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Use of oral fluid samples for the investigation of outbreaks of human parvovirus B19 infection.

ABSTRACT: The use of oral fluid (OF) samples for serological diagnosis of parvovirus B19 infection during outbreaks of erythema infectiosum had already been demonstrated, but the feasibility of using OF for the characterization of B19 genotypes circulating during outbreaks has not been described. The aim of this study was to assess the use of "in-house" PCR-based assays as a powerful tool for a rapid diagnosis and molecular characterization of B19 strains in OF samples during outbreaks. Paired serum and OF samples collected from anti-B19 IgM-positive patients, during two outbreaks of ertythema infectiosum (1999-2000 and 2004-2005), were tested by conventional (cPCR) and quantitative PCR (qPCR). qPCR was more sensitive than cPCR for detecting B19-DNA in both OF and serum. Overall, OF presented lower viral load (9.97 × 10⁶ UI/mL) than serum (2.42 × 10¹⁰ UI/mL) and this difference was statistically significant. All OF samples obtained from patients in the age group < 14 years presented low viral load (< 10⁴ IU/mL). No correlation was found between viral load and the number of days of onset of rash. Sequence analysis from PCR positive OF samples confirmed the circulation of subgenotype 1a (G1a) during these outbreaks. Our findings indicate that PCR-based assays may fail to detect B19-DNA in approximately 50% of OF compared to serum samples. Nevertheless, our study has shown for the first time that the genome sequence of the amplicon from non-invasive clinical sample is useful for molecular genotyping and may be a tool to clarify the genetic diversity of B19 strains circulating in distinct outbreaks.

SUBMITTER: Almada DL

PROVIDER: S-EPMC9679058 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Use of oral fluid samples for the investigation of outbreaks of human parvovirus B19 infection.

Almada Daiana Lima DL Alves Arthur Daniel Rocha ADR Leon Luciane Almeida Amado LAA Macedo Débora Familiar Rodrigues DFR de Oliveira Solange Artimos SA Siqueira Marilda Mendonça MM Brown David D Cubel Garcia Rita de Cássia Nasser RCN

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 20220923 4

The use of oral fluid (OF) samples for serological diagnosis of parvovirus B19 infection during outbreaks of erythema infectiosum had already been demonstrated, but the feasibility of using OF for the characterization of B19 genotypes circulating during outbreaks has not been described. The aim of this study was to assess the use of "in-house" PCR-based assays as a powerful tool for a rapid diagnosis and molecular characterization of B19 strains in OF samples during outbreaks. Paired serum and O ...[more]

PMID: 36149627

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Project description:BackgroundHuman parvovirus B19 (HPV B19) is a single-stranded DNA virus. The detection rate of HPV B19 in the blood of healthy blood donors using PCR technology was reported to be 6.323/100000. However, that among hospitalized patients suspected of being infected with a pathogenic microorganism is unknown.MethodsA retrospective analysis was conducted on 2,182 high-throughput NGS results for 1,484 inpatients admitted to the First Affiliated Hospital of Zhengzhou University from January 2020 to October 2021 who were suspected of being infected with a pathogenic microorganism, as well as on clinical data of some HPV B19-positive patients.ResultsHuman parvovirus B19 was detected in 39 samples from 33 patients. The positivity rate was 2.22% among patients and 1.78% among samples. HPV B19 was detected in 20 cerebrospinal fluid samples, 13 blood samples, 3 alveolar lavage fluid samples, 2 tissue samples, and 1 throat swab. Based on clinical symptoms and NGS results, 16 patients were diagnosed with HPV B19 infection. The number of HPV B19 sequences in these patients was greater than 6, and the patients showed common symptoms such as fever (14 cases), anemia (11 cases), and severe nervous system symptoms such as meningoencephalitis (9 cases) and Guillain-Barré syndrome with peripheral motor and sensory nerve axon damage (4 cases). All 16 patients had experienced events likely to lead to decreased immunity (11 had a history of trauma/surgery/major disease, 4 had a history of precursor infection, and 3 had used immunosuppressants) and 7 had a history of blood transfusion during hospitalization. After treatment with antiviral drugs (12 cases) and intravenous human immunoglobulin (3 cases), of the 16 patients, 14 patients improved.ConclusionThe HPV B19 infection rate in hospitalized patients suspected of microbial infection was 2.22%. Most patients with HPV B19 infection had a history of low immunity and blood transfusion. HPV B19 could be detected in various bodily fluids and tissues (especially cerebrospinal fluid) using NGS. Patients with severe HPV B19 infection may have nervous system damage such as Guillain-Barré syndrome and meningoencephalitis. Early diagnosis using NGS and treatment with antiviral drugs and immunoglobulin can improve prognosis.

Project description:Parvovirus B19 (B19V) infection can cause hematological disorders and fetal hydrops during pregnancy. Currently, no antivirals or vaccines are available for the treatment or prevention of B19V infection. To identify novel small-molecule antivirals against B19V replication, we developed a high-throughput screening (HTS) assay, which is based on an in vitro nicking assay using recombinant N-terminal amino acids 1 to 176 of the viral large nonstructural protein (NS1N) and a fluorescently labeled DNA probe (OriQ) that spans the nicking site of the viral DNA replication origin. We collectively screened 17,040 compounds and identified 2,178 (12.78%) hits that possess >10% inhibition of the NS1 nicking activity, among which 84 hits were confirmed to inhibit nicking in a dose-dependent manner. Using ex vivo-expanded primary human erythroid progenitor cells (EPCs) infected by B19V, we validated 24 compounds that demonstrated >50% in vivo inhibition of B19V infection at 10 μM, which can be categorized into 7 structure scaffolds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, the top 4 compounds were chosen to examine their inhibitions of B19V infection in EPCs at two times of the 90% maximal effective concentration (EC90). A purine derivative (P7) demonstrated an antiviral effect (EC50 = 1.46 μM) without prominent cytotoxicity (CC50 = 71.8 μM) in EPCs and exhibited 92% inhibition of B19V infection in EPCs at 3.32 μM, which can be used as the lead compound in future studies for the treatment of B19V infection-caused hematological disorders. IMPORTANCE B19V encodes a large nonstructural protein, NS1. Its N-terminal domain (NS1N) consisting of amino acids 1 to 176 binds to viral DNA and serves as an endonuclease to nick the viral DNA replication origins, which is a pivotal step in rolling-hairpin-dependent B19V DNA replication. For high-throughput screening (HTS) of anti-B19V antivirals, we miniaturized a fluorescence-based in vitro nicking assay, which employs a fluorophore-labeled probe spanning the terminal resolution site (trs) and the NS1N protein, into a 384-well-plate format. The HTS assay showed high reliability and capability in screening 17,040 compounds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, a purine derivative demonstrated an antiviral effect of 92% inhibition of B19V infection in EPCs at 3.32 μM (two times the EC90). Our study demonstrated a robust HTS assay for screening antivirals against B19V infection.

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Use of oral fluid samples for the investigation of outbreaks of human parvovirus B19 infection.

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Use of oral fluid samples for the investigation of outbreaks of human parvovirus B19 infection.

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