Nucleoprotein-based ELISA for detection of SARS-COV-2 IgG antibodies: Could an old assay be suitable for serodiagnosis of the new coronavirus?

OBJECTIVES: We evaluated the performance of a nucleoprotein-based enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies to SARS-CoV-2. METHODS: The ELISA was based on serum IgG reactivity to a 46-kDa protein derived from the recombinant SARS-CoV2 nucleoprotein. Assay sensitivity was assessed using serum samples from 134 COVID-19 confirmed cases obtained > 15 days after symptom onset. Specificity was determined by testing sera from 94 healthy controls. Cross-reactivity was evaluated with sera from 96 individuals with previous dengue or zika virus-confirmed infections, with 44 sera from individuals with confirmed infections to other respiratory viruses or with bacterial and fungal infections that cause pneumonia and with 40 sera negative for SARS-CoV-2 nucleoprotein by commercial ELISA kits. RESULTS: The majority of subjects were male and ≥ 60 years old. Assay sensitivity was 90.3 % (95 % confidence interval 84.1 %-94.2 %) and specificity was 97.9 % (92.6 %-99.4 %). There was no cross-reactivity with sera from individuals diagnosed with dengue, zika virus, influenza virus, rhinovirus, adenovirus, respiratory syncytial virus, seasonal coronavirus, Mycobacterium tuberculosis, Staphylococcus (S. aureus and coagulase-negative), Streptococcus pneumoniae, Klebsiella pneumoniae and the fungus Aspergillus fumigatus. The level of concordance of our test with results from commercial ELISA kits was 100 %. CONCLUSION: The nucleoprotein-based ELISA was specific for detection of IgG anti-nucleoprotein antibodies to SARS-CoV-2. It utilizes a frequently employed low expense assay protocol and is easier to perform than other currently available commercial SARS-CoV2 antibody detection tests.

Performance of a qualitative rapid chromatographic immunoassay to diagnose COVID-19 in patients in a middle-income country.

OBJECTIVES: We evaluated a rapid chromatographic immunoassay (IgG/IgM antibodies) and an ELISA assay to diagnose COVID-19 in patient sat two Brazilian hospitals. METHODS: A total of 122 subjects with COVID-19 were included: 106 SARS-COV-2 RT-PCR-positive patients and 16 RT-PCR-negative patients with symptoms and chest computed tomography (CT) consistent with COVID-19. Ninety-six historical blood donation samples were used as controls. Demographic and clinical characteristics were retrieved from electronic records. Sensitivity and specificity were calculated, as were their 95% binomial confidence intervals using the Clopper-Pearson method. All analyses were performed in R version 3.6.3. RESULTS: The sensitivity of the chromatographic immunoassay in all RT-PCR-positive patients, irrespective of the timing of symptom onset, was 85.8% (95% binomial CI 77.7% to 91.9%). This increased with time after symptom onset, and at >14 days was 94.9% (85.9% to 98.9%). The specificity was 100% (96.4% to 100%). 15/16 (94%) RT- PCR-negative cases tested positive. The most frequent comorbidities were hypertension and diabetes mellitus and the most frequent symptoms were fever, cough, and dyspnea. All RT-PCR-negative patients had pneumonia. The most frequent thoracic CT findings were ground glass changes (n = 11, 68%), which were bilateral in 9 (56%) patients, and diffuse reticulonodular infiltrates (n = 5, 31%). CONCLUSIONS: The COVID-19 rapid chromatographic immunoassay evaluated in this study had a high sensitivity and specificity using plasma, particularly after 14 days from symptom onset. ELISA and qualitative rapid chromatographic immunoassays can be used for the diagnosis of RT-PCR-negative patients.

Role of organisational factors on the 'weekend effect' in critically ill patients in Brazil: a retrospective cohort analysis.

