Genome Amplification Research Articles

Emergence of a New Pathogen: A Retrospective Study of HPeV-5 Central Nervous System Disease in Alberta, Canada

Human parechoviruses (HPeVs) are known to cause meningo-encephalitis among neonates and infants. We aimed to describe the epidemiology of HPeVs causing central nervous system infections in Alberta from 2014 to 2019 with comparison of known HPeV-3 and emerging HPeV-5. Genomic analysis was completed on a subset of HPeV-5 strains to understand genetic relatedness to other known strains. All cerebrospinal fluid (CSF) samples in Alberta with detection of HPeVs were identified and a case review of medical records was conducted, retrospectively, to gather demographic and clinical details. Descriptive and analytic statistics were used to describe and compare the characteristics of cases affected by HPeV-3 with HPeV-5. Genome amplification was completed on six HPeV-5 samples. During the study period, 18,882 CSF samples were tested; 56 were positive for HPeV-3 or HPeV-5, and 52 patients were included in this study (40 HPeV-3 cases and 12 HPeV-5). A total of 40% of cases occurred in 2016, and 64% of infections occurred in the months of August to October. The mean age of cases was 18 days for HPeV-5 compared with 26 days for HPeV-3 (p = 0.045). Phylogenetic comparison showed similarity to a recombinant strain reported in Australia. HPeV meningo-encephalitis affected neonates/infants, mostly in late summer/early fall, and genomic sequencing of new strains can contribute to understanding the epidemiology of HPeV infections globally.

Next-Generation Sequencing Methods for Near-Real-Time Molecular Epidemiology of HIV and HCV.

The World Health Organisation has set targets of reducing the transmission of new hepatitis C (HCV) infections by 90%, and ending human immunodeficiency virus-1 (HIV) as a public health threat, by 2030. To achieve this, efficient and timely viral surveillance, and effective public health interventions, are required. Traditional epidemiological methods are largely dependent on the recognition of incident cases with symptomatic illness; acute HIV and HCV infections are commonly asymptomatic, which may lead to delays in the recognition of such new infections. Instead, for these viruses, molecular epidemiology may improve the detection of, and response to, clusters of viral transmission. Molecular epidemiology using historical datasets has highlighted key populations that may have benefitted from a timely intervention. Similar analyses performed on contemporary samples are needed to underpin the 2030 targets, but this requires the generation of a cohesive dataset of viral genome sequences in near-real-time. To generate such data, methodologies harnessing next-generation sequencing (NGS) should be utilised. Here we discuss the opportunity presented by NGS for public health surveillance of HIV and HCV, and discuss three methods that can generate sequences for such analysis. These include full-length genome amplification, utilised for analysis of HCV in the research space; tiling PCR, which was the method of choice for many diagnostic laboratories in the SARS-CoV-2 pandemic; and bait-capture hybridisation, which has been utilised in local HIV outbreaks. These techniques could be applied for near-real-time HIV and HCV surveillance, informing public health strategies that will be key to achieving 2030 targets.

Tracking the emergence of a novel genotype of Decapod hepanhamaparvovirus in shrimp using laser microdissection and next generation sequencing.

The prevalence of hepatopancreatic diseases in cultured shrimp has increased in recent years. Decapod Hepanhamaparvovirus 1 (DHPV) infection was identified by histology in samples that could not be detected by PCR-based assay for this virus. Employing Laser Microdissection (LMD), we dissected cells containing intranuclear inclusion bodies pathognomonic for DHPV infection from histological sections. Whole Genome Amplification and NGS were used to generate five complete genomes of the novel DHPV isolate that showed identities ranging from 77% to 98% to previously reported isolates. Phylogenetic analyses revealed the DHPV isolate represents a novel genotype, Genotype V. We developed PCR and in situ hybridization methods tailored for the specific detection of this genotype. Our approach of combining LMD with NGS opens avenues for rapid identification of emerging viral pathogens and retrospective studies to understand origin and evolution of viruses showcasing the transformative potential of the innovative approach used in this study.

