Serum RNA Research Articles

Orthohepevirus C infection as an emerging cause of acute hepatitis in Spain: First report in Europe

Hepatitis E virus (HEV) was considered the only member of the Hepeviridae family with zoonotic potential. Nevertheless, this consideration has been reassessed owing to several reported cases of acute and chronic hepatitis linked to the Orthohepevirus C genus. Because the circulation of Orthohepevirus C in rodents has been described worldwide, the risk of zoonotic transmission is plausibly global. Orthohepevirus C RNA was retrospectively evaluated in 2 cohorts of patients in Spain. The first cohort included patients with acute hepatitis without etiological diagnosis after screening for hepatotropic virus infection. The second cohort included patients diagnosed with acute HEV infection, defined as positivity for anti-HEV-IgM antibodies and/or detectable HEV RNA in serum. Cohort 1 comprised 169 patients (64.4% male, median age 43 years) and cohort 2 comprised 98 individuals (68.3% male, median age 45 years). Of the individuals included in Cohort 1, two (1.18%; 95% CI 0.2-3.8) had detectable Orthohepevirus C RNA in serum. In Cohort 2, of the 98 included patients, 58 showed detectable HEV RNA, while 40 only showed positivity for IgM antibodies. Among those bearing only IgM antibodies, Orthohepevirus C RNA was detected in 1 (2.5%; 95% CI 0.06-13.1) individual. All strains were consistent with genotype C1. The infection resulted in mild self-limiting acute hepatitis in 2 patients. Infection caused severe acute hepatitis in the remaining patient who died as a result of liver and renal failure. We described 3 cases of Orthohepevirus C in patients with acute hepatitis, resulting in the first description of this infection in Europe. The prevalence obtained in our study suggests that Orthohepevirus C could be an emerging disease in Europe. We describe the first cases of acute hepatitis related to rat hepatitis E virus in Europe. The prevalence found in our study suggest that rat hepatitis E virus could be considered an emerging disease in Europe.

Serum RNAs can predict lung cancer up to 10 years prior to diagnosis.

Lung cancer (LC) prognosis is closely linked to the stage of disease when diagnosed. We investigated the biomarker potential of serum RNAs for the early detection of LC in smokers at different prediagnostic time intervals and histological subtypes. In total, 1061 samples from 925 individuals were analyzed. RNA sequencing with an average of 18 million reads per sample was performed. We generated machine learning models using normalized serum RNA levels and found that smokers later diagnosed with LC in 10 years can be robustly separated from healthy controls regardless of histology with an average area under the ROC curve (AUC) of 0.76 (95% CI, 0.68-0.83). Furthermore, the strongest models that took both time to diagnosis and histology into account successfully predicted non-small cell LC (NSCLC) between 6 and 8 years, with an AUC of 0.82 (95% CI, 0.76-0.88), and SCLC between 2 and 5 years, with an AUC of 0.89 (95% CI, 0.77-1.0), before diagnosis. The most important separators were microRNAs, miscellaneous RNAs, isomiRs, and tRNA-derived fragments. We have shown that LC can be detected years before diagnosis and manifestation of disease symptoms independently of histological subtype. However, the highest AUCs were achieved for specific subtypes and time intervals before diagnosis. The collection of models may therefore also predict the severity of cancer development and its histology. Our study demonstrates that serum RNAs can be promising prediagnostic biomarkers in an LC screening setting, from early detection to risk assessment.

Another piece in the COVID-19 treatment puzzle

Another piece in the COVID-19 treatment puzzle

Transmission of yellow fever vaccine virus from breast feeding mothers to their infants: reporting of yellow fever virus (YFV) RNA detection in milk specimens

