Distinct clinical features of transplanted children with Parvovirus B19 infection.

BACKGROUND: The immature and suppressed immune response makes transplanted children a special susceptible group to Parvovirus B19 (PVB19). However, the clinical features of transplanted children with PVB19 infection haven't been comprehensively described. METHODS: We searched the medical records of all the transplant recipients who attended the Children's Hospital of Fudan University from 1 Oct 2020 to 31 May 2023, and reviewed the medical literature for PVB19 infection cases among transplanted children. RESULTS: A total of 10 cases of PVB19 infection were identified in 201 transplanted children at our hospital, and the medical records of each of these cases were shown. Also, we retrieved 40 cases of PVB19 infection among transplanted children from the literature, thus summarizing a total of 50 unique cases of PVB19 infection. The median time to the first positive PVB19 DNA detection was 14 weeks post-transplantation. PVB19 IgM and IgG were detected in merely 26% and 24% of the children, respectively. The incidence of graft loss/dysfunction was as high as 36%. Hematopoietic stem cell transplant (HSCT) recipients showed higher PVB19 load, lower HGB level, greater platelet damage, lower PVB19 IgM/IgG positive rates, and more graft dysfunction than solid-organ transplant (SOT) recipients, indicating a more incompetent immune system. CONCLUSIONS: Compared with the published data of transplanted adults, transplanted children displayed distinct clinical features upon PVB19 infection, including lower PVB19 IgM/IgG positive rates, more graft dysfunction, and broader damage on hematopoietic cell lines, which was even more prominent in HSCT recipients, thus should be of greater concern.

Nano-flow cytometry unveils mitochondrial permeability transition process and multi-pathway cell death induction for cancer therapy.

Mitochondrial permeability transition (mPT)-mediated mitochondrial dysfunction plays a pivotal role in various human diseases. However, the intricate details of its mechanisms and the sequence of events remain elusive, primarily due to the interference caused by Bax/Bak-induced mitochondrial outer membrane permeabilization (MOMP). To address these, we have developed a methodology that utilizes nano-flow cytometry (nFCM) to quantitatively analyze the opening of mitochondrial permeability transition pore (mPTP), dissipation of mitochondrial membrane potential ( Δ Ψm), release of cytochrome c (Cyt c), and other molecular alternations of isolated mitochondria in response to mPT induction at the single-mitochondrion level. It was identified that betulinic acid (BetA) and antimycin A can directly induce mitochondrial dysfunction through mPT-mediated mechanisms, while cisplatin and staurosporine cannot. In addition, the nFCM analysis also revealed that BetA primarily induces mPTP opening through a reduction in Bcl-2 and Bcl-xL protein levels, along with an elevation in ROS content. Employing dose and time-dependent strategies of BetA, for the first time, we experimentally verified the sequential occurrence of mPTP opening and Δ Ψm depolarization prior to the release of Cyt c during mPT-mediated mitochondrial dysfunction. Notably, our study uncovers a simultaneous release of cell-death-associated factors, including Cyt c, AIF, PNPT1, and mtDNA during mPT, implying the initiation of multiple cell death pathways. Intriguingly, BetA induces caspase-independent cell death, even in the absence of Bax/Bak, thereby overcoming drug resistance. The presented findings offer new insights into mPT-mediated mitochondrial dysfunction using nFCM, emphasizing the potential for targeting such dysfunction in innovative cancer therapies and interventions.

Mitochondrial Esterase Activity Measured at the Single Organelle Level by Nano-flow Cytometry.

