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High rate of RNAemia and impaired immunity in patients with immunodeficiency in the vaccination era
IF 4 3区 医学 Q2 VIROLOGY Pub Date : 2025-02-17 DOI: 10.1016/j.jcv.2025.105774
Anne Thierbach , Veronica Di Cristanziano , Kirsten A. Eberhardt , Martin Pirkl , Gertrud Steger , Eva Heger , Rolf Kaiser , Manuel Koch , Florian Klein , Dominic Rauschning , Jakob J. Malin

Background

Immunocompromised individuals, hemato-oncologic diseases or post-transplantation included, are, due to impaired immune response, at increased risk for severe and prolonged COVID-19. Observational Studies showed that SARS-CoV-2 RNAemia has been associated with poorer prognosis and higher disease severity.

Objective

The aim of this study was to investigate the occurrence of RNAemia and its association with anti-SARS-CoV-2 antibodies in immunocompromised COVID-19 patients. Risk factors for RNAemia were included in the analysis.

Study design

A retrospective study was conducted in 55 immunocompromised patients tested positive for SARS-CoV-2, who received treatment with monoclonal antibodies (mAb) between December 2021 and March 2022. Serological and virological tests were performed before mAb administration and clinical data were collected from electronic health records.

Results

Out of 55 patients, 35 % showed SARS-CoV-2 RNAemia. RNAemia was present in the 2 reported fatal cases. It was associated with negative testing for anti-receptor binding domain (RBD) IgG, anti-S2 domain of spike protein (S2) IgG and a lower leukocyte count. No association was found between previous COVID-19 vaccinations and the risk for RNAemia in immunocompromised patients.

Conclusion

The study underscores the importance of humoral response in controlling SARS-CoV-2 replication. RNAemia can serve as a potential biomarker for disease severity in immunocompromised individuals. Therefore, it should be considered in clinical settings for appropriate therapy decisions. Further research is needed to evaluate the pathophysiology and implications of RNAemia in immunodeficient patients with COVID-19.
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引用次数: 0
Detection of diverse HIV strains by the m-PIMATM HIV-1/2 detect point-of-care assay
IF 4 3区 医学 Q2 VIROLOGY Pub Date : 2025-02-07 DOI: 10.1016/j.jcv.2025.105771
Mark Anderson , Lara Teodoro , Fiona Harley , Eduardo Almaraz , Ana Vallari , Carolyn Strobel , Barbara Harris , Todd V. Meyer , Nicaise Ndembi , Dora Mbanya , Linda James , Souleymane Mboup , Jean-Christophe Plantier , Gavin Cloherty , Mary Rodgers

Background

HIV displays exceptionally high virus diversity that can impact detection by diagnostic assays, which rely on sequence conservation.

Methods

We tested the m-PIMA HIV-1/2 Detect point-of-care (POC) assay (Abbott Rapid Diagnostics) against a diverse HIV panel of 340 serum/plasma specimens and diluted cultured virus isolates for which viral load (VL) and classified sequences were known, including HIV-1 groups M, N, O, P, Circulating Recombinant Forms (CRF), and Unique Recombinant Forms (URF), and HIV-2. An in silico inclusivity analysis of 53,503 HIV-1 and 68 HIV-2 sequences from NCBI was performed to predict performance of m-PIMA HIV-1/2 Detect against a broader range of circulating strains.

Results

m-PIMA HIV-1/2 Detect detected HIV in 329/340 (96.8 %) tested samples. The mean VL was 3.80 (2.09–6.14) log copies/mL. Among samples with HIV VL >4000 copies/mL (3.60 log copies/mL; m-PIMA HIV-1/2 Detect design sensitivity), 181/181 (100 %) were detected. Among samples with VL between 3.0 and 3.6 log copies/mL, m-PIMA HIV-1/2 Detect detected 93/96 (96.9 %), and 55/63 (87.3 %) samples with VL below 3.0 log copies/mL were detected. At least one member from each subtype/CRF and all URFs were detected. In silico analysis identified 2/53,503 (0.0037 %) HIV-1 (both group O) and 1/68 (1.47 %) HIV-2 (subtype F) sequences with target region mutations that decreased identity below a 90 % threshold.

