Genetic diversity of norovirus in hospitalised diarrhoeic children and asymptomatic controls in Dar es Salaam,Tanzania

This study investigated and reports norovirus diarrhoea, genetic diversity and associated clinical symptoms, HIV status and seasonality in a paediatric population of Tanzania.Stool specimens and demographic/clinical information, were prospectively collected from 705 hospitalised children with diarrhoea (cases) and 561 children without diarrhoea (controls) between 2010 and 2011. Norovirus detection was done by real-time RT-PCR. Genotype was determined using Gel-based and real time RT-PCR methods and sequencing targeting the polymerase and the capsid region respectively. Norovirus was detected in 14.3%, 181/1266 children. The prevalence of norovirus was significantly higher in cases (18.3%, 129/705) than in controls, (9.2%, 52/561), P < 0.05. Except for one child who had double infection with GI and GII all 129 cases had GII. Among controls, 23.1% had GI and 76.9% had GII. Norovirus GII.4 was significantly more prevalent in cases 87.9% than in controls 56.5%. Other genotypes detected in both cases and controls were GII.21, GII.16 and GII.g. The highest numbers of norovirus were detected in April 2011. The number of norovirus detected was significantly higher during the first than second year of life (109/540, 20.2% vs. 20/165, 12.1%). The prevalence of norovirus in HIV-positive and negative children was (21.2%, 7/33) and (10.3%, 40/390, P = 0.05) respectively, regardless of diarrhoea symptoms. No significant difference in gender, parent’s level of education or nutritional status with norovirus infection was observed within cases or controls. This study confirms the significant role of norovirus infection, especially GII.4 in diarrhoeic children who need hospitalisation and adds knowledge on norovirus epidemiology in the African region.     

Molecular characterization,serotyping, and antibiotic susceptibility profile of Leptospira interrogans serovar Copenhageni isolates from Brazil

Leptospira interrogans serogroup Icterohaemorrhagiae is the major serogroup infecting humans worldwide, and rodents and dogs are the most significant transmission sources in urban environments. Knowledge of the prevalent serovars and their maintenance hosts is essential to understand the epidemiology of leptospirosis. In this study, 20 Leptospira isolates were evaluated by pulsed-field gel electrophoresis (PFGE), variable number tandem-repeat analysis (VNTR), serotyping, and determination of antimicrobial resistance profile. Isolates, originated from bovine, canine, human, and rodent sources, were characterized by microscopic agglutination test with polyclonal and monoclonal antibodies and were identified as L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni. MICs of antimicrobials often used in veterinary medicine were determined by broth microdilution test. Most of tested antibiotics were effective against isolates, including penicillin, ampicillin, and ceftiofur. Higher MIC variability was observed for fluoroquinolones and neomycin; all isolates were resistant to trimethoprim/sulfamethoxazole and sulphadimethoxine. Isolates were genotyped by PFGE and VNTR; both techniques were unable to discriminate between serovars Copenhageni and Icterohaemorrhagiae, as expected. PFGE clustered all isolates in 1 pulsotype, indicating that these serovars can be transmitted between species and that bovine, rodent, and dogs can maintain them in the environment endangering the human population.     

Prediction of spurious HLA class II typing results using probabilistic classification

While modern high-throughput sequence-based HLA genotyping methods generally provide highly accurate typing results, artefacts may nonetheless arise for numerous reasons, such as sample contamination, sequencing errors, read misalignments, or PCR amplification biases. To help detecting spurious typing results, we tested the performance of two probabilistic classifiers (binary logistic regression and random forest models) based on population-specific genotype frequencies. We trained the model using high-resolution typing results for HLA-DRB1, DQB1, and DPB1 from large samples of German, Polish and UK-based donors. The high predictive capacity of the best models replicated both in 10-fold cross-validation for each gene and in using independent evaluation data (AUC 0.820–0.893). While genotype frequencies alone provide enough predictive power to render the model generally useful for highlighting potentially spurious typing results, the inclusion of workflow-specific predictors substantially increases prediction specificity. Low initial DNA concentrations in combination with low-volume PCR reactions form a major source of stochastic error specific to the Fluidigm chip-based workflow at DKMS Life Science Lab. The addition of DNA concentrations as a predictor variable thus substantially increased AUC (0.947–0.959) over purely frequency-based models.     

