Evaluation of a new automated viral RNA extraction platform for hepatitis A virus and human norovirus in testing of berries, lettuce, and oysters.

Fruits, vegetables, and shellfish are often associated with outbreaks of illness caused particularly by human norovirus (HuNoV) and hepatitis A virus (HAV), the leading causative agents of foodborne illness worldwide. The aim of this study was to evaluate a new automated nucleic acid extraction platform (EGENE-UP EASYPREP) for enteric viruses in several at-risk food matrices and to test its limit of detection in comparison to a semi-automated method (EGENE-UP) using Boom methodology for nucleic acid extraction as suggested in the reference method ISO 15216-2:2019. Fresh and frozen raspberries, frozen blackberries, romaine lettuce and oyster digestive glands were artificially contaminated with HAV, HuNoV GII.4 or HuNoV GI.7 at 102, 103 or 104 genome copies/sample. Virus was then recovered from the food matrix using the ISO method. Viral RNA extracted from frozen berry samples by the automated system was purified on a column for additional removal of RT-qPCR inhibitors. For fresh raspberry, oysters, and romaine lettuce, the two extraction platforms were deemed equivalent. For frozen raspberry, the automated platform appeared to be more efficient for viral recovery, particularly for HAV and HuNoV GI at lower concentrations. With frozen blackberries, the two platforms may be considered equivalent for all targeted viruses. However, the automated method led to less sample-associated inhibition of the PCR, 56.5 % of samples versus 95.0 % for the semi-automated. We thus found that the automated extraction can be performed easily by users while obtaining equivalent or even superior results to the ISO 15216-2:2019 method, and therefore appears to be suitable for routine sanitary monitoring in food processing and for tracing outbreaks of illness.

SARS-CoV-2 viral RNA detection using the novel CoVradar device associated with the CoVreader smartphone app.

The COVID-19 pandemic has highlighted the need for innovative approaches to its diagnosis. Here we present CoVradar, a novel and simple colorimetric method that combines nucleic acid analysis with dynamic chemical labeling (DCL) technology and the Spin-Tube device to detect SARS-CoV-2 RNA in saliva samples. The assay includes a fragmentation step to increase the number of RNA templates for analysis, using abasic peptide nucleic acid probes (DGL probes) immobilized to nylon membranes in a specific dot pattern to capture RNA fragments. Duplexes are formed by labeling complementary RNA fragments with biotinylated SMART bases, which act as templates for DCL. Signals are generated by recognizing biotin with streptavidin alkaline phosphatase and incubating with a chromogenic substrate to produce a blue precipitate. CoVradar results are analysed by CoVreader, a smartphone-based image processing system that can display and interpret the blotch pattern. CoVradar and CoVreader provide a unique molecular assay capable of detecting SARS-CoV-2 viral RNA without the need for extraction, preamplification, or pre-labeling steps, offering advantages in terms of time (∼3 h/test), cost (∼€1/test manufacturing cost) and simplicity (does not require large equipment). This solution is also promising for developing assays for other infectious diseases.

Molecular insights on potato yellow vein crinivirus infections in the highlands of Colombia.

Potato yellow vein virus (PYVV) was detected in potatoes grown in the Central highlands, north of Bogotá (~3000 m altitude), Colombia. At this altitude viral whitefly vectors are largely absent, but infection persists because of the use of uncertified tubers. Plants with typical PYVV-induced yellowing symptoms, as well as with atypical yellowing or non-symptomatic symptoms were sampled at three separate geographical locations. PYVV presence was assessed by RT-PCR, and several plants were subjected to high-throughput sequencing (HTS) of their small RNA (sRNA) populations. Complete or almost complete sequences of four PYVV isolates were thus reconstructed, all from symptomatic plants. Three viral isolates infected plants singly, while the fourth co-infected the plant together with a potyvirus. Relative proportions of sRNAs to each of the three crinivirus genomic RNAs were found to remain comparable among the four infections. Genomic regions were identified as hotspots of sRNA formation, or as regions that poorly induced sRNAs. Furthermore, PYVV titres in the mixed versus single infections remained comparable, indicating an absence of synergistic/antagonistic effects of the potyvirus on the accumulation of PYVV. Daughter plants raised in the greenhouse from tubers of the infected, field-sampled plants displayed mild PYVV infection symptoms that disappeared with time, demonstrating the occurrence of recovery and asymptomatic infection phenotypes in this pathosystem.

