Assessing Predictive Value of SARS-CoV-2 Epitope-Specific CD8+ T-Cell Response in Patients with Severe Symptoms.

Specific T cell responses against SARS-CoV-2 provided an overview of acquired immunity during the pandemic. Anti-SARS-CoV-2 immunity determines the severity of acute illness, but also might be related to the possible persistence of symptoms (long COVID). We retrospectively analyzed ex vivo longitudinal CD8+ T cell responses in 26 COVID-19 patients diagnosed with severe disease, initially (1 month) and long-term (10 months), and in a cohort of 32 vaccinated healthcare workers without previous SARS-CoV-2 infection. We used peptide-human leukocyte antigen (pHLA) dextramers recognizing 26 SARS-CoV-2-derived epitopes of viral and other non-structural proteins. Most patients responded to at least one of the peptides studied, mainly derived from non-structural ORF1ab proteins. After 10 months follow-up, CD8+ T cell responses were maintained at long term and reaction against certain epitopes (A*01:01-ORF1ab1637) was still detected and functional, showing a memory-like phenotype (CD127+ PD-1+). The total number of SARS-CoV-2-specific CD8+ T cells was significantly associated with protection against long COVID in these patients. Compared with vaccination, infected patients showed a less effective immune response to spike protein-derived peptides restricted by HLA. So, the A*01:01-S865 and A*24:02-S1208 dextramers were only recognized in vaccinated individuals. We conclude that initial SARS-CoV-2-specific CD8+ T cell response could be used as a marker to understand the evolution of severe disease and post-acute sequelae after SARS-CoV-2 infection.

Postoperative Staphylococcus aureus Infections in Patients With and Without Preoperative Colonization.

Importance: Staphylococcus aureus surgical site infections (SSIs) and bloodstream infections (BSIs) are important complications of surgical procedures for which prevention remains suboptimal. Contemporary data on the incidence of and etiologic factors for these infections are needed to support the development of improved preventive strategies. Objectives: To assess the occurrence of postoperative S aureus SSIs and BSIs and quantify its association with patient-related and contextual factors. Design, Setting, and Participants: This multicenter cohort study assessed surgical patients at 33 hospitals in 10 European countries who were recruited between December 16, 2016, and September 30, 2019 (follow-up through December 30, 2019). Enrolled patients were actively followed up for up to 90 days after surgery to assess the occurrence of S aureus SSIs and BSIs. Data analysis was performed between November 20, 2020, and April 21, 2022. All patients were 18 years or older and had undergone 11 different types of surgical procedures. They were screened for S aureus colonization in the nose, throat, and perineum within 30 days before surgery (source population). Both S aureus carriers and noncarriers were subsequently enrolled in a 2:1 ratio. Exposure: Preoperative S aureus colonization. Main Outcomes and Measures: The main outcome was cumulative incidence of S aureus SSIs and BSIs estimated for the source population, using weighted incidence calculation. The independent association of candidate variables was estimated using multivariable Cox proportional hazards regression models. Results: In total, 5004 patients (median [IQR] age, 66 [56-72] years; 2510 [50.2%] female) were enrolled in the study cohort; 3369 (67.3%) were S aureus carriers. One hundred patients developed S aureus SSIs or BSIs within 90 days after surgery. The weighted cumulative incidence of S aureus SSIs or BSIs was 2.55% (95% CI, 2.05%-3.12%) for carriers and 0.52% (95% CI, 0.22%-0.91%) for noncarriers. Preoperative S aureus colonization (adjusted hazard ratio [AHR], 4.38; 95% CI, 2.19-8.76), having nonremovable implants (AHR, 2.00; 95% CI, 1.15-3.49), undergoing mastectomy (AHR, 5.13; 95% CI, 1.87-14.08) or neurosurgery (AHR, 2.47; 95% CI, 1.09-5.61) (compared with orthopedic surgery), and body mass index (AHR, 1.05; 95% CI, 1.01-1.08 per unit increase) were independently associated with S aureus SSIs and BSIs. Conclusions and Relevance: In this cohort study of surgical patients, S aureus carriage was associated with an increased risk of developing S aureus SSIs and BSIs. Both modifiable and nonmodifiable etiologic factors were associated with this risk and should be addressed in those at increased S aureus SSI and BSI risk.

