RNA Load Research Articles

Engineered human H-chain ferritin with reversed charge of the internal cavity exhibits RNA-mediated spongelike effect for loading RNA/DNA-binding molecules.

Ferritins are globular proteins with an internal cavity that enables the encapsulation of a plethora of low-mass compounds. Unfortunately, the overall negative surface charge of ferritin's internal cavity hampers efficient loading of negatively charged molecules. Therefore, we produced a genetically engineered human H-chain ferritin containing a cationic RKRK domain, reversing the natural net charge of the cavity to positive, thus allowing for efficient encapsulation of negatively charged siRNA. Due to the reversed, positive charge mediated by RKRK domains, the recombinant ferritin produced in E. coli inherently carries a load of bacterial RNA inside its cavity, turning the protein into an effective sponge possessing high affinity for DNA/RNA-binding substances that can be loaded with markedly higher efficiency compared to the wildtype protein. Using doxorubicin as payload, we show that due to its loading through the RNA sponge, doxorubicin is released in a sustained manner, with a cytotoxicity profile similar to the free drug. In summary, this is the first report demonstrating a ferritin/nucleic acid hybrid delivery vehicle with a broad spectrum of properties exploitable in various fields of biomedical applications.

Detached epithelial cell plugs from the upper respiratory tract favour distal lung injury in Golden Syrian hamsters (Mesocricetus auratus) when experimentally infected with the A.2 Brazilian SARS-CoV-2 strain.

The Golden Syrian hamster (Mesocricetus auratus), Ferrets (Mustela putorius furo), and macaques have been described as useful laboratory animals naturally susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To study the mechanism of lung injury, we describe the histopathological features of SARS-CoV-2 infection in Golden Syrian hamsters inoculated intranasally with the A.2 Brazilian strain. Hamsters were intranasally inoculated with the A.2 variant and euthanised at 3-, 5-, 10- and 15-days post-inoculation. The physical examination and body weight were recorded daily. Neutralising antibodies and viral RNA load of the respiratory tract were assessed during necropsies. The coronavirus disease 2019 (COVID-19) model presented body weight loss, high levels of respiratory viral RNA load, severe segmentary pneumonitis, and bronchial fistula besides lymphatic trapping and infiltration, like the human SARS-COV-2 pathogenesis. The presence of subepithelial lymphoeosinophilic infiltrate was highlighted in our results; it contributed to the detachment of SARS-CoV-2 nucleocapsid-positive epithelial cells resulting in the infectious cell plugs. The SARS-CoV-2 caused segmentary pneumonia and vascular damage. In our comprehension, the infectious cell plugs, as being aspirated from the upper respiratory tract into the terminal bronchial lumen, work as a "Trojan horse", thus contributing to the dissemination of the SARS-CoV-2 infection into specific regions of the deep lung parenchyma.

Preservation of functionality, immunophenotype, and recovery of HIV RNA from PBMCs cryopreserved for more than 20 years.

Many research laboratories have long-term repositories of cryopreserved peripheral blood mononuclear cells (PBMC), which are costly to maintain but are of uncertain utility for immunological studies after decades in storage. This study investigated preservation of cell surface phenotypes and in-vitro functional capacity of PBMC from viraemic HIV+ patients and healthy seronegative control subjects, after more than 20 years of cryopreservation. PBMC were assessed by 18-colour flow cytometry for major lymphocyte subsets within T, B, NK, and dendritic cells and monocytes. Markers of T-cell differentiation and activation were compared with original immunophenotyping performed in 1995/1996 on fresh blood at the time of collection. Functionality of PBMC was assessed by culture with influenza antigen or polyclonal T-cell activation, to measure upregulation of activation-induced CD25 and CD134 (OX40) on CD4 T cells and cytokine production at day 2, and proliferative CD25+ CD4 blasts at day 7. RNA was extracted from cultures containing proliferating CD4+ blast cells, and intracellular HIV RNA was measured using short amplicons for both the Double R and pol region pi code assays, whereas long 4-kbp amplicons were sequenced. All major lymphocyte and T-cell subpopulations were conserved after long-term cryostorage, except for decreased proportions of activated CD38+HLA-DR+ CD4 and CD8 T cells in PBMC from HIV+ patients. Otherwise, differences in T-cell subpopulations between recent and long-term cryopreserved PBMC primarily reflected donor age-associated or HIV infection-associated effects on phenotypes. Proportions of naïve, memory, and effector subsets of T cells from thawed PBMC correlated with results from the original flow cytometric analysis of respective fresh blood samples. Antigen-specific and polyclonal T-cell responses were readily detected in cryopreserved PBMC from HIV+ patients and healthy control donors. Intracellular HIV RNA quantitation by pi code assay correlated with original plasma viral RNA load results. Full-length intracellular and supernatant-derived amplicons were generated from 5/12 donors, and sequences were ≥80% wild-type, consistent with replication competence. This unique study provides strong rationale and validity for using well-maintained biorepositories to support immunovirological research even decades after collection.