Evaluation of potential COVID-19 recurrence in patients with late repeat positive SARS-CoV-2 testing.

BACKGROUND: SARS-CoV-2 reinfection and reactivation has mostly been described in case reports. We therefore investigated the epidemiology of recurrent COVID-19 SARS-CoV-2. METHODS: Among patients testing positive for SARS-CoV-2 between March 11 and July 31, 2020 within an integrated healthcare system, we identified patients with a recurrent positive SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) assay ≥60 days after an initial positive test. To assign an overall likelihood of COVID-19 recurrence, we combined quantitative data from initial and recurrent positive RT-PCR cycle thresholds-a value inversely correlated with viral RNA burden- with a clinical recurrence likelihood assigned based on independent, standardized case review by two physicians. "Probable" or "possible" recurrence by clinical assessment was confirmed as the final recurrence likelihood only if a cycle threshold value obtained ≥60 days after initial testing was lower than its preceding cycle threshold or if the patient had an interval negative RT-PCR. RESULTS: Among 23,176 patients testing positive for SARS-CoV-2, 1,301 (5.6%) had at least one additional SARS-CoV-2 RT-PCRs assay ≥60 days later. Of 122 testing positive, 114 had sufficient data for evaluation. The median interval to the recurrent positive RT-PCR was 85.5 (IQR 74-107) days. After combining clinical and RT-PCR cycle threshold data, four patients (3.5%) met criteria for probable COVID-19 recurrence. All four exhibited symptoms at recurrence and three required a higher level of medical care compared to their initial diagnosis. After including six additional patients (5.3%) with possible recurrence, recurrence incidence was 4.3 (95% CI 2.1-7.9) cases per 10,000 COVID-19 patients. CONCLUSIONS: Only 0.04% of all COVID-19 patients in our health system experienced probable or possible recurrence; 90% of repeat positive SARS-CoV-2 RT-PCRs were not consistent with true recurrence. Our pragmatic approach combining clinical and quantitative RT-PCR data could aid assessment of COVID-19 reinfection or reactivation by clinicians and public health personnel.

Efficacy and safety assessment of severe COVID-19 patients with Chinese medicine: A retrospective case series study at early stage of the COVID-19 epidemic in Wuhan, China.