Conclusions

The m-PIMA HIV-1/2 Detect assay detected each of the major circulating HIV strains, including rare divergent strains. In silico analysis predicted that m-PIMA HIV-1/2 Detect would detect the majority of HIV-1 and HIV-2 strains indicating that this assay can detect the full range of HIV viral diversity.
{"title":"Detection of diverse HIV strains by the m-PIMATM HIV-1/2 detect point-of-care assay","authors":"Mark Anderson ,&nbsp;Lara Teodoro ,&nbsp;Fiona Harley ,&nbsp;Eduardo Almaraz ,&nbsp;Ana Vallari ,&nbsp;Carolyn Strobel ,&nbsp;Barbara Harris ,&nbsp;Todd V. Meyer ,&nbsp;Nicaise Ndembi ,&nbsp;Dora Mbanya ,&nbsp;Linda James ,&nbsp;Souleymane Mboup ,&nbsp;Jean-Christophe Plantier ,&nbsp;Gavin Cloherty ,&nbsp;Mary Rodgers","doi":"10.1016/j.jcv.2025.105771","DOIUrl":"10.1016/j.jcv.2025.105771","url":null,"abstract":"<div><h3>Background</h3><div>HIV displays exceptionally high virus diversity that can impact detection by diagnostic assays, which rely on sequence conservation.</div></div><div><h3>Methods</h3><div>We tested the m-PIMA HIV-1/2 Detect point-of-care (POC) assay (Abbott Rapid Diagnostics) against a diverse HIV panel of 340 serum/plasma specimens and diluted cultured virus isolates for which viral load (VL) and classified sequences were known, including HIV-1 groups M, N, O, P, Circulating Recombinant Forms (CRF), and Unique Recombinant Forms (URF), and HIV-2. An <em>in silico</em> inclusivity analysis of 53,503 HIV-1 and 68 HIV-2 sequences from NCBI was performed to predict performance of m-PIMA HIV-1/2 Detect against a broader range of circulating strains.</div></div><div><h3>Results</h3><div>m-PIMA HIV-1/2 Detect detected HIV in 329/340 (96.8 %) tested samples. The mean VL was 3.80 (2.09–6.14) log copies/mL. Among samples with HIV VL &gt;4000 copies/mL (3.60 log copies/mL; m-PIMA HIV-1/2 Detect design sensitivity), 181/181 (100 %) were detected. Among samples with VL between 3.0 and 3.6 log copies/mL, m-PIMA HIV-1/2 Detect detected 93/96 (96.9 %), and 55/63 (87.3 %) samples with VL below 3.0 log copies/mL were detected. At least one member from each subtype/CRF and all URFs were detected. <em>In silico</em> analysis identified 2/53,503 (0.0037 %) HIV-1 (both group O) and 1/68 (1.47 %) HIV-2 (subtype F) sequences with target region mutations that decreased identity below a 90 % threshold.</div></div><div><h3>Conclusions</h3><div>The m-PIMA HIV-1/2 Detect assay detected each of the major circulating HIV strains, including rare divergent strains. In silico analysis predicted that m-PIMA HIV-1/2 Detect would detect the majority of HIV-1 and HIV-2 strains indicating that this assay can detect the full range of HIV viral diversity.</div></div>","PeriodicalId":15517,"journal":{"name":"Journal of Clinical Virology","volume":"177 ","pages":"Article 105771"},"PeriodicalIF":4.0,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143421224","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Cytomegalovirus urinary excretion in children with congenital and postnatally acquired infection 巨细胞病毒尿排泄与先天性和后天获得性感染的儿童。
IF 4 3区 医学 Q2 VIROLOGY Pub Date : 2025-02-01 DOI: 10.1016/j.jcv.2024.105756
Tatiana M. Lanzieri , A. Chantal Caviness , Jill J. Williams , Gail Demmler-Harrison , the Houston Congenital Cytomegalovirus Longitudinal Study Group

Background

Cytomegalovirus (CMV) infection in children is associated with prolonged viral excretion in urine and saliva. This study characterizes CMV urinary excretion in children with congenital (cCMV) and postnatally acquired CMV infection.

Methods

Children with virologically confirmed cCMV (75 symptomatic and 105 asymptomatic at birth) and 51 children without cCMV were followed through median 11, 18 and 17 years of age, respectively. In children with cCMV, duration of CMV excretion was defined as uninterrupted positive results from initial to last positive culture, and recurrent CMV excretion as ≥1 positive following >1 negative result. CMV urinary excretion in children without cCMV was defined as resulting from postnatally acquired CMV infection.

Results

Mean duration of persistent CMV urinary excretion in children with cCMV was 1.9 (maximum 8.7) years for symptomatic and 2.8 (maximum 9.8) years for asymptomatic children (P = 0.011). Mean duration of CMV excretion was not statistically different for 17 symptomatic children treated with ganciclovir (2.4 years) compared with 58 untreated (1.8 years); P = 0.356. Recurrent excretion occurred in 19 (25 %) symptomatic and 21 (20 %) asymptomatic children, at mean age 4.0 and 6.2 years, respectively (P = 0.084). In 16 (31 %) children with postnatally acquired CMV infection, CMV urinary excretion began at mean age 1.8 (range 0.3–7.3) years.

