Comparison of three air samplers for the collection of four nebulized respiratory viruses - Collection of respiratory viruses from air.

Viral respiratory tract infections are a leading cause of morbidity and mortality worldwide. Unfortunately, the transmission routes and shedding kinetics of respiratory viruses remain poorly understood. Air sampling techniques to quantify infectious viruses in the air are indispensable to improve intervention strategies to control and prevent spreading of respiratory viruses. Here, the collection of infectious virus with the six-stage Andersen cascade impactor was optimized with semi-solid gelatin as collection surface. Subsequently, the collection efficiency of the cascade impactor, the SKC BioSampler, and an in-house developed electrostatic precipitator was compared. In an in vitro set-up, influenza A virus, human metapneumovirus, parainfluenza virus type 3, and respiratory syncytial virus were nebulized and the amount of collected infectious virus and viral RNA was quantified with each air sampler. Whereas only low amounts of virus were collected using the electrostatic precipitator, high amounts were collected with the BioSampler and cascade impactor. The BioSampler allowed straight-forward sampling in liquid medium, whereas the more laborious cascade impactor allowed size fractionation of virus-containing particles. Depending on the research question, either the BioSampler or the cascade impactor can be applied in laboratory and field settings, such as hospitals to gain more insight into the transmission routes of respiratory viruses.

Engineering Protease-Resistant Peptides to Inhibit Human Parainfluenza Viral Respiratory Infection.

The lower respiratory tract infections affecting children worldwide are in large part caused by the parainfluenza viruses (HPIVs), particularly HPIV3, along with human metapneumovirus and respiratory syncytial virus, enveloped negative-strand RNA viruses. There are no vaccines for these important human pathogens, and existing treatments have limited or no efficacy. Infection by HPIV is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. A viral fusion protein (F), once activated in proximity to a target cell, undergoes a series of conformational changes that first extend the trimer subunits to allow insertion of the hydrophobic domains into the target cell membrane and then refold the trimer into a stable postfusion state, driving the merger of the viral and host cell membranes. Lipopeptides derived from the C-terminal heptad repeat (HRC) domain of HPIV3 F inhibit infection by interfering with the structural transitions of the trimeric F assembly. Clinical application of this strategy, however, requires improving the in vivo stability of antiviral peptides. We show that the HRC peptide backbone can be modified via partial replacement of α-amino acid residues with ß-amino acid residues to generate α/ß-peptides that retain antiviral activity but are poor protease substrates. Relative to a conventional α-lipopeptide, our best α/ß-lipopeptide exhibits improved persistence in vivo and improved anti-HPIV3 antiviral activity in animals.

Seasonality of distinct respiratory viruses in a tropical city: implications for prophylaxis.

OBJECTIVE: The frequency and seasonality of viruses in tropical regions are scarcely reported. We estimated the frequency of seven respiratory viruses and assessed seasonality of respiratory syncytial virus (RSV) and influenza viruses in a tropical city. METHODS: Children (age ≤ 18 years) with acute respiratory infection were investigated in Salvador, Brazil, between July 2014 and June 2017. Respiratory viruses were searched by direct immunofluorescence and real-time polymerase chain reaction for detection of RSV, influenza A virus, influenza B virus, adenovirus (ADV) and parainfluenza viruses (PIV) 1, 2 and 3. Seasonal distribution was evaluated by Prais-Winsten regression. Due to similar distribution, influenza A and influenza B viruses were grouped to analyse seasonality. RESULTS: The study group comprised 387 cases whose median (IQR) age was 26.4 (10.5-50.1) months. Respiratory viruses were detected in 106 (27.4%) cases. RSV (n = 76; 19.6%), influenza A virus (n = 11; 2.8%), influenza B virus (n = 7; 1.8%), ADV (n = 5; 1.3%), PIV 1 (n = 5; 1.3%), PIV 3 (n = 3; 0.8%) and PIV 2 (n = 1; 0.3%) were identified. Monthly count of RSV cases demonstrated seasonal distribution (b3 = 0.626; P = 0.003). More than half (42/76 [55.3%]) of all RSV cases were detected from April to June. Monthly count of influenza cases also showed seasonal distribution (b3 = -0.264; P = 0.032). Influenza cases peaked from November to January with 44.4% (8/18) of all influenza cases. CONCLUSIONS: RSV was the most frequently detected virus. RSV and influenza viruses showed seasonal distribution. These data may be useful to plan the best time to carry out prophylaxis and to increase the number of hospital beds.