ETHNOPHARMACOLOGICAL RELEVANCE: The coronavirus disease 2019 (COVID-19) has formed a global pandemic since late 2019. Benefitting from the application experience of Chinese Medicine (CM) for influenza and SARS, CM has been used to save patients at the early stage of COVID-19 outbreak in China. AIM OF THE STUDY: In order to evaluate the efficacy and safety of CM, and compare with Western Medicine (WM) for COVID-19, we conducted a retrospective case series study based on the patients in Wuhan Jinyintan Hospital, Wuhan, China. METHODS: The inclusion and exclusion criteria of data extraction were set for this retrospective study. All patients who were admitted by the Wuhan Jinyintan Hospital between January 17th and February 25th 2020 were considered. In addition, patients enrolled met the severe defined by the guidelines released by the National Health Commission of the People's Republic of China. In these cases included in the study, CM or WM treatment was selected according to the wishes of the patients at the beginning of hospitalization. The patients in CM group were treated with Huashi Baidu granule (137 g po, bid) combined with the injections of Xiyanping (100 mg iv, bid), Xuebijing (100 ml iv, bid) and Shenmai (60 ml iv, qd) according to the syndrome of epidemic toxin blocking the lung in the theory of Traditional Chinese Medicine. The WM group received antiviral therapy (including abidor capsule 0.2 g po, tid; Lopinavir-Ritonavir tablets, 500 mg po, bid), antibiotics (such as cefoperazone 2 g iv, bid; moxifloxacin hydrochloride tablets, 0.4 g po, qd) or corticosteroid therapy (such as methylprednisolone succinate sodium 40 mg iv, qd; prednisone, 30 mg po, qd). In addition, patients in both groups received routine supportive treatment, including oxygen inhalation, symptomatic therapy, and/or human intravenous immunoglobulin, and/or serum albumin, and treatment for underlying diseases. The clinical outcomes were evaluated based on changes related with clinical manifestations, computer tomography (CT) scan images, and laboratory examinations before and after the treatment. RESULTS: 55 severe COVID-19 patients, with 23 in CM group and 32 in WM group, were included for analyzed. There was no case of death, being transferred to ICU, or receiving invasive mechanical ventilation in two groups during hospitalization. The median time of SARS-CoV-2 RNA clearance in CM and WM group were 12 days and 15.5 days respectively, the ratio of nucleic acid negative conversion of CM group at different follow-up time points was significantly higher than that of WM group (HR: 2.281, P = 0.018). Further, the chest CT imaging showed more widely lung lesion opacity absorbed in the CM group. The high sensitivity C-reactive protein and serum ferritin decreased significantly in the CM group (P<0.05). There was no significant difference in adverse events in terms of liver function and renal function between the two groups. CONCLUSION: Based on this retrospective analysis from Wuhan Jinyintan Hospital, CM has better effects in SARS-CoV-2 RNA clearance, promoting lung lesion opacity absorbed and reducing inflammation in severe COVID-19 patients, which is effective and safe therapy for treating severe COVID-19 and reducing mortality.

Prevalence of hepatitis B and C virus co-infection in HIV positive patients attending a health institution in southeast Nigeria.

BACKGROUND: The health of people living with HIV/AIDS becomes progressively worse when co-infected with hepatitis B virus (HBV) and hepatitis C virus (HCV), resulting in shortened life span. The modes of transmission of HIV, HBV and HCV are similar. OBJECTIVE: To determine the prevalence of HBV and HCV co-infection in HIV patients. METHOD: This was a retrospective study of serology test results for hepatitis B surface antigen (HBsAg) and antibodies to HCV (anti-HCV) of HIV positive patients registered from 2008-2013 (6years) at the University of Nigeria Teaching Hospital. Adult patients with confirmed HIV seropositivity were included. Ethical approval was obtained and confidentiality of the patient information was maintained. Laboratory records were reviewed to obtain HBsAg, anti-HCV, and CD4 T-lymphocyte results. Prevalence was determined by the number of positive results over total number of patients tested. Chi-square test was used to determine relationships and p<0.05 was considered to be statistically significant. RESULTS: 4663 HIV patient records were included comprising 3024 (65%) females and 1639 (35%) males. Serology results showed 365/4663 (7.8%) tested HBsAg-positive only; 219/4663 (4.7%) tested anti-HCV-positive only; and 27/4663 (0.58%) tested both HBsAg and anti-HCV-positive. Correlation of age and sex were statistically significant with HBV and HCV (p<0.05) but not CD4 count (p>0.05). CONCLUSION: HBV co-infection was more prevalent than HCV, and triple infection was also observed. Screening for these viral infections in the HIV population is necessary for early identification to enable appropriate, holistic management of these patients.

