional strategies to tackle frailty: A multifactorial, double-blind, randomized placebo-controlled trial.

BACKGROUND: Sarcopenia plays a central role in the development of frailty syndrome. Nutrition and exercise are cornerstone strategies to mitigate the transition to frailty; however, there is a paucity of evidence for which dietary and exercise strategies are effective. OBJECTIVE: This large, multifactorial trial investigated the efficacy of different dietary strategies to enhance the adaptations to resistance training in pre-frail and frail elderly. METHODS: This was a single-site 16-week, double-blind, randomized, placebo-controlled trial conducted at the Clinical Hospital, School of Medicine - University of São Paulo, Sao Paulo, Brazil. Four integrated, sub-investigations were conducted to compare: 1) leucine vs. placebo; 2) whey vs. soy vs. placebo; 3) creatine vs. whey vs. creatine plus whey vs. placebo; 4) women vs. men in response to whey. Sub-investigations 1 to 3 were conducted in women, only. Two-hundred participants (154 women/46 men, mean age 72 ± 6 years) underwent a twice-a-week, resistance training program. The main outcomes were muscle function (assessed by dynamic and isometric strength and functional tests) and lean mass (assessed by DXA). Muscle cross-sectional area, health-related quality of life, bone and fat mass, and biochemical markers were also assessed. RESULTS: We observed that leucine supplementation was ineffective to improve muscle mass and function. Supplementation with whey and soy failed to enhance resistance-training effects. Similarly, supplementation with neither whey nor creatine potentiated the adaptations to resistance training. Finally, no sex-based differences were found in response to whey supplementation. Resistance exercise per se increased muscle mass and function in all sub-investigations. There were no adverse effects. CONCLUSION: Neither protein (whey and soy), leucine, nor creatine supplementation enhanced resistance training-induced adaptations in pre-frail and frail elderly, regardless of sex. These findings do not support the notion that some widely used supplement-based interventions can add to the already potent effects of resistance exercise to counteract frailty-related muscle wasting and dynapenia. CLINICAL TRIAL REGISTRY: NCT01890382; https://clinicaltrials.gov/ct2/show/NCT01890382. DATA SHARING: Data described in the manuscript will be made available upon request pending application.

Immunogenicity and safety of the CoronaVac inactivated vaccine in patients with autoimmune rheumatic diseases: a phase 4 trial.

CoronaVac, an inactivated SARS-CoV-2 vaccine, has been approved for emergency use in several countries. However, its immunogenicity in immunocompromised individuals has not been well established. We initiated a prospective phase 4 controlled trial (no. NCT04754698, CoronavRheum) in 910 adults with autoimmune rheumatic diseases (ARD) and 182 age- and sex-frequency-matched healthy adults (control group, CG), who received two doses of CoronaVac. The primary outcomes were reduction of ≥15% in both anti-SARS-CoV-2 IgG seroconversion (SC) and neutralizing antibody (NAb) positivity 6 weeks (day 69 (D69)) after the second dose in the ARD group compared with that in the CG. Secondary outcomes were IgG SC and NAb positivity at D28, IgG titers and neutralizing activity at D28 and D69 and vaccine safety. Prespecified endpoints were met, with lower anti-SARS-Cov-2 IgG SC (70.4 versus 95.5%, P < 0.001) and NAb positivity (56.3 versus 79.3%, P < 0.001) at D69 in the ARD group than in the CG. Moreover, IgG titers (12.1 versus 29.7, P < 0.001) and median neutralization activity (58.7 versus 64.5%, P = 0.013) were also lower at D69 in patients with ARD. At D28, patients with ARD presented with lower IgG frequency (18.7 versus 34.6%, P < 0.001) and NAb positivity (20.6 versus 36.3%, P < 0.001) than that of the CG. There were no moderate/severe adverse events. These data support the use of CoronaVac in patients with ARD, suggesting reduced but acceptable short-term immunogenicity. The trial is still ongoing to evaluate the long-term effectiveness/immunogenicity.