HDAC Inhibitors against SARS-CoV-2

Abstract: Following the coronavirus outbreak, global efforts to find a vaccine and drug affecting Covid- 19 have been widespread. Reusing some of the available drugs has had relatively satisfactory results. One of the classes of drugs studied against SARS-CoV-2 is the HDAC inhibitors collected in this review. Among the most important points of this study can be mentioned: (a) SARS-COV-2 infection can influence the ACE/ACE2-ATR1-Cholesterol-HDAC axis signaling, (b) By limiting endocytosis and decreasing ACE2-spike protein recognition at the same time, Romidepsin may hinder SARS-2-S-driven host cell entry. (c) HDAC inhibitors affect the expression of ABO, ACE2 and TMPRSS2 in epithelial cell lines. (d) Valproic acid may help to reduce ARDS as well as hospitalizations and death. (e) Trichostatin A inhibits antigen expression, viral RNA load and infectious particle production in SARS-CoV-2.

Prevalence and risk factors of transmission of hepatitis delta virus in pregnant women in the Center Region of Cameroon.

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) co-infection has been described as the most severe form of viral hepatitis, and can be co-transmitted from mother-to-child. A seroprevalence of 4.0% of HDV infection was reported in pregnant women in Yaoundé, and 11.9% in the general population in Cameroon. Our objective was to describe the rate of HDV infection in HBsAg-positive pregnant women and to determine risk factors associated with mother-to-child transmission of HDV. A cross-sectional, descriptive study was conducted from January 2019 to July 2022 among pregnant women attending antenatal contacts in seven health structures in the Centre Region of Cameroon. A consecutive sampling (non-probability sampling) was used to select only pregnant women of age over 21 years, who gave a written informed consent. Following an informed consent, an open-ended questionnaire was used for a Knowledge, Attitude and Practice (KAP) survey of these women, and their blood specimens collected and screened for HBsAg, anti-HIV and anti-HCV antibodies by rapid tests and ELISA. HBsAg-positive samples were further screened for HBeAg, anti-HDV, anti-HBs, and anti HBc antibodies by ELISA, and plasma HDV RNA load measured by RT-qPCR. Of 1992 pregnant women, a rate of 6.7% of HBsAg (133/1992) with highest rate in the rural areas, and 3.9% of hepatitis vaccination rate were recorded. Of 130, 42 (32.3%) were anti-HDV antibody-positive, and 47.6% had detectable HDV RNA viraemia. Of 44 anti-HDV-positive cases, 2 (4.5%) were co-infected with HBV and HCV, while 5 (11.4%) with HIV and HBV. Multiple pregnancies, the presence of tattoos and/or scarifications were significantly associated with the presence of anti-HDV antibodies. Of note, 80% of women with negative HBeAg and positive anti-HBe serological profile, had plasma HDV RNA load of more than log 3.25 (>10.000 copies/ml). These results show an intermediate rate of HDV infection among pregnant women with high level of HDV RNA viremia, which suggest an increased risk of vertical and horizontal co-transmission of HDV.

The role of turmeric and black pepper oil nanoemulsion in attenuating cytokine storm triggered by duck hepatitis A virus type I (DHAV-I)-induced infection in ducklings