Evaluation and Clinical Impact of Biofire FilmArray Pneumonia Panel Plus in ICU-Hospitalized COVID-19 Patients.

Microbiological diagnosis by using commercial multiplex quantitative PCR systems provides great advantages over the conventional culture. In this work, the Biofire FilmArray Pneumonia Panel Plus (FAPP+) was used to test 144 low respiratory tract samples from 105 COVID-19 patients admitted to an Intensive Care Unit (ICU), detecting 78 pathogens in 59 (41%) samples. The molecular panel was evaluated by using the conventional culture (CC) as comparator, which isolated 42 pathogens in 40 (27.7%) samples. The overall percentage of agreement was 82.6%. Values of sensitivity (93%), specificity (62%), positive predictive value (50%), and negative predictive value (96%) were obtained. The mean time elapsed from sample extraction to modification of antibiotic treatment was 7.6 h. A change in antimicrobial treatment after the FAPP+ results was performed in 27% of patients. The FAPP+ is a highly sensitive diagnostic method that can be used to significantly reduce diagnostic time and that allows an early optimization of antimicrobial treatment.

Divergent SARS-CoV-2-specific T cell responses in intensive care unit workers following mRNA COVID-19 vaccination.

The cellular immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in response to full mRNA COVID-19 vaccination could be variable among healthy individuals. Studies based only in specific antibody levels could show an erroneous immune protection at long times. For that, we analyze the antibody levels specific to the S protein and the presence of SARS-CoV-2-specific T cells by ELISpot and AIM assays in intensive care unit (ICU) workers with no antecedents of COVID-19 and vaccinated with two doses of mRNA COVID-19 vaccines. All individuals were seronegative for the SARS-CoV-2 protein S before vaccination (Pre-v), but 34.1% (14/41) of them showed pre-existing T lymphocytes specific for some viral proteins (S, M and N). One month after receiving two doses of COVID-19 mRNA vaccine (Post-v1), all cases showed seroconversion with high levels of total and neutralizing antibodies to the spike protein, but six of them (14.6%) had no T cells reactive to the S protein. Specifically, they lack of specific CD8+ T cells, but maintain the contribution of CD4+ T cells. Analysis of the immune response against SARS-CoV-2 at 10 months after full vaccination (Post-v10), exhibited a significant reduction in the antibody levels (p<0.0001) and protein S-reactive T cells (p=0.0073) in all analyzed individuals, although none of the individuals become seronegative and 77% of them maintained a competent immune response. Thus, we can suggest that the immune response to SARS-CoV-2 elicited by the mRNA vaccines was highly variable among ICU workers. A non-negligible proportion of individuals did not develop a specific T cell response mediated by CD8+ T cells after vaccination, that may condition the susceptibility to further viral infections with SARS-CoV-2. By contrast, around 77% of individuals developed strong humoral and cellular immune responses to SARS-CoV-2 that persisted even after 10 months. Analysis of the cellular immune response is highly recommended for providing exact information about immune protection against SARS-CoV-2.

Extensively drug-resistant Acinetobacter baumannii carrying blaOXA-23-like and armA in a hospital after an intervention in the intensive care unit which ended a long-standing endemicity.

The aim of the study was to evaluate for a long time the effectiveness of an intervention designed to reduce carbapenem-resistant Acinetobacter baumannii (CRAB) and its impact on colistin usage in the ICU of a tertiary hospital in Spain. The rate of carbapenem resistance declined drastically during the period of study (2015 to 2018), from 93.57 to 74.65%, especially in the ICU. A significant decrease in colistin usage, from 1.16 to 0.39 DOTs, was observed. Forty-nine CRAB isolates recovered nearly 1 year after starting the intervention were characterized. Most of them were recovered from patients admitted in wards other than ICU and were extensively drug-resistant, carried blaOXA-23-like and armA, and belonged to ST218. Implementation of control measures is crucial to CRAB control in ICUs but must be extended to all wards in order to eradicate CRAB from hospitals.

Spread of OXA-48-producing Klebsiella pneumoniae among COVID-19-infected patients: The storm after the storm.