Conclusions

Both symptomatic and asymptomatic cCMV were associated with persistent long-term CMV excretion in urine, which was significantly longer in asymptomatic cCMV and not influenced by ganciclovir treatment in symptomatic cCMV. CMV urinary excretion was common in young children without cCMV, suggesting rapid CMV acquisition in childhood.
背景:儿童巨细胞病毒(CMV)感染与尿和唾液中病毒排泄时间延长有关。本研究探讨先天性(cCMV)和出生后获得性巨细胞病毒感染儿童巨细胞病毒尿排泄的特点。方法:对病毒学确诊的cCMV患儿(75例出生时有症状,105例出生时无症状)和51例无cCMV患儿进行随访,中位年龄分别为11岁、18岁和17岁。在cCMV患儿中,CMV排泄持续时间定义为从最初到最后一次阳性培养不间断的阳性结果,在>1阴性结果后CMV排泄复发≥1阳性。没有cmmv的儿童的巨细胞病毒尿排泄被定义为出生后获得性巨细胞病毒感染。结果:cCMV患儿持续CMV尿排泄的平均持续时间,有症状者为1.9年(最长8.7年),无症状者为2.8年(最长9.8年)(P = 0.011)。接受更昔洛韦治疗的17名有症状儿童(2.4年)与未接受治疗的58名(1.8年)相比,CMV排泄的平均持续时间无统计学差异;P = 0.356。有症状和无症状儿童分别有19例(25%)和21例(20%)出现反复排泄,平均年龄分别为4.0岁和6.2岁(P = 0.084)。在16例(31%)出生后获得性巨细胞病毒感染的儿童中,巨细胞病毒尿排泄开始于平均年龄1.8岁(范围0.3-7.3岁)。结论:有症状和无症状的cCMV都与CMV在尿液中持续的长期排泄有关,无症状的cCMV明显更长,更昔洛韦治疗对有症状的cCMV没有影响。巨细胞病毒尿排泄在没有cCMV的幼儿中很常见,提示儿童期巨细胞病毒获得迅速。
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引用次数: 0
In memoriam David Brown, December 2024
IF 4 3区 医学 Q2 VIROLOGY Pub Date : 2025-02-01 DOI: 10.1016/j.jcv.2024.105757
Kevin Brown , Sabine Ditrich , John Harris , Miren Iturriza , Cherry Kang , Marion Koopmans , Satu Kurkela , Ben Lopman , Mick Mulders , Sarah O'Brien , Jan Vinjé , Hubert Niesters
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引用次数: 0
Evaluation of a next generation sequencing assay for Hepatitis B antiviral drug resistance on the oxford nanopore system 牛津纳米孔系统对乙型肝炎抗病毒药物耐药性的新一代测序测定的评价。
IF 4 3区 医学 Q2 VIROLOGY Pub Date : 2025-02-01 DOI: 10.1016/j.jcv.2025.105762
Michael Payne , Gordon Ritchie , Tanya Lawson , Matthew Young , Willson Jang , Aleksandra Stefanovic , Marc G. Romney , Nancy Matic , Christopher F. Lowe

Background

Next-generation sequencing (NGS) for Hepatitis B virus (HBV) antiviral resistance (AVR) testing is a highly sensitive diagnostic method, able to detect low-level mutant subpopulations. Our clinical virology laboratory previously transitioned from DNA hybridization (INNO-LiPA) to NGS, initially with the GS Junior System and subsequently the MiSeq. The Oxford Nanopore Technology (ONT) sequencing system was evaluated for HBV resistance testing, with regards to sequencing accuracy and turn-around time.

Methods

We performed amplicon sequencing of the HBV polymerase gene from patient plasma and external quality assessment (EQA) samples on the MiSeq Reagent Nano Kit v2 and GridION ONT with R10.4.1 flowcells. Mutational analysis and genotyping were performed by DeepChek®Assay-HBV (version 2.0).

Results

A total of 49 patient samples and 15 EQA samples were tested on both the MiSeq and ONT. There was high agreement for both patient and EQA samples between the MiSeq and ONT systems, with regards to total drug resistance mutations detected and total patient sample agreement, 68/70 (97 %) and 47/49 (96 %), respectively.