Background: Because of yellow fever’s serious impact on health, vaccination is the principal strategy to control the disease. Administration of the yellow fever vaccine to breastfeeding women should be before they complete 9 months post-delivery, in order to prevent transmission of the yellow fever vaccine virus to their infants through breast feeding. This study aimed to confirm whether the excretion of yellow fever vaccine virus is in milk of vaccinated breastfeeding mothers and to confirm the probable transmission to their infants through breast milk. Methods: Samples were taken as follows: one serum specimen was taken 3-14 days after the date of the vaccination, and breast milk specimens were taken at four different time points between 3-4 days apart. Specimens were obtained from eight nursing mothers, who received the YVF vaccine (17DD). Mothers were asymptomatic before and after the vaccine administration but their infants developed symptoms after administration. Maternal serum samples were tested for YFV specific IgM antibodies through immuno-fluorescent assay (IFA). RNA was extracted from serum and breast milk specimens and YFV RNA screened using real-time polymerase chain reaction (RT-PCR). Results: In total, five mothers (62.5%) were positive for YFV and two mothers (25%) had YFV RNA in serum. Among milk specimens, YFV RNA was detected during the four different mentioned collection times as follows (positive milk specimens/total milk specimens): 3/8 (37.5 %), 4/6 (66.6%) and 1/4(25%). RNA was completely undetectable in the last collection time. Conclusions: YFV transmission from mothers to their babies through breast-feeding was highly probable indicated by the temporal relationship to mother’s YF vaccination.

Serum HBV DNA plus RNA reflecting cccDNA level before and during NAs treatment in HBeAg positive CHB patients.

Background & Aims: Correlations between serum viral markers and intrahepatic cccDNA in patients undergoing long-term nucleos(t)ide analogues (NAs) treatment haven't been fully explored. In this study, we evaluate the correlation between intrahepatic cccDNA and other serum viral markers and intrahepatic HBV DNA in HBeAg positive chronic hepatitis B (CHB) patients during 60-month treatment with NAs.Methods: Fifty-four HBeAg positive CHB patients received long-term NAs treatment were included in this study. Serial serum samples were regularly collected and quantitatively analyzed for HBsAg, HBV DNA, HBV RNA and HBcrAg. Histological samples from liver biopsy at baseline and month 60 were analyzed for intrahepatic HBV DNA and cccDNA.Results: At baseline, serum HBV DNA plus RNA was positively associated with intrahepatic cccDNA in multivariate regression analysis (β=0.205, P<0.001). In the correlation analysis between cccDNA and serum viral markers, HBV DNA plus RNA had the highest correlation coefficient (r=0.698, P<0.001), followed by serum HBV DNA (r=0.641, P<0.001), HBV RNA (r=0.590, P<0.001), and HBcrAg (r=0.564, P<0.001). At month 60, correlations between these serum viral markers and cccDNA were not observed (P>0.05). Multivariate regression analysis showed that only the decreased HBV DNA plus RNA was positively associated with cccDNA decline (β=0.172, P =0.006). Changes of HBV DNA plus RNA (r=0.525, P=0.001) was better correlated with cccDNA decline as compared to HBV RNA (r=0.384, P=0.008), HBV DNA (r=0.431, P=0.003), and HBsAg (r=0.342, P=0.029).Conclusions: Serum HBV DNA plus RNA better correlated with intrahepatic cccDNA than other viral makers before and during NAs treatment in HBeAg positive CHB patients.

Quantitative Real-Time RT-PCR Systems to Detect SARS-CoV-2.

Since the outbreak of coronavirus disease 2019 (COVID-19) on the Diamond Princess cruise ship docked at Yokohama Port on February 3, 2020, real-time reverse transcription-polymerase chain reaction (RT-PCR) testing using nasopharyngeal swab samples from symptomatic and asymptomatic COVID-19 individuals has been the main way to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in almost all clinical laboratories in Japan. With the diffusion of sets containing the primers and probe, the gold standard real-time RT-PCR test has permeated throughout the country. To prevent the spread of infection, real-time RT-PCR testing is important to confirm whether people are positive, asymptomatic, or negative for COVID-19. Now, in addition to pharyngeal swab, saliva and blood samples can be used to detect SARS-CoV-2 RNA. Here, we introduce a clinical laboratory test performed using the High Pure viral nucleic acid kit and subsequent real-time RT-PCR system to detect SARS-CoV-2 RNA in serum, plasma, or whole blood in a hospital in Yokohama, Japan.