The cytokine storm induced by duck hepatitis A virus type 1 (DHAV-1) infection significantly contributes to severe, rapid deaths and economic losses in the duck industry in Egypt. This study aimed to investigate the potential inhibitory effect of a nanoemulsion containing turmeric and black pepper oil on the immune response and pathogenesis of DHAV-1 in ducklings. A total of 105 ducklings from nonvaccinated breeders were divided into 5 experimental groups, each comprising 21 birds. The negative control group (G1) remained noninfected with DHAV-1 and nontreated with nanoemulsion, while the positive control group (G2) was infected with DHAV-1 but not treated with nanoemulsion. The other 2 groups (G3, the supplemented group which was noninfected with DHAV-1), and group 4 (the prophylactic group G4) which was infected with DHAV-1, both received nanoemulsion throughout the experiment. Group 5 (G5, the therapeutic group), on the other hand, which was infected with DHAV-1 received nanoemulsion only from the onset of clinical signs. At 5 days old, the ducklings in the positive control (G2), the prophylactic (G4), and the therapeutic group (G5) were infected with DHAV-1. All the ducklings in the infected groups exhibited depression, anorexia, and opisthotonos, and their livers displayed various degrees of ecchymotic hemorrhage, liver enlargement, and microscopic pathological lesions. Notably, the positive control group (G2) experienced the most severe and pronounced effects compared to the other infected groups treated with the nanoemulsion. Meanwhile, the viral RNA loads were lower in the liver tissues of the infected ducklings treated with the nanoemulsion (G4, and G5) compared to the positive control group G2. Additionally, the nanoemulsion effectively modulated proinflammatory cytokine expression, antioxidant enzymes, liver enzymes, and lipid profile of treated ducklings. In conclusion, the turmeric and black pepper oil nanoemulsion has the potential to be a therapeutic agent for regulating and modulating the immune response, decreasing DHAV-1-induced cytokine storms, and minimizing mortality and economic losses in the duck business. More research is needed to understand how turmeric and black pepper oil nanoemulsion alleviates DHVA-1-induced cytokine storms and lowers duckling mortality.

Clinical, Pathological and Virological Outcomes of Tissue-Homogenate-Derived and Cell-Adapted Strains of Porcine Epidemic Diarrhea Virus (PEDV) in a Neonatal Pig Model.

Porcine epidemic diarrhea virus (PEDV) is characterized by diarrhea, vomiting, dehydration, and high mortality rates in neonatal piglets. Two distinct genogroups, S-INDEL (G1a, G1b) and non-S INDEL (G2a, G2b, and G2c), circulate worldwide and are characterized by varying degrees of virulence. Here, we compared the early pathogenesis of a PEDV S-INDEL strain obtained from intestine homogenate (CALAF-HOMOG) or adapted to cell culture by 22 passages (CALAF-ADAP) and a virulent non-S INDEL strain (PEDV-USA) in newborn piglets. After orogastric inoculation of PEDV strains, body weight, temperature and clinical signs were monitored for 48 hpi. Pathological studies were performed at 48 hpi and RNA extracts from jejunal content (at 48 hpi) and rectal swabs (at 0 and 48 hpi) were tested for the presence of PEDV RNA as well as sequenced and compared to the inoculum. Piglets inoculated with PEDV-USA and CALAF-HOMOG isolates showed more severe weight loss, diarrhea, villi fusion and atrophy compared to CALAF-ADAP inoculated piglets. The viral load of rectal swabs was higher in the PEDV-USA inoculated group, followed by CALAF-HOMOG and CALAF-ADAP isolates. Similarly, viral RNA load in jejunal content was comparable among PEDV-USA and CALAF-HOMOG inoculated piglets and higher than that of CALAF-ADAP ones. The comparison of three full PEDV sequences of the inocula with the corresponding ones of pigs after 48 hpi yielded a nucleotide identity >99.9%. This study highlights variations in virulence among S-INDEL and non-S INDEL strains and between S-INDEL isolates obtained from homogenate and cell culture.

An Atypical Course of Cardiomyopathy Syndrome (CMS) in Farmed Atlantic Salmon (Salmo salar) Fed a Clinical Nutrition Diet.

Cardiomyopathy syndrome (CMS) poses a significant threat to farmed Atlantic salmon (Salmo salar), leading to high mortality rates during the seawater phase. Given that controlled experimental challenge trials with PMCV do not reproduce the mortality observed in severe field outbreaks of CMS, field trials on natural CMS outbreaks are warranted. This field study explored the impact of a clinical nutrition intervention, specifically a diet enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on a severe CMS outbreak in a commercial sea farm. CMS was diagnosed in a single sea cage with high mortality rates. Histopathological analysis, RT-qPCR in situ hybridization for virus detection, and fatty acid composition analysis were used to monitor the impact of disease and the inclusion of EPA and DHA in heart tissue. Following the implementation of clinical nutrition, a decline in mortality rates, regression of CMS-associated changes, and a significant reduction in piscine myocarditis virus (PMCV) RNA load were observed within the salmon population. Fatty acid composition analysis of heart samples demonstrated increased levels of EPA and DHA, reinforcing the association between dietary factors, viral load dynamics, and overall fish health. Although further validation is needed in future studies, as field trials may not be sufficient to establish causation, our results indicate that optimizing the EPA + DHA levels may prove beneficial in severe CMS outbreaks.

Infectious virus shedding duration reflects secretory IgA antibody response latency after SARS-CoV-2 infection.

