Transcription-polymerase Chain Reaction Assay Research Articles

E型肝炎ウイルスの遺伝子型3および4型株迅速鑑別のためのマルチプレックスreal-time RT-PCR法における検出試薬の比較検討

E型肝炎ウイルスの遺伝子型3および4型株迅速鑑別のためのマルチプレックスreal-time RT-PCR法における検出試薬の比較検討

The deployment of mobile diagnostic laboratories for Ebola virus disease diagnostics in Sierra Leone and Guinea.

BackgroundEbola virus emerged in West Africa in December 2013. The ease of mobility, porous borders, and lack of public health infrastructure led to the largest Ebola virus disease (EVD) outbreak to date.InterventionThe 2013 EVD outbreak signalled the need for laboratory diagnostic capabilities in areas without strong public health systems. As part of the United States’ Department of Defense response, MRIGlobal was contracted to design, fabricate, equip, deploy, and operate two mobile diagnostic laboratories (MDLs). The first laboratory analysed blood samples from patients in an adjacent Ebola Treatment Centre (ETC) and buccal swabs from the deceased in the community in Moyamba, Sierra Leone. The second laboratory was deployed to support an ETC in Conakry, Guinea. The Department of Defense provided real-time quantitative reverse transcription polymerase chain reaction assays that were deployed and validated on-site.Lessons LearntPrompt and accurate molecular diagnostics reduced sample turn-around times from over 24 h to under 4 h. Experienced laboratory staff tested up to 110 samples per day and on-site engineering proved necessary for MDL setup and operation. As the Ebola response slowed, the sustainment of the MDLs’ operations was prioritised, including staff training and the transition of the MDLs to local governments. Training programmes for local staff were prepared in Sierra Leone and Guinea.RecommendationsThe MRIGlobal MDL team significantly contributed to establishing increased laboratory capacity during the EVD outbreak in West Africa. Using the MDLs for molecular diagnosis is highly recommended until more sustainable solutions can be provided.

Development of a one-step RT-qPCR detection assay for the newly described citrus viroid VII

An apscaviroid, tentatively named citrus viroid VII (CVd-VII), was recently discovered in citrus in Australia. A diagnostic assay using real-time reverse transcription polymerase chain reaction was developed and validated to detect the viroid in citrus plants. The assay showed a high level of sensitivity, reliably detecting 2000 plasmid copies per reaction, while down to 20 plasmid copies per reaction were occasionally detected. The assay showed high specificity, producing no false positives or cross-reactivity with a range of other citrus graft-transmissible pathogens, including viroids, viruses and bacteria. The real-time assay was also found to be more sensitive than the available end-point reverse transcription polymerase chain reaction assay by a factor of 100,000 and could be a useful tool for the rapid detection of CVd-VII in diagnostic and research environments.

Application of dried blood spot sample pooling strategies for Plasmodium 18S rRNA biomarker testing to facilitate identification of infected persons in large-scale epidemiological studies

BackgroundPlasmodium 18S rRNA is a sensitive biomarker for detecting Plasmodium infection in human blood. Dried blood spots (DBS) are a practical sample type for malaria field studies to collect, store, and transport large quantities of blood samples for diagnostic testing. Pooled testing is a common way to reduce reagent costs and labour. This study examined performance of the Plasmodium 18S rRNA biomarker assay for DBS, improved assay sensitivity for pooled samples, and created graphical user interface (GUI) programmes for facilitating optimal pooling.MethodsDBS samples of varied parasite densities from clinical specimens, Plasmodium falciparum in vitro culture, and P. falciparum Armored RNA® were tested using the Plasmodium 18S rRNA quantitative triplex reverse transcription polymerase chain reaction (qRT-PCR) assay and a simplified duplex assay. DBS sample precision, linearity, limit of detection (LoD) and stability at varied storage temperatures were evaluated. Novel GUIs were created to model two-stage hierarchy, square matrix, and three-stage hierarchy pooling strategies with samples of varying positivity rates and estimated test counts. Seventy-eight DBS samples from persons residing in endemic regions with sub-patent infections were tested in pools and deconvoluted to identify positive cases.ResultsAssay performance showed linearity for DBS from 4 × 107 to 5 × 102 parasites/mL with strong correlation to liquid blood samples (r2 > 0.96). There was a minor quantitative reduction in DBS rRNA copies/mL compared to liquid blood samples. Analytical sensitivity for DBS was estimated 5.3 log copies 18S rRNA/mL blood (28 estimated parasites/mL). Properly preserved DBS demonstrated minimal degradation of 18S rRNA when stored at ambient temperatures for one month. A simplified duplex qRT-PCR assay omitting the human mRNA target showed improved analytical sensitivity, 1 parasite/mL blood, and was optimized for pooling. Optimal pooling sizes varied depending on prevalence. A pilot DBS study of the two-stage hierarchy pooling scheme corroborated results previously determined by testing individual DBS.ConclusionsThe Plasmodium 18S rRNA biomarker assay can be applied to DBS collected in field studies. The simplified Plasmodium qRT-PCR assay and GUIs have been established to provide efficient means to test large quantities of DBS samples.

