Journal of Clinical Virology最新文献

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.

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 %.

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.

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.

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.

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.

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.