Infectious virus shedding from individuals infected with severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is used to estimate human-to-human transmission risk. Control of SARS-CoV-2 transmission requires identifying the immune correlates that protect infectious virus shedding. Mucosal immunity prevents infection by SARS-CoV-2, which replicates in the respiratory epithelium and spreads rapidly to other hosts. However, whether mucosal immunity prevents the shedding of the infectious virus in SARS-CoV-2-infected individuals is unknown. We examined the relationship between viral RNA shedding dynamics, duration of infectious virus shedding, and mucosal antibody responses during SARS-CoV-2 infection. Anti-spike secretory IgA antibodies (S-IgA) reduced viral RNA load and infectivity more than anti-spike IgG/IgA antibodies in infected nasopharyngeal samples. Compared with the IgG/IgA response, the anti-spike S-IgA post-infection responses affected the viral RNA shedding dynamics and predicted the duration of infectious virus shedding regardless of the immune history. These findings highlight the importance of anti-spike S-IgA responses in individuals infected with SARS-CoV-2 for preventing infectious virus shedding and SARS-CoV-2 transmission. Developing medical countermeasures to shorten S-IgA response time may help control human-to-human transmission of SARS-CoV-2 infection and prevent future respiratory virus pandemics.

Treatment-naïve people living with HIV aged 50 years or older in Beijing, China, 2010-2020: joinpoint regression model analysis of surveillance data.

As they age, people living with HIV (PLWH) must face new challenges, such as accelerated ageing and higher rates of comorbidities. This study described the characteristics of HIV acquisition among treatment-naïve PLWH aged ≥50 years and <50 years in Beijing from 2010 to 2020, exploring associated risk factors for comorbidities. In this cross-sectional study, differences in HIV-related and non-HIV-related characteristics were compared using the t-test, Mann-Whitney U test and chi-square test. Temporal trend data were analysed via joinpoint regression. A multivariate logistic regression model was conducted to analyse the associated factors with PLWH having one or more comorbidities. The proportion of PLWH aged ≥50 years has significantly increased since 2013, with a corresponding increase in homosexual transmission in this age group over the past decade. The proportion of individuals with CD4 counts <200 cells/μl significantly decreased from 2010 to 2013 among PLWH aged ≥50 years and from 2010 to 2014 among those aged <50 years. Delayed initiation of antiretroviral therapy (ART) improved for both age groups over the course of the decade, especially from 2014 to 2020. Compared to PLWH aged <50 years, those aged ≥50 years had a higher proportion of CD4 counts <200 cells/μl, higher levels of plasma HIV RNA load and a higher prevalence of non-HIV-related risk factors. Multivariate analysis revealed that PLWH aged ≥50,male, not single, transmission through heterosexual contact or drug injection, WHO Stage IV, coinfection with hepatitis B virus/hepatitis C virus and CD4 counts <200 cells/μl at the initiation of ART were associated with higher risk of the presence of an HIV comorbidity. Due to the persistent burden of HIV-related characteristics or symptoms and the increasing prevalence of coexisting comorbidities among treatment-naïve PLWH aged ≥50 years, physicians should provide the highest-quality screening, prevention, treatment and management of coexisting comorbidities, adopting a multidisciplinary approach.

Selinexor in Combination with R-EPOCH for Patients with Previously Untreated HIV-Associated Diffuse Large B Cell Lymphoma(DLBCL)