INTRODUCTION: Higher mortality for patients admitted to intensive care units (ICUs) during the weekends has been occasionally reported with conflicting results that could be related to organisational factors. We investigated the effects of ICU organisational and staffing patterns on the potential association between weekend admission and outcomes in critically ill patients. METHODS: We included 59 614 patients admitted to 78 ICUs participating during 2013. We defined 'weekend admission' as any ICU admission from Friday 19:00 until Monday 07:00. We assessed the association between weekend admission with hospital mortality using a mixed logistic regression model controlling for both patient-level (illness severity, age, comorbidities, performance status and admission type) and ICU-level (decrease in nurse/bed ratio on weekend, full-time intensivist coverage, use of checklists on weekends and number of institutional protocols) confounders. We performed secondary analyses in the subgroup of scheduled surgical admissions. RESULTS: A total of 41 894 patients (70.3%) were admitted on weekdays and 17 720 patients (29.7%) on weekends. In univariable analysis, weekend admitted patients had higher ICU (10.9% vs 9.0%, P<0.001) and hospital (16.5% vs 13.5%, P<0.001) mortality. After adjusting for confounders, weekend admission was not associated with higher hospital mortality (OR 1.05, 95% CI 0.99 to 1.12, P=0.095). However, a 'weekend effect' was still observed in scheduled surgical admissions, as well as in ICUs not using checklists during the weekends. For unscheduled admissions, no 'weekend effect' was observed regardless of ICU's characteristics. For scheduled surgical admissions, a 'weekend effect' was present only in ICUs with a low number of implemented protocols and those with a reduction in the nurse/bed ratio and not applying checklists during weekends. CONCLUSIONS: ICU organisational factors, such as decreased nurse-to-patient ratio, absence of checklists and fewer standardised protocols, may explain, in part, increases in mortality in patients admitted to the ICU mortality on weekends.

Systemic inflammatory response syndrome criteria and the prediction of hospital mortality in critically ill patients: a retrospective cohort study. / Critérios para síndrome de resposta inflamatória sistêmica e predição de mortalidade hospitalar em pacientes críticos: estudo retrospectivo de coorte.

OBJECTIVE: This study intended to determine whether the systemic inflammatory response syndrome criteria can predict hospital mortality in a Brazilian cohort of critically ill patients. METHODS: We performed a retrospective cohort study at a private tertiary hospital in São Paulo (SP), Brazil. We extracted information from the adult intensive care unit database (Sistema EpimedTM). We compared the SAPS 3 and the systemic inflammatory response syndrome model as dichotomous (≥ 2 criteria: systemic inflammatory response syndrome -positive versus 0 - 1 criterion: systemic inflammatory response syndrome -negative) and ordinal variables from 0 to 4 (according to the number of systemic inflammatory response syndrome criteria met) in the prediction of hospital mortality at intensive care unit admission. Model discrimination was compared using the area under the receiver operating characteristics (AUROC) curve. RESULTS: From January to December 2012, we studied 932 patients (60.4% were systemic inflammatory response syndrome -positive). systemic inflammatory response syndrome -positive patients were more critically ill than systemic inflammatory response syndrome -negative patients and had higher hospital mortality (16.9% versus 8.1%, p < 0.001). In the adjusted analysis, being systemic inflammatory response syndrome -positive independently increased the risk of death by 82% (odds ratio 1.82; 95% confidence interval [CI] 1.12 - 2.96, p = 0.016). However, the AUROC curve for the SAPS 3 model was higher (0.81, 95%CI 0.78 - 0.85) compared to the systemic inflammatory response syndrome model with the systemic inflammatory response syndrome criteria as a dichotomous variable (0.60, 95%CI 0.55 - 0.65) and as an ordinal variable (0.62, 95%CI 0.57 - 0.68; p < 0.001) for hospital mortality. CONCLUSION: Although systemic inflammatory response syndrome is associated with hospital mortality, the systemic inflammatory response syndrome criteria show low accuracy in the prediction of mortality compared with the SAPS 3.

