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The COVID-19 pandemic, emerging/re-emerging infections as well as other non-communicable chronic diseases, highlight the necessity of smart microfluidic point-of-care diagnostic (POC) devices and systems in developing nations as risk factors for infections, severe disease manifestations and poor clinical outcomes are highly represented in these countries. These POC devices are also becoming vital as analytical procedures executable outside of conventional laboratory settings are seen as the future of healthcare delivery. Microfluidics have grown into a revolutionary system to miniaturize chemical and biological experimentation, including disease detection and diagnosis utilizing muPads/paper-based microfluidic devices, polymer-based microfluidic devices and 3-dimensional printed microfluidic devices. Through the development of droplet digital PCR, single-cell RNA sequencing, and next-generation sequencing, microfluidics in their analogous forms have been the leading contributor to the technical advancements in medicine. Microfluidics and machine-learning-based algorithms complement each other with the possibility of scientific exploration, induced by the framework's robustness, as preliminary studies have documented significant achievements in biomedicine, such as sorting, microencapsulation, and automated detection. Despite these milestones and potential applications, the complexity of microfluidic system design, fabrication, and operation has prevented widespread adoption. As previous studies focused on microfluidic devices that can handle molecular diagnostic procedures, researchers must integrate these components with other microsystem processes like data acquisition, data processing, power supply, fluid control, and sample pretreatment to overcome the barriers to smart microfluidic commercialization.Copyright © 2023 by Begell House, Inc.
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SARS-CoV-2 infected cases diagnosis is based on the count of realtime reverse transcription-polymerase chain reaction (RT-PCR). The widely used reverse transcription-polymerase chain reaction (RTPCR) method has some limitations for clinical diagnosis and treatment. However, there are only few reports on the detection of the viral load in the stool and urine samples. While information about other modes of transmission is relatively less, some published literature supporting the possibility of a faecal-oral mode of transmission has been accumulating. Objective(s): The current study's objective was to assess the performance of real-time RT-qPCR assay and a droplet digital RT-PCR (dd RT-PCR) for detecting SARS-CoV-2 in stool and urine specimens. Methodology: One hundred and seven paired samples from 107 COVID-19-confirmed patients were analysed by dd RT-PCR and RTPCR based target gene (N1 and N2). Stool and urine were collected from COVID Care Centers of Pune Region. RNA was isolated using MagMax magnetic beads base procedure for further analysis. Real Time RT-PCR and DD PCR was performed from all the patients. Result(s): In 107 patients, all the stool samples showed 100% positive concordance by both methods, the average of 28.88 cycle threshold (Ct) of RT-PCR was highly correlated with the average copy number of 327.10 copies/mul analyzed in ddPCR. Whereas 27.1% urine samples were tested positive in ddPCR & 1.86% were positive with the average of 36.41 cycle threshold (Ct) in RT-PCR. Using Pangolin COVID-19 Lineage Assigner variants were analyzed and found to be delta prevalent. Conclusion(s): In the context of the COVID-19 pandemic, environmental surveillance for the detection of SARS-CoV-2 has become increasingly important. The findings of this study not only show that SARS-CoV-2 is present in urine and faeces, but they also raise the possibility that low concentrations of the viral target may make it easier to identify positive samples and help resolve situations of inconclusive diagnosis.
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Background: Antigen-driven CD4+ T cell proliferation is a proposed mechanism of HIV-1 reservoir persistence. We previously reported that SARSCoV- 2 infection leads to increased detectable low-level HIV-1 plasm RNA blips months after COVID-19, but the impact of SARS-CoV-2-mediated T cell activation on expansion of HIV-1 reservoirs is not known. We sought to identify if SARSCoV- 2 infection leads to expansion of preferentially HIV-infected CD4+ T cells in people with HIV (PWH) on ART. Method(s): Five PWH with samples collected prior to and approximately two months after SARS-CoV-2 infection were identified. We performed a surface activation induced marker (AIM) assay using a CD4-optimized overlapping SARS-CoV-2 peptide pool to measure OX40/CD137 expression following peptide stimulation and sorted CD4+ T cells based on surface marker expression. ddPCR quantification of genomic HIV-1 DNA was performed on sorted subsets. Result(s): We observed an increase in the frequency of SARS-CoV-2 AIM+ non-naive CD4+ T cells following COVID-19 in samples from 4 of 5 participants (mean AIM+ % 0.13 pre- vs 0.31 post). A large percentage of non-naive AIM+ CD4+ T cells expressed PD1 compared with total non-naive cells before (76% vs 36%) and after (65% vs 19%) COVID-19;PD1 expression was lower following SARS-CoV-2 in both AIM+ and AIM- CD4+ T cell subsets (although very few cells were AIM+ prior to COVID-19). HIV-1 DNA levels in non-naive AIM- CD4+ T cells prior to COVID-19 unexpectedly decreased following infection (mean 3,522 to 766 copies/106 cells). The numbers of AIM+ cells obtained by cell sorting were overall low ( 3,863 mean) and only one participant had detectable DNA in post-COVID AIM+ CD4+ T cells. However, a large majority of this participant's post-COVID AIM+ cells harbored HIV-1 DNA (0.89 copies per cell) whereas HIV DNA in their AIM- cells decreased from 8,387 to not detected following SARSCoV- 2 infection. No HIV-1 DNA was detected in the small number of AIM+ cells obtained prior to COVID-19 in this participant. Conclusion(s): COVID-19 in PWH led to a modest SARS-CoV-2-specific CD4+ cell response approximately two months following acute presentation. One participant may have preferentially expanded HIV-1-infected, SARS-CoV-2- specific CD4+ T cells following COVID-19 but studies involving larger numbers of participants and larger numbers of cells will be needed to fully understand the impact of SARS-CoV-2 on clonal expansion and HIV persistence.