Single-cell somatic copy number variants in brain using different amplification methods and reference genomes

The presence of somatic mutations, including copy number variants (CNVs), in the brain is well recognized. Comprehensive study requires single-cell whole genome amplification, with several methods available, prior to sequencing. Here we compare PicoPLEX with two recent adaptations of multiple displacement amplification (MDA): primary template-directed amplification (PTA) and droplet MDA, across 93 human brain cortical nuclei. We demonstrate different properties for each, with PTA providing the broadest amplification, PicoPLEX the most even, and distinct chimeric profiles. Furthermore, we perform CNV calling on two brains with multiple system atrophy and one control brain using different reference genomes. We find that 20.6% of brain cells have at least one Mb-scale CNV, with some supported by bulk sequencing or single-cells from other brain regions. Our study highlights the importance of selecting whole genome amplification method and reference genome for CNV calling, while supporting the existence of somatic CNVs in healthy and diseased human brain.

Reducing amplification cycles to improve the coverage of influenza A virus genome sequencing in heterosubtypic co-infection

This study delineates the enhancement of a Reverse Transcription Polymerase Chain Reaction (RT-PCR) method for the amplification of the complete genome of the influenza A virus during heterosubtypic co-infection, relying on the amplification of intact gene segments. The precision of the method was assessed using all amplicons, which underwent both capillary electrophoresis and DNA sequencing. Five samples featuring co-infection of Influenza A viruses with H1N1 and H3N2 subtypes were evaluated. The improved strategy successfully amplified all eight segments of H3N2 strains in four samples, and the entire genome of H1N1 strains in three samples.

MITF regulates IDH1, NNT, and a transcriptional program protecting melanoma from reactive oxygen species

Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte function, development and plays a significant role in melanoma pathogenesis. MITF genomic amplification promotes melanoma development, and it can facilitate resistance to multiple therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo. Some of the MITF target genes involved, such as IDH1 and NNT, are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state.

Development and validation of a multi-marker liquid bead array assay for the simultaneous detection of PIK3CA and ESR1 hotspot mutations in single circulating tumor cells (CTCs)

BackgroundPIK3CA and ESR1 mutations are associated with progression and therapy resistance in metastatic breast cancer (MBC). CTCs are highly heterogeneous and their analysis at single cell level can provide unique information for mutational profiling and the existence of different sub-clones related to tumor progression. We have developed a novel multi-marker liquid bead array assay based on combination of an enzymatic mutation enrichment method, multiplex PCR-based assay, and liquid bead array technology for the simultaneous detection of PIK3CA and ESR1 hotspot mutations in liquid biopsy samples. We focus on single CTCs, however the assay can be used for bulk CTC and ctDNA analysis. Materials and methodsSingle CTCs were isolated from an ER+/HER2+ MBC patient from CellSearch® cartridges using the VyCAP Puncher System and subjected to whole genome amplification followed by nuclease-assisted minor-allele enrichment with probe-overlap (NaME-PrO) enrichment. The assay was validated for analytical sensitivity and specificity for the simultaneous detection of PIK3CA (E545K, E542K, H1047R, H1047L) and ESR1 (Y537S, Y537C, Y537N, D538G, L536H) mutations in single CTCs, while its clinical performance was evaluated on 22 single CTCs and three single white blood cells (WBCs). ResultsThe developed multi-marker liquid bead array assay is novel, highly specific and sensitive for both mutation panels. The assay can reliably detect mutation-allelic-frequencies (MAFs) as low as 0.1 %. The presence of PIK3CA and ESR1 mutations was detected in 13.6 % and 72.7 % of single CTCs, respectively. The developed assay is sample-saving since it requires only 2 μL of amplified DNA to check for nine hotspot PIK3CA and ESR1 mutations in a single cell. The developed liquid bead array assay (Luminex, US), based on a 96 microwell plate format, enables the simultaneous analysis of 96 single cells. ConclusionsThe developed novel multi-marker liquid bead array assay for the simultaneous detection of PIK3CA and ESR1 hotspot mutations in single CTCs is highly specific, highly sensitive, high-throughput, and sample-, cost-, and time-saving. This multi-marker liquid bead array assay can be extended to detect up to 100 mutations in many genes at once and can be applied for bulk CTC and ctDNA analysis.