The Mechanism and Clinical Outcome of patients with Corona Virus Disease 2019 Whose Nucleic Acid Test has changed from negative to positive, and the therapeutic efficacy of Favipiravir: A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: A variety of possible mechanisms can make the nucleic acid test of patients who meet the discharge conditions positive again, including reinfection, reactivation of the original virus, lack of strict discharge criteria, new infection, and so on. Different reasons will correspond to different prevention and control measures. We will enroll patients who are discharged after treatment, whose nucleic acid test has changed from negative to positive during the screening visit, regardless of the severity of the symptoms, to investigate the mechanism, clinical outcome and therapeutic efficacy with Favipiravir patients with Corona virus Disease 2019. Favipiravir is an anti-viral agent that selectively and potently inhibits the RNA-dependent RNA polymerase, it has been used for treatment of some life-threatening infections such as Ebola virus, Lassa virus and rabies. Its therapeutic efficacy has been proven in these diseases. TRIAL DESIGN: This is a multi-center, two arm, open label, parallel group, randomized controlled trial. PARTICIPANTS: Eligibility criteria: Inclusion criteria: 1.Adults 18 to 80 years, male or female.2.After the first diagnosis and treatment of COVID-19, the nucleic acid test of respiratory specimens such as sputum or nasopharyngeal swabs, has been negative for two consecutive times (sampling time interval of at least 24 hours), in accordance with the COVID-19's diagnosis and treatment Plan (7th Edition), discharged.3.During screening visit (follow-up after discharge), The nucleic acid test of COVID-19 is positive in any one of the following samples: sputum, throat swabs, blood, feces or other specimens. Regardless of whether or not they had symptoms and the severity of symptoms.4.Volunteer to participate in the research and sign the Informed Consent Form. EXCLUSION CRITERIA: 1.Allergic to Favipiravjr;2.Pregnant or lactating women3.Uncontrolled diseases of the blood and cardiovascular system, liver or kidney.4.History of mental disorders, drug abuse or dependence;5.Researchers consider it inappropriate for adults to participate;6.Participating in other clinical studies. Loss to Follow up: Cases that do not complete the clinical trial program will be regarded as lost to follow up. Including the withdrawal of patients by themselves (such as poor compliance, etc.), or the withdrawal of patients ordered by the researcher (those who need other drugs which affect the judgment of the curative effect, and those who need to stop taking drugs for severe adverse events) Study setting: The participating hospitals are some of the designated hospitals that have been or may be admitting patients who meet the eligibility criteria, mainly in Hubei, Shenzhen, Anhui and Beijing. Participants will be recruited from these 15 hospitals: Wuhan Pulmonary Hospital, Hubei; Jinyintan Hospital of Wuhan, Hubei; Ezhou Central Hospital, Hubei; The Second People's Hospital of Fuyang, Anhui; The First Affiliated Hospital of USTC, Anhui; Beijing Youan Hospital, Beijing; Capital Medical University Beijing Institute of Hepatology, Beijing; Ezhou Hospital of Traditional Chinese Medicine, Hubei; Zhongnan Hospital of Wuhan University, Hubei; The Fifth Hospital of ShiJiazhuang, Hebei; Jinan Infectious Diseases Hospital, Shandong; Public Health Clinical Center of Chengdu, Sichuan; Wuxi No.5 People's Hospital, Jiangsu; The Third People's Hospital of Shenzhen, Guangdong; The First Affiliated Hospital of Bengfu Medical College, AnHui. INTERVENTION AND COMPARATOR: Favipiravir group (experimental): Favipiravir 1600mg each dose, twice a day on the 1st day; 600mg each dose, twice a day from the 2nd to the 7th day, Oral administration, the maximum number of days taken will be no more than 14 days plus routine treatment for COVID-19. Regular treatment group (control): Treatments other than Antiviral drugs can be given. Routine treatment for patients with the corona virus will be administered, this includes oxygen therapy, drugs that reduced phlegm and relieve cough, including thymosin, proprietary Chinese medicine, etc. MAIN OUTCOMES: Primary Outcome Measures: Viral nucleic acid test negative [Time Frame: 5 months]: Subjects who tested negative for nucleic acid from sputum or nasopharyngeal swabs for two consecutive times (sampling time interval of at least 24 hours). SECONDARY OUTCOME MEASURES: Clinical cure [Time Frame: 5 months]: 1.Body temperature returned to normal for more than 3 days;2.Lung image improved.3.Clinical manifestation improved;4.The viral nucleic acid test of respiratory specimens was negative for two consecutive times (sampling time interval of at least 24 hours). RANDOMIZATION: The central randomization system (Interactive Web Response Management System), will be used to randomly divide the subjects into the experimental group and the control group according to the ratio of 2:1. In this study, block randomization will be used, in blocks of 6. BLINDING (MASKING): This is an open label trial. Trial participants, investigators, care givers, outcome assessors, and date analysts are not blinded to group assignment. NUMBERS TO BE RANDOMISED: 210 patients are expected to be enrolled and allocated according to the ratio of 2 (Favipiravir group, n=140): 1(regular treatment group, n=70). TRIAL STATUS: Protocol version number 3.0, 10th April 2020 First Patient, first visit 17th March 2020; recruitment end date anticipated June 1, 2020. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04333589, April 3, 2020. Registered April 3, 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