Genetic diversity and evolutionary analysis of human respirovirus type 3 strains isolated in Kenya using complete hemagglutinin-neuraminidase (HN) gene.

Human respirovirus type 3 (HRV3) is a leading etiology of lower respiratory tract infections in young children and ranks only second to the human respiratory syncytial virus (HRSV). Despite the public health importance of HRV3, there is limited information about the genetic characteristics and diversity of these viruses in Kenya. To begin to address this gap, we analyzed 35 complete hemagglutinin-neuraminidase (HN) sequences of HRV3 strains isolated in Kenya between 2010 and 2013. Viral RNA was extracted from the isolates, and the entire HN gene amplified by RT-PCR followed by nucleotide sequencing. Phylogenetic analyses of the sequences revealed that all the Kenyan isolates grouped into genetic Cluster C; sub-clusters C1a, C2, and C3a. The majority (54%) of isolates belonged to sub-cluster C3a, followed by C2 (43%) and C1a (2.9%). Sequence analysis revealed high identities between the Kenyan isolates and the HRV3 prototype strain both at the amino acid (96.5-97.9%) and nucleotide (94.3-95.6%) levels. No amino acid variations affecting the catalytic/active sites of the HN glycoprotein were observed among the Kenyan isolates. Selection pressure analyses showed that the HN glycoprotein was evolving under positive selection. Evolutionary analyses revealed that the mean TMRCA for the HN sequence dataset was 1942 (95% HPD: 1928-1957), while the mean evolutionary rate was 4.65x10-4 nucleotide substitutions/site/year (95% HPD: 2.99x10-4 to 6.35x10-4). Overall, our results demonstrate the co-circulation of strains of cluster C HRV3 variants in Kenya during the study period. This is the first study to describe the genetic and molecular evolutionary aspects of HRV3 in Kenya using the complete HN gene.

Changes in the etiology of viral lower respiratory tract infections in hospitalized children in Wenzhou, China: 2008-2017.

This study investigated the seasonality and secular trends in the etiology of viral lower respiratory tract infections (LRTIs) among hospitalized children in Wenzhou, southeastern China. A retrospective review was conducted concerning viral LRTIs in children hospitalized at a university hospital between January 1, 2008 and December 31, 2017. Direct immunofluorescence was used to detect respiratory syncytial virus (RSV), adenovirus (AdV), influenza A virus (Inf A), influenza B virus (Inf B), and human parainfluenza virus types 1 to 3 (hPIV1-3). Of 89 898 children tested, at least one viral respiratory pathogen was identified in 25.6% and multiple pathogens were identified in 0.4%. RSV (17.6%), hPIV3 (4.0%), and AdV (2.2%) were the most frequently detected pathogens. The proportion of positive samples varied with age and was the highest in children aged <6 months (36.2%). Seasonal differences were observed in RSV, AdV, Inf A, Inf B, hPIV1, and hPIV3 infections. There was a declining trend in the proportion of positive samples over time, primarily due to a decrease in RSV and hPIV3 infections. RSV, hPIV3, and AdV were the most common viral respiratory pathogens identified among hospitalized children with LRTIs. The distribution of viruses varied with age and season.

Characteristics of human parainfluenza virus type 4 infection in hospitalized children in Korea.