Evaluation of co-cultured spermatogonial stem cells encapsulated in alginate hydrogel with Sertoli cells and their transplantation into azoospermic mice.

An in vitro spermatogonial stem cell (SSC) culture can serve as an effective technique to study spermatogenesis and treatment for male infertility. In this research, we compared the effect of a three-dimensional alginate hydrogel with Sertoli cells in a 3D culture and co-cultured Sertoli cells. After harvest of SSCs from neonatal mice testes, the SSCs were divided into two groups: SSCs on a 3D alginate hydrogel with Sertoli cells and a co-culture of SSCs with Sertoli cells for 1 month. The samples were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays and bromodeoxyuridine (BrdU) tracing, haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining after transplantation into an azoospermic testis mouse. The 3D group showed rapid cell proliferation and numerous colonies compared with the co-culture group. Molecular assessment showed significantly increased integrin alpha-6, integrin beta-1, Nanog, Plzf, Thy-1, Oct4 and Bcl2 expression levels in the 3D group and decreased expression levels of P53, Fas, and Bax. BrdU tracing, and H&E and PAS staining results indicated that the hydrogel alginate improved spermatogenesis after transplantation in vivo. This finding suggested that cultivation of SSCs on alginate hydrogel with Sertoli cells in a 3D culture can lead to efficient proliferation and maintenance of SSC stemness and enhance the efficiency of SSC transplantation.

Why has permanent control of cassava brown streak disease in Sub-Saharan Africa remained a dream since the 1930s?

Effective control of ipomoviruses that cause cassava brown streak disease (CBSD) in Africa has remained problematic despite eight remarkable decades (1930-2021) of research efforts. Molecular mechanisms underlying resistance breakdown in genetically improved cassava are still unknown. The vast genetic diversity of cassava brown streak viruses, which is crucial for the improvement of routine reverse transcription polymerase chain reaction (RT-qPCR) assays in CBSD-endemic regions of Africa, is controversial and underrepresented. From a molecular epidemiology viewpoint, this review discusses the reasons for why permanent control of CBSD is difficult in the modern era, even with the presence of diverse in silico and omics tools, recombinant DNA, and high throughput next-generation sequencing technologies. Following an extensive nucleotide data search in the National Centre for Biotechnology Information (NCBI) database and a literature review in PubMed and Scopus, we report that genomic data of 87.62% (474/541) strains of cassava brown streak virus are missing due to poor sequencing capacity in Africa. The evolution dynamics of viral virulence and pathogenicity has not yet been fully explored from the available 67 (12.38%) genomic sequences, owing to poor bioinformatics capacity. Tanzania and Zambia have the highest and lowest disease inoculum pressure, correspondingly. Knowledge gaps in molecular biology and the overall molecular pathogenesis of CBSD viruses impede effective disease control in Africa. Recommendations for possible solutions to the research questions, controversies, and hypotheses raised in this study serve as a roadmap for the invention of more effective CBSD control methods.

Analysis of COVID-19 in Professionals Working in Geriatric Environment: Multicenter Prospective Study.