RT-qPCR Detection of Low-Copy HIV RNA with Yin-Yang Probes.

Accurate monitoring of low levels of viral load (the number of viral particles per milliliter of plasma) in HIV-infected patients is important in terms of evaluation of the progress of antiretroviral therapy. The general approach for detection of low copy HIV RNA is reverse transcription combined with quantitative real-time PCR based on fluorescence detection. The selection of primers and the structure of fluorogenic oligonucleotide probes are crucial for sensitivity and accuracy of the assay. In this chapter, we report the RT-qPCR protocol for detection of low copy HIV RNA using double stranded Yin-Yang DNA probes containing identical fluorescent dyes on each strand of the probe. Dye residues attached to the 3'-end of an oligonucleotide and 5'-end of the complementary oligonucleotide form a self-quenched aggregate in a Yin-Yang duplex probe, and display fluorescence light up upon probe strand displacement with the target sequence amplified in the course of PCR. Among several fluorescent dyes tested (R6G, ROX, Cy5) the ROX labeled Yin-Yang probes showed better fluorescence increase and lower Ct values. All the homo Yin-Yang probes were superior to corresponding dye-quencher probes and allowed reliable detection of 10-10,000 copies of HIV RNA per mL.

Optimization of an isothermal recombinase polymerase amplification method for real-time detection of Potato virus Y O and N types in potato.

Potato virus Y (PVY) is a global challenge for potato production and the leading cause of seed crop downgrading and rejection for certification. Accurate and timely diagnosis is key to effective control of PVY. Here we optimized the isothermal recombinase polymerase amplification (RPA) for accurate detection of different PVY O and N types that were tested, present in different tissues of potato plants including tubers with a primer set that specifically targets the highly conserved pipo region within the viral genome. Combined with a simplified preparation of the template by tissue homogenization, we established a rapid RPA procedure, which can allow real time detection in less than 10 min with a fluorescent probe. Specificity of the reaction was determined by the lack of cross-reactivity with other common potato viruses. Although RPA reagents remain more expensive than PCR reagents, RPA technology is equivalent in that results can be visualized by gel electrophoresis or with a fluorescent probe with greater sensitivity; and it is superior to the common PCR-based assay in its versatility, speed, and lack of need for a highly purified RNA template.

Development of CdSe⁻ZnO Flower-Rod Core-Shell Structure Based Photoelectrochemical Biosensor for Detection of Norovirous RNA.

In this work, the CdSe⁻ZnO flower-rod core-shell structure (CSZFRs) was prepared by ion-exchange method. The surface of CSZFRs was modified by 3-mercaptopropionic acid (MPA), and then the DNA probe was immobilized on the surface via chemical bond between -NH2 of DNA probe and -COOH of MPA. Finally, the target norovirous (NV) RNA was combined with the probe according to the principle of complementary base pairing, resulting in a decrease of the photocurrent. The results show that the absorbance spectrum of visible light is enhanced for CSZFRs compared with pure ZnO. Under visible light irradiation, the photocurrent of CSZFRs is up to 0.1 mA, which can improve the sensitivity of the photoelectrochemical (PEC) biosensor. In the measurement range of 0⁻5.10 nM, the measured concentrations (c) have a good linear relationship with the output photocurrent of the biosensor. The linear regression equation is expressed as I = 0.03256 - 0.0033c (R² = 0.99, S/N = 3) with a detection limit of 0.50 nM. Therefore, this work realizes a rapid and sensitive method for the detection of NV RNA.