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Background: Metformin has in vitro activity against SARS-CoV-2. In a published phase 3, quadruple-blinded, placebo-controlled randomized trial of outpatient COVID-19 therapy, metformin resulted in a 42% reduction in ER visits/hospitalizations/deaths by day 14, 58% reduction in hospitalizations/ death by day 28, and 42% reduction in Long Covid through 10 months. This analysis presents the results of viral load sampling performed during that clinical trial. Method(s): Covid-Out trial (NCT04510194) enrolled adults aged 30 to 85 within 3 days of a documented SARS-CoV-2 infection and < 7 days after symptom onset. The trial randomized 1323 participants to metformin (1000mg/day days 2-5;1500mg/day days 6 to 14), ivermectin, fluvoxamine, and/or exact-matching placebo in a 2x3 factorial trial design. Nasal swabs for viral load were an optional component, self-collected from the anterior nares on day 1, 5, and 10. Viral loads were measured via RT-qPCR using N1 and N2 targets in the SARSCoV- 2 nucleocapsid protein, with relative Ct values converted to absolute copy number via calibration to droplet digital PCR. A linear Tobit regression model was used to assess change over time while accounting for left censoring due to the viral load limit of detection. Results were adjusted for other treatment allocations within the factorial design, vaccination status, and baseline viral load. Repeated measures were accounted for using clustered standard errors within participants. Result(s): Samples were available from n = 945, 871, and 775 participants on days 1, 5, and 10, respectively. The mean change from baseline to followup was -0.64 log10 copies/mL (95%CI, -1.16 to -0.13) for metformin versus placebo, which equates to a 4.4-fold greater decrease. The mean change in SARS-CoV-2 from baseline to day 5 was -0.48 log10 copies/mL, and was -0.81 log10 copies/mL from baseline to day 10. The anti-viral effect increased with increased metformin dosing days 6-14. The antiviral effect was larger in those unvaccinated (mean -0.95 log copies/mL) than vaccinated (mean -0.39 log copies/mL). There was no change in viral load vs. placebo for ivermectin or fluvoxamine. Conclusion(s): Metformin lowered SARS-CoV-2 viral load in this quadrupleblinded, randomized clinical trial. The temporal relationship to dose titration suggests a dose-dependent effect. The magnitude of antiviral effect was similar to nirmatrelvir at day 5, greater than nirmatrelvir at day 10. Metformin is safe, widely available, and has few contraindications.
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Circulating tumor DNA is a liquid biomarker that offers a highly specific method to assess HPV-associated tumor burden via a blood draw. It has the potential for many clinical applications in cancer care, including prognostication, monitoring treatment response, and surveillance for disease recurrence. In this case report, we present a case of recurrent HPV-associated hypopharyngeal squamous cell carcinoma first detected by circulating tumor HPV DNA that demonstrates the role of circulating tumor HPV DNA tests in posttreatment surveillance and the utility of HPV testing in all HPV-mediated tumors, regardless of subsite.Copyright © 2023 Elsevier Inc.
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Polymerase chain reaction (PCR) amplifies specific fragment of DNA molecules and has been extensively applied in fields of pathogens and gene mutation detection, food safety and clinical diagnosis which on the other hand, holds the drawbacks of large size instrument, high heat dissipation etc. It has been demonstrated that microfluidics technique coupling with PCR reaction exhibits characteristics of integration, automatization, miniaturization, and portability. Meanwhile, various designed fabrication of microchip could contribute to diverse applications. In this review, we summarized major works about a variety of microfluidic chips equipped with several kinds of PCR techniques (PCR, RT-PCR, mPCR, dPCR) and detection methods like fluorescence, electrochemistry, and electrophoresis detection. The development and application of PCR-based microfluidic chip in pathogen and gene mutation detection, diseases prevention and diagnosis, DNA hybridization and low-volume sample treatment were also discussed. Copyright © 2022 Elsevier B.V.
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INTRODUCTION: While the COVID-19 syndrome is triggered by infection and expansion of the SARS-CoV2 RNA virus, secondary opportunistic infections can be a significant contributor to morbidity. In prior studies, our group employed RNA sequencing of whole blood RNA to identify RNA biomarkers of COVID-19 infection and severity. In the present studies, those biomarkers were expanded. METHOD(S): We performed a single-center prospective cohort study of SARS-COV2 infected ICU patients (n=20) during the peak of the Omicron wave (Jan-Feb 2022). Participants were consented for a venous blood draw into an RNA preservative. Samples were stored at -80degree C. Stored blood was used for RNA purification and droplet digital PCR quantitation of 6 novel RNA biomarkers for bacterial (DEFA1), biofilm (ALPL, IL8RB/CXCR2), and viral infections (IFI27, RSAD2). Viral titer in blood was analyzed in parallel by ddPCR for SARS-CoV2 sequences (BioRad, EUA). RESULT(S): Among clinical biomarkers, Pearson correlational analysis with SOFA scores identified lactate (r=0.24), BMI (r=0.34), creatinine (r=0.58), and LDH (r=0.68), as the best predictors. Viremic titer was not associated with SOFA scores (r=-0.07). Among the RNA biomarkers ALPL (r=0.48), a biofilm marker, showed the best correlation with SOFA score. The RNA biomarkers of viral infection IFI27 (r=0.72) and RSAD2 (r=0.42) were positively correlated with SARSCoV2 viral titer, suggesting that the host immune response is proportional to the viremia of COVID-19. CONCLUSION(S): Collectively, the results suggest that whole blood RNA transcripts involved in the host immune response can indicate the presence and severity of infection, including unexpected comorbidities. Furthermore, these biomarkers can distinguish between viremia, biofilms, and other types of infections that may undermine recovery from COVID-19.