Monitoring mitochondrial esterase activity is crucial not only for investigating mitochondrial metabolism but also for assessing the effectiveness of mitochondrial-targeting prodrugs. However, accurately detecting esterase activity within mitochondria poses challenges due to its ubiquitous presence in cells and the uncontrolled localization of fluorogenic probes. To overcome this hurdle and reveal variations among different mitochondria, we isolated mitochondria and preserved their activity and functionality in a buffered environment. Subsequently, we utilized a laboratory-built nano-flow cytometer in conjunction with an esterase-responsive calcein-AM fluorescent probe to measure the esterase activity of individual mitochondria. This approach enabled us to investigate the influence of temperature, pH, metal ions, and various compounds on the mitochondrial esterase activity without any interference from other cellular constituents. Interestingly, we observed a decline in the mitochondrial esterase activity following the administration of mitochondrial respiratory chain inhibitors. Furthermore, we found that mitochondrial esterase activity was notably higher in the presence of a high concentration of ATP compared to that of ADP and AMP. Additionally, we noticed a correlation between elevated levels of complex IV and increased mitochondrial esterase activity. These findings suggest a functional connection between the mitochondrial respiratory chain and mitochondrial esterase activity. Moreover, we detected an upsurge in mitochondrial esterase activity during the early stages of apoptosis, while cellular esterase activity decreased. This highlights the significance of analyzing enzyme activity within specific organelle subregions. In summary, the integration of a nano-flow cytometer and fluorescent dyes introduces a novel method for quantifying mitochondrial enzyme activity with the potential to uncover the alterations and unique functions of other mitochondrial enzymes.

Emergence and high prevalence of unusual rotavirus G8P[8] strains in outpatients with acute gastroenteritis in Shanghai, China.

Group A rotavirus (RVA) is considered an important cause of acute gastroenteritis (AGE) in all age groups, especially in children. We investigated the epidemiology of RVA in outpatients aged ≤ 16 years at the Children's Hospital of Fudan University, Shanghai, China. In this study, 16.6% (246/1482) were infected with RVA. The detection rate of RVA was significantly higher in the year of 2021 (20.3%, 147/725) compared to the year of 2020 (14.5%, 77/531) and 2022 (9.7%, 22/226) (p = 0.000). RVA infection was prevalent in all seasons from 2020 to 2022, with a different monthly distribution observed in different years. Among 246 RVA-positive samples, 14 different RVA genotypes were detected with different frequencies. Overall, G9P[8] (45.5%, 112/246) was the most common RVA genotype, followed by G8P[8] (37.4%, 92/246) and G3P[8] (4.1%, 10/246). The prevalence of G/P combinations varied from 2020 to 2022. G9P[8] was the most prevalent circulating genotype in 2020 (68.2%, 15/22) and 2021 (57.8%, 85/147). However, G8P[8] (68.8%, 53/77) suddenly became the most prevalent genotype in 2022 after being first identified in 2020 and prevalent in 2021. The G8 strains detected in the study were all clustered to DS-1-like G8 strains with the closest genetic distance to strains circulating in Southeast Asia. Our study demonstrated the diversity of circulating RVA genotypes in Shanghai. The sudden emergence and high prevalence of unusual G8P[8] strains deserve more concern and indicate the need for continuous surveillance of RVA in children with AGE in the future to refine future vaccine strategy.

Diversity of classic and novel human astrovirus in outpatient children with acute gastroenteritis in Shanghai, China.

Introduction: Human astrovirus (HAstV) is an important pathogen of acute gastroenteritis (AGE) in children. This study was aimed at investigating the diversity and epidemiology of classic and novel HAstV in outpatient children aged 0-16 years old with AGE in Shanghai. Methods: From May 2020 to December 2022, a total of 1,482 stool samples were collected from children diagnosed as AGE from the Children's Hospital of Fudan University. HAstV was identified using pan-astrovirus consensus primers by Reverse transcription PCR. Results: During the study period, 3.3% (49/1,482) of specimens were identified as HAstV, with a detection rate of 2.5% (37/1,482) for classic HAstV and 0.8% (12/1,482) for novel HAstV. Among the 12 novel HAstV strains, 11 (91.7%) belonged to the HAstV-MLB and 1 (8.3%) was HAstV-VA. Genotyping revealed six circulating genotypes. Strain HAstV-1 was predominant in the study population with a detection rate of 1.8% (26/1,482) followed by HAstV-MLB1 (0.7%, 10/1,482) and HAstV-4 (0.6%, 9/1,482). Of note, all the HAstV-4 strains detected in this study were close to one astrovirus strain isolated from Bactrian camels with 99.0-100.0% amino acid sequences identity. In this study, HAstV was detected in all age groups with the highest detection rate of HAstV-positive specimens observed in children older than 73 months (5.7%, 12/209). Discussion: This study provided useful information and contributed to the molecular epidemiology of both classic and novel HAstV, which were simultaneously characterized and reported for the first time in Shanghai.