Introduction : The incidence of HIV-positive lymphoma remains significantly higher than HIV-negative patients in the era of combined antiretroviral therapy(cART). DLBCL is the most common histological type of HIV-positive non-Hodgkin's lymphoma(NHL). Exportin 1 protein(XPO1) inhibitor has demonstrated preliminary anti-HIV activity and synergy with anti-lymphoma drug(e.g. etoposide, doxorubicin, prednisone). Methods: The study was designed to evaluate safety and efficacy of selinexor (60mg Days 1, 8, 15) combined with R-EPOCH(rituximab 375 mg/m2d1, etoposide 50 mg/m2 d1-d4, doxorubicin 10mg/m2 d1-d4, vinblastine 0.4mg/m2 d1-d4, prednisone 60mg/m2 d1-d5 and cyclophosphamide 750mg/m2 d5) every 3 weeks (4-6 cycles), followed by selinexor maintenance (60 mg weekly) or transplantation as per physician's choice in patients with untreated HIV-positive DLBCL. All patients received cART concomitantly during chemotherapy. This study was registered at Chictr.org.cn(ChiCTR2300069941). Results: From Feb 24, 2023 to July 17, 2023, 6 patients were enrolled and received at least one cycles of study treatment. Median age was 55 years (range 43-67) with four patients(66.7%) was male. All six patients were in advanced stage according to Ann Arbor staging system, with aaIPI intermediate to high risk or IPI low to intermediate risk. Four patients(66.7%) were germinal center B-cell-like lymphoma (GCB) subtype and three patients(50%) had an Elevated lactate dehydrogenase level. Median HIV RNA load was 74450(10000-100000) copies/mL and Median CD4 cell count was 233 cells/μL (233 cells/mm3) with one patient having a CD4 cell count less than 200 cells/μL (200 cells/mm3). Two patients were HBV-positive and one was EB virus(EBV) coinfection at baseline. Three patients achieved PR after two treatment cycles, one patient of which had completed five treatment cycles and achieved complete response at the end of the fourth cycle (Figure 1). One patients after interim evaluation with PR stopped treatment because of loss to follow-up. The EBV DNA load of the patient with EBV coinfection at baseline turned to normal level after one treatment cycle. Similar result was observed in two HBV-positive patients, the one achieved HBV-negative after two cycles and the other's HBV DNA load level declined from 7.96×10^4 IU/mL to 1.12×10^3 after completing first cycle. HIV RNA load of the patient with CR had declined to &amp;lt;20 copies/mL from 58300 copies/mL after the fourth cycle, another patient likewise became HIV-negative before the initiation of the third cycle. The change of CD4+T cell count during treatment was presented in Figure 2. All patients experienced at least one treatment-emergent adverse event (TEAE), 3 (50%) patients with at least one Grade≥3 TEAE. The most common grade 3 or 4 adverse events were hematological toxicities including lymphopenia(83.3%), neutropenia (50%), leukopenia(33.3%), thrombocytopenia (33.3%). Two patients experienced Febrile neutropenia. The non-hematological toxicities were grade 1 or 2, including nausea(66.7%), vomit(50%), constipation(33.3%), cough(33%). One patient stopped treatment for one month due to pulmonary infection. All toxicities were manageable by supportive care. Conclusion: Selinexor plus R-EPOCH regimen demonstrated a favorable Interim efficacy and manageable safety profile in patients with HIV-associated DLBCL. The change of HIV RNA load and CD4+ cell count still requires further evaluation.

Spike Protein Genetic Evolution in Patients at High Risk of Severe Coronavirus Disease 2019 Treated by Monoclonal Antibodies.

High-risk patients, often immunocompromised and not responding to vaccine, continue to experience severe coronavirus disease 2019 (COVID-19) and death. Monoclonal antibodies (mAbs) were shown to be effective to prevent severe COVID-19 for these patients. Nevertheless, concerns about the emergence of resistance mutations were raised. We conducted a multicentric prospective cohort study, including 264 patients with mild to moderate COVID-19 at high risk for progression to severe COVID-19 and treated early with casirivimab/imdevimab, sotrovimab, or tixagevimab/cilgavimab. We sequenced the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome during follow-up and searched for emerging spike mutations. Immunocompromised patients have a 6-fold increased risk of developing mutations, which are associated with a prolonged duration of viral clearance but no clinical worsening. Emerging P337S/R/L/H, E340D/K/A/Q/V/G, and K356T/R substitutions in patients treated with sotrovimab are associated with higher viral RNA loads for up to 14 days post-treatment initiation. Tixagevimab/cilgavimab is associated with a 5-fold increased risk of developing mutations. R346K/I/T/S and K444R/N/M substitutions associated with tixagevimab/cilgavimab have been identified in multiple SARS-CoV-2 lineages, including BQ.1 and XBB. The probability of emerging mutations arising in response to mAbs is significant, emphasizing the crucial need to investigate these mutations thoroughly and assess their impact on patients and the evolutionary trajectory of SARS-CoV-2.

Correlation between HBeAg and Hepatitis B Virus DNA and RNA Levels in Diverse Liver Disease Cohorts

Objective: To investigate the disparities and associations between HBV DNA and HBV RNA in various liver disease groups with respect to HBeAg status. Methods: Between September 2020 and September 2023, 90 patients diagnosed with chronic hepatitis B (CHB), 74 patients diagnosed with liver cirrhosis (LC), and 102 patients diagnosed with hepatocellular carcinoma (HCC) from the Department of Gastroenterology or Infection at the First Affiliated Hospital of Xi’an Jiaotong University were selected. HBV DNA, HBV RNA, and HBeAg quantitative tests were conducted using serum samples from the same patients. Results: In the three groups of cases, the HBV RNA load was higher when HBeAg was positive than when HBeAg was negative, and this difference was statistically significant. Only in the HCC group was the HBV DNA load significantly higher when HBeAg was positive than when HBeAg was negative. Additionally, there was a positive correlation between HBV DNA and HBV RNA regardless of HBeAg status. Conclusion: During HBeAg conversion, HBV RNA demonstrates a more sensitive response than HBV DNA. As CHB progresses to LC or HCC, HBV RNA exhibits better diagnostic value than HBV DNA.