A method for detecting microfilaraemia, filarial specific antigens and antibodies and typing of parasites for drug resistance and genotypes using finger prick blood sample

Monitoring and evaluation of programme to eliminate lymphatic filariasis (LF) depends on epidemiological assessment using appropriate indicators. Minimum efforts using reliable tests are necessary to guide the programme managers in decision-making. Impact of Mass Drug Administration (MDA) towards filariasis elimination can be assessed by the detection of microfilariae (mf) or parasite DNA (infective), filarial antigens (infected) and antibodies (exposure). It is also important to monitor drug resistance and variation in genetic structure of parasite populations using molecular markers. We developed a method to carry out parasitological, molecular, immunological and genetic analysis from a minimum volume of blood sample (about 150 μl) drawn from finger tip of an individual residing in LF endemic area. The method involves separation of sera for immunological assays and isolation of mf of Wuchereria bancrofti from the blood clots for counting, which were then used for W. bancrofti specific PCR, screening for albendazole sensitivity/resistance alleles by AS-PCR, RAPD profiling and ITS 2 PCR for genotyping. A protocol is also suggested for the separation of sera for assays to detect antigen and antibodies and isolation of mf from clots for genetic analysis. The protocol developed has shown potential application in monitoring several immunological, parasitological and molecular parameters from a limited amount of blood sample collected by finger prick, in large-scale operations.     

Predicted S and s phenotypes from genotyping results among Thai populations to prevent transfusion-induced alloimmunization risks

Background

S and s antigens of the MNS system are of clinical importance because alloanti-S and -s have usually caused delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Various red cell genotyping has been established to predict the phenotypes to solve serological test limitations.

Serum Carboxypeptidase Activity and Genotype-Stratified CA19-9 to Detect Early-Stage Pancreatic Cancer

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安徽及周边省份绵羊和山羊隐孢子虫分子特性分析

目的　调查安徽及周边省份绵羊和山羊隐孢子虫流行情况及分子特性。方法　选择安徽省及其周边的河南、江苏和山东部分地区的7个规模化绵羊场和10个规模化山羊场，分别采集832份和781份新鲜绵羊和山羊粪便样品，利用隐孢子虫SSU rDNA基因特异的巢氏PCR技术对所有样品进行检测，调查上述地区绵羊和山羊隐孢子虫感染和虫种分布；对获得的微小隐孢子虫和泛在隐孢子虫进行gp60基因扩增与分析，以鉴定其基因亚型。结果　安徽及周边省份绵羊和山羊隐孢子虫感染率分别为5.8%（48/832）和8.7%（68/781）。SSU rDNA基因分析显示，绵羊感染的隐孢子虫为肖氏隐孢子虫和泛在隐孢子虫，山羊感染的隐孢子虫为微小隐孢子虫。gp60基因分析显示，泛在隐孢子虫基因亚型均为XIIa亚型2，微小隐孢子虫基因亚型均为IIdA19G1。结论　人兽共患泛在隐孢子虫XIIa亚型2和微小隐孢子虫 IIdA19G1基因亚型的鉴定，提示绵羊和山羊可能为人隐孢子虫感染的潜在来源。     

Recommendations for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

ObjectivesGenotyping of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in monitoring viral evolution and transmission during the pandemic. The quality of the sequence data obtained from these genotyping efforts depends on several factors, including the quantity/integrity of the input material, the technology, and laboratory-specific implementation. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in genomic epidemiology studies. We aimed to establish clear and broadly applicable recommendations for reliable virus genotyping.MethodsWe established and used a sequencing data analysis workflow that reliably identifies and removes technical artefacts; such artefacts can result in miscalls when using alternative pipelines to process clinical samples and synthetic viral genomes with an amplicon-based genotyping approach. We evaluated the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination.ResultsWe found that at least 1000 viral genomes are necessary to confidently detect variants in the SARS-CoV-2 genome at frequencies of ≥10%. The broad applicability of our recommendations was validated in over 200 clinical samples from six independent laboratories. The genotypes we determined for clinical isolates with sufficient quality cluster by sampling location and period. Our analysis also supports the rise in frequencies of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was facilitated by travel during the summer of 2020.ConclusionsWe present much-needed recommendations for the reliable determination of SARS-CoV-2 genome sequences and demonstrate their broad applicability in a large cohort of clinical samples.