Evaluation of two RT-PCR techniques for SARS-CoV-2 RNA detection in serum for microbiological diagnosis

Presence of SARS-CoV-2 RNA in serum (viremia) of COVID-19 patients has been related to poor prognosis and death. The aim of this study was to evaluate both the ability to detect viremia in COVID-19 patients of two commercial reverse real-time-PCR (rRT-PCR) tests, Cobas® and TaqPath™, comparing them with a gold standard method, and their implementation in microbiology laboratories.This retrospective cohort study included 303 adult patients (203 diagnosed with COVID-19 and 100 non-COVID-19 patients) admitted to a tertiary hospital, with at least one serum sample collected within the first 48 h from admission. A total of 365 serum samples were included: 100 from non-COVID patients (pre-pandemic and pandemic control groups) and 265 from COVID-19 patients. Serum samples were considered positive when at least one target was detected. All patients in control groups showed negative viremia. Cobas® and TaqPath™ tests showed specificity and Positive Predictive Value over 96%. Nevertheless, sensitivity (53.72 and 73.63, respectively) and Negative Predictive Value (64.78 and 75) were lower. Viremia difference between ICU and non-ICU patients was significant (p ≤ 0.001) for both techniques.Consequently, SARS-CoV-2 viremia detection by both rRT-PCR tests should be considered a good tool to stratify COVID-19 patients and could be implemented in microbiology laboratories.

372. Detection of SARS-CoV-2 RNAemia in Deceased Tissue Donors

BackgroundTissue donors are evaluated for communicable disease in order to minimize the risk of transmission to recipients. Although there are data suggesting SARS-CoV-2 viremia across a wide spectrum of illness, prevalence in deceased tissue donors and the potential for transplant transmission are unknown.MethodsEight tissue banks participated in a retrospective analysis of samples from eligible deceased tissue donors from Oct 2019 through June 2020, one participant in Canada and the remainder located in the United States. All four Census regions of the continental US and all major racial-ethnic groups were represented. EDTA or sodium citrate plasma aliquots were tested in singlicate with the Research Use Only Procleix SARS-CoV-2 Assay on the Procleix Panther System, which uses transcription-mediated nucleic acid amplification (TMA) technology for detection of the SARS-CoV-2 RNA. Plasma (or if unavailable, serum) aliquots were sent to Grifols for an alternate SARS-CoV-2 nucleic acid amplification (NAT) test to verify reactivity and also sent for antibody testing using the emergency use authorization Ortho VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total test. The VITROS assay uses immunometric technology for qualitative measurement of total antibody (IgG, IgA and IgM) to SARS-CoV-2. The proportion of donors with confirmed RNAemia (i.e., presence of SARS-CoV-2 RNA in plasma or serum) and 95% confidence intervals were computed.ResultsOf 3,455 donor samples with valid final results, 26 (0.76%) were initially positive for SARS-CoV-2 RNA; of these, 3 were confirmed by alternate NAT. Of donor samples collected in 2019 0.00% (95% CI: 0.00%,0.43%) were confirmed RNAemic, while of those collected in 2020, 0.12% (0.04%,0.34%) were confirmed RNAemic. One of 26 initial positive, and none of the three samples confirmed by alternate NAT, tested positive for anti-SARS-CoV-2 Spike antibodies by serology. Infectivity studies are pending on one sample with sufficient available volume.ConclusionThe rate of SARS-CoV-2 RNAemia in deceased tissue donors is approximately 1 per 1,000, and it is unknown whether this RNAemia reflects the presence of infectious virus. Given these results, the risk of transmission through tissue is most likely to be low.Disclosures Melissa Greenwald, MD, Alamo Biologics (Consultant)Eurofins VRL Laboratories (Consultant)Right Cell Biologics (Consultant, Consultant Medical Director) Eduard Grebe, PhD, Gilead Sciences (Consultant)Sedia Biosciences Corporation (Consultant, Grant/Research Support)Vitalant (Employee) Alyce Linthurst Jones, PhD, LifeNet Health (Employee) Matthew Kuehnert, MD, American Association of Tissue Banks (Board Member)ICCBBA (Board Member)Musculoskeletal Transplant Foundation (Employee)