Changing predominance of norovirus strains in children with acute gastroenteritis in Shanghai, 2018-2021.

Norovirus (NoV) is a major pathogen that causes acute gastroenteritis (AGE) in people of all ages, especially in children. In this study, we investigated the molecular epidemiological characteristics of NoV in children with AGE in Shanghai from 2018 to 2021. The overall detection rate of NoV was 11.9% (181/1545), with annual detection rates of 9.4% (36/381), 13.6% (29/213), 5.8% (13/226) and 14.2% (103/725), respectively. Of note, the prevalence of NoV in 2020 was significantly lower than that in 2018-2019 (10.9%, 65/594) (P â€‹= â€‹0.023) and 2021 (14.2%, 103/725) (P â€‹= â€‹0.000). The 181 NoV strains identified in this study were classified into the GI group (1.1%, 2/181), GII group (98.3%, 178/181) and GIX group (0.6%, 1/181) according to the VP1 gene. The most common NoV VP1 genotype was GII.4 Sydney_2012 (63.5%, 115/181), followed by GII.3 (19.9%, 36/181) and GII.2 (9.4%, 17/181). For P genotypes, 174 strains were sequenced successfully according to the RdRp gene, and the predominant genotype was GII.P16 (44.8%, 78/174), followed by GII.P31 (25.9%, 45/174) and GII.P12 (21.3%, 37/174). Among the 174 cases, GII.4 Sydney_2012[P16] (36.8%, 64/174) was the dominant genotype, followed by GII.4 Sydney_2012[P31] (25.3%, 44/174), GII.3[P12] (20.1%, 35/174) and GII.2[P16] (8.0%, 14/174). In particular, the dominant genotypes in Shanghai changed from GII.4 Sydney_2012[P31] in 2018-2019 to GII.4 Sydney_2012[P16] in 2020-2021. This is the first report to describe the epidemiological changes in NoV infection before and during the COVID-19 pandemic in Shanghai. These data highlight the importance of continuous surveillance for NoV in children with AGE in Shanghai.

Surveillance and epidemiological characterization of human adenovirus infections among outpatient children with acute gastroenteritis during the COVID-19 epidemic in Shanghai, China.

BACKGROUND: Human adenovirus (HAdV) has been recognized as one of the common enteric viruses associated with acute gastroenteritis (AGE) in children. The aim of this study was carried out to illustrate the epidemiological characterization of HAdV Infections among children younger than 15 years in Shanghai during COVID-19. METHODS: During May 2020 and April 2022, 1048 fecal samples were collected from children ≤ 15 years diagnosed with AGE in the Children's Hospital of Fudan University. HAdV was identified by PCR and sequenced with specific primers. All the obtained sequences were analyzed by MEGA (version 6.0). Demographic information and clinical features data were also collected and analyzed. RESULTS: In total, 97 (9.3%, 97/1048) samples were detected to be HAdV during May 2020 and April 2022. We found an atypical upsurge in HAdV infection in the year 2021 after a major suppression in the year 2020. Approximately 84.5% (82/97) of HAdV-infected children were aged 0-60 months. Among the 97 HAdV-positive samples, only two species and five genotypes were detected. HAdV-F (88.7%, 86/97) was the most prevalent species and HAdV-F41 (87.6%, 85/97) was the most common genotype. Diarrhea, vomiting, and fever were the main clinical manifestations in children infected with HAdV. The children aged from 0 to 12 months showed simpler patterns of clinical presentation than those of children older than 13 months. CONCLUSIONS: Our findings described the epidemiological changes of HAdV infection in children with AGE during the COVID-19, which further underscored the importance of continuous surveillance of HAdV at both local and global scales.

Distribution of CMV envelope glycoprotein B, H and N genotypes in infants with congenital cytomegalovirus symptomatic infection.