Conclusion

The ONT NGS platform provided accurate HBV AVR results, with improved turn-around times. Sequencing error rates at AVR codons were below 1 %.
背景:下一代测序(NGS)用于乙型肝炎病毒(HBV)抗病毒药物耐药性(AVR)检测是一种高度敏感的诊断方法,能够检测低水平突变亚群。我们的临床病毒学实验室之前从DNA杂交(INNO-LiPA)过渡到NGS,最初使用GS初级系统,随后使用MiSeq。我们评估了牛津纳米孔技术(ONT)测序系统在HBV耐药检测方面的测序准确性和周转时间。方法:采用MiSeq Reagent Nano Kit v2和GridION ONT,采用R10.4.1流式细胞对患者血浆和外部质量评估(EQA)样品中的HBV聚合酶基因进行扩增子测序。采用DeepChek®Assay-HBV(2.0版)进行突变分析和基因分型。结果:共有49例患者样本和15例EQA样本在MiSeq和ONT上进行了检测。MiSeq和ONT系统在检测到的总耐药突变和总患者样本一致性方面对患者和EQA样本均有很高的一致性,分别为68/70(97%)和47/49(96%)。结论:ONT NGS平台提供了准确的HBV AVR结果,缩短了周转时间。AVR密码子的测序错误率低于1%。
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引用次数: 0
Early insights of dengue virus serotype 3 (DENV-3) re-emergence in São Paulo, Brazil
IF 4 3区 医学 Q2 VIROLOGY Pub Date : 2025-02-01 DOI: 10.1016/j.jcv.2025.105763
Lívia Sacchetto , Victória Bernardi , Marini L. Brancini , Beatriz de C. Marques , Andreia Negri , Nikos Vasilakis , Cassia F. Estofolete , Maurício L. Nogueira

Background

In dengue hyperendemic regions, the evolution of the virus is marked by frequent virus introduction/reintroduction and clade replacement events, occasionally linked to an epidemic outbreak. From 2023 onwards, an increase in the detection of DENV-3 cases has been reported in different regions of Brazil. Thus, molecular and genomic surveillance of circulating DENV strains is crucial for public health preparedness and response efforts for the disease.

Objectives

This work aimed to characterize and provide preliminary insights into dengue virus serotype 3 (DENV-3) re-emergence in São Paulo state, Brazil.

Study design

We conducted active arbovirus molecular surveillance on samples from patients with acute febrile illness combined with next-generation sequencing and phylogenetic analyses.

Results

We detected and characterized DENV-3 circulation in São Paulo, Brazil, since late 2023. The genomes clustered within genomes recently (2022–2024) identified in Florida, the Caribbean region, and Brazil.

Conclusions

Our results demonstrate the resurgence of DENV-3 in the region since 2009, raising concerns about a potential outbreak in regions with a high epidemic history.
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引用次数: 0
Molecular epidemiology of Hepatitis E virus among humans in the Niger Republic, 2017–2023 2017-2023年尼日尔共和国人间戊型肝炎病毒分子流行病学研究
IF 4 3区 医学 Q2 VIROLOGY Pub Date : 2025-02-01 DOI: 10.1016/j.jcv.2025.105761
Adamou Lagare , Issifi Kollo Abdoulkader , Gamou Fall , Hadiza Ousmane , Wilfried Hounkanrin , Balki Aoula , Bacary Djilocalisse Sadio , Bassira Issaka , Zaneidou Maman , Ousmane Faye , Sabo Haoua Seini , Martin Faye
Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis, responsible for large outbreaks in resource limited countries. The virus belongs to the genus Orthohepevirus which is subdivided into eight distinct genotypes (HEV-1 to HEV-8). Human disease transmission is mostly through the faecal-oral route. However, zoonotic transmission could also occur. In the Niger Republic, the first documented HEV outbreak was recorded in 2017 around displaced persons camps in the Diffa region across the Lake Chad basin, resulting in 736 confirmed cases and 38 (1.9%) deaths. Since then, sporadic cases were annually reported despite the lack of specific surveillance. Overall, from 2017 to 2023, a total of 2820 HEV suspected cases were sampled, out of which 906 (32.12%) were confirmed positive by either qRT-PCR and/or IgM ELISA. Out of the 21 characterized isolates, we identified 15 sequences belonging to the genotype 1e and 6 sequences to the genotype 2b. The newly characterized sequences from Niger clustered with those circulating in neighbouring countries, suggesting a cross-border virus circulation. The co-circulation of HEV genotypes 1 and 2 is an indicator of the probable virus transmission through contaminated water sources. Thus, there is a crucial need to improve the preparedness and implement an active and integrated community-based surveillance. This should include field testing for rapid detection and characterization of HEV as well as actions for disease containment, strengthening of hygiene measures and community-based sensitization for behavioural changes.
戊型肝炎病毒(HEV)是急性病毒性肝炎的最常见病因,在资源有限的国家造成大规模暴发。该病毒属于正疱疹病毒属,可细分为八个不同的基因型(HEV-1至HEV-8)。人类疾病主要通过粪口途径传播。然而,人畜共患传播也可能发生。在尼日尔共和国,2017年在整个乍得湖盆地迪法地区的流离失所者营地周围记录了首次有记录的戊肝病毒疫情,导致736例确诊病例和38例(1.9%)死亡。从那时起,尽管缺乏具体的监测,每年仍报告零星病例。总体而言,2017 - 2023年共采集了2820例HEV疑似病例,其中906例(32.12%)经qRT-PCR和/或IgM酶联免疫吸附试验证实为阳性。在21个特征分离株中,我们鉴定出15个序列属于基因型1e, 6个序列属于基因型2b。来自尼日尔的新特征序列与在邻国传播的序列聚集在一起,表明存在跨境病毒传播。HEV基因型1和2的共循环是病毒可能通过受污染水源传播的一个指标。因此,迫切需要改进准备工作并实施积极和综合的基于社区的监测。这应包括为快速检测和确定戊肝病毒特征而进行的现场试验,以及为控制疾病采取的行动,加强卫生措施和以社区为基础的行为改变宣传。
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引用次数: 0
Highly-multiplex detection of plasma cell-free human papillomavirus-16 DNA in oropharyngeal carcinoma 口咽癌无浆细胞人乳头瘤病毒-16 DNA的高度多重检测。
IF 4 3区 医学 Q2 VIROLOGY Pub Date : 2025-02-01 DOI: 10.1016/j.jcv.2025.105760
Payton Clark , Natalya Karasik , Shauna R. Campbell , Neil M. Woody , Jamie A. Ku , Natalie Silver , Danielle Bottalico , Brandon L. Prendes , Eric D. Lamarre , Joseph Scharpf , Tamara A. Sussman , Jessica L. Geiger , Hannah Wang , Timothy A. Chan , Shlomo A. Koyfman , Jacob A. Miller