Detection of increased serum miR-122-5p and miR-455-3p levels before the clinical diagnosis of liver cancer in people with type 2 diabetes

People with type 2 diabetes (T2D) have increased cancer risk. Liver cancer (LC) has a high prevalence in East Asia and is one of the leading causes of cancer death globally. Diagnosis of LC at early stage carries good prognosis. We used stored serum from patients of Hong Kong Diabetes Register before cancer diagnosis to extract RNA to screen for microRNA markers for early detection of LC in T2D. After screening with Affymetrix GeneChip microarray with serum RNA from 19 incident T2D LC (T2D-LC), 20 T2D cancer free (T2D-CF) and 20 non-T2D non-cancer patients, top signals were validated in a 3-group comparison including 1888 T2D-CF, 127 T2D-LC, and 487 T2D patients with non-liver cancer patients using qPCR. We detected 2.55-fold increase in miR-122-5p and 9.21-fold increase in miR-455-3p in the T2D-LC group. Using ROC analysis, miR-122-5p and miR-455-3p jointly predicted LC with an area under the curve of 0.770. After adjustment for confounders, each unit increase of miR-455-3p increased the odds ratio for liver cancer by 1.022. Increased serum levels of miR-122-5p and miR-455-3p were independently associated with increased risk of incident LC in T2D and may serve as potential biomarkers for early detection of LC in T2D.

Immunization with recombinant ORF2 p551 protein protects common marmosets (Callithrix jacchus) against homologous and heterologous hepatitis E virus challenge

BackgroundHepatitis E virus (HEV) is a major causative agent of acute hepatitis worldwide, prompting continuous HEV vaccine efforts. Vaccine development is hampered by the lack of convenient animal models susceptible to infection with different HEV genotypes. We produced recombinant open reading frame 2 protein (pORF2; p551) of HEV genotype (GT) 3 and assessed its immunogenicity and protectivity against HEV challenge in common marmosets (Callithrix jacchus, CM). Methodsp551 with consensus sequence corresponding to amino acid residues 110–660 of HEV GT3 pORF2 was expressed in E. coli and purified by affinity chromatography. CMs were immunized intramuscularly with 20 μg of p551 VLPs with alum adjuvant (n = 4) or adjuvant alone (n = 2) at weeks 0, 3, 7 and 19. At week 27, p551-immunized and control animals were challenged with HEV GT1 or GT3 and thereafter longitudinally screened for markers of liver function, anti-HEV IgG and HEV RNA in feces and sera. ResultsPurified p551 formed VLPs with particle size of 27.71 ± 2.42 nm. Two immunizations with p551 induced anti-HEV IgG mean titer of 1:1810. Immunized CMs challenged with homologous and heterologous HEV genotype did not develop HEV infection during the follow-up. Control CMs infected with both HEV GT1 and GT3 demonstrated signs of HEV infection with virus shedding and elevation of the levels of liver enzymes. High levels of anti-HEV IgG persisted in vaccinated CMs and control CMs that resolved HEV infection, for up to two years post challenge. ConclusionsCMs are shown to be a convenient laboratory animal model susceptible to infection with HEV GT1 and GT3. Immunization with HEV GT3 ORF2/p551 triggers potent anti-HEV antibody response protecting CMs from homologous and heterologous HEV challenge. This advances p551 in VLPs as a prototype vaccine against HEV.

Circadian Oscillation of Natural Antisense Transcripts Related to Human Core Clock Genes