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Background. We sought to compareWWSARS-CoV-2 RNA detection across a range of sites and scales using RTqPCR and RTddPCR. Figure. Methods. Composite-24hWW was collected from aWWtreatment plant (WTP;n=18), a neighborhood (Nb1;n=12) and three hospitals;H-1, H-2, and H-3 (3-sites;A-C)(n=84). RNA was extracted using the 4S-silica column method. RTqPCR (QuantStudio5, Thermo Fisher) and RTddPCR (C1000 Thermal Cycler and QX200 Droplet Reader, BioRad) quantified SARS-CoV-2 RNA nucleocapsid (N2, US CDC) and envelope (E Sarbeco, Corman et al 2020) in triplicate. Fisher's exact test was used to compare assay sensitivity. Correlations between modalities and RNA - clinically-confirmed COVID-19 cases (defined by postal code of primary residence using 5-day rolling average) was assessed using Persons correlation. Results. 114 samples were tested (02/23/2021-04/22/2021). SARS-CoV-2-N2 was identified in 90/114 (79%) by RTqPCR and 89/114 (78%) by ddPCR (p=1). SARS-CoV-2 E was found in 72/114 (63%) by RTqPCR and 90/114 (79%) by ddPCR, p=0.01. Correlations between modalities were strongest for N2 relative to E across all sites (see Table). N2 correlated with clinically diagnosed cases for both modalities greater at the level of the WTP (RTqPCR;r=0.8972, p< 0.0001and ddPCR;0.933, p< 0.0001) relative to neighborhood (RTqPCR;r=0.6, p=0.04 and ddPCR;0.60, p=0.04). E correlated to a lesser degree with cases at WTP (RTqPCR;r=0.65, p=0.0035 and ddPCR;0.88, p=< 0.001) and neighborhoods (RTqPCR;r=0.40, p=0.20 and ddPCR;r=0.43, p=0.16). Conclusion. SARS-CoV-2 detection of N2 was similar between RTqPCR and RTddPCR across a range of sites and scales in the sewershed, and this correlated best with clinical cases whereas E detection was superior with ddPCR.
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Background. Droplet digital PCR (ddPCR) has been shown to be more sensitive and precise in the quantification of SARS-CoV-2 when compared to traditional quantitative RT-PCR. Multiple studies have explored associations between SARS-CoV-2 viral load and patient outcomes;however, few have used ddPCR technology. Here we investigated the associations between viral load measured using ddPCR and clinical presentation and outcomes. Methods. We performed a retrospective observational study of individuals who tested positive for COVID-19 at the VA San Diego between August 2020 and December 2021. SARS-CoV-2 viral load from nasopharyngeal swabs was determined using ddPCR. Baseline demographics, past medical history, clinical course, and laboratory data were ed from the chart. Results. A total of 696 individuals were included, 86% (n=603) of whom were male. The average age was 50-years-old [range: 19-98]. Three-quarters of individuals (76%, n=528) were unvaccinated at diagnosis. Frequency of comorbidities are shown in Table 1. The majority of individuals developed symptoms with 75% (n=516) reporting respiratory symptoms, 47% (n=317) fever, 34% (n=230) GI symptoms, and 23% (n=161) loss of taste and/or smell. A total of 24% of veterans were evaluated only in the emergency department, 21% (n=149) were admitted to the hospital;9% (n=60) required ICU level of care, 33% of these (n=20) required intubation, and 16 individuals died during hospitalization. SARS-CoV-2 log10 viral load was not associated with age, and only a weak correlation was seen with time from onset of symptoms (r2=-0.1, p=0.04). No association was observed between viral load and peak CRP, ferritin, d-dimer, or nadir absolute lymphocyte count. Mean viral load was significantly higher in veterans reporting fever (5.0 vs 5.4, p=0.02) and respiratory symptoms (4.7 vs 5.3, p=0.01). Interestingly, vaccinated veterans also had higher viral loads(5.8 vs 5.0, p< 0.0001). Conclusion. Fever and respiratory symptoms were associated with higher viral loads as expected. The association of vaccination with higher viral load may reflect selection bias for infections in the delta wave. Future work will include multivariate analyses to adjust for medical history and timing of sampling.
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Purpose : To systematically investigate ocular changes in autopsied eyes from fatal cases of Coronavirus disease 2019 (COVID-19) and to investigate the localization of severe acute respiratory syndrome coronavirus (SARS-CoV-2) within ocular structures. Methods : Macroscopic and microscopic histopathological evaluation was performed and the localization of SARS-CoV-2 RNA within ocular tissues investigated using an in situ hybridization (ISH) technique in 13 eyes. Contralateral eyes were freshly dissected, and droplet digital polymerase chain reaction (ddPCR) assay was performed on ocular fluids and tissues to quantify SARS-CoV-2 RNA. Results : A total of 21 fatal COVID-19 cases were included (mean age, 60.2 years [range, 27- 91 years];23.8% female). Histopathological abnormalities include vascular changes (61.9%), cytoid bodies (52.4%), and retinal edema (23.8%) with minimal inflammation (0.09%) were observed. Non-CMV viral inclusions were identified in one eye. No CMV positivity was detected. Of the 21 contralateral eyes tested by ddPCR, 14 tested positive for SARS-CoV-2. Using ddPCR and ISH, SARS-CoV-2 localization was observed in the following ocular tissues and fluid: cornea (27.3%), aqueous (26.3%), lens (54.5%), vitreous (15.0%), retina (22.2%), choroid/sclera (47.4%), and optic nerve (50.0%). The choroid/sclera, optic nerve and lens were the most frequent ocular structures found to be ddPCR positive. Evidence of replication was detected in four cases. Conclusions : Our results suggest that SARS-CoV-2 localizes to intraocular tissues. However, histological changes observed are likely a secondary hemodynamic change rather than primary effect of the virus.