OBJETIVO: Determinar se os critérios para definição de síndrome de resposta inflamatória sistêmica podem predizer a mortalidade hospitalar em uma coorte brasileira de pacientes críticos. MÉTODOS: Conduzimos um estudo retrospectivo de coorte em um hospital terciário privado localizado na cidade de São Paulo (SP). Extraímos as informações da base de dados de uma unidade de terapia intensiva para adultos (Sistema EpimedTM). Comparamos o SAPS 3 e o modelo da síndrome de resposta inflamatória sistêmica de forma dicotomizada (≥ 2 critérios, para síndrome de resposta inflamatória sistêmica positiva, em comparação com zero a um critério, para síndrome de resposta inflamatória sistêmica negativa) e variáveis ordinais de zero a 4 (segundo o número de critérios preenchidos para síndrome de resposta inflamatória sistêmica) para predição de mortalidade hospitalar por ocasião da admissão à unidade. A discriminação do modelo foi comparada com uso da área sob a curva receiver operating characteristics (ASCROC). RESULTADOS: Entre janeiro e dezembro de 2012, estudamos 932 pacientes (60,4% deles eram síndrome de resposta inflamatória sistêmica positiva). Os pacientes positivos para síndrome de resposta inflamatória sistêmica estavam em estado crítico mais grave do que os negativos, e tiveram mortalidade hospitalar mais elevada (16,9% versus 8,1%; p < 0,001). Na análise ajustada, ser síndrome de resposta inflamatória sistêmica positivo aumentou de forma independente o risco de óbito em 82% (OR 1,82; IC95% 1,12 - 2,96; p = 0,016). Entretanto, a ASCROC para os critérios do modelo SAPS 3 foi mais elevada (0,81; IC95% 0,78 - 0,85) em comparação ao modelo síndrome de resposta inflamatória sistêmica, tendo os critérios para síndrome de resposta inflamatória sistêmica de forma dicotomizada (0,60; IC95% 0,55 - 0,65) e como variável ordinal (0,62; IC95% 0,57 - 0,68; p < 0,001) para mortalidade hospitalar. CONCLUSÃO: Embora a síndrome de resposta inflamatória sistêmica se associe com mortalidade hospitalar, os critérios para esta síndrome tiveram baixa acurácia para predição da mortalidade, quando comparados ao SAPS 3.

Admission factors associated with intensive care unit readmission in critically ill oncohematological patients: a retrospective cohort study.

OBJECTIVE: The purpose of our study was to determine the admission factors associated with intensive care unit readmission among oncohematological patients. METHODS: Retrospective cohort study using an intensive care unit database from a tertiary oncological center. The participants included 1,872 critically ill oncohematological patients who were admitted to the intensive care unit from January 2012 to December 2014 and who were subsequently discharged alive. We used univariate and multivariate analysis to identify the admission risk factors associated with later intensive care unit readmission. RESULTS: One hundred seventy-two patients (9.2% of 1,872 oncohematological patients discharged alive from the intensive care unit) were readmitted after intensive care unit discharge. The readmitted patients were sicker compared with the non-readmitted group and had higher hospital mortality (32.6% versus 3.7%, respectively; p < 0.001). In the multivariate analysis, the independent risk factors for intensive care unit readmission were male sex (OR: 1.5, 95% CI: 1.07 - 2.12; p = 0.019), emergency surgery as the admission reason (OR: 2.91, 95%CI: 1.53 - 5.54; p = 0.001), longer hospital length of stay before intensive care unit transfer (OR: 1.02, 95%CI: 1.007 - 1.035; p = 0.003), and mechanical ventilation (OR: 2.31, 95%CI: 1.57 - 3.40; p < 0.001). CONCLUSIONS: In this cohort of oncohematological patients, we identified some risk factors associated with intensive care unit readmission, most of which are not amenable to interventions. The identification of risk factors at intensive care unit discharge might be a promising approach.

Antifibrotic effect of tamoxifen in a model of progressive renal disease.