Background: Cytomegalovirus (CMV) is the leading cause of congenital infections worldwide and contributes to long-term sequelae in neonates and children. CMV envelope glycoproteins play a vital role in virus entry and cell fusion. The association between CMV polymorphisms and clinical outcomes remains controversial. The present study aims to demonstrate the distribution of glycoprotein B (gB), H (gH) and N (gN) genotypes in congenitally CMV (cCMV) infected symptomatic infants and attempts to figure out the association between viral glycoprotein genotypes and clinical outcomes. Methods: Genotyping of gB, gH and gN was performed in 42 cCMV symptomatic infants and 149 infants with postnatal CMV (pCMV) infection in Children's hospital of Fudan university. Nested PCR, gene sequencing and phylogenetic analyses were used to identify the genotypes. Results: Our study demonstrated that: 1. The CMV gB1, gH1 and gN1 were the predominant genotypes among symptomatic cCMV infected infants, while gB1, gH1 and gN3a were more prevalent in pCMV group. gH1 genotype has a significant association with symptomatic cCMV infection (p = 0.006). 2. No significant correlation was found between CMV genotypes and hearing impairment. However, gH1 was more prevalent among cCMV infected infants with moderate/severe hearing loss although without statistical difference (p = 0.130). 3. gB3 was more prevalent among infants with skin petechiae (p = 0.049) and found to be associated with an increased risk of skin petechiae (OR = 6.563). The gN4a subtype was significantly associated with chorioretinitis due to cCMV infection (p = 0.007). 4. Urine viral loads were not significantly associated with different genotypes or hearing impairment among symptomatic cCMV infected infants. Conclusions: Our findings demonstrated the overall distribution of gB, gH and gN genotypes in infants with symptomatic cCMV infection in Shanghai for the first time. The findings in our study may suggest a possible association between gH1 genotype and early infancy hearing loss. gB3 genotype was associated with a 6.5-fold increased risk of petechiae while gN4a strongly correlated with chorioretinitis due to cCMV infection. No significant correlation was found between urine viral loads and CMV genotypes or hearing impairment in cCMV infected infants.

Development of Spectral Nano-Flow Cytometry for High-Throughput Multiparameter Analysis of Individual Biological Nanoparticles.

Correlated analysis of multiple biochemical parameters at the single-particle level and in a high-throughput manner is essential for insights into the diversity and functions of biological nanoparticles (BNPs), such as bacteria and subcellular organelles. To meet this challenge, we developed a highly sensitive spectral nano-flow cytometer (S-nFCM) by integrating a spectral recording module to a laboratory-built nFCM that is 4-6 orders of magnitude more sensitive in side scattering detection and 1-2 orders of magnitude more sensitive in fluorescence detection than conventional flow cytometers. An electron-multiplying charge-coupled device (EMCCD) was used to acquire the full fluorescence spectra of single BNPs upon holographic grating dispersion. Up to 10,000 spectra can be collected in 1 min with 2.1 nm resolution. The precision, linearity, and sensitivity were examined. Complete discernment of single influenza viruses against the background signal, discrimination of different strains of marine cyanobacteria in a mixed sample based on their spectral properties of natural fluorescence, classification of bacterial categories exhibiting different patterns of antigen expression, and multiparameter analysis of single mitochondria for drug discovery were successfully demonstrated.

Resurgence of Respiratory Syncytial Virus Infection During COVID-19 Pandemic Among Children in Shanghai, China.

Respiratory syncytial virus (RSV) is the most common pathogen causing acute lower respiratory tract infection (LRTI) in children. RSV usually peaks in winter and declines by early spring in China. The outbreak of coronavirus disease 2019 (COVID-19) was reported to bring changes to the transmission pattern of respiratory pathogens including RSV. Here in this paper, we analyzed RSV-positive nasopharyngeal aspirates from inpatients in the Children's Hospital of Fudan University from October 2019 to October 2021 and compared the clinical features of the RSV-positive patients before and during COVID-19. We found an atypical upsurge of RSV infection in the late summer of 2021 after a major suppression in 2020. RSV B was the main subtype spreading among children throughout the study. Phylogenetic analysis revealed that all RSV A strains belonged to ON1 genotype and all RSV B strains were BA9 genotype. Deduced amino acid analysis displayed different substitutions in the RSV strains observed before and during COVID-19. Demographic analysis suggested that males and infants aged under 5 months were the main populations infected with RSV by gender and age, respectively. Less severe clinical outcomes were observed in patients during COVID-19 than before the pandemic, especially in RSV B-positive patients. Our findings described the epidemiological changes in RSV infection brought by COVID-19, which further underscored the importance of continuous surveillance of RSV in the shadow of COVID-19 at both local and global scales.