GoEnrich: creating high quality genomic DNA resources from limited voucher specimen tissues or museum specimens of at-risk species for conservation-friendly use in the validation of environmental DNA assays

ObjectiveEnvironmental DNA (eDNA) methods are crucial for monitoring populations, particularly rare and cryptic species. For confident eDNA application, rigorous assay validation is required including specificity testing with genomic DNA (gDNA). However, this critical step is often difficult to achieve as obtaining fresh tissue samples from at-risk species can be difficult, highly limited, or impossible. Natural history museum collections could serve as a valuable and ethical voucher specimen resource for eDNA assay validation. The present study demonstrates the effectiveness of whole genome amplification (WGA) in providing enough gDNA to assemble high quality mitogenomes from which robust targeted eDNA assays can be designed.ResultsUsing fresh and historical museum tissue samples from six species spanning fish, birds, and mammals, we successfully developed a WGA method with an average yield of 380 to 1,268 ng gDNA per 20 µL reaction. This gDNA was used for whole genome shotgun sequencing and subsequent assembly of high quality mitogenomes using mtGrasp. These mitogenomes were then used to develop six new robust, targeted quantitative real time polymerase chain reaction-based eDNA assays and 200 ng WGA-enriched yielded satisfactory Cq values and near 100% detection frequencies for all assays tested. This approach offers a cost-effective and non-invasive alternative, streamlining eDNA research processes and aiding in conservation efforts.

Technically feasible solutions to challenges in preimplantation genetic testing for thalassemia: experiences of multiple centers between 2019 and 2022.

In clinical practice, the success of preimplantation genetic testing for monogenic diseases (PGT-M) for thalassemia was hindered by the absence of probands, incomplete family members, or failure in detecting embryonic gene mutation sites. This study aimed to address these issues. This retrospective study included 342 couples undergoing PGT-M for α- or β-thalassemia at three reproductive medicine centers from 2019 to 2022. Various methods were used to construct parental haplotypes. A total of 1778 embryos were analyzed and selected for transfer based on chromosomal ploidy and PGT-M results. Follow-up involved amniocentesis results and clinical outcomes. Haplotypes were established using DNA samples from probands or parents, as well as sibling blood samples, single sperm, and affected embryos, achieving an overall success rate was 99.4% (340/342). For α-thalassemia and β-thalassemia, the concordance between embryo single nucleotide polymorphism (SNP) haplotype analysis results and mutation loci detection results was 93.8% (1011/1078) and 98.2% (538/548), respectively. Multiple annealing and looping-based amplification cycles (MALBAC) showed a higher whole genome amplification success rate than multiple displacement amplification (MDA) (98.8% (1031/1044) vs. 96.2% (703/731), p < 0.001). Amniocentesis confirmed PGT-M outcomes in 100% of cases followed up (99/99). This study summarizes feasible solutions to various challenging scenarios encountered in PGT-M for thalassemia, providing valuable insights to enhance success rate of PGT-M in clinical practice.

Characterization of Flower’s Color based on CHS Gene Structure in &lt;i&gt;Phalaenopsis&lt;/i&gt; ‘OX Queen’ and &lt;i&gt;Dendrobium&lt;/i&gt; ‘Cheddi Jagan’ Orchids