Background

Plasma cell-free Human Papillomavirus DNA (cfHPVDNA) is a biomarker for oropharyngeal carcinoma. Existing diagnostics may be limited by inadequate sensitivity or high cost/complexity for longitudinal monitoring.

Objectives

We hypothesized that sensitive and specific plasma cfHPVDNA detection may be achieved via a highly-multiplex qPCR method.

Study Design

We designed and validated a single-tube one-step genotype-specific qPCR assay for detection of cfHPV16DNA in human plasma using >8,000 genomes spanning 18 genotypes. Amplicons were optimized for cfHPVDNA fragment size.

Results

The cfHPV16DNA qPCR amplicons spanned 16 % of the HPV16 genome. Amplicons were conserved in a median of 99.0 % of 3,944 genomes in silico. The 95 % lower limit of detection was 0.35 genome copies/reaction and the limit of blank was 0. Multiplexing achieved a tenfold improvement in sensitivity compared with single amplicons using in silico simulations of cfHPVDNA fragmentation, which was in close agreement with experimental observations. An assay was replicated for HPV18 with similar observations.
Among 36 patients with head/neck mucosal carcinomas (26 HPV-positive, 12 HPV-negative), there was 100 % concordance with tissue HPV status and with NavDx digital PCR. Pre-treatment specimens with sub-genomic cfHPVDNA concentration were detected. False negatives were observed with single amplicons but not with this multiplexed method. Among 17 patients with post-treatment landmark specimens, there was 100 % PPV and 100 % NPV for recurrence.

