High prevalence of co-infecting enteropathogens in suspected rotavirus vaccine breakthrough cases

Despite the global use of rotavirus vaccines, vaccine breakthrough cases remain a pediatric health problem. In this study, we investigated suspected rotavirus vaccine breakthrough cases using next-generation sequencing (NGS) based viral metagenomics (n=102) and a panel of semi-quantitative reverse transcription PCR (RT-qPCR) (n=92) targeting known enteric pathogens. Overall, we identified co-infections in 80% of the cases. Enteropathogens such as Adenovirus (32%), Enterovirus (15%), diarrheagenic Escherichia coli (1-14%), Astrovirus (10%), Blastocystis spp. (10%), Parechovirus (9%), Norovirus (9%), Clostridium difficile (9%), Dientamoeba fragilis (9%), Sapovirus (8%), Campylobacter jejuni (4%) and Giardia lamblia (4%) were detected. Except for a few reassortant rotavirus strains, unusual genotypes or genotype combinations were not present. However, in addition to well-known enteric viruses, divergent variants of enteroviruses and non-classic astroviruses were identified using NGS. We estimated that in 31.5% of the patients, rotavirus was likely not the cause of gastroenteritis and in 14.1% of the patients, it contributed together with another pathogen(s) to disease. The remaining 54.4% of the patients likely had a ‘true vaccine breakthrough infection’. The high prevalence of alternative enteropathogens in the suspected rotavirus vaccine breakthrough cases suggests that gastroenteritis is often the result of a co-infection, and the rotavirus vaccine effectiveness might be underestimated in clinical and epidemiological studies.

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An Update on Zoonotic Cryptosporidium Species and Genotypes in Humans

Animals ◽

10.3390/ani11113307 ◽

2021 ◽

Vol 11 (11) ◽

pp. 3307

Author(s):

Una Ryan ◽

Alireza Zahedi ◽

Yaoyu Feng ◽

Lihua Xiao

Keyword(s):

Next Generation Sequencing ◽

Genome Sequencing ◽

Molecular Detection ◽

Epidemiological Studies ◽

Cryptosporidium Species ◽

Transmission Dynamics ◽

Whole Genome ◽

Zoonotic Pathogen ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

The enteric parasite, Cryptosporidium is a major cause of diarrhoeal illness in humans and animals worldwide. No effective therapeutics or vaccines are available and therefore control is dependent on understanding transmission dynamics. The development of molecular detection and typing tools has resulted in the identification of a large number of cryptic species and genotypes and facilitated our understanding of their potential for zoonotic transmission. Of the 44 recognised Cryptosporidium species and >120 genotypes, 19 species, and four genotypes have been reported in humans with C. hominis, C. parvum, C. meleagridis, C. canis and C. felis being the most prevalent. The development of typing tools that are still lacking some zoonotic species and genotypes and more extensive molecular epidemiological studies in countries where the potential for transmission is highest are required to further our understanding of this important zoonotic pathogen. Similarly, whole-genome sequencing (WGS) and amplicon next-generation sequencing (NGS) are important for more accurately tracking transmission and understanding the mechanisms behind host specificity.

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ERBB2 Pathway in Biliary Tract Carcinoma: Clinical Implications of a Targetable Pathway

Oncology Research and Treatment ◽

10.1159/000511919 ◽

2020 ◽

pp. 1-8

Author(s):

Oded Jacobi ◽

Jeffrey S. Ross ◽

Tal Goshen-Lago ◽

Riad Haddad ◽

Assaf Moore ◽

...

Keyword(s):

Median Overall Survival ◽

Tissue Samples ◽

Genomic Landscape ◽

High Prevalence ◽

Before And After ◽

Disease Stabilization ◽

Poor Outcomes ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing ◽

Copy Number Amplification

Background/Aims: Current chemotherapy regimens for cholangiocarcinoma (CCA) yield poor outcomes, with a median overall survival of <12 months. Recent data on the genomic landscape of CCAs have created opportunities for targeted therapy. Yet, data regarding its efficacy are scarce. We aimed to describe the genomic landscape of a CCA patient cohort using next-generation sequencing (NGS), focusing on the ERBB/EFGR pathway and assessing response to anti-HER2 agents. Methods: Tissue samples of intrahepatic CCA (IHCC) and extrahepatic CCA (EHCC) underwent NGS for somatic aberrations. The clinical outcomes for patients treated with anti-HER2 agents were evaluated. Results: A total of 1,863 CCA cases (1,615 IHCCs and 248 EHCCs) underwent NGS, and they revealed a high prevalence of ERBB alterations (IHCC, 4.2%; EHCC, 9.7%). Among these, 23.8% of the IHCCs and 53.6% of the EHCCs had a point mutation in ERBB2, and 66.6% of the IHCCs and 41.2% of the EHCCs had ERBB copy number amplification. Three EHCC patients were diagnosed at our institute with ERBB/EGFR aberrations; 2 patients were treated with neratinib and 1 patient with a chemotherapy-trastuzumab combination. All 3 achieved disease stabilization and a clinical benefit. One patient underwent a liquid biopsy before and after 3 months of treatment, demonstrating disappearance of the ERBB2 clone and emergence of a Myc-mutated clone after treatment. Conclusions: The genomic landscape of CCAs may harbor targetable alterations, especially in the ERBB/EGFR pathway. These alterations may have clinical significance in everyday practice.