Circadian clocks are autonomous intracellular oscillators that synchronize metabolic and physiological processes with the external signals. So, misalignment of environmental and endogenous circadian rhythms leads to disruption of biological activities in living organisms. Noncoding transcripts including antisense RNAs are an important component of the molecular clocks. Commonly, the antisense transcripts are involved in the regulation of gene expression. PER2AS and CRY1AS are the only known Natural Antisense Transcripts (NAT) among the core clock genes, which overlap with the PER2 and CRY1 genes, respectively. In this study, we hypothesized that PER2AS and CRY1AS like the other clock genes, exhibit the oscillatory behavior in a 24-hour period and affect the expression of PER2 and CRY1. First, the A549 cell line was cultured under standard conditions. After horse serum shock, RNA extraction and cDNA synthesis was performed; then the expression fluctuations of PER2AS, CRY1AS, PER2, and CRY1 were measured with Real-time PCR. Our result showed that PER2AS and CRY1AS had similar oscillation patterns with their sense strand during 24-hour period. Therefore, we suggested that PER2AS and CRY1AS transcripts probably by preventing the interaction of miRNAs with PER2 and CRY1 mRNAs, influence the expression of them, positively.

Circulating non-coding RNAs as a diagnostic and management biomarker for breast cancer: current insights.

Cancer biomarkers can be used to determine the molecular status of a tumor or its metastases, which either release them directly into body fluids or indirectly through disruption of tumor/metastatic tissue. New minimally invasive and repeatable sample collection methods, such as liquid biopsy, have been developed in the last decade to apply cancer knowledge and track its progression. Circulating non-coding RNAs, which include microRNAs, long non-coding RNAs, and PIWI-interacting RNAs, are increasingly being recognized as potential cancer biomarkers. The growing understanding of cancer's molecular pathogenesis, combined with the rapid development of new molecular techniques, encourages the study of early molecular alterationsassociated with cancer development in body fluids. Specific genetic and epigenetic changes in circulating free RNA (cf-RNA) in plasma, serum, and urine could be used as diagnostic biomarkers for a variety of cancers. Only a subset of these cf-RNAs have been studied in breast cancer, with the most extensive research focusing on cf-miRNA in plasma. These findings pave the way for immediate use of selected cf-RNAs as biomarkers in breast cancer liquid biopsy, as well as additional research into other cf-RNAs to advance.

Efficacy of Cladribine Tablets as a Treatment for People With Multiple Sclerosis: Protocol for the CLOBAS Study (Cladribine, a Multicenter, Long-term Efficacy and Biomarker Australian Study).

BackgroundCladribine tablets (marketed as Mavenclad) are a new oral therapy, which has recently been listed on the pharmaceutical benefits scheme in Australia for the treatment of relapsing multiple sclerosis (MS). The current dosing schedule is for 2 courses given a year apart, which has been shown to be effective for treatment of MS for up to 4 years in 75% of patients (based on annualized relapse rate). However, the reinitiation of therapy after year 4 has not been studied.ObjectiveThis study aims to evaluate the safety and efficacy of cladribine tablets over a 6-year period, according to no evidence of disease activity 3.MethodsThis will be a multicenter, 6-year, phase IV, low interventional, observational study that incorporates clinical, hematological, biochemical, epigenetic, radiological and cognitive biomarkers of disease. Participants considered for treatment with cladribine as part of their routine clinical care will be consented to take part in the study. They will be monitored at regular intervals during the initial course of medication administration in years 1 and 2. After year 3, patients will have the option of redosing, if clinically indicated, or to switch to another disease-modifying therapy. Throughout the duration of the study, we will assess blood-based biomarkers including lymphocyte subsets, serum neurofilament light chain, DNA methylation, and RNA analysis as well as magnetic resonance imaging findings (brain volume and/or lesion load) and cognitive performance.ResultsThis study has been approved by the Hunter New England Local Health District Human Research Ethics Committee. Recruitment began in March of 2019 and was completed by June 2021.ConclusionsThis will be the first long-term efficacy trial of cladribine, which offers reinitiation of therapy in the 3rd year, based on disease activity, after the initial 2 courses. We expect that this study will indicate whether any of the assessed biomarkers can be used to predict treatment efficacy or the need for future reinitiation of cladribine in people with MS.Trial RegistrationThis study is registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12619000257167) with Universal Trial Number (U1111-1228-2165).International Registered Report Identifier (IRRID)DERR1-10.2196/24969

Utility of miR-155, miR-21 and miR-16 in plasma as biomarkers of atrial fibrillation