Conclusions

This assay is specific for plasma cfHPVDNA detection and prognostic for recurrence. Sub-genomic sensitivity was in close agreement with in silico simulations. The format might be more accessible than dPCR or NGS for longitudinal testing.
背景:无浆细胞人乳头瘤病毒DNA (cfHPVDNA)是口咽癌的生物标志物。现有的诊断方法可能受到纵向监测灵敏度不足或成本高/复杂程度的限制。目的:我们假设通过高多重qPCR方法可以实现敏感和特异性的血浆cfHPVDNA检测。研究设计:我们设计并验证了一种单管一步基因型特异性qPCR检测方法,用于检测人血浆中cfHPV16DNA,该方法使用了跨越18个基因型的bbb8000个基因组。扩增子对cfHPVDNA片段大小进行优化。结果:cfHPV16DNA qPCR扩增子覆盖了16%的HPV16基因组。扩增子在3,944个基因组中保守的中位数为99.0%。95%检测下限为0.35个基因组拷贝/反应,空白下限为0。使用cfHPVDNA片段的硅模拟,与单个扩增子相比,多路复用的灵敏度提高了10倍,这与实验观察结果非常吻合。对HPV18进行了类似的实验。在36例头颈部粘膜癌患者中(HPV阳性26例,HPV阴性12例),与组织HPV状态和NavDx数字PCR的一致性为100%。检测预处理标本中cfHPVDNA亚基因组浓度。单扩增观察到假阴性,但这种多路扩增方法没有观察到假阴性。在17例治疗后地标标本中,复发的PPV为100%,NPV为100%。结论:该方法对血浆cfHPVDNA检测和复发预后具有特异性。亚基因组敏感性与计算机模拟结果非常一致。对于纵向测试,这种格式可能比dPCR或NGS更容易获得。
{"title":"Highly-multiplex detection of plasma cell-free human papillomavirus-16 DNA in oropharyngeal carcinoma","authors":"Payton Clark ,&nbsp;Natalya Karasik ,&nbsp;Shauna R. Campbell ,&nbsp;Neil M. Woody ,&nbsp;Jamie A. Ku ,&nbsp;Natalie Silver ,&nbsp;Danielle Bottalico ,&nbsp;Brandon L. Prendes ,&nbsp;Eric D. Lamarre ,&nbsp;Joseph Scharpf ,&nbsp;Tamara A. Sussman ,&nbsp;Jessica L. Geiger ,&nbsp;Hannah Wang ,&nbsp;Timothy A. Chan ,&nbsp;Shlomo A. Koyfman ,&nbsp;Jacob A. Miller","doi":"10.1016/j.jcv.2025.105760","DOIUrl":"10.1016/j.jcv.2025.105760","url":null,"abstract":"<div><h3>Background</h3><div>Plasma cell-free Human Papillomavirus DNA (cfHPVDNA) is a biomarker for oropharyngeal carcinoma. Existing diagnostics may be limited by inadequate sensitivity or high cost/complexity for longitudinal monitoring.</div></div><div><h3>Objectives</h3><div>We hypothesized that sensitive and specific plasma cfHPVDNA detection may be achieved via a highly-multiplex qPCR method.</div></div><div><h3>Study Design</h3><div>We designed and validated a single-tube one-step genotype-specific qPCR assay for detection of cfHPV16DNA in human plasma using &gt;8,000 genomes spanning 18 genotypes. Amplicons were optimized for cfHPVDNA fragment size.</div></div><div><h3>Results</h3><div>The cfHPV16DNA qPCR amplicons spanned 16 % of the HPV16 genome. Amplicons were conserved in a median of 99.0 % of 3,944 genomes <em>in silico</em>. The 95 % lower limit of detection was 0.35 genome copies/reaction and the limit of blank was 0. Multiplexing achieved a tenfold improvement in sensitivity compared with single amplicons using <em>in silico</em> simulations of cfHPVDNA fragmentation, which was in close agreement with experimental observations. An assay was replicated for HPV18 with similar observations.</div><div>Among 36 patients with head/neck mucosal carcinomas (26 HPV-positive, 12 HPV-negative), there was 100 % concordance with tissue HPV status and with NavDx digital PCR. Pre-treatment specimens with sub-genomic cfHPVDNA concentration were detected. False negatives were observed with single amplicons but not with this multiplexed method. Among 17 patients with post-treatment landmark specimens, there was 100 % PPV and 100 % NPV for recurrence.</div></div><div><h3>Conclusions</h3><div>This assay is specific for plasma cfHPVDNA detection and prognostic for recurrence. Sub-genomic sensitivity was in close agreement with <em>in silico</em> simulations. The format might be more accessible than dPCR or NGS for longitudinal testing.</div></div>","PeriodicalId":15517,"journal":{"name":"Journal of Clinical Virology","volume":"176 ","pages":"Article 105760"},"PeriodicalIF":4.0,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142965161","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Limited utility of Epstein–Barr virus (EBV) surveillance for predicting post-transplant lymphoproliferative disorders in adult EBV seropositive lung transplant recipients EBV血清反应阳性的成人肺移植受者移植后淋巴增生性疾病的EBV监测作用有限。
IF 4 3区 医学 Q2 VIROLOGY Pub Date : 2025-02-01 DOI: 10.1016/j.jcv.2024.105758
Jordan K. Mah , Patrick C.K. Tam , Yeh-Chung Chang , Jennifer H. Saullo , Arthur W. Baker , Eileen K. Maziarz , Julia A. Messina , Beatrice Sim , Lana Abusalem , Sandrine Hanna , Matthew R. Pipeling , Laurie D. Snyder , John M. Reynolds , Cameron R. Wolfe , Mark J. Lee , Barbara D. Alexander , Madeleine R. Heldman

Background

EBV DNAemia surveillance, with reduction of immunosuppression at certain viral load (VL) thresholds, is a common practice for mitigating progression from EBV DNAemia to post-transplant lymphoproliferative disorder (PTLD) in lung transplant recipients (LTRs). The utility of EBV surveillance in adult EBV seropositive LTRs is unknown.