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Background: Although it prevents severe forms of the disease, vaccination does not completely protect against the occurrence of COVID19 disease. If, DMARDs used have been associated with variable humoral response to SARS-CoV-2 vaccination, the impact of their use after SARS-CoV-2 natural infection have been poorly studied. Objectives: To characterize humoral response after SARS-CoV-2 infection and viral persistence in the nasopharyngeal sphere (NP), stools and blood of patients with rheumatic disease under DMARDs, and compared to healthy controls. Methods: Prospective monocentric longitudinal study including patients with rheumatoid arthritis or spondyloarthritis under DMARDs and with a confrmed SARS-CoV-2 infection (positive NP PCR and/or positive serology and/or pathognomonic thoracic tomography (CT)) during the frst or second wave of the COVID pandemic. Patients were followed up until one year after infection and humoral response was assessed before vaccination. Serum IgG and IgA antibodies against spike (S) and nucleocapsid (N) proteins were measured at every visit. Viral persistence was assessed at the early visit in the NP and stools using conventional RT-PCR and in the blood using a high sensitive technique (droplet digital PCR). Results: Between June 2020 and July 2021, we include 96 patients (50 SpA and 46 RA) with a mean age of 53 +/-14 years and 20 healthy controls (mean age 49 ± 16 years) corresponding to relatives of patients (spouses, children) living together and infected at the same time. The immune responses were analyzed according to 6 treatment groups: methotrexate (MTX)/salazopyrine (SLZ) monotherapy (n=17/2);anti-TNF monotherapy (n=24), anti-TNF + MTX (n=23);rituximab (RTX) (n=11);anti-IL17 or-23 (n=8);others (n=11). Visits were made at 1 month (29 ±13 days;n=18), 3 months (110 ±23 days;n=67), 6 months (231 ±35 days;n=48) and 12 months (368 ± 19 days;n=19) after infection. The anti-S and anti-N IgG Ab titers were not signifcantly different in the 6 treatment groups and the control population at 3 months. A signifcant decrease in anti-S IgA Ab titers was noted in the group treated with RTX (p=0.007) and with molecules targeting the IL17/23 pathway (p=0.007). A similar but non-signifcant trend was observed in these same treatment groups for anti-N IgA Ab (p=0.07). The titers of anti-SARS-CoV-2 antibodies at M3, was not associated with a severe COVID disease. Detection of SARS-Cov-2 RNA in stools and serum was negative for all samples taken at 1 month or 3 months. 4 patients (2 RA treated with abatacept/RTX and 2 SpA treated with anti-TNF/secukinumab) had a positive RT-PCR NP with low to very low viral load at the 1-month visit (mean Ct 36). None of these 4 patients had had a severe form of COVID19 infection. Conclusion: DMARDs-treated patients with previous proven COVID-19 did not seem to alter IgG Ab response but RTX and anti-IL17/-IL-23 might alter IgA humoral response. This lower immune response was not associated with a more severe disease. In these patients, new infection may not be considered as a full boost for the immune system. DMARDs did not induce viral persistence in the serum, the NP or in the stool.
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Objectives: Universal testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within birthing units is an effective strategy to contain infection and estimate community prevalence. Given the high-prevalence of COVID-19 cases in Ontario, the objective of this study was to determine the prevalence of active and recovered SARS-CoV-2 infection among pregnant individuals in Ottawa through universal SARS-CoV-2 and serology testing. Methods: From October 19th to November 27th, 2020, pregnant individuals admitted to triage assessment units at The Ottawa Hospital (TOH) were consented for SARS-CoV-2 testing. Swab and serology samples were analyzed using digital droplet polymerase chain reaction (ddPCR) and enzyme-linked immunosorbent assays, respectively. SARS-CoV-2 seropositivity was defined as a positive result for immunoglobulin (Ig) G, either alone or in combination with IgM and/or IgA. Results: From the 395 enrolled participants, 284 swab and 353 serology samples were collected. We found that 18 of 395 (4.6%) participants had evidence of SARS-CoV-2 exposure: 2/284 (0.70%) were positive for SARS-CoV-2 and 16/353 (4.5%) were positive for anti–SARS-CoV-2 IgG. Seropositive participants were similar to seronegative participants in terms of demographics, clinical characteristics, and pregnancy outcomes. Conclusions: The prevalence of SARS-CoV-2 ddPCR positivity and seropositivity in the obstetrical population at TOH was 0.70% and 4.5%, respectively in the fall of 2020. According to local public health data, the infection rate peaked at 0.6% during the study time period. Universal SARS-CoV-2 testing programs may help approximate community prevalence, however, justification of this strategy depends on testing capabilities and the local context of COVID-19 infection. Keywords: pregnancy;COVID-19;SARS-CoV-2;universal testing;seroprevalence
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Background: The kinetics and functional profiles (granzyme-B production) of HIV-specific T-cell responses support that those targeting the early viral gene product Nef disproportionately recognize residual antigen expression during long-term antiretroviral therapy (ART). Here, we leveraged this insight to test whether SARS-CoV2 mRNA vaccines-which activate TLR and inflammatory signaling pathways-would reactivate latent HIV, stimulating T-cell responses with these characteristics. Methods: T-cell responses to individual HIV gene products were measured by IFN-g or granzyme B ELISPOT, and by activation induced marker (AIM) assays at baseline and ∼2 weeks after SARS-CoV-2 mRNA vaccine prime and boost, in 13 long-term ART treated adults. Total and unspliced HIV mRNA, as well as intact and defective (IPDA) HIV DNA were measured in parallel by digital droplet PCR (ddPCR). Results: We observed transient increases Nef-specific T-cell responses following vaccine prime by granzyme B ELISPOT (3.1-fold increase, p=0.002) and a trend by AIM assay (1.5-fold increase, p=0.06). Such increases were not observed in granzyme B responses to late gene products nor in any IFN-g responses. Both unspliced and total HIV mRNA decreased significantly across the study, unspliced-1.6-fold decrease p = 0.03;total-1.5-fold decrease p = 0.05. Changes in total HIV mRNA correlated inversely with Nef-specific granzyme B-producing (spearman's ρ =-0.73, p = 0.006) and Nef-specific CD8+ AIM T-cell responses (ρ =-0.76, p = 0.006) following vaccine prime. These reductions in HIV RNA were not accompanied by significant changes in total or intact HIV DNA. Conclusion: Consistent with our hypothesis, a restricted profile of HIV-specific T-cell responses showed significant increases following SARS-CoV-2 vaccine prime, each of which were then correlated with reductions in HIV RNA. This supports that vaccination promoted productive interactions between Nef-specific CTL and HIV-infected cells in vivo. We propose three scenarios for why this was not reflected in reductions in intact or total HIV DNA: i) meaningful depletions in inducible proviruses occurred but were lost against the background of non-inducible proviruses ii) interactions with CTL involved only a fraction of inducible proviruses, or iii) substantive proviral depletions occurred, but were counterbalanced by clonal expansion of HIV-infected cells.