Abstract Background Atrial fibrillation (AF) is associated with increased morbidity and mortality. MicroRNAs (miRs) are post-transcriptional regulators that could be useful in the study of AF. Purpose To investigate whether miR-16, miR-21 and miR-155 may differentiate between AF and sinus rhythm. Methods 29 controls and 42 AF (June 2019-May 2020). Lipid profile, hsCRP were measured. Serum RNA extraction was performed. Hsa-miR-21, hsa-miR-16, hsa-miR-155, and cel-miR-39 were amplified RT-PCR. Quantitative results for miR levels were generated according to the 2-ΔCt method using cel-miR-39 for normalization. Correlations were assessed by Spearman rank test. Logistic regression analysis was adjusted by age, sex, and BMI vs miR's. A p&amp;lt;0.05 was set for significance. Results (controls vs AF): age (56 [52–63] vs 60 [64–67] years, p=0.122), BMI (30.52 [27.69–32.67] vs 29.65 [26.3–32.9] kg/m2 p=0.779), female 17 (58.6%) vs 19 (45.2%), p=0.268; total cholesterol (198.4 [17.2–215] vs 171.3 [150.3–216.8] mg/dL, p=0.066), LDL (126.6 [114.7–141.6] vs 99.2 [80.7–124] mg/dL, p=0.005) HDL (40.4 [36.8–45.8] vs 43.8 [36.9–48.5] p=0.326) and CRP (2.69 [1.22–4.72] vs 2.92 [1.4–4.76] mg/L p=0.835). The CHA2DS2VASC score in AF was &amp;lt;2=11 (45.2%) and ≥2= 26 (54.8%). MicroRNA levels (controls vs AF): miR-21 0.0195 (0.0095–0.0275) vs 0.042 (0.028–0.098) p&amp;lt;0.001; miR-155 0.0178 (0.0108–0.0309) vs 0.01 (0.0056–0.0169) p&amp;lt;0.001, and miR-16 0.5238 (0.2618–1.992) vs 0.1652 (0.0765–0.3816) p&amp;lt;0.001. In the logistic regression miR-21 had a positive correlation with AF, c=0.851 p=0.002, Wald=9.89. miR-16 and miR-155 a negative relation (c=0.76,0.72; p=0.036, 0.043, Wald=4.39, 4.085; respectively) (Fig. 1A, B, C). For multivariable analysis we used miR-16, miR-21, and miR-155 as previous analysis demonstrated significance with an overall model c=0.90 p=0.028 (Wald=14.2) and significance was seen in the miR-21 Wald= 9.33 (p=0.002); and miR-155 Wald=4.7 (p=0.03); miR-16 was not significant (Wald=2.7, p=0.1). A ternary plot was performed using the predicted outputs of the model. (Fig. 2) Conclusion miR-16, miR-21 and miR-155 are useful for the discrimination of AF vs sinus rhythm and may be part of important physiopathological pathways in AF. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Instituto Nacional de Cardiología Ignacio Chávez Figure 1. Logistic regression curvesFigure 2. Multivariate ternary plot of miRs

Detection of Occult Hepatitis C Virus Infection in Patients Who Achieved a Sustained Virologic Response to Direct-Acting Antiviral Agents

Abstract Background Occult infection with hepatitis C virus (HCV) is defined as the presence of the HCV genome in either liver tissue or peripheral blood monocytes, despite constant negative results from tests for HCV RNA in serum. Objectives The aim of the study to detect the prevalence of occult hepatitis C Virus infection in patients who achieved a sustained virologic response (SVR) to direct-acting antiviral agents and to outline predictors of OCI. Patients and Methods This study included 100 patients with chronic HCV infection without liver cirrhosis attending to hepatitis C clinics at Ain Shams University Hospital, Ahmed Maher Teaching hospital and Elgomhorya Teaching Hospital.who received sofosbuvir (400mg) plus daclatasvir (60mg) daily for 12 weeks with or without ribavirin according to National committee to combat viral hepatitis (NCCVH) protocol. Results We tested peripheral blood for HCV RNA in PBMCs to detect OCI. Occult HCV was found positive in 12% of the studied cases. Occult HCV was positive more in male cases. Positive cases had significantly lower age, and higher total bilirubin, direct bilirubin, AST and ALT levels. Age had significant moderate diagnostic performance in predicting occult HCV, while direct bilirubin has significant low diagnostic performance in predicting occult HCV. Conclusion OCI following direct antiviral therapy may be present in some cases, and this may require further testing of patients with SVR particularly in younger male patients with persistantly high liver enzymes.