Comparison of Respiratory Pathogens in Children With Lower Respiratory Tract Infections Before and During the COVID-19 Pandemic in Shanghai, China.

Objectives: This study aimed to assess the impact of COVID-19 on the prevalence of respiratory pathogens among hospitalized children with lower respiratory tract infections (LRTIs) in Shanghai. Methods: Respiratory specimens were collected from children with LRTIs in Children's Hospital of Fudan University from February 2019 to January 2021 and common respiratory pathogens were detected using multiplex PCR. The data of 13 respiratory pathogens were analyzed and compared between the year of 2020 (from February 2020 to January 2021) and 2019 (from February 2019 to January 2020). Results: A total of 1,049 patients were enrolled, including 417 patients in 2019 and 632 patients in 2020. In 2020, 27.53% of patients were tested positive for at least one pathogen, which was significantly lower than that in 2019 (78.66%). The top three pathogens were Mycoplasma pneumoniae (Mp), human adenovirus (ADV) and human rhinovirus (RV) in 2019, whereas RV, human respiratory syncytial virus (RSV) and human parainfluenza virus (PIV) were the predominant ones in 2020. The positive rates of Mp, ADV, RV, PIV, Influenza virus B (InfB), H3N2, and H1N1 were significantly decreased in 2020. RV was the most detectable respiratory pathogen in 2020, and become the most frequent pathogen in all five age groups. PIV had a high prevalence from October to December 2020 which was even higher than that in 2019. Influenza virus A (InfA) was not detected in 2020. Co-infection was significantly less frequent in 2020. Conclusion: The public health interventions aiming to eliminate COVID-19 have great impact on the prevalence of common respiratory pathogens. The prevalence of RV and PIV reminds us a possible resurgence of some pathogens.

Nitrogen-Embedded Quintuple [7]Helicene: A Helicene-Azacorannulene Hybrid with Strong Near-Infrared Fluorescence.

Herein, a nitrogen-embedded quintuple [7]helicene (N-Q7H) with an azapentabenzocorannulene core, which can be considered to be a helicene/azacorannulene hybrid π-system, was synthesized from azapentabenzocorannulene in a three-step process. N-Q7H is the first example of a multiple helicene with an azabuckybowl core. Single-crystal X-ray diffractometry unambiguously confirmed the structure of the propeller-shaped hybrid π-system. Owing to nitrogen-atom doping in the multiple helicenes and effective hybridization between the helicene and azacorannulene, N-Q7H exhibits considerably redshifted absorption and emission (yellow-to-green color change and green-to-near-infrared fluorescence change) relative to the azapentabenzocorannulene core. The broad absorption from the ultraviolet-visible to the NIR region is ascribable to the allowed transition between the highest occupied molecular orbital and the lowest unoccupied molecular orbital after symmetry breaking, as revealed by density functional theory calculations. Compared to previous propeller-shaped multiple helicenes with corannulene or hexabenzocoronene (etc.) as cores, N-Q7H demonstrates a significantly higher NIR fluorescence quantum efficiency of 28%. Additionally, the chiral-resolution and redox properties of N-Q7H were investigated. The excellent photophysical and inherent chiral properties of N-Q7H suggest that azapentabenzocorannulene can be used as an outstanding nitrogen-embedded core to construct novel multiple helicenes with wide application potential, including as NIR fluorescent bio-probes.

Improving Thermal and Photostability of Polymer Solar Cells by Robust Interface Engineering.