Association of Viral Control and Maternal Antibody Titers in Congenital Zika Virus Infection

Abstract Background Exposure to Zika virus (ZIKV) in utero can result in a wide spectrum of outcomes, ranging from asymptomatic to severe neurological defects or fetal demise. The presence of virus in the fetus or in the mother’s blood for a prolonged period of time may lead to worse outcomes, which means that limiting the duration of fetal exposure to virus may improve outcomes. One factor that may be related to control of maternal viremia and presence of virus in the fetus is the maternal IgG antibody response. We utilized a rhesus macaque model of prenatal ZIKV to quantify the viral RNA present in the maternal blood and maternal-fetal interface (MFI) tissues and determine the association of viral RNA loads with maternal IgG antibody titers over time. Methods Dams were infected with either African lineage ZIKV-DAKAR (n=9) or Asian lineage ZIKV-PRVABC59 (PR, n=10) isolate in the first trimester, then all fetal and MFI tissues were removed either 7 or 14 days post-infection (dpi) and viral RNA loads were measured. Maternal plasma was tested for viral RNA and IgG throughout pregnancy. Results A greater proportion of animals infected with the DAKAR isolate had viral RNA present in the MFI tissues compared with the animals infected with the PR isolate, at both 7 and 14 dpi. DAKAR animals also tested positive for ZIKV-specific IgG at 14 dpi or earlier, while IgG was typically negative at 14 dpi for PR animals. Viral RNA was detected in the fetal and MFI tissues in a higher proportion of dams that still had ZIKV RNA positive plasma at 14 dpi had viral RNA. We found no difference in IgG antibody titers between dams that were ZIKV positive or negative at 14 dpi. Conclusions The presence of ZIKV in the MFI tissues and earlier detection of ZIKV-specific maternal IgG responses were both associated with the DAKAR isolate, suggesting that it may be a more virulent isolate. A higher proportion of dams that had virus in their plasma at 14 dpi also had virus present in the fetal and MFI tissues, but there was no difference in long term IgG response within either isolate between animals that tested positive or negative at 14 dpi. This suggests that the dam clearing viral RNAfrom their plasma by 14 dpi may impact whether or not the virus infects the MFI tissues, but that maternal IgG response likely does not play a role in this difference.

Digital PCR to Measure SARS-CoV-2 RNA, Variants, and Outcomes in Youth.

The role of SARS-CoV-2 viral load in predicting contagiousness, disease severity, transmissibility, and clinical decision-making continues to be an area of great interest. However, most studies have been in adults and have evaluated SARS-CoV-2 loads using cycle thresholds (Ct) values, which are not standardized preventing consistent interpretation critical to understanding clinical impact and utility. Here, a quantitative SARS-CoV-2 reverse-transcription digital PCR (RT-dPCR) assay normalized to WHO International Units was applied to children at risk of severe disease diagnosed with COVID-19 at St Jude Children's Research Hospital between March 28, 2020, and January 31, 2022. Demographic and clinical information from children, adolescents and young adults treated at St Jude Children's Research Hospital were abstracted from medical records. Respiratory samples underwent SARS-CoV-2 RNA quantitation by RT-dPCR targeting N1 and N2 genes, with sequencing to determine the genetic lineage of infecting virus. Four hundred and sixty-two patients aged 0-24 years (median 11 years old) were included during the study period. Most patients were infected by the omicron variant (43.72%), followed by ancestral strain (22.29%), delta (13.20%) and alpha (2.16%). Viral load at presentation ranged from 2.49 to 9.14 log10 IU/ml, and higher viral RNA loads were associated with symptoms (OR 1.32: CI95% 1.16-1.49) and respiratory disease (OR 1.23: CI95% 1.07-1.41). Viral load did not differ by SARS-CoV-2 variant, vaccination status, age, or baseline diagnosis. SARS-CoV-2 RNA loads predict the presence of symptomatic and respiratory diseases. The use of standardized, quantitative methods is feasible, allows for replication, comparisons across institutions, and has the potential to facilitate consensus quantitative thresholds for risk stratification and treatment.