The relationship between hepatitis B virus serum DNA, RNA and quantitative hepatitis B surface antigen, and the predictive value for mother-to-child transmission: an observational cohort study.

To explore the relationships between hepatitis B virus (HBV) DNA, HBV RNA and hepatitis B surface antigen (HBsAg) and to evaluate their predictive value for mother-to-child transmission of HBV. An observational cohort study. First Hospital of Jilin University. HBsAg-positive and hepatitis B e antigen (HBeAg) -positive pregnant women were recruited. Blood samples were collected from mothers before delivery, and HBV infection of infants was evaluated at 7 months of age. Overall, 268 mothers and 271 infants were enrolled. HBV DNA and HBsAg levels were correlated (rs = 0.699; P < 0.001), and HBV DNA (rs = 0.500; P < 0.001) and HBsAg (rs = 0.372; P < 0.001) were both correlated with HBV RNA. The areas under the curve for HBV DNA, HBsAg and HBV RNA for prediction of infection were 0.69 (95% CI 0.57-0.82), 0.63 (95% CI 0.51-0.76) and 0.65 (95% CI 0.52-0.78), respectively. Higher HBV DNA (odds ratio [OR] 4.77, 95% CI 1.44-15.86), higher HBsAg (OR 4.13, 95% CI 1.12-15.25) and higher HBV RNA (OR 3.19, 95% CI 1.09-9.32) were risk factors for HBV infection. Analysis of the HBV DNA-RNA-HBsAg Score revealed that it was an independent predictive factor for mother-to-child transmission (the OR of Score 3 was 8.81, 95% CI 2.79-27.82). HBV DNA, HBV RNA and HBsAg were correlated in HBeAg-positive pregnant women. HBsAg could be considered as a substitute marker of HBV DNA for HBeAg-positive pregnant women in low-income regions. We should pay special attention to pregnant women with high levels of all three markers. HBsAg could be considered as a substitute marker of HBV DNA for HBeAg-positive pregnant women in low-income regions. Special attention should be given to pregnant women with high levels of all three markers (HBV DNA, HBV RNA and HBsAg).

SARS-CoV-2 RNAemia with a higher nasopharyngeal viral load is strongly associated with disease severity and mortality in patients with COVID-19.

This study aimed to determine the frequency of SARS‐CoV‐2 RNA in serum and its association with the clinical severity of COVID‐19. This retrospective cohort study performed at Toyama University Hospital included consecutive patients with confirmed COVID‐19. The prevalence of SARS‐CoV‐2 RNAemia and the strength of its association with clinical severity variables were examined. Fifty‐six patients were included in this study. RNAemia was detected in 19.6% (11/56) patients on admission, and subsequently in 1.0% (1/25), 50.0% (6/12), and 100.0% (4/4) moderate, severe, and critically ill patients, respectively. Patients with RNAemia required more frequent oxygen supplementation (90.0% vs. 13.3%), ICU admission (81.8% vs. 6.7%), and invasive mechanical ventilation (27.3% vs. 0.0%). Among patients with RNAemia, the median viral loads of nasopharyngeal (NP) swabs that were collected around the same time as the serum sample were significantly higher in critically ill (5.4 log10 copies/μl; interquartile range [IQR]: 4.2–6.3) than in moderate‐severe cases (2.6 log10 copies/μl; [IQR: 1.1–4.5]; p = 0.030) and were significantly higher in nonsurvivors (6.2 log10 copies/μl [IQR: 6.0–6.5]) than in survivors (3.9 log10 copies/μl [IQR: 1.6–4.6]; p = 0.045). This study demonstrated a relatively high proportion of SARS‐CoV‐2 RNAemia and an association between RNAemia and clinical severity. Moreover, among the patients with RNAemia, the viral loads of NP swabs were correlated with disease severity and mortality, suggesting the potential utility of combining serum testing with NP tests as a prognostic indicator for COVID‐19, with higher quality than each separate test.