Healthcare workers (HCWs) are exposed to a higher risk of coronavirus disease (COVID-19) contamination. This prospective multicenter study describes the characteristics of HCWs tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) while working in a geriatric environment. We also compared HCWs with a positive reverse transcription polymerase chain reaction (RTPCR) assay (RTPCR+ group) and those with a negative test result (RTPCR− group). Between 15/5/2020 and 15/9/2020, 258 HCWs, employed in the acute geriatric unit (AGU), geriatric rehabilitation unit (GRU) or nursing home of three hospitals in Burgundy (France) were invited to complete an online survey. Among the 171 respondents, 83 participants, with mean age 42 years and 87.9% female, were tested for SARS-CoV-2 infection. Among these 83 participants, COVID-19 was confirmed in 38 cases (RTPCR+ group) of which 36 were symptomatic, and the RTPCR assay was negative in 45 cases (RTPCR− group) of which 20 participants were symptomatic. A total of 22.9% (of 83) had comorbidities, 21.7% were active smokers, and 65.1% had received the flu vaccine. A total of 37.3% worked in AGU, 19.3% in GRU and 16.9% in nursing homes. The most common symptom described was headache (23.2%), followed by fatigue or cough (12.5% each), and fever or myalgia (10.7% each). There were more participants with normal body mass index (p = 0.03) in the RTPCR+ group. In contrast, there were more users of non-steroidal anti-inflammatory drugs (p = 0.01), active smokers (p = 0.03) and flu vaccinated (p = 0.01) in the RTPCR− group. No difference was found between the two groups for the type of work (p = 0.20 for physicians and p = 0.18 for nurses). However, acquiring COVID-19 was significantly associated with working in AGU (p < 0.001) and nursing homes (p = 0.001). There were significantly more users of surgical masks (p = 0.035) in the RTPCR+ group and more filtering facepiece-2 mask users (p = 0.016) in the RTPCR− group. Our results reflect the first six months of the COVID-19 pandemic in France. Further studies are needed to evaluate and track the risks and consequences of COVID-19 in HCWs.

Influenza vaccine effectiveness within prospective cohorts of healthcare personnel in Israel and Peru 2016–2019

BackgroundThere are limited data on influenza vaccine effectiveness (IVE) in preventing laboratory-confirmed influenza illness among healthcare personnel (HCP). MethodsHCP with direct patient contact working full-time in hospitals were followed during three influenza seasons in Israel (2016–2017 to 2018–2019) and Peru (2016 to 2018). Trivalent influenza vaccines were available at all sites, except during 2018–2019 when Israel used quadrivalent vaccines; vaccination was documented by electronic medical records, vaccine registries, and/or self-report (for vaccinations outside the hospital). Twice-weekly active surveillance identified acute respiratory symptoms or febrile illness (ARFI); self-collected respiratory specimens were tested by real-time reverse transcription polymerase chain reaction (PCR) assay. IVE was 100 × 1-hazard ratio (adjusted for sex, age, occupation, and hospital). ResultsAmong 5,489 HCP who contributed 10,041 person-seasons, influenza vaccination coverage was 47% in Israel and 32% in Peru. Of 3,056 ARFIs in Israel and 3,538 in Peru, A or B influenza virus infections were identified in 205 (7%) in Israel and 87 (2.5%) in Peru. IVE against all viruses across seasons was 1% (95% confidence interval [CI] = −30%, 25%) in Israel and 12% (95% CI = −61%, 52%) in Peru. ConclusionEstimates of IVE were null using person-time models during six study seasons in Israel and Peru.

LINC00852 promotes the proliferation and invasion of ovarian cancer cells by competitively binding with miR-140-3p to regulate AGTR1 expression