Orchids (Orchidaceae) are ornamental plants known for their high aesthetic value attributed to the shapes, colours, and fragrances of their flowers. Two types of hybrid orchids with attractive flowers, namely the Phalaenopsis 'OX Queen' orchid and the Dendrobium 'Cheddi Jagan' boast attractive flowers were used in this research, because of the beauty of its flower colour. The objective of this research is to characterise the morphology of flower colour and CHS (Chalcone Synthase) gene content that induces flower colour. The method used in this research analyzing the flower’s colour by using the RHS (Royal Horticultural Society) colour chart and molecular analysis by DNA genomic isolation and PCR amplification of gDNA for CHS gene specific primers. The results showed that purple colour is observed through the RHS, with P. 'OX Queen' coded as Deep Purple Pink (N73A) and D. 'Cheddi Jagan' coded as Strong Reddish Purple (N72C). The CHS gene can be amplified in P. ’OX Queen’ 1,287 bp and D. ’Cheddi jagan’ 3,731 bp. In both orchids, the results of amplification showed CHS motifs with conserved domains PLN03172 and PLN03170. The research results show that there is a significant difference in the morphology of the flowers of orchids. Purple colour is observed through the RHS, with P. 'OX Queen' coded as N73A and D. 'Cheddi Jagan' coded as N73C. The results showed that gDNA can be isolated by using CTAB method according to Murray and Thomson, and the CHS gene can be amplified by using CHS primers, resulting 1200 bp of P. 'OX Queen' and 2500 bp for D. 'Cheddi Jagan'. Through this study, preliminary data is expected to be obtained for future research, which is the formation of variegated flowers through editing the CRISPR/Cas9 genome in the CHS gene. This research is intended to support further studies on the formation of variegated flower patterns in P. 'OX Queen' and D. 'Cheddi Jagan’, focusing on the CHS gene using CRISPR/Cas9 technique.

Clinical-grade whole genome sequencing-based haplarithmisis enables all forms of preimplantation genetic testing

High-throughput sequencing technologies have increasingly led to discovery of disease-causing genetic variants, primarily in postnatal multi-cell DNA samples. However, applying these technologies to preimplantation genetic testing (PGT) in nuclear or mitochondrial DNA from single or few-cells biopsied from in vitro fertilised (IVF) embryos is challenging. PGT aims to select IVF embryos without genetic abnormalities. Although genotyping-by-sequencing (GBS)-based haplotyping methods enabled PGT for monogenic disorders (PGT-M), structural rearrangements (PGT-SR), and aneuploidies (PGT-A), they are labour intensive, only partially cover the genome and are troublesome for difficult loci and consanguineous couples. Here, we devise a simple, scalable and universal whole genome sequencing haplarithmisis-based approach enabling all forms of PGT in a single assay. In a comparison to state-of-the-art GBS-based PGT for nuclear DNA, shallow sequencing-based PGT, and PCR-based PGT for mitochondrial DNA, our approach alleviates technical limitations by decreasing whole genome amplification artifacts by 68.4%, increasing breadth of coverage by at least 4-fold, and reducing wet-lab turn-around-time by ~2.5-fold. Importantly, this method enables trio-based PGT-A for aneuploidy origin, an approach we coin PGT-AO, detects translocation breakpoints, and nuclear and mitochondrial single nucleotide variants and indels in base-resolution.

Exome Sequencing Starting from Single Cells.

Single-cell genomic analysis enables researchers to gain novel insights across diverse research areas, including developmental biology, tumor heterogeneity, and disease pathogenesis. Conducting single-cell genomic analysis using next-generation sequencing (NGS) methods has traditionally been challenging as the amount of genomic DNA present in a single cell is limited. Advancements in multiple displacement amplification (MDA) technologies allow the unbiased amplification of limited quantities of DNA under conditions that maintain its integrity. This method of amplification results in high yield and facilitates the generation of high-complexity NGS libraries that ensure the highest coverage to effectively allow variant calling. With the introduction of new sequencing platforms and chemistry, whole genome sequencing became a more cost-effective application, but enrichment of specific regions of interest further reduces the amount of required sequencing output and associated costs. There are two enrichment methods, polymerase chain reaction (PCR)-based and hybrid-capture-based methods. PCR-based methods are very flexible and highly effective but focus on specific loci, typically known to be associated with disease. Inherited diseases of unknown genetic origin require a more comprehensive approach to capture the genetic variation that is not yet associated with a specific disease. Hybrid capture enrichment methods require considerable amounts of DNA such that exome enrichment from single cells is only possible after preamplification of this limited material. This article describes the complete workflow from single cells and small quantities of DNA to exome-NGS libraries for Illumina sequencing instruments and includes the following protocols: © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Whole genome amplification from single cells or small amounts of gDNA Basic Protocol 2: NGS library generation of MDA-amplified material Basic Protocol 3: Exome enrichment.