In vitro screening of selected antiviral drugs against betanodavirus.

The inhibitory effects of ammonium chloride (NH4Cl) and chlorpromazine hydrochloride on betanodavirus were evaluated on Sahul Indian sea bass kidney (SISK) cell line. The cytotoxicity of different concentrations of NH4Cl (0.1 mM, 1 mM, 10 mM, 100 mM and 500 mM) and chlorpromazine hydrochloride (1 µM, 10 µM, 100 µM, 200 µM and 500 µM) were assessed in SISK cells using different cytotoxic assays. Among the selected concentrations, 0.1 mM, 1 mM and 10 mM of NH4Cl and chlorpromazine hydrochloride at the dose of 1 µM, 10 µM and 100 µM were found to be non-toxic to the SISK cell line and same were chosen for the trials against nodavirus. The presence of nodavirus in the infected cells was confirmed by cytopathic effect (CPE) and RT-PCR (Reverse transcriptase PCR). NH4Cl of 1 mM and 10 mM, and chlorpromazine hydrochloride of 10 µM and 100 µM could successfully inhibit betanodavirus infection in SISK cells, which was confirmed by indirect ELISA and real-time PCR analysis. The result further suggested that the chlorpromazine hydrochloride drug could be more effective in inhibiting the betanodavirus with much lower dose than NH4Cl which was more effective at a higher dose. The present study thus suggested that NH4Cl and chlorpromazine hydrochloride drugs could be successfully used for controlling the nodavirus infection in aquaculture.

Time-Course Microarray Analysis Reveals Differences between Transcriptional Changes in Tomato Leaves Triggered by Mild and Severe Variants of Potato Spindle Tuber Viroid.

Viroids are small non-capsidated non-coding RNA replicons that utilize host factors for efficient propagation and spread through the entire plant. They can incite specific disease symptoms in susceptible plants. To better understand viroid-plant interactions, we employed microarray analysis to observe the changes of gene expression in "Rutgers" tomato leaves in response to the mild (M) and severe (S23) variants of potato spindle tuber viroid (PSTVd). The changes were analyzed over a time course of viroid infection development: (i) the pre-symptomatic stage; (ii) early symptoms; (iii) full spectrum of symptoms and (iv) the so-called 'recovery' stage, when stem regrowth was observed in severely affected plants. Gene expression profiles differed depending on stage of infection and variant. In S23-infected plants, the expression of over 3000 genes was affected, while M-infected plants showed 3-fold fewer differentially expressed genes, only 20% of which were specific to the M variant. The differentially expressed genes included many genes related to stress; defense; hormone metabolism and signaling; photosynthesis and chloroplasts; cell wall; RNA regulation, processing and binding; protein metabolism and modification and others. The expression levels of several genes were confirmed by nCounter analysis.

The Use of High-Throughput Sequencing for the Study and Diagnosis of Plant Viruses and Viroids in Pollen.

This protocol details the wet lab preparation, extraction of fruit pollen samples, and analysis of the sequencing data following Illumina NextSeq small and total RNA sequencing. The protocol was developed for virus and viroid detection using NGS sequencing and was based on the results of a comparison between different extraction methods followed by yield, RNA purity, and integrity assessment. Moreover, the advantage of an additional ribosomal (r)RNA depletion step to the total RNA extraction protocol was evaluated. The smallRNA procedure is the preferred method of choice. If the total RNA protocol is chosen, the use of the mirVana kit followed by an rRNA depletion step is the best option. The library preparation and sequencing steps were outsourced. As a final step in the data analysis, the VirusDetect software was used to detect the viruses and viroids in the pollen samples.

Mechanism of Action of the Anti-Influenza Virus Active Kampo (Traditional Japanese Herbal) Medicine, Hochuekkito.