As the power conversion efficiency (PCE) of organic photovoltaics (OPVs) approaches 19%, increasing research attention is being paid to enhancing the device's long-term stability. In this study, a robust interface engineering of graphene oxide nanosheets (GNS) is expounded on improving the thermal and photostability of non-fullerene bulk-heterojunction (NFA BHJ) OPVs to a practical level. Three distinct GNSs (GNS, N-doped GNS (N-GNS), and N,S-doped GNS (NS-GNS)) synthesized through a pyrolysis method are applied as the ZnO modifier in inverted OPVs. The results reveal that the GNS modification introduces passivation and dipole effects to enable better energy-level alignment and to facilitate charge transfer across the ZnO/BHJ interface. Besides, it optimizes the BHJ morphology of the photoactive layer, and the N,S doping of GNS further enhances the interaction with the photoactive components to enable a more idea BHJ morphology. Consequently, the NS-GNS device delivers enhanced performance from 14.5% (control device) to 16.5%. Moreover, the thermally/chemically stable GNS is shown to stabilize the morphology of the ZnO electron transport layer (ETL) and to endow the BHJ morphology of the photoactive layer grown atop with a more stable thermodynamic property. This largely reduces the microstructure changes and the associated charge recombination in the BHJ layer under constant thermal/light stresses. Finally, the NS-GNS device is demonstrated to exhibit an impressive T80 lifetime (time at which PCE of the device decays to 80% of the initial PCE) of 2712 h under a constant thermal condition at 65 °C in a glovebox and an outstanding photostability with a T80 lifetime of 2000 h under constant AM1.5G 1-sun illumination in an N2 -controlled environment.

The changing pattern of common respiratory and enteric viruses among outpatient children in Shanghai, China: Two years of the COVID-19 pandemic.

Nonpharmaceutical interventions (NPIs) taken to combat the coronavirus disease 2019 (COVID-19) pandemic have not only decreased the spread of severe acute respiratory syndrome coronavirus 2 but also have had an impact on the prevalence of other common viruses. This study aimed to investigate the long-term impact of NPIs on common respiratory and enteric viruses among children in Shanghai, China, as NPIs were relaxed after June 2020. The laboratory results and clinical data of outpatient children with acute respiratory tract infections (ARTI) and acute gastroenteritis (AGE) were analyzed and compared between the post-COVID-19 period (from June 2020 to January 2022) and pre-COVID-19 period (from June 2018 to January 2020). A total of 107 453 patients were enrolled from June 2018 to January 2022, including 43 190 patients with ARTI and 64 263 patients with AGE. The positive rates of most viruses decreased during the post-COVID-19 period, with the greatest decrease for influenza A (-0.94%), followed by adenoviruses (AdV) (-61.54%), rotaviruses (-48.17%), and influenza B (-40%). However, the positive rates of respiratory syncytial virus (RSV) and enteric AdV increased during the post-COVID-19 period as the NPIs were relaxed. Besides this, in the summer of 2021, an unexpected out-of-season resurgence of RSV activity was observed, and the resurgence was more prominent among children older than 5 years. The effectiveness of the current relaxed NPIs in control of common respiratory and enteric viruses was variable. Relaxation of NPIs might lead to the resurgence of common viruses.

Prevalence and genotypes distribution of group A rotavirus among outpatient children under 5 years with acute diarrhea in Shanghai, China, 2012-2018.