Turning the needle into the haystack: Culture-independent amplification of complex microbial genomes directly from their native environment.

High-throughput sequencing (HTS) has revolutionized microbiology, but many microbes exist at low abundance in their natural environment and/or are difficult, if not impossible, to culture in the laboratory. This makes it challenging to use HTS to study the genomes of many important microbes and pathogens. In this review, we discuss the development and application of selective whole genome amplification (SWGA) to allow whole or partial genomes to be sequenced for low abundance microbes directly from complex biological samples. We highlight ways in which genomic data generated by SWGA have been used to elucidate the population dynamics of important human pathogens and monitor development of antimicrobial resistance and the emergence of potential outbreaks. We also describe the limitations of this method and propose some potential innovations that could be used to improve the quality of SWGA and lower the barriers to using this method across a wider range of infectious pathogens.

Genomic Amplification of TBC1D31 Promotes Hepatocellular Carcinoma Through Reducing the Rab22A-Mediated Endolysosomal Trafficking and Degradation of EGFR.

Hepatocellular carcinomas (HCCs) are characterized by a vast spectrum of somatic copy number alterations (CNAs); however, their functional relevance is largely unknown. By performing a genome-wide survey on prognosis-associated focal CNAs in 814 HCC patients by an integrative computational framework based on transcriptomic data, genomic amplification is identified at 8q24.13 as a promising candidate. Further evidence is provided that the 8q24.13 amplification-driven overexpression of Rab GTPase activating protein TBC1D31 exacerbates HCC growth and metastasis both in vitro and in vivo through activating Epidermal growth factor receptor (EGFR) signaling. Mechanistically, TBC1D31 acts as a Rab GTPase activating protein to catalyze GTP hydrolysis for Rab22A and then reduces the Rab22A-mediated endolysosomal trafficking and degradation of EGFR. Notably, overexpression of TBC1D31 markedly increases the resistance of HCC cells to lenvatinib, whereas inhibition of the TBC1D31-EGFR axis can reverse this resistance phenotype. This study highlights that TBC1D31 at 8q24.13 is a new critical oncogene, uncovers a novel mechanism of EGFR activation in HCC, and proposes the potential strategies for treating HCC patients with TBC1D31 amplification or overexpression.

Break-induced replication drives large-scale genomic amplifications in cancer cells.

DNA double-strand breaks (DSBs) are highly toxic lesions that underly the efficacy of ionizing radiation (IR) and a large number of cytotoxic chemotherapies 1-3 . Yet, abnormal repair of DSBs is associated with genomic instability and may contribute to cancer heterogeneity and tumour evolution. Here, we show that DSBs induced by IR, by DSB-inducing chemotherapeutics, or by the expression of a rare-cutting restriction endonuclease induce large-scale genomic amplification in human cancer cells. Importantly, the extent of DSB-induced genomic amplification (DIGA) in a panel of melanoma cell lines correlated with the degree of cytotoxicity elicited by IR, suggesting that DIGA contributes significantly to DSB-induced cancer cell lethality. DIGA, which is mediated through conservative DNA synthesis, does not require origin re-licensing, and is enhanced by the depletion or deletion of the methyltransferases SET8 and SUV4-20H1, which function sequentially to mono- and di-methylate histone H4 lysine 20 (H4K20) at DSBs to facilitate the recruitment of 53BP1-RIF1 and its downstream effector shieldin complex to DSBs to prevent hyper-resection 4-11 . Consistently, DIGA was enhanced in cells lacking 53BP1 or RIF1, or in cells that lacked components of the shieldin complex or of other factors that help recruit 53BP1 to DSBs. Mechanistically, DIGA requires MRE11/CtIP and EXO1, factors that promote resection and hyper-resection at DSBs, and is dependent on the catalytic activity of the RAD51 recombinase. Furthermore, deletion or depletion of POLD3, POLD4, or RAD52, proteins involved in break-induced replication (BIR), significantly inhibited DIGA, suggesting that DIGA is mediated through a RAD51-dependent BIR-like process. DIGA induction was maximal if the cells encountered DSBs in early and mid S-phase, whereas cells competent for homologous recombination (in late S and G2) exhibited less DIGA induction. We propose that unshielded, hyper-resected ends of DSBs may nucleate a replication-like intermediate that enables cytotoxic long-range genomic DNA amplification mediated through BIR.