When the Kampo medicine, Hochuekkito (Hochu), was administered to normal mice for 2 weeks, influenza virus titer was reduced. The mechanism of action of Hochu was examined using the plaque assay method. It was suggested that Hochu may either obstruct the first stage of the infection process (adsorption and entry) or may directly target viral particles. Using the plaque assay method, these 2 modes of action could not be differentiated. Virus RNA in the infected cell was verified by quantitative real-time polymerase chain reaction. An equal inhibition effect was obtained when Hochu was preprocessed for normal cells and when they were made to act simultaneously with virus adsorption. The viral load at the cell surface following UV irradiation was higher in the Hochu-administered group as compared with that of the control. Moreover, the affinity of Hochu for the influenza virus was hundred times higher than its affinity for the host cell. The effect of entry obstruction by Hochu was observed via image analysis, where the amount of virus nucleocapsid protein (NP) invading the cell was visualized with FITC-labeled NP antibody. Hochu does not seem to have an effect on nucleic acid synthesis, viral release from infected cells, and on the subsequent second round of infection. In conclusion, Hochu binds to viral particles and forms complexes that can obstruct the entry of influenza virus into cells.

[Primary central nervous system diffuse large B cell lymphoma: a clinicopathologic and molecular study].

Objective: To investigate clinicopathologic characteristics, immunophenotype and EB virus-related molecular genetic alterations in primary central nervous system diffuse large B cell lymphoma (DLBCL) along with correlation with clinical prognosis. Methods: A total of 30 cases of primary central nervous system DLBCL were retrospectively studied by retrieving clinical data, histological evaluation and immunophenotyping by EnVision two steps methods. The expression of EBER mRNA was detected by in situ hybridization and bcl-2, bcl-6 and C-MYC gene abnormalities were analyzed by interphase fluorescence in situ hybridization. Results: The cases included 18 males and 12 females (sex ratio of 1.5∶1.0) with an age ranging from 24 to 78 years (average age of 52 years, the median age of 53 years). The single primary clinical presentation was focal neurologic deficits. Tumor locations were supratentorial (21 cases), subtentorial (7 cases), involving both locations in 2 cases. Diffuse growth pattern was observed with large lymphoid cells mostly resembling centroblasts with abundant basophilic cytoplasm with oval to round, vesicular nuclei containing fine chromatin. An angiocentric and angiodestructive growth pattern was also present. Other features included perivascular space invasion. Immunohistochemical staining using a panel of CD10, bcl-6 and MUM1, six cases were germinal center-like (GCB) and 24 cases were non-germinal central-like (non-GCB). The positive rates of bcl-2, bcl-6 and C-MYC were 53.3% (16/30), 80.0% (24/30) and 20.0% (6/30), respectively. Genetic alterations were detected by FISH and the gene arrangement rates of bcl-2, bcl-6 and C-MYC were 3.3% (1/30), 16.7% (5/30) and 3.3% (1/30), respectively. There were 19 cases in stage 0-1 disease and 11 cases had stage 2-3 disease. Postoperative follow-up for average 13.6 months showed the median survival of 10 months, one-year survival of 46.7% and 16 patients died within a year. Conclusions: The clinical prognosis of primary central nerve system DLBCL depends on age, clinical performence status score, IPI score, immune classification and treatment. Patients typically progress rapidly with the high mortality within one year of diagnosis. Surgical resection combined with high-dose methotrexate or cytarabine chemotherapy offer the best treatment option.

Efficacy of traditional Chinese medicine for cooling blood and eliminating toxins and strengthening and purifying spleen in the treatment of refractory hepatitis C.

This study was designed to observe the comprehensive efficacy of traditional Chinese medicine (TCM) combined with interferon in the treatment of chronic hepatitis C (CHC), compare this combined therapy with interferon therapy alone and investigate its possible mechanism to provide a basis for the development of an integrated traditional Chinese and Western medicine for the treatment of CHC. According to patient contraindications for antiviral treatment, patients who were suitable for interferon therapy and willing to use TCM were enrolled as combined traditional Chinese and Western medicine group, and 21 CHC patients were selected as Western medicine control group; patients who had contraindications for antiviral treatment were included in the TCM group. The three groups of patients were all diagnosed with positive hepatitis C virus - ribose nucleic acid (HCV-RNA). The treatment course lasted for one year and the patients were followed up for 12 months. Patients’ demographic data, course of disease, chronic liver disease questionnaire (CLDQ), genetic typing, biochemical indexes, HCV-RNA and side effects were compared between the groups. The efficacy, incidence of side effects and improvement in quality of life were analyzed in each group. Results showed that the combination of TCM and interferon could protect liver, reduce side effects and also improve quality of life of the patients, while the antiviral activity of TCM alone was not obvious.