Weight and metabolic changes among virally suppressed people with HIV who switched to co-formulated bictegravir/emtricitabine/tenofovir alafenamide

We aimed to investigate the evolution of weight, lipid profiles, and glucose homeostasis among virally suppressed people with HIV (PWH) who switched to bictegravir/emtricitabine/tenofovir alafenamide (BIC/FTC/TAF). PWH with viral suppression who switched to BIC/FTC/TAF in Taiwan between October 2019 and May 2021 were followed for 96 weeks to examine changes in weight, lipid profiles (total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG)), and glycated hemoglobin (HbA1c) levels. 889 PWH with an average weight of 72.1 kg at baseline were included. At week 96, more than 95% of PWH consistently maintained plasma HIV RNA load <50 copies/mL at each 24-week interval of follow-up, while the weight change was small (+0.7 kg, P < 0.0001), although statistically significant. Baseline levels of TC, LDL-C, HDL-C, TG, and HbA1c were 191.8 mg/dL, 114.2 mg/dL, 48.9 mg/dL, 174.3 mg/dL, and 5.31%, respectively. After 96 weeks, changes were observed in TC (-11.6 mg/dL, P < 0.0001), LDL-C (-3.4 mg/dL, P = 0.0084), HDL-C (+0.6 mg/dL, P = 0.1089), TG (-30.2, P < 0.0001), and HbA1c (+0.12%, P < 0.0001). A 5% or more weight gain was associated with age of 30-40 years, normal weight at baseline, and prior use of non-integrase inhibitors or tenofovir disoproxil fumarate. Obesity was associated with development of both dyslipidaemia and diabetes mellitus after switch. Stable switch to BIC/FTC/TAF maintained high rates of viral suppression and had a small effect on weight and metabolic changes in virally suppressed PWH. Follow-up of the weight and metabolic changes is warranted in PWH on long-term antiretroviral therapy.

Dual targeted extracellular vesicles regulate oncogenic genes in advanced pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) tumours carry multiple gene mutations and respond poorly to treatments. There is currently an unmet need for drug carriers that can deliver multiple gene cargoes to target high solid tumour burden like PDAC. Here, we report a dual targeted extracellular vesicle (dtEV) carrying high loads of therapeutic RNA that effectively suppresses large PDAC tumours in mice. The EV surface contains a CD64 protein that has a tissue targeting peptide and a humanized monoclonal antibody. Cells sequentially transfected with plasmid DNAs encoding for the RNA and protein of interest by Transwell®-based asymmetric cell electroporation release abundant targeted EVs with high RNA loading. Together with a low dose chemotherapy drug, Gemcitabine, dtEVs suppress large orthotopic PANC-1 and patient derived xenograft tumours and metastasis in mice and extended animal survival. Our work presents a clinically accessible and scalable way to produce abundant EVs for delivering multiple gene cargoes to large solid tumours.

Comparison of clinical efficacy and safety of baloxavir marboxil versus oseltamivir as the treatment for influenza virus infections: A systematic review and meta-analysis

IntroductionBaloxavir marboxil (BXM), a newly developed cap-dependent endonuclease inhibitor, is widely used to treat influenza virus infections in inpatients and outpatients. A previous meta-analysis included only outpatients and patients suspected of having an influenza virus infection based on clinical symptoms. However, whether BXM or oseltamivir is safer and more effective for inpatients remains controversial. Therefore, we conducted a systematic review and meta-analysis validating the effectiveness and safety of BXM versus oseltamivir in inpatients with influenza virus. MethodsThe Scopus, EMBASE, PubMed, Ichushi, and CINAHL databases were systematically searched for articles published until January 2023. The outcomes were mortality, hospitalization period, incidence of BXM- or oseltamivir-related adverse events, illness duration, and changes of virus titers and viral RNA load in patients with influenza virus infections. ResultsTwo randomized controlled trials with 1624 outpatients and two retrospective studies with 874 inpatients were enrolled. No deaths occurred in outpatients treated with BXM or oseltamivir. Among inpatients, BXM reduced mortality (p = 0.06) and significantly shortened hospitalization period (p = 0.01) compared to oseltamivir. In outpatients, BXM had a significantly lower incidence of adverse events (p = 0.03), reductions in influenza virus titers (p < 0.001) and viral RNA loads (p < 0.001), and a tendency to be a shorter illness duration compared with that of oseltamivir (p = 0.27). ConclusionsOur meta-analysis showed that BXM was safer and more effective in patients than oseltamivir; thus, supporting the use of BXM for the initial treatment of patients with proven influenza virus infection.

Comparison of analytical sensitivity of DNA-based and RNA-based nucleic acid amplification tests for reproductive tract infection pathogens: implications for clinical applications.