High prevalence of HIV-associated neurocognitive disorders (HAND) in São Paulo City, Brazil / Alta prevalência de desordem neurocognitiva associada ao HIV (HAND) na Cidade de São Paulo, Brazil

INTRODUCTION: HIV-associated neurocognitive disorders (HAND) are the subject of many studies, some of them reporting a prevalence of up to 50 percent. OBJECTIVES: To determine the prevalence and factors associated with HIV neurocognitive disorders (HAND) in a cohort of HIV-1-infected patients in São Paulo city, Brazil. METHODOLOGY: Descriptive cross-sectional study including 106 HIV-1-infected patients, employing direct interview and neuropsychological tests, applied by trained neuro-psychologists with expertise in the tests. Other, similar assessment tools we used were Brief Neurocognitive Questionnaire, International HIV Dementia Scale, Lawton Instrumental Activities of Daily Living, Hospital Anxiety and Depression Scale, Social Support Scale for People with HIV/Aids, Assessment of Adherence to Antiretroviral Therapy Questionnaire, and a complex neuropsychological assessment. RESULTS: We included 106 patients from May 2015 to April 2018. We found a high prevalence of HAND in our patients (45%), with 27.5% presenting asymptomatic neurological impairment (ANI) and 17.5% mild neurological dysfunction (MND); only one patient presented HIV-associated dementia (HAD) (0.9%). Women were more likely to have MND (52.9%) and the only case of HAD was also female. The high prevalence of neurocognitive disorders was independent of the immunological status, use of efavirenz, or virological control. CONCLUSIONS: This study may mirror the national and international scenarios, showing a high prevalence of HAND (45%) and the prevalence of some risk factors, in special among women

INTRODUÇÃO: As doenças neurocognitivas associadas ao HIV (HAND), são o assunto de muitos estudos, alguns deles relatando uma prevalência de até 50 por cento. OBJETIVOS: Determinar a prevalência e os fatores associados aos distúrbios neurocognitivos do HIV (HAND) em uma coorte de pacientes infectados pelo HIV-1 na cidade de São Paulo, Brasil. METODOLOGIA: Estudo transversal descritivo incluindo 106 pacientes infectados pelo HIV-1, utilizando entrevista direta e testes neuropsicológicos, aplicados por neuropsicólogos treinados com experiência nos testes. Foram utilizados também: Questionário Neurocognitivo Breve, Escala Internacional de Demência do HIV, Atividades Instrumentais de Vida Diária de Lawton, Escala Hospitalar de Ansiedade e Depressão, Escala de Apoio Social para Pessoas com HIV / Aids, Avaliação da Adesão à Terapia Antiretroviral Questionário e uma bateria de avaliação neuropsicológica complexa. RESULTADOS: Foram avalaidos 106 pacientes de maio de 2015 a abril de 2018. Foi observado uma alta prevalência de HAND em nossos pacientes (45%), com 27,5% apresentando comprometimento neurológico assintomático (ANI) e 17,5% comprometimento cognitive leve (MND); apenas um paciente apresentou demência associada ao HIV (DAH) (0,9%). As mulheres eram mais propensas a ter MND (52,9%) e o único caso de HAD também era do sexo feminino. A alta prevalência de distúrbios neurocognitivos foi independente do estado imunológico, uso de efavirenz ou controle virológico. CONCLUSÕES: Este estudo pode espelhar o cenário nacional e internacional, mostrando uma alta prevalência de HAND (45%) e a prevalência de alguns fatores de risco, em especial entre as mulheres

AIDS incidence and survival in a hospital-based cohort of HIV-positive patients from São Paulo, Brazil: The role of IFN-λ4 polymorphisms.

Few studies have reported the prognosis of human immunodeficiency virus (HIV)-positive patients followed for a long time in Brazil, particularly those including pre and post-HAART eras. The polymorphisms of interferon (IFN)-λ4 have been postulated as possibly associated with the pathogenesis of HIV infection. The aim of this study was to describe the incidence and mortality from a cohort of HIV-positive patients as well as whether IFN-λ4 gene polymorphisms (SNP rs8099917 and SNP rs12979860) were associated with HIV/acquired immune deficiency syndrome (AIDS) progression. We followed 402 patients for up to 30 years; 347 of them began follow-up asymptomatic, without any AIDS-defining opportunistic disease and/or a lymphocytes T CD4+ count of 350 cells/mm3 or lower. We determined the probability of the asymptomatic subjects to remain AIDS-free, and the risk of death for those entering the study already with an AIDS diagnosis, as well as for subjects developing AIDS during follow-up. We compared the prognosis of patients with two different polymorphisms for the genes encoding for IFN-λ4, variants rs8099917 and rs12979860. The follow-up time of the 347 asymptomatic-at-entry subjects was 3687 person-years. IFN-λ4 rs8099917 polymorphisms were not associated with AIDS progression, but IFN-λ4 rs12979860 wild type genotype (CC) was associated with higher mortality compared to CT and TT, with an increased probability of death from AIDS (P = .01). In conclusion, genetic variations in IFN-λ4 on rs12979860 polymorphisms in HIV-infected patients may drive mortality risk.