The impact of secondary infections by multidrug-resistant bacteria in COVID-19- infected patients has yet to be evaluated. Here, we report the clinical and molecular features of an outbreak of seven patients carrying CTX-M-15- and OXA-48-producing Klebsiella pneumoniae belonging to ST326 during COVID-19 pandemic in an ICU in northern Spain. Those patients were admitted to beds close to each other, two of them developed ventilator-associated pneumonia (VAP), one exhibited primary bacteremia and the remaining four were considered to be colonized. None of them was colonized prior to admission to the ICU an all, except one of those who developed VAP, were discharged. Hydroxychloroquine and lopinavir/ritonavir were administered to all of them as COVID-19 therapy and additionally, three of them received tocilizumab and corticosteroids, respectively. Reusing of personal protective equipment due to its initial shortage, relaxation in infection control measures and negative-pressure air in ICU rooms recommended for the protection of health care workers (HCWs), could have contributed to this outbreak. Maximization of infection control measures is essential to avoid secondary infections by MDR bacteria in COVID-infected patients.

SARS-CoV-2 analysis on environmental surfaces collected in an intensive care unit: keeping Ernest Shackleton's spirit.

BACKGROUND: Intensive care unit workers are at high risk of acquiring COVID-19 infection, especially when performing invasive techniques and certain procedures that generate aerosols (< 5 µm). Therefore, one of the objectives of the health systems should implement safety practices to minimize the risk of contagion among these health professionals. Monitoring environmental contamination of SARS-CoV-2 may help to determine the potential of the environment as a transmission medium in an area highly exposed to SARS-CoV-2, such as an intensive care unit. The objective of the study was to analyze the environmental contamination by SARS-CoV-2 on surfaces collected in an intensive care unit, which is dedicated exclusively to the care of patients with COVID-19 and equipped with negative pressure of - 10 Pa and an air change rate of 20 cycles per hour. Furthermore, all ICU workers were tested for COVID-19 by quantitative RT-PCR and ELISA methods. RESULTS: A total of 102 samples (72 collected with pre-moistened swabs used for collection of nasopharyngeal exudates and 30 with moistened wipes used in the environmental microbiological control of the food industry) were obtained from ventilators, monitors, perfusion pumps, bed rails, lab benches, containers of personal protective equipment, computer keyboards and mice, telephones, workers' shoes, floor, and other areas of close contact with COVID-19 patients and healthcare professionals who cared for them. The analysis by quantitative RT-PCR showed no detection of SARS-CoV-2 genome in environmental samples collected by any of the two methods described. Furthermore, none of the 237 ICU workers was infected by the virus. CONCLUSIONS: Presence of SARS-CoV-2 on the ICU surfaces could not be determined supporting that a strict cleaning protocol with sodium hypochlorite, a high air change rate, and a negative pressure in the ICU are effective in preventing environmental contamination. These facts together with the protection measures used could also explain the absence of contagion among staff inside ICUs.

Evaluation of a commercial multiplex PCR for diagnosis of central nervous system (CNS) nosocomial infections.

Nosocomial Central Nervous System (CNS) infections are often serious complications of neurosurgical procedures. Their diagnosis is complex and frequently based on microbiological culture. The aim of this work was to evaluate the effectiveness of the FilmArray® Blood Culture Identification (BCID) panel, a multiplex PCR designed to identify the most common etiologic agents of sepsis involved with nosocomial CNS infections. A total of ninety samples were analyzed with the BCID panel. The sensitivity and specificity achieved were 77.4% and 100% respectively, when compared with the reference method (culture). Based on the analysis of the melting curves, another cut-off was established improving sensitivity to 83.9% whilst maintaining 98.3% specificity. The BCID panel seems to be a helpful tool for the prompt diagnosis of CNS nosocomial infections. The cut-off proposed here can increase sensitivity, but further studies are required to confirm its effectiveness and its applicability in clinical microbiology laboratories.

Clock Genes Disruption in the Intensive Care Unit.