ERBB2/ ERBB3-mutated S100/ SOX10-positive unclassified high-grade uterine sarcoma: first detailed description of a novel entity.

With the increasing use of innovative next generation sequencing (NGS) platforms in routine diagnostic and research settings, the genetic landscape of uterine sarcomas has been dynamically evolving during the last two decades. Notably, the majority of recently recognized genotypes in uterine sarcomas represent gene fusions, while recurrent oncogene mutations of diagnostic and/ or therapeutic value have been rare. Recently, a distinctive aggressive uterine sarcoma expressing S100 and SOX10, but otherwise lacking diagnostic morphological, immunophenotypic and molecular features of other uterine malignancies has been presented in a scientific abstract form (USCAP, 2023), but detailed description and delineation of the entity is still missing. We herein describe two high-grade unclassified uterine sarcomas characterized by spindle to round cell morphology and diffuse expression of S100 and SOX10, originating in the uterine body and cervix of 53- and 45-year-old women and carrying an ERBB3 (p.Glu928Gly) and an ERBB2 (p.Val777Leu) mutation, respectively. Both tumors harbored in addition genomic HER2 amplification, ATRX mutation and CDKN2A deletion. Methylation studies revealed a methylome most similar to MPNST-like tumors, but distinct from melanoma, MPNST, clear cell sarcoma, and endometrial stromal sarcoma. Case 1 died of progressive peritoneal metastases after multiple trials of chemotherapy 47months after diagnosis. Case 2 is a recent case who presented with a cervical mass, which was biopsied. This study defines a novel heretofore unrecognized aggressive uterine sarcoma with unique phenotypic and genotypic features. Given the potential value of targeting HER2, recognizing this tumor type is mandatory for appropriate therapeutic strategies and for better future delineation of the entity.

From Cell to Gene: Deciphering the Mechanism of Heart Failure With Single-Cell Sequencing.

Heart failure (HF) is a prevalent cardiovascular disease with significant morbidity and mortality rates worldwide. Due to the intricate structure of the heart, diverse cell types, and the complex pathogenesis of HF, further in-depth investigation into the underlying mechanismsis required. The elucidation of the heterogeneity of cardiomyocytes and the intercellular communication network is particularly important. Traditional high-throughput sequencing methods provide an average measure of gene expression, failing to capture the "heterogeneity" between cells and impacting the accuracy of gene function knowledge. In contrast, single-cell sequencing techniques allow for the amplification of the entire genome or transcriptome at the individual cell level, facilitating the examination of gene structure and expression with unparalleled precision. This approach offers valuable insights into disease mechanisms, enabling the identification of changes in cellular components and gene expressions during hypertrophy associated with HF. Moreover, it reveals distinct cell populations and their unique roles in the HF microenvironment, providing a comprehensive understanding of the cellular landscape that underpins HF pathogenesis. This review focuses on the insights provided by single-cell sequencing techniques into the mechanisms underlying HF and discusses the challenges encountered in current cardiovascular research.

Comprehensive analysis of flavohemoprotein copy number variation in Giardia intestinalis: exploring links to metronidazole resistance