Methods

We performed a retrospective cohort study of EBV seropositive adult LTRs who underwent lung transplant between 1/1/19 and 12/31/20 and received whole blood (WB) EBV PCR surveillance. We compared peak WB EBV VLs among 3 groups: 1) asymptomatic LTRs who developed PTLD, before PTLD was clinically suspected, 2) LTRs who developed PTLD, after PTLD was clinically suspected, and 3) LTRs who did not develop PTLD. We calculated the positive predictive value (PPV) of moderate-grade DNAemia (2840 to 11,360 IU/mL) and high-grade DNAemia (≥ 11,360 IU/mL) for identifying active or future PTLD.

Results

Six (2.6 %) of 229 LTRs developed PTLD. Among LTRs who developed PTLD, median peak EBV VL was significantly higher after PTLD was suspected than before clinical signs of PTLD were present (16,004 IU/mL vs. ≤568 IU/mL, p = 0.016). Median peak EBV VLs were similar between asymptomatic LTRs who later developed PTLD and LTRs who did not develop PTLD (median peak EBV VL ≤568 IU/mL vs. ≤568 IU/mL, p = 0.62). The PPVs for moderate- and high-grade DNAemia were 14.7 % and 33.3 %, respectively.

Conclusions

EBV surveillance did not accurately identify EBV seropositive LTRs at risk for progressing to PTLD. EBV PCR testing in asymptomatic EBV seropositive transplant recipients may represent an opportunity for diagnostic stewardship.
背景:EBV dna血症监测,在一定病毒载量(VL)阈值下降低免疫抑制,是缓解肺移植受者(lts)从EBV dna血症发展为移植后淋巴细胞增生性疾病(PTLD)的一种常见做法。EBV监测在成人EBV血清阳性ltr中的应用尚不清楚。方法:我们对19年1月1日至20年12月31日期间接受肺移植并接受全血(WB) EBV PCR监测的EBV血清阳性成人ltr进行回顾性队列研究。我们比较了3组患者的WB EBV峰值VLs: 1)无症状ltr,在临床怀疑PTLD之前,2)ltr,在临床怀疑PTLD后,3)ltr未发生PTLD。我们计算了中度DNAemia (2840 - 11360 IU/mL)和高度DNAemia(≥11360 IU/mL)的阳性预测值(PPV),用于识别活性或未来的PTLD。结果:229例ltr患者中有6例(2.6%)发生PTLD。在发生PTLD的ltr中,怀疑PTLD后EBV VL中位峰明显高于出现PTLD临床症状前(16,004 IU/mL vs.≤568 IU/mL, p = 0.016)。无症状晚期发展为PTLD的ltr与未发展为PTLD的ltr的中位峰值VL相似(EBV的中位峰值VL≤568 IU/mL vs≤568 IU/mL, p = 0.62)。中度和高度dna血症的ppv分别为14.7%和33.3%。结论:EBV监测并不能准确识别EBV血清阳性的ltr进展为PTLD的风险。在无症状的EBV血清阳性移植受者中进行EBV PCR检测可能代表诊断管理的机会。
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引用次数: 0
The molecular epidemiology of respiratory syncytial virus in Ontario, Canada from 2022–2024 using a custom whole genome sequencing assay and analytics package 使用定制的全基因组测序测定和分析包研究2022-2024年加拿大安大略省呼吸道合胞病毒的分子流行病学。
IF 4 3区 医学 Q2 VIROLOGY Pub Date : 2025-02-01 DOI: 10.1016/j.jcv.2024.105759
Henry Wong , Calvin P. Sjaarda , Brittany Rand , Drew Roberts , Kyla Tozer , Ramzi Fattouh , Robert Kozak , Prameet M. Sheth

Background

Respiratory Syncytial Virus (RSV) infections are a cause of significant morbidity and mortality in children and the elderly. Despite the clinical burden of disease, very little is known about the inter- and intra-seasonal genomic variability of RSV. Furthermore, the recent approval of vaccines and monoclonal antibody therapies will likely lead to higher selective pressure on RSV. Genomic surveillance will be essential to monitor viral changes and inform future therapeutic developments and public health responses. Here, we describe the development of an amplicon-based whole-genome sequencing assay for RSV to enable genomic surveillance.

