Journal of Clinical Virology最新文献

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS).

A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline.

Overall, viral pathogens with loads down to 10⁴ copies/ml (corresponding to C_T values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of C_T values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively.

A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.

Background

Measles, mumps, and rubella(MMR) vaccination is critical to measles outbreak responses. However, vaccine reactions and detection of measles vaccine RNA in recently immunized persons may complicate case classification especially in those presenting with another respiratory viral illness. We aim to characterize cases of measles vaccine shedding in recently vaccinated children presenting with respiratory viral symptoms.

Methods

Children who were tested with a multiplex respiratory panel <30 days after receiving MMR were identified. Remnant nasopharyngeal(NP) samples were tested for measles vaccine by PCR. Medical records were reviewed for demographics, presenting symptoms, and test results.

Results

From January 2022 to March 2023, 127 NP from children who received MMR were tested. Ninety-six NP were collected after the first dose, of which 33(34.4 %) were positive for vaccine RNA. The median interval between MMR and detection was 11.0 days. Thirty-one NP were collected after the second MMR and 1(3.2 %) was positive; time between the vaccination and detection was 18.9 days. Median cycle threshold(Ct) value of the measles PCR for vaccine shedding was significantly higher than median Ct in children with wild-type infection.

Conclusion

Shedding of measles vaccine RNA is not uncommon and vaccine RNA can be detected up to 29 days post MMR; the amount of vaccine RNA shedding is low indicated by high Ct values. Clinicians and public health officials should consider performing measles vaccine testing on those testing positive for measles within one month of MMR vaccination, especially if the Ct value is high and definitive epidemiological links are absent.

Background

Community-acquired pneumonia (CAP) is a major global cause of death and hospitalization. Bacteria or community-acquired viruses (CARVs) cause CAP. COVID-19 associated restrictions effectively reduced the circulation of CARVs.

Objectives

The aim of this study was to analyze the proportion of CARVs in adult patients with CAP from mid-2020 to mid-2023. Specifically, we aimed to compare the rate of influenza virus, SARS-CoV-2, and RSV detections in patients aged 18–59 years and ≥60 years.

Study design

We analyze the proportion of 21 community-acquired respiratory viruses (CARVs) and three atypical bacteria (Bordetella pertussis, Legionella pneumophila, and Mycoplasma pneumoniae) in nasopharyngeal swab samples using molecular multiplex methods within the prospective, multicentre, multinational study of the German study Group CAPNETZ. We used stringent inclusion criteria throughout the study.

Results

We identified CARVs in 364/1,388 (26.2 %) patients. In detail, we detected SARS-CoV-2 in 210/1,388 (15.1 %), rhino-/enterovirus in 64/1,388 (4.6 %), influenza virus in 23/1,388 (1.6 %) and RSV in 17/1,388 (1.2 %) of all patients. We detected RSV and influenza more frequently in patients ≥60 years, especially in 22/23 compared to the previous season. None of the atypical bacteria were detected.

Conclusions

Beginning in 2023, we demonstrate a re-emergence of CARVs in CAP patients. Effective vaccines or specific antiviral therapies for more than two thirds of the detected viral infections are currently available. High detection rates of vaccine-preventable viruses in older age groups support targeted vaccination campaigns.

Background

: Viral respiratory Infections pose a health risk, especially to vulnerable patient populations. Effective testing programs can detect and differentiate these infections at an early stage, which is particularly important for high-risk clinical departments. The objective of this study was to develop and validate a multiplex PCR-panel for 16 different respiratory viruses on a fully-automated high-throughput platform.

Methods

Three multiplex-PCR assays were designed to run on the cobas5800/6800/8800 systems, consolidating 16 viral targets: RESP1: SARS-CoV-2, influenza-A/B, RSV; RESP2: hMPV, hBoV, hAdV, rhino-/ENV; RESP3: HPIV-1–4, hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1. Analytic performance was evaluated using digital-PCR based standards and international reference material. Clinical performance was determined by comparing results from clinical samples with reference assays.

Results

Analytical sensitivity (i.e. lower limit of detection (LoD), 95 % probability of detection) was determined as follows: SARS-CoV-2: 29.3 IU/ml, influenza-A: 179.9 cp/ml, influenza-B: 333.9 cp/ml and RSV: 283.1 cp/ml. LoDs of other pathogens ranged between 9.4 cp/ml (hCoV-NL63) and 21,419 cp/ml (HPIV-2). Linearity was verified over 4–7 log-steps with pooled standard differentials (SD) ranging between 0.18–0.70ct. Inter-/intra-run variability (precision) was assessed for all targets over 3 days. SDs ranged between 0.13–0.74ct. Positive agreement in clinical samples was 99.4 % and 95 % for SARS-CoV-2 and influenza-A respectively. Other targets were in the 80–100 % range. Negative agreement varied between 96.3–100 %.