BackgroundDysregulation of long non-coding RNAs (lncRNAs) has been identified in ovarian cancer. However, the expression and biological functions of LINC00852 in ovarian cancer are not understood.MethodsThe expressions of LINC00852, miR-140-3p and AGTR1 mRNA in ovarian cancer tissues and cells were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. Gain- and loss-of-function assays were performed to explore the biological functions of LINC00852 and miR-140-3p in the progression of ovarian cancer in vitro. The bindings between LINC00852 and miR-140-3p were confirmed by luciferase reporter gene assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay.ResultsWe found that LINC00852 expression was significantly up-regulated in ovarian cancer tissues and cells, whereas miR-140-3p expression was significantly down-regulated in ovarian cancer tissues. Functionally, LINC00852 knockdown inhibited the viability, proliferation and invasion of ovarian cancer cells, and promoted the apoptosis of ovarian cancer cells. Further investigation showed that LINC00852 interacted with miR-140-3p, and miR-140-3p overexpression suppressed the viability, proliferation and invasion of ovarian cancer cells. In addition, miR-140-3p interacted with AGTR1 and negatively regulated its level in ovarian cancer cells. Mechanistically, we found that LINC00852 acted as a ceRNA of miR-140-3p to promote AGTR1 expression and activate MEK/ERK/STAT3 pathway. Finally, LINC00852 knockdown inhibited the growth and invasion ovarian cancer in vivo.ConclusionLINC00852/miR-140-3p/AGTR1 is an important pathway to promote the proliferation and invasion of ovarian cancer.

Real-time Reverse Transcription PCR-based SARS-CoV-2 Detection in Khwaja Yunus Ali Medical College Hospital, Enayetpur, Sirajganj, Bangladesh

Background: Coronavirus disease 2019 (COVID-19) is a global pandemic declared on 11 March 2020 by World Health Organization (WHO). It is a highly pathogenic viral infection caused by SARS-CoV-2 which has caused global health concern. The emergence of SARS-CoV-2 infection has triggered a global need for accurate diagnostic assays. Detection of viral RNA in clinical specimens is the hallmark of diagnosis. Objectives: The aim of present study was to determine the prevalence of SARS-CoV-2 infection in some districts of North Bengal area of Bangladesh according to age, sex and area distribution. Materials and Methods: This cross sectional descriptive study was carried out in the Department of Laboratory Services, KYAMCH, Enayetpur, Sirajganj, Bangladesh. Real-Time reverse transcription Polymerase chain reaction assay was used for detection of virus. Results: A total of 1760 cases in the month from March to May 2021 were tested, among which 310 (17.6%) cases were detected positive for SARS-CoV-2. Out of 310 positive cases 211 (68.06%) were male and 99 (31.94%) were female. Maximum positive cases were from Sirajganj followed by Pabna, Natore, Naogaon, Bogura, Gaibandha districts and some parts of Rajshahi district respectively. Conclusion: This is the first study for genetic detection of SARS-CoV-2 done in KYAMCH, Enayetpur, Sirajganj. Strategies of using rRT-PCR for detection of SARS-CoV-2 may facilitate early and confirmatory diagnosis of COVID-19 KYAMC Journal.2021;12(02): 59-65

Integration of traditional Chinese medicine and nibble debridement and dressing method reduces thrombosis and inflammatory response in the treatment of thromboangiitis obliterans.

BackgroundThromboangiitis obliterans (TAO), also known as Buerger’s disease, is an occlusive arterial disease; however, the pathogenesis of TAO is still unclear. Research has shown that traditional Chinese medicine (TCM) has significant advantages in the treatment of TAO. Our purpose was to explore the underlying roles of TCM in combination with nibble debridement and dressing method (NDDM) in a TAO rat model.MethodsWe administered rats with 10 mg/mL sodium laurate to establish a TAO model, and then the TAO model rats were treated with notoginseng powder (NP), maifusheng (MFS), or the combination of NP or MFS and NDDM. Gangrene classification and blood rheology were evaluated; the pathological characteristics of rat limbs were examined by hematoxylin and eosin (H&E) staining and Masson staining; and cluster of differentiation 3+ (CD3+) and cluster of differentiation 20+ (CD20+) levels were measured by immunohistochemistry (IHC) and flow cytometry. In addition, inflammation-associated cytokines were analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blot, and enzyme-linked immunosorbent assay (ELISA).ResultsIntegration of NP or MFS and NDDM dramatically reduced the gangrene classification and affected blood rheology parameters of TAO model rats compared with NP and MFS alone. Meanwhile, NP or MFS in combination with NDDM decreased CD3+CD20+ T cells, reduced thrombosis and inflammatory cell infiltration, and dramatically decreased the levels of inflammation-associated cytokines.ConclusionsOur results suggested that integration of NP or MFS and NDDM could relieve the symptoms of TAO model rats induced by sodium laurate, which might provide a new management strategy for TAO.