Diagnostic performance of the Xpert Carba-R™ assay directly from rectal swabs for active surveillance of carbapenemase-producing organisms in the largest Brazilian University Hospital.

BACKGROUND: The global spread of carbapenemase-producing organisms (CPO) has been considered by international health authorities as a critical public health concern. Brazil has a high CPO prevalence according to distinct publications but many routine microbiology laboratories have only phenotypic resources to evaluate this epidemiological situation, which is time-consuming and detects only carbapenem-resistant isolates missing CPO susceptible expressing a slightly decreased susceptibility. New molecular platforms can detect CPO faster but a local evaluation is essential. AIM: To evaluate the performance of CPO detection direct from rectal swabs with the Xpert Carba-R™ assay (Cepheid, Sunnyvale, CA) in the largest Brazilian University Hospital. METHODS: A prospective diagnostic accuracy study of CPO was performed with the collection of rectal swabs from patients admitted into the Intensive Care Unit (ICU) and into the Emergency Department (ED) between April and July 2016. The Xpert Carba-R™ assay results were compared with carbapenem-resistant Enterobacterales (CRE) surveillance cultures plus in-house PCR carbapenemase detection (reference method). In case of discordant results between methods, additional tests were performed. The limit of detection (LoD) for the CRE culture and the Xpert Carba-R™ assay were performed with contrived isolates of known carbapenemases genes. RESULTS: A total of 921 clinical rectal swabs were analyzed being 21% (196/921) from the ICU and 79% (725/921) from the ED. Overall, the Xpert Carba-R™ assay detected 9.9% (91/921) of CPOs being 9.5% (87/921) positive only for blaKPC and 0.4% (4/921) positive only for blaNDM. The reference method detected 9.1% (84/921) CPO being 77 (8.4%) blaKPC, 5 blaVIM (0.5%) and 2 blaNDM (0.2%). No IMP or OXA-48 like gene was detected. Overall, twelve samples, 1.3% (10 blaKPC, 2 blaNDM) were Xpert Carba-R™ positive but negative by the reference method. Five isolates (0.5%) were positive for blaVIM only by in-house PCR and confirmed to be blaVIM-2 by DNA sequencing. The Kappa value, sensitivity, specificity, positive/negative predictive values and accuracy of the Xpert Carba-R™ assay were; 0.893 (95% confidence interval [CI], 0.842-0.944), 94% (86.7-98.0), 98.6% (97.5-99.3), 86.8% (78.1-93.0), 99.4% (98.6-99.8) and 98.2% (97.3-99.1), respectively. The LoD for blaKPC of the Xpert Carba-R™ assay and the CRE cultures were 101 CFU/swab. CONCLUSION: The Xpert Carba-R™ assay is an accurate test to detect CPO directly from the rectal swabs with significant lower turnaround time (TAT) when compared to the reference method (CRE culture plus in-house PCR). Xpert Carba-R™ may, therefore, be regarded as a good and fast epidemiological tool.

Expansion and suppressive capacity of regulatory T cells isolated from patients across the leprosy spectrum: a pilot study.

Knowledge of the role of Tregs in the immunopathogenesis of the different clinical outcomes within the leprosy spectrum remains limited due to the lack of studies directly assessing their suppression capacity. We thus tested a protocol to expand Tregs from the peripheral blood of patients across the leprosy spectrum and analyzed their suppressive capacity in autologous TCD4+ responses. Results of these pilot assays show that Tregs can be expanded and exert suppressive capacity, but also that their rate of expansion and suppressive capacity are influenced by the patient's clinical classification, suggesting that they possibly retain some in vivo characteristics.