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Background Human parechovirus type 3 (HPeV3) is an emerging virus that causes sepsis and meningoencephalitis in neonates and young infants. Correct diagnosis of HPeV3 infection is critical in determining appropriate management and predicting patients’ clinical course. Real-time reverse transcription PCR (RT-PCR) analysis of serum and/or cerebrospinal fluid (CSF) has been used to diagnose HPeV3 infection; however, the assay detection limits have not been fully evaluated. Objectives We tested the hypothesis that droplet digital RT-PCR (RT-ddPCR)—a novel technique that precisely quantitates low-copy target genes by diluting and partitioning samples into compartments—increases the detection rate of HPeV3 RNA as compared with real-time RT-PCR. Study design Using samples with predetermined HPeV3 copy numbers, we evaluated one-step and two-step RT-ddPCR. Then, we tested two-step RT-ddPCR and real-time RT-PCR, using clinical samples with low copy numbers. Finally, we used two-step RT-ddPCR to evaluate clinical samples obtained from HPeV3-infected patients with positive serum but negative CSF, as determined by real-time RT-PCR. Results Two-step RT-ddPCR was less variable and more specific than one-step RT-ddPCR. Two-step RT-ddPCR detected HPeV3 RNA in all six CSF samples; four samples (67%) were reproducibly positive and the other two samples (33%) were positive at least once in four replicates. Finally, no nonspecific droplet was positive by two-step RT-ddPCR. Conclusions Two-step RT-ddPCR may enhance the rate of HPeV3 RNA detection from samples with low viral loads, thereby improving diagnosis and management of HPeV3-infected patients. [ABSTRACT FROM AUTHOR]

Background Human parechovirus type 3 (HPeV3) epidemics occur worldwide and can lead to severe disease in neonates and young infants. Little is known about the source of HPeV3 infection. Objectives To investigate the source of HPeV3 infection and the role of asymptomatic children in the families of infected children. Study design During a 2014 HPeV3 epidemic in Niigata, Japan, we analyzed (1) clinical information on sick contacts for 43 neonates and young infants with HPeV3-related disease diagnosed by PCR analysis of serum and/or cerebrospinal fluid and (2) stool samples from symptomatic and asymptomatic siblings/cousins of index patients. To confirm transmission, the P1 (VP0, VP3, and VP1) and 3D pol regions of HPeVs were sequenced and analyzed. Results Sick contact with family members was confirmed for 51% ( n = 22) of patients. Among the 30 symptomatic family members, 67% ( n = 20) were siblings, 20% ( n = 6) were mothers, and 13% ( n = 4) were other relatives. Stool samples from symptomatic and asymptomatic siblings/cousins of 4 HPeV3-infected patients yielded positive results for HPeVs on PCR analysis. Furthermore, the P1 and 3D pol nucleotide sequences of family members were 100% identical to those of the respective index cases. Conclusions Identification of genetically identical virus from HPeV3-infected patients and asymptomatic children in their families suggests that the latter are a source of infection in neonates and young infants with HPeV3-related diseases. [ABSTRACT FROM AUTHOR]

• HPeV-3 and EV are commonly detected in febrile neonates and infants. • Serum and CSF were PCR-positive for 100 % and 88 % of HPeV-3-infected patients, respectively. • Serum and CSF were PCR-positive for 83 % and 71 % of EV-infected patients, respectively. • In EV-infected patients, detection rates for serum and CSF varied by EV genotype. • Collection of both serum and CSF samples improves viral diagnosis in this population. Human parechovirus 3 (HPeV-3) and enteroviruses (EV) are commonly detected viruses in febrile neonates and young infants and are usually diagnosed by PCR. However, in this population, data on detection rates for samples from different anatomical sites are limited. To determine PCR detection rates for HPeV-3 and EVs in serum and cerebrospinal fluid (CSF) samples from febrile neonates and young infants. This prospective study identified viruses in serum and CSF samples collected from febrile neonates and young infants (age <4 months) in Niigata, Japan, during 2014−2018. HPeV-3 or EV infection was defined as a positive quantitative real-time PCR result for the virus in serum or CSF. Genotypes were identified by sequence analyses of the viral protein 1 region. Among 216 patients, we identified 56 HPeV-3-infected (26 %) and 48 EV-infected patients (22 %). All (56/56; 100 %) HPeV-3-infected patients had a positive PCR result for serum, and 49/56 (88 %) had a positive result for CSF. In EV-infected patients, 40/48 (83 %) were positive for serum, and 34/48 (71 %) were positive for CSF, and 22/48 (46 %) were positive for serum (n = 14) or CSF (n = 8). If only a CSF sample had been obtained, 7 (12 %) HPeV-3 infections and 14 (29 %) EV infections would have been undiagnosed. Detection rates in serum and CSF differed by genotype in EV-infected patients. Viral RNA detection rates differed between serum and CSF in HPeV-3- and EV-infected neonates/infants. Combined evaluation of serum and CSF samples is important for accurate viral diagnosis in this population. [ABSTRACT FROM AUTHOR]

• PeV-A3 and EVs are the most common viruses causing severe diseases in young infants. • PeV-A3 patients had some severer symptoms and signs compared to EVs patients. • PeV-A3 patients tended to have a lower WBC count in blood and no CSF pleocytosis. • The scoring system with these findings were successful to distinguish 2 infections. Parechovirus-A3 (PeV-A3) and the enteroviruses (EVs) are the most common viral pathogens responsible for sepsis and meningoencephalitis in neonates and young infants; however, differences in the clinical presentations of two infections are not well described. To describe the clinical presentations of PeV-A3- and EVs-related diseases and develop a novel scoring system to differentiate two diseases. This prospective study used real-time PCR and genetic sequencing to evaluate viral etiologies of febrile neonates and infants <4 months with suspected sepsis or meningoencephalitis in Niigata area, Japan, in 2014-2016. The clinical manifestations of PeV-A3- and EVs-infected patients were compared, and a novel scoring system was developed after identifying the most distinguishable clinical findings, followed by the external cohort validation. In 210 patients evaluated, we identified 56 PeV-A3-infected (27%) and 43 EVs-infected (20%) patients. The following clinical manifestations were significant in PeV-A3-infected patients, as compared with EVs-infected patients; a higher body temperature (38.9°C vs. 38.5°C, P <.01) and heart rate (181/min vs. 168/min, P =.01), cold extremities (72% vs. 34%, P <.01) and skin mottling (65% vs. 23%, P <.01), lower white blood cell count (5,200/μL vs. 8,900/μL, P <.01) and incidence of cerebrospinal fluid (CSF) pleocytosis (2% vs. 63%, P <.01). Using some of these significant findings, the scoring system successfully distinguished the diseases (accuracy: 86% and 83% for the derivative and external validation cohorts, respectively). We found significant clinical manifestations in PeV-A3-infected patients compared to EVs-infected patients. The scoring system may be helpful to distinguish two infections, especially at onset of outbreak. [ABSTRACT FROM AUTHOR]