Tamoxifen, a selective estrogen receptor modulator, has antifibrotic properties; however, whether it can attenuate renal fibrosis is unknown. In this study, we tested the effects of tamoxifen in a model of hypertensive nephrosclerosis (chronic inhibition of nitric oxide synthesis with L-NAME). After 30 days, treated rats had significantly lower levels of albuminuria as well as lower histologic scores for glomerulosclerosis and interstitial fibrosis than untreated controls. Tamoxifen was renoprotective despite having no effect on the sustained, severe hypertension induced by L-NAME. Tamoxifen prevented the accumulation of extracellular matrix by decreasing the expression of collagen I, collagen III, and fibronectin mRNA and protein. These renoprotective effects associated with inhibition of TGF-ß1 and plasminogen activator inhibitor-1, and with a significant reduction in α-smooth muscle actin-positive cells in the renal interstitium. Furthermore, tamoxifen abrogated IL-1ß- and angiotensin-II-induced proliferation of fibroblasts from both kidney explants and from the NRK-49F cell line. Tamoxifen also inhibited the expression of extracellular matrix components and the production and release of TGF-ß1 into the supernatant of these cells. In summary, tamoxifen exhibits antifibrotic effects in the L-NAME model of hypertensive nephrosclerosis, likely through the inhibition of TGF-ß1, suggesting that it may have therapeutic use in CKD treatment.

Early brief treatment with losartan plus mycophenolate mofetil provides lasting renoprotection in a renal ablation model.

BACKGROUND: Inflammatory events antecede established renal injury in rats with 5/6 renal ablation (Nx), as indicated by the beneficial effects of early, uninterrupted treatment with mycophenolate mofetil (MMF). Angiotensin II also exerts a major pathogenic role at this initial phase. We investigated whether losartan (L) or L+MMF treatment, started early, and L+MMF treatment, started late, would exert lasting renoprotection in Nx even after being discontinued. METHODS: Adult male Munich-Wistar rats underwent Nx and were divided into three groups: Nx (untreated), Nx(L) (given L), and Nx(LMMF) (given L and MMF). Protocol 1: treatments began on day 1, and ceased on day 30, after Nx. Protocol 2: L+MMF treatment began on day 30 and ceased on day 60. RESULTS: Protocol 1: on day 30, hypertension, albuminuria and renal injury were strongly attenuated in Groups Nx(L) and Nx(LMMF). On day 120, these abnormalities were still attenuated in group Nx(LMMF). Protocol 2: on day 120, all parameters were similar between this late Nx(LMMF) group and untreated Nx. CONCLUSION: In Nx, temporary suppression of early, transitory hemodynamic/inflammatory phenomena affords relatively durable renoprotection even after treatment discontinuation. This effect is not obtained with similar temporary treatment initiated later in the course of renal disease.

Association of the MCP-1 -2518 A/G polymorphism and no association of its receptor CCR2 -64 V/I polymorphism with lupus nephritis.

OBJECTIVE: To evaluate whether the A/G polymorphism at position -2518 in the regulatory region of the monocyte chemoattractant protein-1 (MCP-1) or the V/I polymorphism at position -64 of the receptor, CCR2, are associated with lupus nephritis (LN) or any clinical characteristics of the disease or with renal survival in a patient population. METHODS: We selected 197 patients with lupus nephritis and 220 matched healthy controls for study. MCP-1 and CCR2 genotyping was performed by polymerase chain reaction. Clinical and laboratory data were compiled from patients' charts over followup that ranged from 6 months to 10 years. RESULTS: The G/G genotype of MCP-1 was more common in LN patients (p = 0.019), while the A allele was associated with healthy controls (p = 0.007) as was the V allele of CCR2 (p = 0.046) compared to LN patients. Clinical index measures [SLE Disease Activity Index (SLEDAI)], immunological markers, renal histology, renal function at enrollment, and renal survival were not influenced by these polymorphisms. A less aggressive renal disease, measured by renal SLEDAI index, was associated with the V allele of the CCR2 gene polymorphism. CONCLUSION: These findings support that MCP-1 -2518 G/G is associated with LN but there was no association of this genotype with renal function or renal survival. When studying CCR2 -64 V/I polymorphism we showed a positive association of the V allele with healthy controls but no association of the genotype with LN patients.