BACKGROUND: Intensive care unit (ICU) environment disrupts the circadian rhythms due to environmental and other nonphotic synchronizers. The main purpose of this article is to establish whether critically patients have desynchronization at the molecular level after 1 week of stay in the ICU. METHODS: The rhythm of Clock, Bmal1, Cry1, and Per2 genes in neuro-ICU patients (n = 11) on the first day after admission in the unit (1 day) and 1 week later (1 week) was studied, 4 time points throughout the day, at 6, 12, 18, and 24 hours. Human whole blood samples were obtained from neuro-ICU patients. The total RNA was isolated and each sample was reverse transcribed to complementary DNA and quantitative polymerase chain reaction (PCRq) was performed. The possible rhythm was studied using Fourier Series. RESULTS: After 1 week, the clock gene rhythmicity completely disappeared. Messenger RNA (mRNA) expression for the 4 clock genes was shown rhythmicity at the first day after admission in the ICU. Circadian rhythmicity for none of them was observed but rather, ultradian rhythmicity was found. The expression of Clock, Bmal1, and Per2 mRNA after 1 week was similar in the 4-time point studies without significant fluctuation among the 4 time points analyzed. DISCUSSION: Rhythmic mRNA expression is present at the first day after admission in the ICU. However, ICU stay during 1 week affects the molecular machinery of the biological clock generating chronodisruption. Circadian disruption is associated with the risk of several pathologies, thus, it seems to be clear that ICU stay in constant conditions could adversely affect patient evolution and probably, circadian resynchronization restoring clock gene expression could lead to a better clinical evolution of the patient. CONCLUSIONS: Clock genes disruption is observed in neuro-ICU patients. Light therapy as well as melatonin treatment could reduce the impact of ICU stay period in biological clock, thereby improving patients' recovery.

Summary of Spanish recommendations on intensive care to facilitate organ donation.

With the aim of consolidating recommendations about the practice of initiating or continuing intensive care to facilitate organ donation (ICOD), an ad hoc working group was established, comprising 10 intensivists designated by the Spanish Society of Intensive Care and Coronary Units (SEMICYUC) and the Spanish National Transplant Organization (ONT). Consensus was reached in all recommendations through a deliberative process. After a public consultation, the final recommendations were institutionally adopted by SEMICYUC, ONT, and the Transplant Committee of the National Health-Care System. This article reports on the resulting recommendations on ICOD for patients with a devastating brain injury for whom the decision has been made not to apply any medical or surgical treatment with a curative purpose on the grounds of futility. Emphasis is made on the systematic referral of these patients to donor coordinators, the proper assessment of the likelihood of brain death and medical suitability, and on transparency in communication with the patient's family. The legal and ethical aspects of ICOD are addressed. ICOD is considered a legitimate practice that offers more patients the opportunity of donating their organs upon their death and helps to increase the availability of organs for transplantation.

Organ Donation and Elective Ventilation: A Necessary Strategy.

Organ transplantation is the sole treatment to improve or save the life of patients with final-stage organ failure. The shortage of available organs for transplantation constitutes a universal problem, estimating that 10% of patients on waiting lists die. Brain death is an undesirable result; nevertheless, it has beneficial side-effects since it is the most frequent source of organs for transplantation. However, this phenomenon is relatively uncommon and has a limited potential. One of the options that focuses on increasing organ donation is to admit patients with catastrophic brain injuries (with a high probability of brain death and nontreatable) to the Intensive Care Unit, with the only purpose of donation. To perform elective nontherapeutic ventilation (ENTV), a patient's anticipated willingness to donate organs and/or explicit acceptance by his/her relatives is required. This process should focus exclusively on those patients with catastrophic brain injuries and imminent risk of death which, due to its acute damage, are not considered treatable. This article defends ENTV as an effective strategy to improve donation rate, analyzing its ethical and legal basis.

[For an open-door, more comfortable and humane intensive care unit. It is time for change]. / Por una UCI de puertas abiertas, más confortable y humana. Es tiempo de cambio.

The Intensive Care Unit is a wonderful place where lives are saved, but it is also a very harsh and unpleasant place where critically ill patients face terrible diseases in very adverse environmental conditions. We must change the design of the ICU and its organization; we must improve privacy, welfare and comfort of patients and families, following their personal and emotional demands. To free up the visiting hours and to improve family care are among our most urging matters, which we should delay no further. We must equip the ICUs with modern monitors and respirators but we must also invest in organization, design, environmental comfort and humanization. We need to redesign clinical practise so that ICU care becomes more agreeable and humane. We should put off this change no longer, since it is an imperative social and professional demand.

Repair of long-bone pseudoarthrosis with autologous bone marrow mononuclear cells combined with allogenic bone graft.