Diarrheagenic pathogens in adults attending a hospital in Singapore.

BACKGROUND: Singapore's diarrhoeal notification system is based on specific pathogens. Official data may thus be skewed towards notifiable diseases. Limited information is available on the profiles of aetiological agents responsible for acute gastroenteritis (AGE) cases, especially among the adult population. To understand the frequency and distribution of potential causative agents of diarrheal disease in Singapore, we screened adults' stool samples collected from a large public hospital. METHODS: The stool samples were screened for 18 diarrheagenic pathogens using a combination of commercial multiplex polymerase chain reaction (PCR), in-house singleplex PCR and immunochromatographic assays. One hundred adult faecal samples that were collected from October 2013 to January 2014 for routine diagnostic purposes and submitted for culture at Tan Tock Seng Hospital, Singapore were used. RESULTS: Pathogens were detected in 32% of the samples. The predominant organisms encountered were norovirus genogroup II (11%), Aeromonas spp. (9%) and Campylobacter spp. (5%). One sample was positive for both verocytotoxigenic E. coli (VTEC) and E. coli O157:H7. Two other samples were positive for VTEC only, and one other sample was positive for E. coli O157:H7 only. Astrovirus, C. perfringens, Shigella spp. and toxigenic C. difficile were each detected in 2% of the samples. Cryptosporidium parvum, Giardia lamblia, group A rotavirus, Salmonella spp. and Vibrio spp. were each detected in 1% of the samples. No L. monocytogenes, Y. enterocolitica, enteric adenovirus, or norovirus genogroup I were detected. CONCLUSION: Our preliminary findings suggest that pathogens causing non-notifiable diseases might have contributed considerably to the adult hospitalised AGE cases. However, as the samples were from an adult hospital, the data obtained may not be representative of the whole community. Thus, a larger study to collect clinical samples and risk exposure data from primary healthcare clinics and children hospital is planned for, to gain a more holistic perspective on the epidemiology of AGE in Singapore. A larger study may also offer valuable insights for improving the approach of microbiological surveillance of food, as well as strategizing inspection efforts along the food supply chain by public health authorities.

Human liver chimeric mice as a new model of chronic hepatitis E virus infection and preclinical drug evaluation.

BACKGROUND & AIMS: Hepatitis E virus (HEV) is a major cause of acute hepatitis as well as chronic infection in immunocompromised individuals; however, in vivo infection models are limited. The aim of this study was to establish a small animal model to improve our understanding of HEV replication mechanisms and permit the development of effective therapeutics. METHODS: UPA/SCID/beige mice repopulated with primary human hepatocytes were used for infection experiments with HEV genotype (GT) 1 and 3. Virological parameters were determined at the serological and intrahepatic level by real time PCR, immunohistochemistry and RNA in situ hybridization. RESULTS: Establishment of HEV infection was achieved after intravenous injection of stool-derived virions and following co-housing with HEV-infected animals but not via inoculation of serum-derived HEV. GT 1 infection resulted in a rapid rise of viremia and high stable titres in serum, liver, bile and faeces of infected mice for more than 25 weeks. In contrast, viremia in GT 3 infected mice developed more slowly and displayed lower titres in all analysed tissues as compared to GT 1. HEV-infected human hepatocytes could be visualized using HEV ORF2 and ORF3 specific antibodies and HEV RNA in situ hybridization probes. Finally, six-week administration of ribavirin led to a strong reduction of viral replication in the serum and liver of GT 1 infected mice. CONCLUSION: We established an efficient model of HEV infection to test the efficacy of antiviral agents and to exploit mechanisms of HEV replication and interaction with human hepatocytes in vivo.