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Background: The number of cases of SARS-CoV-2 infection after BNT162b2 mRNA vaccination is significantly higher in elderly people, which has been associated to lower frequencies of SARS-CoV-2 neutralizing antibodies. Our objective was to investigate the differences in the cellular response in old and young people after the SARS-CoV-2 vaccination. Methods: Young (24-53 years, n=20) and old (70-76 years, n=20) healthy subjects vaccinated with BNT162b2 SARS-CoV-2 mRNA vaccine were studied before vaccination, two weeks after the first dose and two months after the second dose. SARS-CoV-2 (spike) specific T cell response, TLR-4 dependent monocyte response and TLR-3 dependent myeloid dendritic cell (DC) response and DC, monocyte and T-cell immunophenotype, were studied by multiparametric flow cytometry. TLR-9 dependent interferon-α (IFNα) production by PBMCs was measured by ELISA and thymic function assayed by sj/β TREC ratio using droplet digital PCR. Results: The SARS-CoV-2 specific T cell response was lower and less polyfunctional in old people. Most of the differences in CD4+ and CD8+ T cell subsets were found in degranulation (CD107a), cytokine (IFN-γ) and cytotoxic (perforin) profile (eg, Memory CD8+ perforin+;p=0.0016). The lower SARS-CoV-2 specific T cell response was associated with lower thymic function levels (eg, Memory CD4+ perforin+, r=0.631;p=0.0001). The vaccination induced a higher activation and proliferation (eg, CM CD4 HLA-DR+ p=0.002, Ki67+ p=0.019) of T cells in young people than in old ones, in addition to a higher level of homing makers to different tissues and inflammatory sites (eg, CD1c mDC integrin β7+ p=0.001, intermediate monocytes CCR2+ p=0.0003) in DCs and monocytes. Moreover, after the vaccination, old subjects showed a higher production of proinflammatory cytokines by monocytes in response to LPS (eg, IL6+;p=0.015), while young people showed a higher production of IFNα by plasmacytoid DCs after CpG-A stimulation (p=0.0009). Conclusion: The magnitude and polyfunctionality of SARS-CoV-2 specific T cell response is lower in old people, associated to a lower thymic function. In old people, the vaccination induced less immune activation and homing and the myeloid TLR-dependent response is directed towards a proinflammatory response, while in young people prevails IFNα production, related to a more effective antiviral response. These results support the additional boosting strategies in this vulnerable population.
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Pharmaceutical products contaminated with Burkholderia cepacia complex (BCC) strains constitute a serious health issue for susceptible individuals. New detection methods to distinguish DNA from viable cells are required to ensure pharmaceutical product quality and safety. In this study, we have assessed a droplet digital PCR (ddPCR) with a variant propidium monoazide (PMAxx) for selective detection of live/dead BCC cells in autoclaved nuclease-free water after 365 days, in 0.001% chlorhexidine gluconate (CHX), and in 0.005% benzalkonium chloride (BZK) solutions after 184 days. Using 10 μM PMAxx and 5 min light exposure, a proportion of dead BCC was quantified by ddPCR. The detection limit of culture-based method was 104 CFU/mL, equivalent to 9.7 pg/μL for B. cenocepacia J2315, while that of ddPCR was 9.7 fg/μL. The true positive rate from nuclease-free water and CHX using PMAxx-ddPCR assay was 60.0% and 38.3%, respectively, compared to 85.0% and 74.6% without PMAxx (p < 0.05), respectively. However, in BZK-treated cells, no difference in the detection rate was observed between the ddPCR assay on samples treated with PMAxx (67.1%) and without PMAxx (63.3%). This study shows that the PMAxx-ddPCR assay provides a better tool for selective detection of live BCC cells in non-sterile pharmaceutical products.