Serotype and genotype diversity of dengue viruses circulating in India: a multi-centre retrospective study involving the Virus Research Diagnostic Laboratory Network in 2018

A retrospective study was undertaken to investigate the circulating dengue virus (DENV) serotypes and genotypes in India in 2018. In total, 4963 samples referred to virus research diagnostic laboratories (n=21), the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV) and ICMR-NIV field units (n=2) for diagnosis of dengue in 2018 were tested using a real-time reverse transcription polymerase chain reaction assay for the presence of DENV serotypes. Representative samples were sequenced for the envelope (E) gene. Regional diversity was observed with regard to the dominant circulating serotypes. DENV-2 was found to be the most common serotype in many states. Thrombocytopenia, petechiae and malaise were associated with DENV-2 infection. Phylogenetic analyses of DENV E gene sequences revealed the circulation of genotypes I and V of DENV-1, two lineages of DENV-2 genotype IV, DENV-3 genotype III and DENV-4 genotype I. This study found regional differences in the prevalence of circulating DENV serotypes in India, and provides baseline data for continuous molecular surveillance. Molecular surveillance may have implications for predicting large-scale outbreaks of dengue if regional shifts in the predominantly circulating serotypes and genotypes are detected during the early phase of the dengue season.

Mesenchymal adipose stem cells maintain the capacity for differentiation and survival in culture beyond the long term

ABSTRACT Mesenchymal cells (MSCs) are considered to be cellular populations of common embryological origin. For clinical research applications, MSCs are expanded and increased with cells obtained from a primary culture. By extracting cells from tissue and encouraging them to reproduce, the stem cell population ends up dominating the culture due to a high proliferation rate and self-renewal. The first subcultures between the third and sixth are chosen in order to obtain the maximum number of cells with optimal differentiation capacity. However, few studies have reported long-term cultivation of MSCs. The objective of this study was to advance the knowledge on the characteristics of MSCs by assessing their capacity for self-renewal and phenotypic maintenance beyond 50 cell subcultures, which is defined as the normal limit for cellular survival. Rat subcutaneous adipose tissue was the source of mesenchymal adipose stem cells (MASCs) cultured over 175 subcultures. Early 1 to 5 and late 25 to 30 subcultures were used to induce cellular differentiation to become adipogenic, chondrogenic and osteogenic connective tissue cells. MASCs characteristics were studied using flow cytometry, transmission electron microscopy (TEM), and immunohistochemical and reverse transcription polymerase chain reaction (RT-qPCR) assays. The MASCs maintained cell differentiation capacity for more than 30 subcultures but lost potentiality starting at 60 up to 175 subcultures. MASCs showed the embryonic phenotypes OCT3/4 and Nanog indefinitely, and developed compensatory mechanisms, such as autophagy, to achieve cell survival over a long time period. Therefore, long-term subcultures showed that MASCs could maintain their potential for clinical research use.

Expression profiles of long noncoding RNAs in human corneal epithelial cells exposed to fine particulate matter

PurposeThe aim of this study was to investigate the expression profiles of long noncoding RNAs (lncRNAs) in human corneal epithelial cells (HCECs) exposed to fine particulate matter (PM2.5) and to identify potential biological pathways involved in PM2.5-induced toxicity in HCECs. MethodsUsing RNA sequencing (RNA-seq) and hierarchy clustering analysis, lncRNA expression profiles in PM2.5-treated and untreated HCECs were examined. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to predict the role of altered lncRNAs in biological processes and pathways. A quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was conducted to verify the RNA-seq results in HCECs and human corneal epithelial cell sheets. ResultsIn total, 65 lncRNAs were altered in the PM2.5-treated HCECs, including 41 upregulated and 24 downregulated lncRNAs. The results of the qRT-PCR assay were consistent with those of the RNA-seq analysis. The expression of two significantly upregulated lncRNAs was confirmed in human corneal epithelial cell sheets. The GO analysis demonstrated that altered lncRNAs in the PM2.5-treated HCECs were significantly enriched in three domains: cellular component, molecular function, and biological process. The KEGG pathway analysis revealed enriched pathways of lncRNA co-expressed mRNAs, including cancer, RNA transport, and Rap1 signaling. ConclusionsOur results suggest that lncRNAs are involved in the pathogenesis of PM2.5-induced ocular diseases, exerting their effects through biological processes and pathogenic pathways. Among the altered lncRNAs, RP3-406P24.3 and RP11-285E9.5 may play significant roles in PM2.5-induced ocular surface injury.