BackgroundGiardiasis, caused by the protozoan parasite Giardia intestinalis, often presents a treatment challenge, particularly in terms of resistance to metronidazole. Despite extensive research, markers for metronidazole resistance have not yet been identified.MethodsThis study analysed 28 clinical samples of G. intestinalis from sub-assemblage AII, characterised by varying responses to metronidazole treatment. We focussed on copy number variation (CNV) of the multi-copy flavohemoprotein gene, analysed using digital polymerase chain reaction (dPCR) and next generation sequencing (NGS). Additionally, chromosomal ploidy was tested in 18 of these samples. Flavohemoprotein CNV was also assessed in 17 samples from other sub-assemblages.ResultsAnalyses revealed variable CNVs of the flavohemoprotein gene among the isolates, with no correlation to clinical metronidazole resistance. Discrepancies in CNVs detected from NGS data were attributed to biases linked to the whole genome amplification. However, dPCR helped to clarify these discrepancies by providing more consistent CNV data. Significant differences in flavohemoprotein CNVs were observed across different G. intestinalis sub-assemblages. Notably, Giardia exhibits a propensity for aneuploidy, contributing to genomic variability within and between sub-assemblages.ConclusionsThe complexity of the clinical metronidazole resistance in Giardia is influenced by multiple genetic factors, including CNVs and aneuploidy. No significant differences in the CNV of the flavohemoprotein gene between isolates from metronidazole-resistant and metronidazole-sensitive cases of giardiasis were found, underscoring the need for further research to identify reliable genetic markers for resistance. We demonstrate that dPCR and NGS are robust methods for analysing CNVs and provide cross-validating results, highlighting their utility in the genetic analyses of this parasite.Graphical

Characterizing HIV drug resistance in cases of vertical transmission in the VESTED randomized antiretroviral treatment trial.

VESTED (NCT03048422) compared the safety and efficacy of three antiretroviral treatment (ART) regimens in pregnant and postpartum women: dolutegravir+emtricitabine/tenofovir alafenamide fumarate; dolutegravir+emtricitabine/tenofovir disoproxil fumarate (TDF); efavirenz/emtricitabine/TDF. Vertical HIV transmission (VT) occurred to 4/617 (0.60%) live-born infants, who were evaluated for HIV drug resistance (HIVDR) and other risk factors. In 2018-2020, pregnant (weeks-14-28) women living with HIV and ≤14 days of ART were enrolled at 22 international sites and followed with their infants through 50 weeks postpartum. HIV sequences derived by single genome amplification (SGA) from longitudinally collected specimens were assessed from VT Cases for HIVDR in protease, reverse transcriptase, integrase, and the nef 3'polypurine tract (3'PPT). The four Case mothers were prescribed efavirenz-based-ART for 1-7 days prior to randomization to study ART. Their infants received postnatal nevirapine+/-zidovudine prophylaxis and were breastfed. A total of 833 SGA sequences were derived. The "major" (Stanford HIVDR Score ≥60) non-nucleoside reverse transcriptase inhibitor (NNRTI) mutation (K103N) was detected persistently in one viremic mother, and likely contributed to VT of HIVDR. Major NNRTI HIVDR mutations were detected in all three surviving infants. No integrase, nor high frequencies of 3'PPT mutations conferring dolutegravir HIVDR were detected. The timing of HIV infant diagnosis, plasma HIV RNA levels and HIVDR suggests one in utero, one peripartum, one early, and one late breastfeeding transmission. VT was rare. New-onset NNRTI HIVDR in Case mothers was likely from efavirenz-ART prescribed prior to study dolutegravir-ART, and in one case appeared transmitted to the infant despite nevirapine prophylaxis.

Hepatitis E Virus Genotype 3 among Hemodialysis Patients in Mexico: First Identification of Chronic Infection.

The global distribution of hepatitis E virus (HEV) is attributed to its capacity to spread through several routes of transmission; hemodialysis has gained increased amounts of attention in recent years. Although Mexico is considered a hyperendemic region for hepatitis E, no HEV surveillance is performed in the country. The frequency of HEV in hemodialysis (HD) patients has not been determined. Herein, we conducted a cross-sectional single-center analytical study including 67 serum samples from HD patients. Anti-HEV IgG and IgM antibodies and the viral genome were determined; partial regions within the HEV genome were sequenced for further phylogenetic analysis. Globally, 14.9% of the tested patients exhibited reactivity for IgG antibodies against HEV, and none showed reactivity to IgM. A total of 5.9% of the samples showed HEV genome amplification, and sequencing confirmed the identity of genotype 3; subsequent analysis of positive cases revealed two acute cases and chronic hepatitis E infection in one patient. Notably, the chronic patient was negative for anti-HEV IgG antibodies. Our findings highlight the importance of viral genome testing in HD patients and the need to establish guidelines for HEV detection in Mexico.