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Objetivos: Correlacionar os valores de dados laboratoriais – D-Dímero do primeiro dia (DD -ng/dL), Tempo de Protrombina (TP –segundos), Proteína C Reativa (PCR - mg/dL) – de pacientes COVID-19 positivo, com o desfecho de internação (alta hospitalar ou óbito), a necessidade de internação em Unidade de Terapia Intensiva (UTI), a necessidade de ventilação mecânica invasiva (VMI), a ocorrência de Trombose Venosa Profunda (TVP), Tromboembolismo Pulmonar (TEP), Acidente Vascular Cerebral Isquêmico (AVCI), Infarto Agudo do Miocárdio (IAM), a idade dos pacientes, a presença ou não de comorbidades e o gênero. Material e método: Trata-se de um estudo retrospectivo transversal observacional de análise sequencial e sigilosa de prontuários médicos. Foram incluídos no estudo os pacientes com COVID-19 confirmados por RT-PCR e com nível de D-Dímero acima de 1000 ng/mL, no período de março de 2020 a março de 2021. Para a análise dos dados foram utilizados os testes estatísticos Shapiro-Wilk, Mann-Whitney, Kruskal-Wallis, Teste de Dunn e Correlação de Spearman. Resultados: Foram incluídos 1220 pacientes, destes 42.5% tinham ao menos uma comorbidade, 31.6% necessitaram de ventilação mecânica invasiva, 17.6% necessitaram de internação em UTI, 57.4% tiveram alta hospitalar e 42.6% evoluíram para óbito. Dentre os pacientes analisados 2.5% apresentaram TVP, 2.6% TEP, 1.7% AVCI, 0.8% IAM. Resultado dos valores significativos: o grupo com alta hospitalar obteve intervalo interquartil (IQ) de DD = 1236.95-2742.87;IQ de TP = 11.1-12.7;IQ de PCR = 6.475-21.95. O grupo que evoluiu para óbito obteve IQ de DD = 1295.95-5376.3;IQ de TP = 11.4-13.5;IQ de PCR=8.75-27.15. O grupo que evoluiu com TEP apresentou IQ de DD = 2139.1-7252.3, já o que não evoluiu com TEP apresentou IQ de DD=1243.97-3558.12. O grupo submetido à VMI apresentou IQ de TP = 11.5-13.3;IQ de PCR=13.375-29.75, já o que não foi submetido apresentou IQ de TP = 11.1-12.9;IQ de PCR = 6.5-22.2. O grupo que necessitou de internação em UTI apresentou IQ de TP = 11.5-13.42;IQ de PCR=9.3-27.8, o que não necessitou apresentou IQ de TP=11.17-13;IQ de PCR = 6.975-23.5. Os pacientes do gênero feminino apresentaram IQ de TP=11.1-12.8, IQ de PCR = 6.8-22.6;os do gênero masculino apresentaram IQ de TP = 11.4-13.1, IQ de PCR = 7.8-25.4. Discussão: Conforme os valores encontrados, os pacientes com idade avançada apresentaram valores de DD, TP maiores e evoluíram mais para óbito;os do gênero masculino apresentaram maior mortalidade, PCR e TP comparado com o gênero feminino;os com comorbidades apresentaram PCR maior e evoluíram mais para óbito;os que tiveram alta apresentaram menor DD, PCR e idade e maior TP;os que necessitaram de VMI tiveram maior TP, PCR e óbitos;os que necessitaram de internação em UTI apresentaram maior valor de TP, PCR e mais óbitos;os que necessitaram mais dias de internação apresentaram menor TP, PCR e mais óbitos;os que evoluíram com TEP apresentaram DD maior;os que evoluíram com AVCi apresentaram maior TP;os que evoluíram com TVP, IAM e AVCi não apresentaram significância estatística de DD. Conclusão: Neste estudo, valores de DD maiores estiveram relacionados à maior mortalidade e à ocorrência de TEP. Valores de TP elevados relacionaram-se a maior mortalidade, internação em UTI, necessidade de VMI e presença de AVCi. Valores de PCR elevados estiveram relacionados à maior mortalidade, presença de comorbidade, internação em UTI e necessidade de VMI. Demais correlações não apresentaram relevância estatística.
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Introdução: A transfusão de plasma convalescente tem sido utilizada como terapêutica alternativa no tratamento de COVID19 nos últimos meses. Avaliamos o impacto dos anticorpos neutralizantes produzidos pelos pacientes e dos anticorpos presentes nas unidades transfundidas na redução da carga viral em pacientes em tratamento hospitalar de COVID19. Materiais e métodos: Foram avaliados consecutivamente 29 pacientes admitidos para tratamento hospitalar de COVID19 em um único centro. Doses de 300 a 600 ml de plasma convalescente foram administradas ao longo de 2 dias. Foram coletados swabs nasais a cada 48 h a partir do D0 (dia de transfusão de plasma convalescente) até a alta hospitalar, a fim de determinar a carga viral por digital droplet PCR (ddPCR) dos alvos N1 e N2 do gene N (nucleocapsídeo) para análise de redução de carga viral, sendo considerado o número de cópias virais por 1000 células presente na amostra. Mensuramos os títulos de anticorpos neutralizantes (cytopathic effect-based virus neutralization test -SARS-CoV-2 GenBank MT126808.1) dos pacientes (NAbsP) antes da transfusão (D0) e títulos de anticorpos neutralizantes das unidades de plasma transfundidas (NAbsT). Para análise de associação entre NAbsP e redução de carga viral, os pacientes foram divididos em dois grupos de acordo com o status de NAbsP no D0: título de NabsP inferior a 80 e título de NabsP igual ou superior a 80. Para esta análise, foi utilizado o teste de Mann-Whitney. Para verificar a associação entre NAbsT e redução de carga viral, os pacientes foram divididos em três grupos: aqueles que receberam transfusão de plasma convalescente com título de NAbsT até 160, título de NAbsT entre 160-640 e NAbsT superior a 640. Para esta análise, foi utilizado o teste de Kruskall-Wallis. Resultados: Pacientes com baixos títulos de neutralizantes à admissão (NAbsP inferior a 80) apresentaram redução de carga viral significativamente maior (p=0,009) que pacientes com NAbsP igual ou superior a 80. Com relação ao impacto da transfusão de plasma, observou-se que quanto maior o título de anticorpos neutralizantes transfundidos maior foi a redução da carga viral;porém, tal achado não apresentou significância estatística (p = 0,528). Discussão: O combate e eliminação da viremia através dos anticorpos neutralizantes presentes no plasma convalescente compreende uma das justificativas da sua aplicação como terapia alternativa para COVID19. Contudo, estudos prévios demonstraram resultados contraditórios em relação ao seu impacto no clearance viral. Na presente casuística, pacientes com baixos títulos de anticorpos neutralizantes apresentaram maior redução de carga viral após a transfusão de plasma convalescente do que pacientes com altos títulos. Pacientes que já apresentavam títulos elevados de neutralizantes parecem não se beneficiar da transfusão de plasma no que se refere à redução de carga viral. A estratificação dos pacientes de acordo com os níveis basais de anticorpos neutralizantes parece ser um ponto importante a ser discutido no tratamento de COVID19 com plasma convalescente e fornece uma explicação plausível para os resultados controversos previamente observados.