Micromechanical force promotes aortic valvular calcification

ObjectiveCalcified aortic valvular disease is known as an inflammation-related process related to force. The purpose of this study was to determine whether micromechanical force could induce valve calcification of porcine valvular interstitial cells and to examine the role of integrin αvβ3 in valvular calcification by using a novel method: magnetic twisting cytometry. MethodsPorcine valvular interstitial cells were cultured in vitro, and micromechanical force was applied to porcine valvular interstitial cells using magnetic twisting cytometry. Changes in calcification-related factors osteopontin and RUNX2 were detected. By using the calcification medium, the optimal magnetic twisting cytometry parameters for inducing valvular interstitial cell calcification were determined, and a magnetic twisting cytometry calcification promotion model was established. The role of αvβ3 in calcification was studied by using αvβ3 antagonists to block the function of αvβ3. ResultsReverse transcription polymerase chain reaction assays showed that the expression of osteopontin was enhanced 30 minutes after 25G-1Hz 5 minutes of stimulation. Western blotting assays showed that the expression of osteopontin and RUNX2 was upregulated 24 hours after 25G-1Hz 5 minutes of stimulation. The optimal magnetic twisting cytometry parameter for inducing porcine valvular interstitial cell calcification was 25G-2Hz for 10 minutes. The expression of osteopontin and RUNX2 decreased significantly after the addition of αvβ3 antagonist. Clinically, patients with bicuspid aortic valves had high expression of RUNX2 and β3 in the aortic valve, and β3 significantly correlated with RUNX2. ConclusionsBy using magnetic twisting cytometry, we established a porcine valvular interstitial cell calcification model by micromechanical force stimulation and obtained the optimal parameters. Integrin αvβ3 plays a key role in the aortic valve calcification process.

Systematic beach monitoring as a health assessment tool: Cetacean morbillivirus under non-epizootic circumstances in stranded dolphins.

Cetacean morbillivirus (CeMV) was identified as the etiologic agent of several epizootic episodes worldwide. Most of these studies are based on unusual mortality events or identification of new viral strains. We investigated the occurrence of CeMV under non-epizootic circumstances at a world heritage in Southern Brazil by a combination of pathologic, immunohistochemical and molecular assays. From 325 stranded cetaceans, 40 were included. Guiana dolphin (Sotalia guianensis) was the most frequent species. Interstitial pneumonia and non-suppurative encephalitis were the main pathologic findings associated with CeMV infection. Intracytoplasmic immunolabelling anti-CeMV was observed mainly in lungs and lymph nodes. All samples were negative in reverse transcription polymerase chain reaction assay. Diagnosis of CeMV is challenging in areas where epizootic episodes have not been recorded and due to post-mortem changes. We observed a CeMV prevalence of 27.5%. The results described here increase the knowledge about CeMV under non-epizootic conditions in Brazil and worldwide.

Cycle threshold values are inversely associated with poorer outcomes in hospitalized patients with COVID-19: a prospective, observational cohort study conducted at a UK tertiary hospital

This single-centre observational study demonstrated that lower cycle threshold (Ct) values (indicating higher viral loads) on admission to hospital were associated with poorer outcomes in unvaccinated, hospitalized patients with coronavirus disease 2019 (COVID-19). Demographic and outcome data were collected prospectively for all adult patients who tested positive for severe acute respiratory syndrome coronavirus-2 on admission to the University Hospitals North Midlands NHS Trust between 1 February and 1 July 2020. Nasopharyngeal swab samples were obtained, and a valid Ct value was determined for all patients using the Viasure reverse transcription polymerase chain reaction assay, validated by Public Health England, on admission to hospital. Multi-variable logistic regression results based on data from 618 individuals demonstrateda significant inverse relationship between the odds of death and Ct values (adjusted odds ratio 0.95, 95% confidence interval 0.92-0.98, P=0.001).The association remained highly significant after adjusting for known clinical risk factors for COVID-19.