Role of p21 and oxidative stress on renal tubular resistance after acute ischaemic injury.

BACKGROUND: Subsequent ischaemic episodes may induce renal resistance. P21 is a cell cycle inhibitor that may be induced by oxygen-free radicals and may have a protective effect in ischaemic acute kidney injury (AKI). This study aimed at evaluating the role of oxidative stress and p21 on tubular resistance in a model of acquired resistance after renal ischaemia and in isolated renal tubules. METHODS: Wistar rats were divided into: Group 1--sham; Group 2--sham operated and after 2 days submitted to 45-min ischaemia; and Group 3--45-min ischaemia followed after 2 days by a second 45-min ischaemia. Plasma urea was evaluated on Days 0, 2 and 4. Serum creatinine, creatinine clearance and oxidants (thiobarbituric acid-reactive substances) were determined 48 h after the second procedure (Day 4). Histology, immunohistochemistry for lymphocytes (CD3), macrophages (ED1), proliferation (PCNA) and apoptosis (TUNEL) were also evaluated. Rat proximal tubules (PTs) were isolated by collagenase digestion and Percoll gradient from control rats and rats previously subjected to 35 min of ischaemia. PTs were submitted to 15-min hypoxia followed by 45-min reoxygenation. Cell injury was assessed by lactate dehydrogenase release and hydroperoxide production (xylenol orange). RESULTS: Ischaemia induced AKI in Group 2 and 3 rats. Subsequent ischaemia did not aggravate renal injury, demonstrating renal resistance (Group 3). Renal function recovery was similar in Group 2 and 3. Plasma and urine oxidants were similar among in Group 2 and 3. Histology disclosed acute tubular necrosis in Group 2 and 3. Lymphocyte infiltrates were similar among all groups whereas macrophages infiltrate was greater in Group 3. Cell proliferation was greater in Group 2 compared with Group 3. Apoptosis was similar in groups 2 and 3. The p21 expression was increased only in Group 3 whereas it was similar in groups 1 and 2. PTs from the ischaemia group were sensitive to hypoxia but resistant to reoxygenation injury which was followed by lower hydroperoxide production compared to control PT. CONCLUSION: Renal resistance induced by ischaemia was associated with cell mechanism mediators involving oxidative stress and increased p21 expression.

Acute effects of intermittent hemodialysis and sustained low-efficiency hemodialysis (SLED) on the pulmonary function of patients under mechanical ventilation.

The effects of hemodialysis (HD) on pulmonary function are still controversial. The objective of this study was to evaluate the effect of intermittent hemodialysis (IHD) and sustained low-efficiency dialysis (SLED) on the respiratory mechanics of ICU patients under invasive mechanical ventilation. We prospectively studied 31 patients. Laboratory and respiratory evaluation (static and dynamic compliance and resistance) was performed pre- and post-HD. Forty HD sessions were studied and grouped in: SLED (n = 17; Qa = 200-250 mL/min, Qd = 300 mL/min) and IHD (n = 23; Qa = 250-300 mL/min, Qd = 500 mL/min). There was no difference between the groups according to age, gender, comorbidities, APACHE II, and cause of mechanical ventilation, but pre-HD, patients in the IHD group had higher levels of plasma creatinine (5.4 +/- 2.0 vs. 4.2 +/- 1.3 mg/dL, p = 0.048) and platelets (286 +/- 186 vs. 174 +/- 95 10(3)/mm(2), p = 0.032) and lower arterial pH (7.37 +/- 0.07 vs. 7.42 +/- 0.05, p = 0.02). The efficiency of the treatment was similar (p > 0.05) with both types of HD regarding fluid removal, urea reduction rate, and decrease in plasma creatinine. Pre-HD, the ventilatory conditions of both groups were similar (p > 0.05) except for pressure support ventilation and airflow resistance. There were no changes (pre- versus post-HD p > 0.05) induced either by IHD or SLED in the ratio PaO(2)/FiO(2) or in any measured ventilatory parameter. In conclusion, neither IHD nor SLED modifies the pulmonary function of patients under mechanical ventilation.