Discussion

Lab-developed tests are a key factor for effective clinical diagnostics. The multiplex panel presented in this study demonstrated high performance and provides an easily scalable high-throughput solution for respiratory virus testing, e.g. for testing in high-risk patient populations.

Background

A global mpox outbreak occurred in 2022, and a domestic outbreak started in South Korea in April 2023. This study aimed to evaluate the clinical characteristics, viral shedding, and immune response of mpox in South Korea.

Methods

Patients hospitalized with mpox in the National Medical Center between September 2022 and June 2023 were included in this study. Oropharyngeal (OP), anogenital lesion (AL), and skin lesion (SL) swabs and blood samples were collected, and monkeypox virus (MPXV) DNA using real-time polymerase chain reaction (RT-PCR) and culture assays were performed. Neutralizing antibodies (NAbs) against MPXV A.2.1, B.1.1, and B.1.3 were detected using plaque reduction neutralization tests.

Results

Eighteen patients were enrolled, of whom 17 (94.4 %) were male, with a median (IQR) age of 32.5 (24–51) years. While nine (50 %) were HIV-infected individuals, none of them revealed CD4+ counts less than 200 cells/μL. MPXV DNA was detected in 87.3 % and 82.7 % of patient's ALs and SLs, respectively, until 2 weeks after symptom onset. While MPXV was isolated for up to 15 days in all three sample types, the culture positivity decreased to 53.8 % and 42.9 % in ALs and SLs after 10 days, respectively, and 28.6 % and 22.2 %, respectively, after 2 weeks from symptom onset. The NAb titers against MPXV A.2.1 were significantly lower than those against B.1.1 and B.1.3.

Conclusions

Infectious MPXV was isolated from various anatomical sites up to 15 days after symptom onset. The MPXV NAb response was varied among different lineages, and this implies limited cross-lineage protection.

Respiratory pathogens, such as SARS-CoV-2 and influenza A/B, can cause severe illnesses in susceptible individuals. This research evaluated a novel digital microfluidic point-of-care testing platform designed to detect 23 pathogens, comparing its performance to conventional laboratory-based nucleic acid tests. The platform integrates nucleic acid extraction and amplification processes for rapid detection with only 2 min of hands-on time. Performance assays demonstrated that the platform has high sensitivity (87 %-100 %) and specificity (99 %-100 %) for the detection of the evaluated 3 viruses. Additionally, the platform can be adapted for the detection of other respiratory pathogens, aiding in the early diagnosis of respiratory diseases, identifying the source of an outbreak or epidemic, and curbing the spread of the disease.

Background

Cytomegalovirus (CMV) induces multi-organ pathogenesis in hematopoietic stem cell transplant (HSCT) and kidney transplant (KT) recipients. Effective management involves systematic monitoring for CMV reactivation by quantitative real-time PCR, allowing timely preemptive intervention. However, the optimal blood compartment for CMV surveillance remains undetermined.

Objective

The aim of the study was to compare the quantification of CMV DNA in paired plasma and whole blood samples.

Study design

From June and October 2022, we conducted a prospective study with 390 sets of paired plasma and whole blood specimens collected from 60 HSCT and 24 KT recipients. CMV DNA levels were compared between the cobas® CMV assay on the automated cobas® 6800 system for plasma and the reference assay, Abbott RealTime CMV assay on the m2000 RealTime platform for whole blood.

Results

The sensitivity and specificity of CMV quantification in plasma using the cobas® CMV assay were 90.0 % (95 %CI: 81.5 to 95.9) and 94.8 % (95 %CI: 91.8 to 96.8), respectively, compared to whole blood quantification with the Abbott assay. The overall agreement between these two strategies was 0.89 (95 %CI: 0.86–0.91). In samples with quantifiable results, a correlation was observed between the two methods (R² = 0.62, 95 %CI: 0.65–0.87, p < 0.0001). CMV loads were significantly higher in whole blood, with a mean bias of 0.42 log₁₀ IU/mL (95 %CI: -0.32–1.15).

Conclusion

The cobas® CMV assay in plasma showed significant concordance with the Abbott RealTime CMV assay in whole blood, confirming the relevance of plasma samples for CMV monitoring in HSCT and KT recipients.