Activation and Kinetics of Circulating T Follicular Helper Cells, Specific Plasmablast Response, and Development of Neutralizing Antibodies following Yellow Fever Virus Vaccination.

A single dose of the replication-competent, live-attenuated yellow fever virus (YFV) 17D vaccine provides lifelong immunity against human YFV infection. The magnitude, kinetics, and specificity of B cell responses to YFV 17D are relatively less understood than T cell responses. In this clinical study, we focused on early immune events critical for the development of humoral immunity to YFV 17D vaccination in 24 study subjects. More specifically, we studied the dynamics of several immune cell populations over time and the development of neutralizing Abs. At 7 d following vaccination, YFV RNA in serum as well as several antiviral proteins were detected as a sign of YFV 17D replication. Activation of Th1-polarized circulating T follicular helper cells followed germinal center activity, the latter assessed by the surrogate marker CXCL13 in serum. This coincided with a plasmablast expansion peaking at day 14 before returning to baseline levels at day 28. FluoroSpot-based analysis confirmed that plasmablasts were specific to the YFV-E protein. The frequencies of plasmablasts correlated with the magnitude of neutralizing Ab titers measured at day 90, suggesting that this transient B cell subset could be used as an early marker of induction of protective immunity. Additionally, YFV-specific memory B cells were readily detectable at 28 and 90 d following vaccination, and all study subjects tested developed protective neutralizing Ab titers. Taken together, these studies provide insights into key immune events leading to human B cell immunity following vaccination with the YFV 17D vaccine.

HCV RNA in serum and liver samples of patients undergoing living donor liver transplantation.

ObjectiveTo compare hepatitis C virus (HCV) RNA levels from serum and explanted native liver samples from patients undergoing living donor liver transplantation (LDLT).MethodsThis was a prospective observational study. Serum and liver samples were collected from consecutive serum anti-HCV-positive transplant recipients between February 2016 to August 2019. HCV RNA was extracted from liver samples and subjected to one-step reverse-transcription qPCR. using the TopScript One Step qRT-PCR Probe Kit with HCV qPCR probe assay and human GAPDH qPCR probe assay on a ViiA7 Real-Time PCR System.ResultsAmong the 80 patients, 36% (29/80) were HCV RNA positive in serum and 85% (68/80) had positive hepatic HCV RNA. Post-liver transplantation, 4% (3/80) patients were serum positive.ConclusionsOur study suggests that pre-transplant serum HCV RNA levels may give an underestimate of the number of positive HCV RNA cases and that hepatic HCV RNA data may be more accurate.

Phylogenetic analysis of the 5\u02b9 untranslated region of HCV from cirrhotic patients in Khyber Pakhtunkhwa, Pakistan

Hepatitis C virus (HCV), a small, single-stranded RNA virus with a 9.6 kb genome, is one of the most common causes of liver diseases. Sequencing of the 5ʹ untranslated region (UTR) is usually used for HCV genotyping, but it is less important in numerous subtypes due to its scarce sequence variations. This study aimed to identify genotypes using the 5ʹ UTR of HCV from cirrhotic patients of Khyber Pakhtunkhwa (KP). Serum RNA samples (44) were screened by real time PCR to determine the HCV viral load. Nested PCR was performed to identify cDNA and the 5ʹ UTR. The HCV 5′ UTR was sequenced using the Sanger method. MEGA-7 software was used to analyze evolutionary relatedness. After 5ʹ UTR sequencing, 26 samples (59%) were identified as genotype 3, and 2 samples (6%) were identified as genotypes 1, 2 and 4. The most predominant genotype was 3a, and genotype 4 was rarely reported in the phylogenetic tree. Analysis of the HCV 5ʹ UTR is an efficient alternative method for confirmation of various genotypes. Phylogenetic analysis showed that genotype 3 was dominant in the area of KP, Pakistan.