Methods

A 750-bp overlapping amplicon design was developed to co-amplify RSV-A/-B directly from patient samples collected during two respiratory illness seasons (2022/23, 2023/24) for whole-genome sequencing. RSV subtype, clade, and F-protein antigenic site sequences were determined with a custom analytical pipeline.

Results

Of the 429 specimens included in the study 410 (95.6 %) samples met acceptability. Our data demonstrated co-circulation of both RSV subtypes, with increasing predominance of RSV-A since 2022. There were seven genomic clades of RSV-A, while >95 % of RSV-B belonged to a single clade. 1.5 % of samples had amino acid changes within the binding sites of the current RSV therapeutics Palivizumab or Nirsevimab.

Conclusions

Continuous monitoring of RSV genotypes and mutations will be critical for understanding the impact of new therapeutics and vaccines on RSV epidemiology and detecting emergence of vaccine-escape and/or antiviral resistant mutations.
背景:呼吸道合胞病毒(RSV)感染是儿童和老年人发病和死亡的重要原因。尽管存在疾病的临床负担,但对RSV的季节间和季节内基因组变异性知之甚少。此外,最近批准的疫苗和单克隆抗体疗法可能会导致对RSV的更高选择压力。基因组监测对于监测病毒变化和为未来的治疗发展和公共卫生反应提供信息至关重要。在这里,我们描述了基于扩增子的RSV全基因组测序测定的发展,以实现基因组监测。方法:建立750 bp重叠扩增子设计,直接从两个呼吸道疾病季节(2022/23和2023/24)收集的患者样本中共扩增RSV-A/-B,用于全基因组测序。RSV亚型、进化支和f蛋白抗原位点序列用定制的分析管道测定。结果:纳入研究的429份样本中,410份(95.6%)样本符合可接受性。我们的数据显示两种RSV亚型共循环,自2022年以来RSV- a的优势越来越大。RSV-A有7个基因组分支,而RSV-B有95%属于单一分支。1.5%的样本在当前RSV治疗药物帕利维珠单抗或尼瑟维单抗的结合位点有氨基酸变化。结论:持续监测RSV基因型和突变对于了解新疗法和疫苗对RSV流行病学的影响以及检测疫苗逃逸和/或抗病毒耐药突变的出现至关重要。
{"title":"The molecular epidemiology of respiratory syncytial virus in Ontario, Canada from 2022–2024 using a custom whole genome sequencing assay and analytics package","authors":"Henry Wong ,&nbsp;Calvin P. Sjaarda ,&nbsp;Brittany Rand ,&nbsp;Drew Roberts ,&nbsp;Kyla Tozer ,&nbsp;Ramzi Fattouh ,&nbsp;Robert Kozak ,&nbsp;Prameet M. Sheth","doi":"10.1016/j.jcv.2024.105759","DOIUrl":"10.1016/j.jcv.2024.105759","url":null,"abstract":"<div><h3>Background</h3><div>Respiratory Syncytial Virus (RSV) infections are a cause of significant morbidity and mortality in children and the elderly. Despite the clinical burden of disease, very little is known about the inter- and intra-seasonal genomic variability of RSV. Furthermore, the recent approval of vaccines and monoclonal antibody therapies will likely lead to higher selective pressure on RSV. Genomic surveillance will be essential to monitor viral changes and inform future therapeutic developments and public health responses. Here, we describe the development of an amplicon-based whole-genome sequencing assay for RSV to enable genomic surveillance.</div></div><div><h3>Methods</h3><div>A 750-bp overlapping amplicon design was developed to co-amplify RSV-A/-B directly from patient samples collected during two respiratory illness seasons (2022/23, 2023/24) for whole-genome sequencing. RSV subtype, clade, and F-protein antigenic site sequences were determined with a custom analytical pipeline.</div></div><div><h3>Results</h3><div>Of the 429 specimens included in the study 410 (95.6 %) samples met acceptability. Our data demonstrated co-circulation of both RSV subtypes, with increasing predominance of RSV-A since 2022. There were seven genomic clades of RSV-A, while &gt;95 % of RSV-B belonged to a single clade. 1.5 % of samples had amino acid changes within the binding sites of the current RSV therapeutics Palivizumab or Nirsevimab.</div></div><div><h3>Conclusions</h3><div>Continuous monitoring of RSV genotypes and mutations will be critical for understanding the impact of new therapeutics and vaccines on RSV epidemiology and detecting emergence of vaccine-escape and/or antiviral resistant mutations.</div></div>","PeriodicalId":15517,"journal":{"name":"Journal of Clinical Virology","volume":"176 ","pages":"Article 105759"},"PeriodicalIF":4.0,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142894852","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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Journal of Clinical Virology
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