The A756T Mutation of the ERG11 Gene Associated With Resistance to Itraconazole in Candida Krusei Isolated From Mycotic Mastitis of Cows.

Candida krusei (C. krusei) has been recently recognized as an important pathogen involved in mycotic mastitis of cows. The phenotypic and molecular characteristics of 15 C. krusei clinical isolates collected from cows with clinical mastitis in three herds of Yinchuan, Ningxia, were identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry. In addition to sequencing analysis, the ERG11 gene that encodes 14α-demethylases, the expression of the ERG11 gene, and efflux transporters ABC1 and ABC2 in itraconazole-susceptible (S), itraconazole-susceptible dose dependent (SDD), and itraconazole-resistant (R) C. krusei isolates was also quantified by a quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay. Sequencing analysis revealed three synonymous codon substitutions of the ERG11 gene including T939C, A756T, and T642C in these C. krusei clinical isolates. Among them, T642C and T939C mutations were detected in itraconazole-resistant and -susceptible C. krusei isolates, but the A756T substitution was found only in itraconazole-resistant isolates. Importantly, the expression of the ERG11 gene in itraconazole-resistant isolates was significantly higher compared with itraconazole-SDD and itraconazole-susceptible isolates (p = 0.052 and p = 0.012, respectively), as determined by the qRT-PCR assay. Interestingly, the expression of the ABC2 gene was also significantly higher in itraconazole-resistant isolates relative to the itraconazole-SDD and itraconazole-susceptible strains. Notably, the expression of ERG11 was positively associated with resistance to itraconazole (p = 0.4177 in SDD compared with S, p = 0.0107 in SDD with R, and p = 0.0035 in S with R, respectively). These data demonstrated that mutations of the ERG11 gene were involved in drug resistance in C. krusei. The A756T synonymous codon substitution of the ERG11 gene was correlated with an increased expression of drug-resistant genes including ERG11 and ABC2 in itraconazole-resistant C. krusei isolates examined in this study.

Surveillance of the rabies-related lyssavirus, Mokola in non-volant small mammals in South Africa.

The reservoir host of Mokola virus (MOKV), a rabies-related lyssavirus species endemic to Africa, remains unknown. Only sporadic cases of MOKV have been reported since its first discovery in the late 1960s, which subsequently gave rise to various reservoir host hypotheses. One particular hypothesis focusing on non-volant small mammals (e.g. shrews, sengis and rodents) is buttressed by previous MOKV isolations from shrews (Crocidura sp.) and a single rodent (Lophuromys sikapusi). Although these cases were only once-off detections, it provided evidence of the first known lyssavirus species has an association with non-volant small mammals. To investigate further, retrospective surveillance was conducted in 575 small mammals collected from South Africa. Nucleic acid surveillance using a pan-lyssavirus quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay of 329 brain samples did not detect any lyssavirus ribonucleic acid (RNA). Serological surveillance using a micro-neutralisation test of 246 serum samples identified 36 serum samples that were positive for the presence of MOKV neutralising antibodies (VNAs). These serum samples were all collected from Gerbilliscus leucogaster (Bushveld gerbils) rodents from Meletse in Limpopo province (South Africa). Mokola virus infections in Limpopo province have never been reported before, and the high MOKV seropositivity of 87.80% in these gerbils may indicate a potential rodent reservoir.

Türkiye‘den İlk Yenidoğan Koronavirüs Hastalığı 2019 (COVID-19) Vakası

Newborn cases commonly have milder COVID-19 disease. However, this is not enough to define knowledge about clinical manifestation and prognosis of this disease in newborns. A 17-day-old male neonate was admitted to the emergency room with fever and refusal to breastfeed at March 12, 2020. His pharyngeal swab testing was positive with real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for SARS-CoV-2. Except his father, all his close contacts were asymptomatic and screened for SARS-CoV-2 and all his close contacts have revealed as positive. During this very contagious outbreak, the COVID-19 disease should be considered in newborns with fever or refusal of feeding.