BACKGROUND AIMS: Long-bone pseudoarthrosis is a major orthopedic concern because of numerous factors such as difficulty of the treatment, high recurrence, high costs and the devastating effects on the patients' quality of life, which sometimes ends in amputation. Although the "gold standard" for the treatment of this pathology is autologous bone grafting, which has high osteogenic, osteoconductive and osteoinductive properties, this treatment presents some restrictions such as the limited amount of bone that can be taken from the patient and donor site morbidity. Bone marrow mononuclear cells (BM-MNCs) comprise progenitor and stem cells with pro-angiogenic and pro-osteogenic properties. Allogenic cancellous bone graft is a natural and biodegradable osteoconductive and osteoinductive scaffold. Combination of these two components could mimic the advantages of autologous bone grafting while avoiding its main limitations. METHODS: Long-bone pseudoarthrosis was treated in seven patients with autologous BM-MNCs from iliac crest combined with frozen allogenic cancellous bone graft obtained from the tissue bank. RESULTS: All patients showed complete bone consolidation 5.3 ± 0.9 months (range, 2-9 months) after cell transplantation. Moreover, limb pain disappeared in all of them. The mean follow-up was 35.8 ± 4.6 months after transplantation (range, 24-51 months) without pseudoarthrosis recurrence or pain reappearing. CONCLUSIONS: Combination of autologous BM-MNCs and allogenic bone graft could constitute an easy, safe, inexpensive and efficacious attempt to treat long-bone pseudoarthrosis and non-union by reproducing the beneficial properties of autologous bone grafting while restricting its disadvantages.

Diagnosing brain death by CT perfusion and multislice CT angiography.

INTRODUCTION: Although the diagnosis of brain death (BD) is usually based on clinical criteria, in sedated patients, ancillary techniques are needed. This study was designed to assess the accuracy of cerebral multislice computed tomographic angiography (CTA) and CT perfusion (CTP) in diagnosing BD. METHODS: Prospective observational study in 27 BD patients. RESULTS: All patients were diagnosed as BD based on clinical and electroencephalogram findings. After BD diagnosis, CTP was performed followed by 64-detector multislice CTA from the aortic arch to the vertex. Images were reconstructed from 0.5 mm sections. In 24 patients, a lack of cerebral blood flow (CBF) was detected by CTP, and CTA revealed luminal narrowing of the internal carotid artery in the neck and absence of anterior and posterior intracranial circulation (sensitivity 89%). CTA detected CBF exclusively in extracranial portions of the internal carotid and vertebral arteries. Two patients with anoxic brain injury and decompressive craniectomy showed CBF in the CTA such that the CTP results were considered false negatives, given BD had been confirmed by clinical and EEG findings, along with evoked potentials. In one clinically BD patient, in whom an alpha rhythm was detected in the electroencephalogram, CBF was only observed in the intracranial internal carotid with no posterior circulation noted. This patient was therefore considered exclusively brain stem dead. CONCLUSIONS: The radiological protocol used shows a high sensitivity and excellent specificity for detecting the cerebral circulatory arrest that accompanies BD. As a rapid, non-invasive, and widely available technique it is a promising alternative to conventional 4-vessel angiography.

[Brain death diagnosis]. / Diagnóstico de muerte encefálica.

Brain death has been recognized by the scientific community as the person's death, and accepted in the legislation of different countries. Brain death is defined as the irreversible ending of the functions of all the intracranial neurological structure in both the brain and brain stem. This clinical situation appears when intracranial pressure exceeds the patient's systolic blood pressure, leading to brain circulatory arrest. The most frequent are cerebral hemorrhage and cranioencephalic trauma. Clinical diagnostic must be done by doctors with expertise in neurocritical patient treatment. This diagnosis is based on a systematic, complete and extremely rigorous clinical examination that confirms a non-reactive coma, absence of brain stem reflex, and absence of spontaneous breathing. Instrumental tests may be obligatory in some cases, this depending on each country. Electroencephalogram and evoked potentials are the electrophysiological tests used. In patients treated with sedative drugs, cerebral blood flow evaluation tests, such as cerebral angiography, transcranial Doppler or 99Tc-HMPAO scintigraphy, will be used. More than 92% of the transplants performed in Spain are performed with brain death donor organs. Brain death confirmation is a high responsibility act, with medical, ethical and legal significance since it requires removal of all artificial support, or organs extraction for transplant. Extensive knowledge on its diagnostic and correct decision making avoid unnecessary use of resources and improves management of organs for transplant.