Currently, DNA-based nucleic acid amplification tests (NAATs) and RNA-based NAATs are employed to detect reproductive tract infection (RTI) pathogens including Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Ureaplasma urealyticum (UU). Although evaluations of DNA-based NAATs have already existed, the comparison of the two methods is scarce. Thus, we compared the limits of detection (LODs) of DNA-based and RNA-based NAATs on the same experimental conditions. Inactivated culture supernatants of CT, NG, and UU with determined pathogen DNA and RNA load were used to evaluate LODs of seven DNA kits and one RNA kit. The LODs of the seven DNA kits for CT, NG, and UU ranged between 38-1,480, 94-20,011, and 132-2,011 copies/mL, respectively. As for RNA kits, they could detect samples at RNA concentrations of 3,116, 2,509, and 2,896 copies/mL, respectively. The RNA concentrations of CT, NG, and UU were 40, 885, and 42 times that of corresponding pathogen DNA concentrations in the employed supernatants, so RNA kits could detect pathogen DNA concentrations as low as 78 copies/mL, 3 copies/mL, and 69 copies/mL, respectively, but the level of pathogen load that the RNA tests could detect was primarily dependent on the infectious phase and transcriptional level of RNA. Thus, a schematic of bacterial dynamics during the period of reproductive tract infections was provided, which suggests that in terms of the analytical sensitivity of pathogen detection, RNA tests are more suitable for detecting active infection and recovery phase, while DNA tests are more suitable for detection in the early stage of infection. IMPORTANCE Reproductive tract infections have considerable effects on the health of humans. CT, NG , and UU are common pathogens. Although evaluation of DNA-based tests has already existed, the comparison between DNA-based and RNA-based tests is rare. Therefore, this study compared the limits of detection of the two tests on the same experimental conditions. Results suggested that most DNA-based NAATs could detect CT, NG, and UU at DNA concentrations lower than 1,000 copies/mL, while RNA-based NAATs could detect bacteria at RNA concentrations around 3,000 copies/mL. Considering the copy number of RNA per bacterium is dynamic through the growth cycle, further comparison is combined with a schematic of bacterial dynamics. Results suggested that in terms of the analytical sensitivity of pathogen detection, RNA tests are more suitable for detecting active infection and recovery phase, while DNA tests are more suitable for detection in the early stage of infection.

Distribution of Opportunistic Pathogens in People Living with HIV at a University Hospital in Istanbul over a One-Year Treatment Period and Its Association with CD4 T Cell Counts.

Among sexually transmitted diseases, HIV causes very serious clinical manifestations that can lead to death. As a result, millions of people have to live with this problem that threatens their health. The virus attacks the immune system of the host, especially CD4+ T lymphocytes, causing the suppression of the immune system. CD4, CD8 counts, and HIV RNA viral loads are monitored in HIV-infected patients with antiretroviral treatment, and CD4 counts play an important role in determining the effectiveness of the treatment. Despite the advances in treatment in the present day, opportunistic infections are the main cause of morbidity and mortality in these patients, and the evaluation of immunological parameters is valuable for the prognosis of the disease in this process. In the present study, the purpose was to investigate the opportunistic infections faced by naive HIV-positive patients who applied to our laboratory and were diagnosed between 2019 and 2022 during their one-year treatment period, and the correlation of the immunological parameters was also evaluated retrospectively using the hospital automation system and laboratory data. A total of 107 opportunistic causative microorganisms were identified in 87 of the 230 HIV-positive patients over one year. T. pallidum was detected in 43 (18.6%) of these patients, Cytomegalovirus (CMV) in 32 (13.9%), Epstein-Barr virus (EBV) in 9 (3.9%), Hepatitis B virus (HBV) in 10 (4.3%), C. albicans in 7 (3%), M. tuberculosis in 3 (1.3%), Hepatitis C virus (HCV) in 2 (0.8%), and C. glabrata in 1 (0.4%) patient. Although mono-agent co-infections were determined in 69 of 87 people living with HIV, two-agent co-infections were detected in 16 HIV patients, and three-agent co-infections were identified in two HIV patients. Considering the correlation between the CD4/CD8 ratio and infection positivity, a moderate negative correlation was determined with HIV RNA viral load and CMV infection. The CD4/CD8 ratio had a low negative correlation with EBV and C. albicans infections. It was also found that the follow-up of HIV RNA load in the diagnosis of T. pallidum, CMV, EBV, and C. albicans may be meaningful. Opportunistic infections mainly affect immunosuppressed patients and can be prevented with effective treatment. Although it is already known that HIV patients may face different infections during their treatment, it was concluded that more attention should be paid to T. pallidum, CMV, EBV, and C. albicans agents. These infections should be routinely monitored with HIV viral load and the CD4/CD8 ratio.