ABSTRACT
Introduction: Treatment-free remission (TFR) in chronic myeloid leukemia (CML) is demonstrated to be achievable and recommended for patients (pts) in sustained deep molecular response (sDMR) who can discontinue tyrosine kinase inhibitor (TKI) treatment and maintain responses in ~50% of cases. While the feasibility and safety of TKI cessation have been largely demonstrated, the strategies of TFR optimization are yet to be clarified. Studies (eg. DESTINY) investigating de-escalation, mainly after imatinib, suggested that a stepwise approach may favor TFR outcome. We present the interim results of the phase 2, prospective, multicenter DANTE study (NCT03874858) evaluating de-escalation and TFR in Italian pts with CML in chronic phase (CML-CP) treated with nilotinib (NIL). Methods: Adults with CML-CP treated with NIL 300 mg twice daily (bid) in first-line for ≥3 years who achieved sDMR for ≥1 year (≥MR 4.0;BCR-ABL level ≤0.01% IS) were enrolled in 27 centers. The study consisted of 4 phases: screening (week [wk] −4-0), consolidation (wk 0-48), TFR (wk 48-144), and follow-up (until wk 144). Ongoing treatment with ≥400 mg/day dose was allowed at study entry. During consolidation, pts were treated with NIL 300 mg once daily (qd). At the end of consolidation phase, pts with sDMR entered TFR phase and discontinued NIL;indeed, pts with at least major molecular response (MMR;BCR-ABL ≤0.1% IS), but without sDMR, continued NIL 300 mg qd. At any time, pts with loss of MMR returned to NIL 300 mg bid. During TFR phase, BCR-ABL levels were monitored monthly from wk 52-96, and then every 3 months. Pts on half-dose or full-dose NIL were monitored every 3 months. The primary endpoint is the percentage of pts in full treatment-free remission (FTFR) 96 wks after the start of consolidation phase. FTFR is defined as pts with MMR or better, including those who remained in discontinuation during TFR phase and those who are treated with half the standard dose. Key secondary endpoints include percentage of pts with sDMR at wk 48;TFR rate at wk 96 and 144;BCR-ABL kinetics and safety. The predictive role of digital droplet PCR is also evaluated as an exploratory objective. Results: Overall, 113 pts were screened and 107 entered consolidation phase. This interim analysis included 52 pts who reached the end of consolidation phase by data cut-off period (February 8, 2021). Of these 52 pts, 49 (94.2%) were ongoing by data cut-off and 3 (5.8%) discontinued the study (1 patient due to adverse event (AE) and 2 per patient's decision). Median age at study entry was 49.5 years. Median time from diagnosis was 5.6 years and median dose of prior NIL treatment was 600 mg/day for all pts except one who was on NIL 450 mg/day at baseline. Median duration of last sustained MR4 and MR4.5 were 30 and 16.5 months, respectively. Further details are listed in Table 1. At screening, molecular response categories were MR4.0 in 13.7%, MR4.5 in 23.1% and undetectable MR4.5 in 63.5% of pts. During consolidation phase, 5 (9.6%) pts discontinued prematurely: 2 pts restarted NIL full dose (3.8%) for MMR loss, 2 (3.8%) discontinued for AEs and 1 (1.9%) for pt decision. Overall, 47 pts completed consolidation: of them 40 (76.9%) sustained DMR and 7 (13.5%) maintained MMR but not sDMR. Of the 7 pts not sustaining DMR during consolidation, 6 regained DMR after a median of 4.4 months, while 1 pt was still in MMR by data cutoff. The 2 pts who lost MMR after 5 and 8 months regained MMR and 1 regained DMR by data cutoff after increasing NIL to 300 mg bid. Median time spent in consolidation phase was 11.7 months, and the evolution of response categories over time is shown in Figure 1. During consolidation phase, AEs were observed in 16 pts (30.8%), of them 2 (3.8%) pts had serious AEs: 1 patient had skin ulcers and COVID-19 related pneumonia, while 1 patient had unstable angina. No deaths and disease progressions were observed. Conclusions: DANTE is the first study that showed the safety and feasibility of NIL de-escalation before TFR in CML-CP pts with sDMR. Inter m results suggest that loss of MMR during de-escalation is rare. De-escalation strategy may lead to further improvement of TFR outcome and tolerability and may also preemptively support the identification of pts who may not be ready for discontinuation, with a tailored approach. To date, accuracy in predicting TFR outcome is still low, and the de-escalation setting may sharpen biological and clinical predictive factors, including the potential role of digital PCR. [Formula presented] Disclosures: Breccia: Abbvie: Honoraria;Pfizer: Honoraria;Novartis: Honoraria;Incyte: Honoraria;Bristol Myers Squibb/Celgene: Honoraria. Abruzzese: Incyte: Consultancy, Honoraria;Novartis: Consultancy, Honoraria;Pfizer: Consultancy, Honoraria;Bristol Myers Squibb: Consultancy, Honoraria. Stagno: InCyte: Consultancy, Honoraria;Pfizer: Consultancy, Honoraria, Other: Support for attending meetings and/or travel;Novartis: Consultancy, Honoraria, Other: Support for attending meetings and/or travel, Research Funding. Iurlo: Incyte: Speakers Bureau;Novartis: Speakers Bureau;Pfizer: Speakers Bureau;Bristol Myers Squibb: Speakers Bureau. Sportoletti: AstraZeneca: Consultancy, Honoraria;Janssen: Consultancy, Honoraria;AbbVie: Consultancy, Honoraria. Lemoli: Jazz, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau;AbbVie, Daiichi Sankyo, Servier: Honoraria, Speakers Bureau;Celgene: Other: Support for attending meetings and/or travel. Siragusa: Novartis, CSL, Behring, Amgen, Novonoridsk, SOBI, Bayer: Consultancy, Honoraria, Speakers Bureau. Chiodi: Novartis: Current Employment.