BACKGROUND: The characteristics of human parainfluenza virus type 4 (hPIV4) infection are not thoroughly understood. We therefore clarified the characteristics of hPIV4 in Korea. METHOD: From January 2013 to December 2017, children admitted with respiratory tract infection at the Department of Pediatrics in Chung-Ang University Hospital were enrolled in the study. Nasopharyngeal aspirate specimens were obtained from patients and tested for hPIV types by multiplex reverse transcription polymerase chain reaction. We retrospectively reviewed subject medical records, focusing on epidemiological and clinical characteristics. RESULTS: Of the 12 423 NPA specimens, 8,406 were positive by multiplex reverse transcription polymerase chain reaction for nine respiratory viruses, and 1,018 were positive for one of the four types of hPIV: 1,018 specimens led to the detection of 1,029 hPIVs; 3ss (31.3%) were positive for hPIV1, 120 (11.7%) were positive for hPIV2, 356 (34.6%) were positive for hPIV3, and 231 (22.4%) were positive for hPIV4. Of the hPIV-positive patients, the mean age was 2.3 years (range, 0.1-12.7 years), 225 (97.4%) had no underlying disease, and 178 (77.1%) had a fever with a duration of 4.1 ± 2.3 days and a peak temperature of 39.0 ± 0.7 ℃. The most common diagnosis in hPIV4 infection was pneumonia (44.2%), followed by bronchiolitis (26.0%) and upper respiratory tract infection (24.3%). Only 2.2% of patients were diagnosed with croup. Although the most prevalent overall type of hPIV was hPIV3, hPIV4 generally caused acute respiratory tract infection in summer and early fall in an irregular annual pattern. CONCLUSIONS: Human parainfluenza virus type 4 is an important common pathogen of respiratory tract infections in pediatric patients in Korea.

Molecular epidemiology of an outbreak of human parainfluenza virus 3 among oncology patients.

A hospital outbreak of human parainfluenza virus type 3 (HPIV-3) in haematologic oncology patients is described in 12 patients over a four-week period. Exposure histories and molecular analysis of HPIV-3 isolates suggest that both community-acquired and nosocomially transmitted infections occurred during this outbreak. Molecular analysis of HPIV-3 isolates indicated that a chain of transmission occurred among multiple patients in an oncology ward. This transmission was later determined to be associated with the movement of fomites, visitors, and activities in the unit. The infection prevention team stopped nosocomial spread of HPIV-3 through interventions including advanced cleaning procedures.

Detecting respiratory viruses in children with protracted bacterial bronchitis.

OBJECTIVE: This study is to investigate the status and clinical significance of respiratory viruses in bronchoalveolar lavage fluid (BALF) of children with PBB. METHODS: Sixty-eight children with PBB aged from 3 months to 5 years were enrolled and retrospectively reviewed from January 2014 to December 2017. Thirty-five children with persistent pneumonia or chronic pneumonia were matched as controls. Bronchoalveolar lavage fluid samples were collected for respiratory virus detection and bacterial culture. RESULTS: The detection rate of bacteria in BALF of children with PBB was 61.8%, which was significantly higher than that of control group (20%) (P < 0.001). The detection rate of virus in BALF of children with PBB was 23.5%, including 6 (8.8%) of rhinovirus, 4 (5.9%) of parainfluenza virus type 3, 2(2.9%) of bocavirus, 2 (2.9%) of respiratory syncytial virus 1 (1.5%) of human metapneumonia virus and 1 (1.5%) of influenza virus A. 10 cases (28.6%) of virus were detected in the control group, including 3 (8.6%) respiratory syncytial virus, 3 (8.6%) rhinovirus and 2 (5.7%) bocavirus. There was no significant difference of viral detection rate between the two groups (P = 0.577). CONCLUSION: Respiratory viruses can be detected in BALF of children with PBB, However, there is no evidence that PBB is virus-induced.

Simultaneous outbreaks of respiratory disease in wild chimpanzees caused by distinct viruses of human origin.