BACKGROUND: Group A rotavirus (RVA) remains the main causative agent of acute diarrhea among children under five years in countries that have not yet introduced the RVA vaccine worldwide. Long-term and continuous monitoring data on RVA infection in outpatient children were lacking in Shanghai. We investigated the prevalence and distribution of RVA genotypes in outpatient children with acute diarrhea in Shanghai from 2012 to 2018. METHODS: Stool specimens of outpatient children under five years were collected from the Children's Hospital of Fudan University in Shanghai, China. All the samples enrolled in this study were detected and characterized for the P and G genotypes of RVA were determined using the semi-multiplex RT-PCR technique. RESULTS: Of 1814 children enrolled with acute diarrhea and having specimens collected, 246 (13.6%) were infected with RVA. The highest frequency of RVA was observed in children younger than two years old (87.0%, 214/246). Year-round RVA transmission was observed and the RVA detection rate peaked every winter and troughed in summer. In this study, 12 different RVA strains were identified in children. G9P[8] (49.2%, 121/246) was detected as the most prevalent genotype, followed by G-P[8] (22.8%, 56/246), G3P[8] (11.4%, 28/246), and G9P- (4.9%, 12/246). Although RVA strains detected in this study varied with the time, G9P[8] has been the most predominant circulating genotype since 2012. Furthermore, 12.2% (30/246) RVA positive samples were co-infected with other diarrhea viruses. CONCLUSION: The present analysis showed that RVA was still a major cause of children with acute diarrhea in Shanghai from 2012 to 2018. A great diversity of RVA strains circulated in children with acute diarrhea with G9P[8] being the predominant genotype since 2012. Long-term and continuous monitoring of RVA genotypes is therefore indispensable to refine future vaccine strategy in Shanghai.

Development of a multiplex droplet digital PCR assay for detection of enterovirus, parechovirus, herpes simplex virus 1 and 2 simultaneously for diagnosis of viral CNS infections.

BACKGROUND: Enterovirus (EV), parechovirus (HPeV), herpes simplex virus 1 and 2 (HSV1/2) are common viruses leading to viral central nervous system (CNS) infections which are increasingly predominant but exhibit deficiency in definite pathogen diagnosis with gold-standard quantitative PCR method. Previous studies have shown that droplet digital PCR (ddPCR) has great potential in pathogen detection and quantification, especially in low concentration samples. METHODS: Targeting four common viruses of EV, HPeV, HSV1, and HSV2 in cerebrospinal fluid (CSF), we developed a multiplex ddPCR assay using probe ratio-based multiplexing strategy, analyzed the performance, and evaluated it in 97 CSF samples collected from patients with suspected viral CNS infections on a two-channel ddPCR detection system. RESULTS: The four viruses were clearly distinguished by their corresponding fluorescence amplitude. The limits of detection for EV, HPeV, HSV1, and HSV2 were 5, 10, 5, and 10 copies per reaction, respectively. The dynamic range was at least four orders of magnitude spanning from 2000 to 2 copies per reaction. The results of 97 tested clinical CSF specimens were identical to those deduced from qPCR/qRT-PCR assays using commercial kits. CONCLUSION: The multiplex ddPCR assay was demonstrated to be an accurate and robust method which could detect EV, HPeV, HSV1, and HSV2 simultaneously. It provides a useful tool for clinical diagnosis and disease monitoring of viral CNS infections.

Human rhinoviruses prevailed among children in the setting of wearing face masks in Shanghai, 2020.

BACKGROUND: Human rhinovirus (HRV) is the predominant etiological agent of the common cold in children and adults. A recent study showed that the inhibitory effect of face masks on viral shedding of HRV was less prominent than that on other respiratory viruses. Considering that most Chinese people have worn face masks in public area since the outbreak of coronavirus disease 2019, we aimed to find out whether HRV prevailed among children in 2020 and demonstrate the details of the epidemiological features of HRV under such a special circumstance. METHODS: We summarized the incidences of various respiratory virus infections in patients who visited the Children's Hospital of Fudan University during 2018-2020, and genotyped HRV positive nasopharyngeal specimens collected from 316 inpatients and 72 outpatients that visited the hospital in 2020. RESULTS: There was a major prevalence of HRV among children in the latter half of 2020, with a clear seasonality that HRV-As prevailed in summer while HRV-Cs in autumn. HRV-As were more prone to cause severe lower respiratory tract infections (LRTI), while HRV-Cs were closely associated with childhood wheezing. The predominant genotypes were A11, A28, A47, A82, A101, C40 and C43. Notably, A21, A82 and A101 took up larger proportions in severe cases than in non-severe cases. CONCLUSIONS: Our findings described a major prevalence of HRVs among children in 2020, which highlight the unique transmitting pattern of HRV and help to narrow the targets for antiviral strategies.