Simvastatin attenuates renal inflammation, tubular transdifferentiation and interstitial fibrosis in rats with unilateral ureteral obstruction.

BACKGROUND: The pleiotropic actions of statins have been largely explored. These drugs have been tested in several models of progressive renal disease, most of them accompanied by hypertension. We sought to investigate more closely the effects of simvastatin on renal interstitial fibrosis due to unilateral ureteral obstruction (UUO). METHODS: Munich-Wistar rats were submitted to UUO and studied after 14 days. Animals were divided into two groups: vehicle (VH) or simvastatin (SIMV) 2 mg/kg b.i.d. by gavage. At sacrifice kidneys were harvested for morphology, mRNA and protein analysis. RT-PCR was done to assess expression of collagen I and III, fibronectin, MCP-1, TGF-beta1 and bFGF. Protein expression was assessed by western blot (TGF-beta) and immunostaining (macrophage, lymphocyte, PCNA, vimentin and alpha-smooth muscle actin). Contralateral kidneys (CL) were used as controls. RESULTS: SIMV-treated animals had less severe renal inflammation. MCP-1 was markedly expressed in obstructed kidneys and diminished with SIMV (48.9+/- 2.5 vs 64.3+/-3.1 OD in VH, P<0.01). Interstitial fibrosis (IF) was significantly attenuated with SIMV (8.2+/-1.3 vs 13.2+/-0.6%, P<0.01 SIMV vs VH), which was confirmed by a decrease in collagen I and fibronectin renal expression. Vimentin, a marker of dedifferentiation, was expressed in tubular cells of VH and decreased with SIMV treatment. alpha-SMA, a marker of myofibroblast-type cells, was increased in renal interstitium of VH rats and SIMV significantly reduced its expression. PCNA was increased in the UUO kidneys, but SIMV did not decrease tubular or interstitial proliferating cells. TGF-beta1, which was highly induced in the obstructed kidneys, decreased at the post-transcriptional level with SIMV treatment (5.35+/-0.75 vs 13.10+/-2.9 OD in VH, P<0.05). bFGF mRNA was also overexpressed in the obstructed kidneys, although SIMV treatment did not significantly decrease its expression. CONCLUSIONS: SIMV had an evident protective effect on renal interstitial inflammation and fibrosis. It is conceivable that by attenuating inflammation, SIMV prevented tubular activation and transdifferentiation, two processes largely involved in the renal fibrosis of the UUO model.

Evidence for the existence of two distinct functions for the inducible NO synthase in the rat kidney: effect of aminoguanidine in rats with 5/6 ablation.

The functional role of the NO synthase (NOS) isoforms in the normal or diseased kidney is uncertain. This study examined the renal expression of the endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) isoforms by both immunohistochemistry and Western blot analyses in sham-operated rats (S) and in rats subjected to 5/6 nephrectomy (Nx). Primary antibodies from two different sources were used to detect iNOS. Additional S and Nx rats were chronically treated with aminoguanidine (AG), a selective iNOS inhibitor. All three isoforms were clearly expressed in S kidney. Their renal abundance, evaluated by Western blot analysis, fell in Nx rats. With the use of anti-iNOS antibodies from two distinct sources, the immunohistochemical analysis showed the presence of what appeared to be two distinct iNOS fractions: a "tubular" fraction, present in S and with decreased intensity in Nx; and an "interstitial" fraction, observed only in inflamed areas of Nx rats. AG treatment greatly attenuated renal injury in Nx rats by a direct antiinflammatory effect, likely related to iNOS inhibition, rather than to amelioration of renal hemodynamics or to reduced protein glycation. These observations suggest that: (1) the functional role of the renal iNOS isoform may vary dramatically under different physiologic conditions; (2) caution should be taken in the interpretation of immunohistochemical iNOS data, because antibodies from different sources may detect different iNOS fractions; and (3) AG treatment may become useful in the treatment of human progressive nephropathies, even those not associated with diabetes or aging.