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Reproducibility and repeatability of six high-throughput 16S rDNA sequencing protocols for microbiota profiling

10.1101/210880 ◽

2017 ◽

Author(s):

Sajan C. Raju ◽

Sonja Lagström ◽

Pekka Ellonen ◽

Willem M. de Vos ◽

Johan G. Eriksson ◽

...

Keyword(s):

Large Scale ◽

Alpha Diversity ◽

Epidemiological Studies ◽

Molecular Techniques ◽

16S Rdna Sequencing ◽

High Reproducibility ◽

Rdna Sequencing ◽

Culture Independent ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

AbstractCulture-independent molecular techniques and advances in next generation sequencing (NGS) technologies make large-scale epidemiological studies on microbiota feasible. A challenge using NGS is to obtain high reproducibility and repeatability, which is mostly attained through robust amplification. We aimed to assess the reproducibility of saliva microbiota by comparing triplicate samples. The microbiota was produced with simplified in-house 16S amplicon assays taking advantage of large number of barcodes. The assays included primers with Truseq (TS-tailed) or Nextera (NX-tailed) adapters and either with dual index or dual index plus a 6-nt internal index. All amplification protocols produced consistent microbial profiles for the same samples. Although, in our study, reproducibility was highest for the TS-tailed method. Five replicates of a single sample, prepared with the TS-tailed 1-step protocol without internal index sequenced on the HiSeq platform provided high alpha-diversity and low standard deviation (mean Shannon and Inverse Simpson diversity was 3.19 ± 0.097 and 13.56 ± 1.634 respectively). Large-scale profiling of microbiota can consistently be produced by all 16S amplicon assays. The TS-tailed-1S dual index protocol is preferred since it provides repeatable profiles on the HiSeq platform and are less labour intensive.

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OR15-03 High Prevalence of Gene Variants in Boys of Prepubertal Age with Clinical Suspicion of Central Hypogonadism and Low AMH

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1462 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Franco Brunello ◽

E Gabriela Sansó ◽

Paula Scaglia ◽

María Esnaola Azcoiti ◽

Ariel Berenstein ◽

...

Keyword(s):

Genetic Diagnosis ◽

Clinical Suspicion ◽

Gene Variants ◽

High Prevalence ◽

History Of ◽

Potential Pathogenicity ◽

Next Generation Sequencing Ngs ◽

Prepubertal Age ◽

Generation Sequencing ◽

Low Serum

Abstract Introduction: In boys of prepubertal age, the diagnosis of central hypogonadism may be difficult to ascertain since gonadotropins and testosterone are normally low. Sertoli cell markers, like AMH and inhibin B, may be useful. In recent years, with the development of next generation sequencing (NGS) technology, the number of genes associated with central hypogonadism has had an exponential increase. However, even with these advanced techniques, the gene variants with potential pathogenicity can be found at present in only 30-50% of the patients. Hypothesis of the study: Low serum AMH is an appropriate screening biomarker to select patients for NGS, in order to make a genetic diagnosis in boys of prepubertal age with suspected central hypogonadism. Patients and methods: All patients aged 1-10 yr referred between 2001 and 2018 with clinical suspicion of central hypogonadism (micropenis and cryptorchidism and/or microorchidism), with low serum AMH (<10th centile) were included. Serum AMH was determined by ELISA (Beckman-Coulter), and LH, FSH and testosterone (T) by ECLIA (Roche). NGS was performed with the TruSight™ One Sequencing Panel in a NextSeq® 500 sequencer (Illumina). Results are expressed as medians (range). Results: 13 patients were included. Age at first visit was 4.4 (0.1-9.2) yr. Cryptorchidism was present in all of them, micropenis in 10 and microorchidism in 11. Orchiopexy was required in 11 boys and the other 2 responded to hCG treatment. 4 patients had olfactory disturbances, 1 had sensory deafness and 1 had piebaldism. 2 patients had a family history of olfactory disturbances and/or central hypogonadism. 7 patients could be followed up to pubertal age, and the diagnosis of central hypogonadism was clinically confirmed. At age 6.1 yr (1.2-10), AMH was 159 pmol/L (65-363), LH was <0.1 IU/L in all, FSH was 0.61 IU/L (<0.1-1.9). 17 variants in 9 genes associated with central hypogonadism were found in 10 of 13 patients. 5 boys had 1 gene variant, while 4 had 2 gene variants and 1 had 3 gene variants indicating probable oligogenicity, in the following genes: FGFR1 (n:4), CHD7 (n:3), PROKR2 (n:2), SOX10 (n:2), AXL (n:2), HS6ST1 (n:1), AMHR2 (n:1), NSMF (n:1), DCC (n:1). Conclusion: A high prevalence of gene variants was found in boys of prepubertal age with a suspicion of central hypogonadism based on micropenis and cryptorchidism and/or microorchidism with low serum AMH.