ABSTRACT
Background. Treatment-free remission (TFR) has become a new treatment goal for chronic myeloid leukemia (CML) patients. However, usually abrupt tyrosine kinase inhibitors (TKIs) therapy discontinuation has been successful only in about half of eligible patients and it can cause burdening TKI withdrawal syndrome (TWS) in about 30% of them. Moreover, any robust clinical or biological factor predictive for successful TFR has not been identified yet. On top of that, sustainable deep molecular response (DMR) as the main prerequisite for TKI discontinuation attempt is achieved only in 20-40% of patients. The majority of CML patients, therefore, need to be treated with the effective and well-tolerated drug for a long time or even life-long. Study design and methods. With the recognition of all these aspects, we designed a nationwide prospective investigator-initiated phase II clinical trial HALF (ClinicalTrials.gov NCT04147533) in order to evaluate efficacy and safety of TKI discontinuation after previous two-step dose reduction in patients with CML in DMR (Fig. 1). Step-wise TKI dose reduction, i.e. half of the standard during the first 6 months after study entry, and the same dose given alternatively (every other day) during the next 6 months, was derived from pharmacokinetics and experimental data as well as from clinical trials' results. We assume that the step-wise and eventually meaningful TKI dose reduction enables a higher rate of patients achieving successful TFR with less pronounce TWS, or even would represent a more reasonable and safer alternative to the complete and sudden TKI interruption. This unique nationwide academic project has been facilitated by hematological patients care centralization in the Czech Republic. A primary study objective is to evaluate the proportion of patients in major molecular response (MMR) at 6 and 12 months and in TFR at 18, 24, and 36 months after the study enrollment, respectively, and molecular recurrence-free survival at all mentioned time points as well. Main secondary and exploratory objectives are: to evaluate the proportion of patients loosing MMR and in whom MMR and MR4.0 would be re-achieved after TKI re-introduction, time to MMR and MR4.0 re-achievement, FFS, PFS, OS, TWS, and QoL assessment, predictive factors for successful TFR identification, quantification of BCR-ABL1 using digital droplet PCR at both the DNA and mRNA levels, immunological profiling, BCR-ABL1 kinetics mathematical modeling, assessment of TKI pharmacokinetics, clonal hematopoiesis and pharmaco-economics. Results. The study was launched in December 2019;however, due to the COVID-19 outbreak, patients' recruitment started on June 16, 2020. Here, characteristics of the first 74 patients included in the study until April 2021 are presented. There were 37 males and 37 females, with median age at the time of diagnosis of 53 years (range, 23-74) and at the time of the study entry of 67 years (range, 35-86). A median time of CML disease, TKI treatment, and DMR duration before the study initiation was as follows: 9.9 years (range, 4.4-22.5), 9.8 years (range, 4.2-20.2), and 7.3 years (range, 3.2-18.3), respectively. The ELTS score was low, intermediate, high and unknown in 62.2%, 21.6%, 13.5%, and 2.7% of patients, respectively. At the time of study entry, 58 patients (79.5%) were treated with imatinib, 10 (13.7%) with nilotinib, and 5 (6.8%) with dasatinib, respectively, whereas in 63 patients (86.3%) it was in the first line of therapy. With almost half of patients (48.6%), the TKI dose was already reduced at the time of study entry. With 10 (13.5%) patients, interferon-α treatment preceded TKI administration. At the time of this preparation, on July 26, 2021, altogether 102 patients (from planned 150) have been enrolled in the study;48 of them (47.1%) have already moved to the second de-escalation phase and 9 (8.8%) patients to the TFR phase. There were 2 cases of confirmed MMR loss (both in month 8 after the study entry) and no patient experienced symptoms resembling TWS. Conclusions. Despite the COVID-19 pandemic, the HALF study was successfully launched and initiated in the majority of centers, with 102 already included patients and continuing intensive enrolment. Based on our very preliminary results, the step-wise dose reduction seems to be an effective and safe approach. More included patients, longer follow-up and further analyses are needed in order to reach all set up objectives. [Formula presented] Disclosures: Žácková: Angelini: Consultancy, Speakers Bureau;Novartis: Speakers Bureau. Faber: Angelini: Consultancy, Other: conference fees, Research Funding, Speakers Bureau;Bristol-Myers Squibb: Consultancy, Other: conference fees, Research Funding, Speakers Bureau;Novartis: Consultancy, Other: conference fees, Research Funding, Speakers Bureau;Pfizer: Other: conference fees;TERUMO: Other: conference fees. Bělohlávková: Novartis: Consultancy;BMS/Celgene: Consultancy. Horňák: Angelini: Honoraria. Svobodník: Roche: Speakers Bureau;Janssen-Cilag: Speakers Bureau. Machová Poláková: Incyte: Consultancy;Angelini: Consultancy;Novartis: Research Funding. Mayer: Principia: Research Funding.