[Evaluation and maintenance of the lung donor]. / Evaluación y mantenimiento del donante pulmonar.

Spain has a rate of 34.4 donors per million population, making it the country with the highest rate of organ donation all over the world. In spite of these values, need of lungs for transplantation is always greater than the number of organs obtained. The adrenergic storming produced during brain herniation and the initiation of a brain death condition entail a series of pathophysiological alterations that can endanger the lung viability. In this paper, we review the evaluation of lung donor and its maintenance. In all the potential lung donors, the Apnea Test with CPAP should be done and the criteria for the donation of the lung under ideal conditions and with expanded criteria lung donors are reviewed. The protocol of aggressive maintenance including a protective ventilation strategy, restricted fluid intake, use of prophylactic antibiotics, corticosteroids and the systematic use of measures aimed at avoiding alveolar collapse together with maneuvers aimed at achieving better lung recruitment can improve the management of the lung donor and obtain a greater number of organs for transplantation.

Identifying the most suitable endogenous control for determining gene expression in hearts from organ donors.

BACKGROUND: Quantitative real-time reverse transcription PCR (qRT-PCR) is a useful tool for assessing gene expression in different tissues, but the choice of adequate controls is critical to normalise the results, thereby avoiding differences and maximizing sensitivity and accuracy. So far, many genes have been used as a single reference gene, without having previously verified their value as controls. This practice can lead to incorrect conclusions and recent evidence indicates a need to use the geometric mean of data from several control genes. Here, we identified an appropriate set of genes to be used as an endogenous reference for quantifying gene expression in human heart tissue. RESULTS: Our findings indicate that out of ten commonly used reference genes (GADPH, PPIA, ACTB, YWHAZ, RRN18S, B2M, UBC, TBP, RPLP and HPRT), PPIA, RPLP and GADPH show the most stable gene transcription levels in left ventricle specimens obtained from organ donors, as assessed using geNorm and Normfinder software. The expression of TBP was found to be highly regulated. CONCLUSION: We propose the use of PPIA, RPLP and GADPH as reference genes for the accurate normalisation of qRT-PCR performed on heart tissue. TBP should not be used as a control in this type of tissue.

Differences in the deflation limb of the pressure-volume curves in acute respiratory distress syndrome from pulmonary and extrapulmonary origin.

OBJECTIVE: To assess the differences in the deflation pressure-volume (PV) curves between acute respiratory distress syndrome from pulmonary (ARDSp) and extrapulmonary (ARDSe) origin. DESIGN: . Prospective study. SETTING: Twenty-bed intensive care unit in an university hospital. PATIENTS: Ten patients within the first 24 h from meeting ARDS criteria, classified as ARDSp or ARDSe in a clinical basis. INTERVENTIONS: A deflation PV curve was recorded by means of decreasing steps of continuous positive airway pressure (CPAP) from 35 to 0 cmH(2)O. RESULTS: The simultaneous recording of pressure at the airway opening (Pao), esophageal pressure (Pes) and volumes (V) allows us to trace the Pao-V, Pes-V and transpulmonary pressure (Ptp)-V curves. These data were fitted to a sigmoid model and ARDSp and ARDSe groups were compared. ARDSp has lower lung compliance and higher chest wall compliance than ARDSe (35.9+/-11.3 vs. 77.2+/-50.6 and 199.6+/-44.4 vs. 125.5+/-16.5 ml/cmH(2)O, respectively, P<0.05). The Pao-V curve in ARDSp is shifted down and right with respect to ARDSe. The Ptp-V curve shows a similar displacement. The Pes-V curve in the ARDSp group is, however, shifted to the left. When relative values (percentage to the maximum volume achieved at 35 cmH(2)O) are considered, these differences persist, but, in the Ptp-V curves, are only significant in the low-pressure range. CONCLUSIONS: Differences between ARDSp and ARDSe PV curves are present all along the pressure axis and are related to differences not only in the Pes-V curve, but also in the Ptp-V curve.