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Targeted Hybridization Capture of SARS-CoV-2 and Metagenomics Enables Genetic Variant Discovery and Nasal Microbiome Insights

Microbiology Spectrum ◽

10.1128/spectrum.00197-21 ◽

2021 ◽

Author(s):

Dorottya Nagy-Szakal ◽

Mara Couto-Rodriguez ◽

Heather L. Wells ◽

Joseph E. Barrows ◽

Marilyne Debieu ◽

...

Keyword(s):

Genetic Variant ◽

Viral Metagenomics ◽

Research Use ◽

The Novel ◽

Emerging Pathogens ◽

Hybrid Capture ◽

Hybridization Capture ◽

Variant Discovery ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

This is the first FDA emergency-use-authorized hybridization capture-based next-generation sequencing (NGS) assay to detect the SARS-CoV-2 genome. Viral metagenomics and the novel hybrid capture NGS-based assay, along with its research-use-only analysis, can provide important genetic insights into SARS-CoV-2 and other emerging pathogens and improve surveillance and early detection, potentially preventing or mitigating new outbreaks.

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Usher syndrome: diagnostic approach, differential diagnoses and proposal of an updated function-based genetic classification

Medizinische Genetik ◽

10.1515/medgen-2020-2023 ◽

2020 ◽

Vol 32 (2) ◽

pp. 131-140

Author(s):

Hanno J. Bolz

Keyword(s):

Usher Syndrome ◽

Later Life ◽

Short Review ◽

Differential Diagnoses ◽

Genetic Classification ◽

Biochemical Basis ◽

New Genes ◽

High Prevalence ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

Abstract Usher syndrome (USH) manifests with congenital and apparently isolated hearing loss, followed by retinal degeneration in later life. Therefore, and because of its high prevalence in the congenitally hearing-impaired population, USH is one of the most relevant deafness syndromes. Next-generation sequencing (NGS)-based testing can now provide most analyzed USH patients with a molecular diagnosis, based on mutations in 11 genes. Given the availability of several excellent articles on the clinical and biochemical basis of USH, this short review focuses on critical assessment of new genes announced as USH genes, clinical and genetic differential diagnoses and therapeutic developments. Because obsolete loci, disproved USH genes and the inclusion of genes whose mutations cause similar phenotypes have increasingly blurred genetic classification, a revision based on phenotype restricted to genes related to the Usher protein complex is proposed.

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Implementation of an in-house real-time reverse transcription-PCR assay for the rapid detection of the SARS-CoV-2 Marseille-4 variant

10.1101/2021.02.03.21250823 ◽

2021 ◽

Author(s):

Marielle Bedotto ◽

Pierre-Edouard Fournier ◽

Linda Houhamdi ◽

Anthony Levasseur ◽

Jeremy Delerce ◽

...

Keyword(s):

Immune Responses ◽

Real Time ◽

Reverse Transcription ◽

Geographical Area ◽

Viral Escape ◽

Pcr Assay ◽

Reverse Transcription Pcr ◽

One Step ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

ABSTRACTIntroductionThe SARS-CoV-2 pandemic has been associated with the occurrence since summer 2020 of several viral variants that overlapped or succeeded each other in time. Those of current concern harbor mutations within the spike receptor binding domain (RBD) that may be associated with viral escape to immune responses. In our geographical area a viral variant we named Marseille-4 harbors a S477N substitution in this RBD.Materials and methodsWe aimed to implement an in-house one-step real-time reverse transcription-PCR (qPCR) assay with a hydrolysis probe that specifically detects the SARS-CoV-2 Marseille-4 variant.ResultsAll 6 cDNA samples from Marseille-4 variant strains identified in our institute by genome next-generation sequencing (NGS) tested positive using our Marseille-4 specific qPCR, whereas all 32 cDNA samples from other variants tested negative. In addition, 39/42 (93%) respiratory samples identified by NGS as containing a Marseille-4 variant strain and 0/26 samples identified as containing non-Marseille-4 variant strains were positive. Finally, 1,585/2,889 patients SARS-CoV-2-diagnosed in our institute, 10/277 (3.6%) respiratory samples collected in Algeria, and none of 207 respiratory samples collected in Senegal, Morocco, or Lebanon tested positive using our Marseille-4 specific qPCR.DiscussionOur in-house qPCR system was found reliable to detect specifically the Marseille-4 variant and allowed estimating it is involved in more than half of our SARS-CoV-2 diagnoses since December 2020. Such approach allows the real-time surveillance of SARS-CoV-2 variants, which is warranted to monitor and assess their epidemiological and clinical characterics based on comprehensive sets of data.

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