Background

The increasing incidence of hand, foot, and mouth disease (HFMD) associated with Coxsackievirus A6 (CVA6) has become a very significant public health problem. The aim of this study is to investigate the recombination, geographic transmission, and evolutionary characteristics of the global CVA6.

Methods

From 2019 to 2022, 73 full-length CVA6 sequences were obtained from HFMD patients in China and analyzed in combination with 1032 published whole genome sequences. Based on this dataset, the phylogenetic features, recombinant diversity, Bayesian phylodynamic characteristics, and key amino acid variations in CVA6 were analyzed.

Results

The four genotypes of CVA6, A, D, E, and F, are divided into 24 recombinant forms (RFs, RF-A – RF-X) based on differences in the P3 coding region. The eastern China region plays a key role in the dissemination of CVA6 in China. VP1–137 and VP1–138 are located in the DE loop on the surface of the CVA6 VP1 protein, with the former being a highly variable site and the latter having more non-synonymous substitutions.

Conclusions

Based on whole genome sequences, this study contributes to the CVA6 monitoring, early warning, and the pathogenic mechanism by studying recombination diversity, geographical transmission characteristics, and the variation of important amino acid sites.

Background

Following the pandemic restrictions, the epidemiology of respiratory syncytial virus (RSV) has changed, leading to intense hospitalization peaks.

Objectives

This study, conducted at multiple sites in Italy, aimed to describe the temporal dynamics of two post-COVID-19 RSV epidemics. Additionally, the circulating RSV-A and -B lineages were characterized and compared to those found in 2018 and 2019.

Study design

Respiratory specimens and data were collected from RSV-positive patients, both inpatients, and outpatients, of all ages at three sites in north-central Italy. To analyze these samples, roughly one-sixth were sequenced in the attachment glycoprotein G gene and subjected to phylogenetic and mutational analyses, including pre-pandemic sequences from north-central Italy.

Results

The first post-pandemic surge of RSV cases was quite intense, occurring from October 2021 to early January 2022. The subsequent RSV epidemic (from November 2022 to early March 2023) also had a high impact, characterized by a rise in elderly patient cases. Post-pandemic cases of RSV-A were caused by various strains present in Italy prior to COVID-19. In contrast, a distinct RSV-B lineage, which was concurrently spreading in other countries, was identified as the main cause of the surge in 2022–2023 but remained undetected in Italy before the pandemic.

Conclusions

This study describes the temporal dynamics of post-pandemic RSV subgroups and uncovers a lineage of RSV-B with high genetic divergence that may have increased the impact of decreased population immunity.

Background

HDV antibody testing is recommended for universal screening and as the first line in an HDV double reflex testing strategy for effectively identifying patients with active infection for therapeutic treatments.

Objective

The aim of this study is to evaluate the performance of a newly developed ARCHITECT HDV Total Ig (ARCHITECT HDV Ig) prototype assay.

Study design

Performance characteristics were determined for the ARCHITECT HDV Ig and a reference test, LIAISON XL Anti-HDV using a well-characterized specimen panel, comprising HDV RNA positive (n = 62) and negative (n = 70) samples, and healthy US blood donors.

Results

Healthy US blood donors (n=200) showed 99.5% (199/200, 95%CI=97.65–99.98) specificity with ARCHITECT HDV Ig and 98.5 % (197/200, 95 %CI = 96.10–99.64) with LIAISON Anti-HDV. Among known HDV RNA positive samples, ARCHITECT HDV Ig detected 59/62 demonstrating 95.2 % sensitivity while LIAISON Anti-HDV sensitivity was 90.3 % (56/62). Among 101 HBV positive samples, 70 were reactive in the ARCHITECT test, 59 of which tested positive for HDV RNA for a positive predictive value (PPV) for the presence of HDV RNA was 84.3 %. For LIAISON Anti-HDV, 79 specimens were reactive and 56 contained HDV RNA: PPV for HDV RNA was 70.9 %. Among 70 HDV RNA negative samples, 39 were HBV positive. ARCHITECT HDV Ig negative predictive value (NPV) was 71.8 % and LIAISON Anti-HDV NPV was 41 % for the HBV positive group, respectively.

Conclusion

When compared to the LIASON Anti-HDV test, the ARCHITECT HDV Ig assay demonstrated enhanced sensitivity and specificity and better NPV and PPV values for HDV RNA status. The ARCHITECT HDV Ig assay represents a promising tool for universal screening of all HBsAg-positive persons.