Mycoplasma pneumoniae and Adenovirus Coinfection Cause Pediatric Severe Community-Acquired Pneumonia.

Consolidation is one complication of pediatric severe community-acquired pneumonia (SCAP) that can respond poorly to conservative medical treatment. We investigated the pathogens that cause pediatric SCAP including cases with persistent consolidation that need bronchoscopy intervention. Alveolar lavage fluid (ALF) samples collected from cases admitted to Children's Hospital of Fudan University with SCAP during January 2019 to March in 2019 were retrospectively tested by the RespiFinder 2SMART multiplex PCR (multi-PCR) assay targeting 22 respiratory pathogens. A total of 90 cases and 91 samples were enrolled; 80.0% (72/90) of the cases had pulmonary consolidation and/or atelectasis. All samples were positive with targeted pathogens tested by multi-PCR, and 92.3% (84/91) of the samples were co-detected with pathogens. Mycoplasma pneumoniae (MP) and adenovirus (ADV) as the two dominant pathogens, with the positive rates of 96.7% (88/91) and 79.1% (72/91), respectively. Most of the samples were positive with MP and ADV simultaneously. As a control, 78.0% (71/91) of the samples were positive by conventional tests (CT), in which MP had the detection rate of 63.9% (55/86) by a traditional real-time PCR assay, while ADV were positive in 13.1% (12/91) of the samples by a direct immunofluorescence assay (DFA). In cases with persistent pulmonary consolidation, the positive rates of pathogens by multi-PCR and CT were 100% (72/72) and 81.9% (59/72), respectively. There were no significant differences of MP or ADV positive rates between cases with and without pulmonary consolidation. MP and ADV most prevalent in pediatric SCAP cases required fiberscope intervention, and presented with coinfections dominantly. IMPORTANCE Pathogens that cause pediatric severe community-acquired pneumonia (SCAP) requiring bronchoscopy intervention are understudied. Through this study, we explore the etiology of SCAP form alveolar lavage fluid (ALF) samples by the RespiFinder 2SMART multi-PCR assay. It is observed that high mixed detection rates of Mycoplasma pneumoniae and adenovirus in ALF samples collected from hospitalized SCAP children experienced bronchoscopy intervention. Eighty percent of the cases had pulmonary consolidation and/or atelectasis. The presence of possible coinfection of these two pathogens might contribute to poor clinical anti-infection response. The results of this study might be helpful for the selection of clinical strategies for the empirical treatment of such pediatric SCAP cases.

Epidemiology of Viruses Causing Pediatric Community Acquired Pneumonia in Shanghai During 2010-2020: What Happened Before and After the COVID-19 Outbreak?

INTRODUCTION: Since the global outbreak of COVID-19, there has been a significant reduction in pediatric outpatient and emergency visits for infectious diseases. The purpose of this study was to analyze the changes in respiratory viruses in children with community-acquired pneumonia (CAP) in Shanghai in the past 10 years, especially in the first year after COVID-19. METHODS: We conducted a retrospective, observational study; the results for eight common respiratory viruses (respiratory syncytial virus (RSV), influenza virus A and B, parainfluenza virus 1-3 (PIV), adenovirus (ADV) and human metapneumovirus) tested by direct fluorescent antibody assays in hospitalized CAP cases in Children's Hospital of Fudan University during 2010-2020 were analyzed. RESULTS: Of the 5544 hospitalized CAP patients included in this study, 20.2% (1125/5544) were positive for the eight respiratory viruses. The top three pathogens were RSV, PIV3 and ADV, detected from 9.8% (543/5544), 5.3% (294/5544) and 2.0% (111/5544) of the samples, respectively. RSV had the highest positive rates among children < 2 years old. In 2020, the detection rate of all viruses showed a sharp decline from February to August compared with the previous 9 years. When the Shanghai community reopened in August 2020, the detection rate of eight viruses rebounded significantly in September. CONCLUSIONS: These eight respiratory viruses, especially RSV and PIV, were important pathogens of CAP in Shanghai children in the past 10 years. The COVID-19 pandemic had a significant impact on the detection rates for eight respiratory viruses in children with CAP in Shanghai.