Respiratory viruses of human origin infect wild apes across Africa, sometimes lethally. Here we report simultaneous outbreaks of two distinct human respiratory viruses, human metapneumovirus (MPV; Pneumoviridae: Metapneumovirus) and human respirovirus 3 (HRV3; Paramyxoviridae; Respirovirus, formerly known as parainfluenza virus 3), in two chimpanzee (Pan troglodytes schweinfurthii) communities in the same forest in Uganda in December 2016 and January 2017. The viruses were absent before the outbreaks, but each was present in ill chimpanzees from one community during the outbreak period. Clinical signs and gross pathologic changes in affected chimpanzees closely mirrored symptoms and pathology commonly observed in humans for each virus. Epidemiologic modelling showed that MPV and HRV3 were similarly transmissible (R0 of 1.27 and 1.48, respectively), but MPV caused 12.2% mortality mainly in infants and older chimpanzees, whereas HRV3 caused no direct mortality. These results are consistent with the higher virulence of MPV than HRV3 in humans, although both MPV and HRV3 cause a significant global disease burden. Both viruses clustered phylogenetically within groups of known human variants, with MPV closely related to a lethal 2009 variant from mountain gorillas (Gorilla beringei beringei), suggesting two independent and simultaneous reverse zoonotic origins, either directly from humans or via intermediary hosts. These findings expand our knowledge of human origin viruses threatening wild chimpanzees and suggest that such viruses might be differentiated by their comparative epidemiological dynamics and pathogenicity in wild apes. Our results also caution against assuming common causation in coincident outbreaks.

A 28-year study of human parainfluenza in Rio Grande do Sul, Southern Brazil.

PROBLEM: Human parainfluenza virus (hPIV) is an important pathogen in respiratory infections, however the health burden of hPIV is underestimated. This study describes the infections by hPIV1-3 in Rio Grande do Sul, Brazil, from 1990 to 2017, providing data of the frequency and seasonality of cases and associated clinical symptoms. METHOD OF STUDY: Nasopharyngeal samples of patients with respiratory infection were collected, clinical data were analyzed, and immunofluorescence was used to detect hPIV. RESULTS: Respiratory viruses were detected in 33.63% of respiratory infections. In a total of 11 606 cases of viral respiratory infection, 781 were positive for hPIV; hPIV prevalence ranged from 2.14% to 27% of viral respiratory infections. hPIV1 circulates mainly during fall; hPIV3 circulation, in turn, starts in fall and peaks during spring; and cases of hPIV2 are reported along the year, with peaks in fall and early spring. The most affected age group was children, with hPIV prevalence of 74.23% in patients for less than 1 year. A higher proportion of girls were infected than boys, however, no difference by sex was observed considering all age groups. The most frequent type was hPIV3, especially in hospitalized patients. Both hPIV1 and 3 were associated with dyspnea, while hPIV2 caused mild symptoms mainly in nonhospitalized patients. Nineteen fatalities occurred, 89.5% of them associated with risk factors (prematurity; chronic diseases; age, <1 or >60 years). CONCLUSION: hPIV causes a high number of respiratory infections, leading to hospitalization especially in children; epidemiological and surveillance studies are important for the control and management of respiratory infections.

Epidemiological investigation and phylogenetic analysis of caprine parainfluenza virus type 3 in sheep of China.

Caprine parainfluenza virus type 3 (CPIV3) is a new member of the Respirovirus genus in the Paramyxivirudae family, mainly causing respiratory disease. Up to now, accumulating evidence has focused on CPIV3 infection in goats, with little understood about its epidemiology in sheep. To that end, we collected 1,163 sheep sera samples from nine provinces/autonomous regions in 2012 and 1,863 samples from six provinces/autonomous regions during 2016-2017, with serological prevalence of 50.3% (95% CI: 47.5, 53.3) and 64.9% (95% CI: 62.9, 67.2), respectively. Pathogenic detection by qRT-PCR was also performed on serum samples collected in 2016-2017, and the percentage of CPIV3 positive samples was 21.5% (95% CI: 19.7, 23.5). Sequence alignment and phylogenetic analyses revealed 11 novel CPIV3 strains based on the M gene sequences. The M gene and full-length sequences of CPIV3 strains derived from sheep shared high nucleotide similarity with goat-origin strains, indicating conserved genome characteristics between the viruses. Furthermore, sequence evolution and epidemiological analysis show that CPIV3 is widespread throughout China. This is the first report describing CPIV3 infection in sheep in China, showing a high sero-prevalence and contributes to the assessment of the epidemiology of CPIV3 in China.

Identfication of viral and bacterial etiologic agents of the pertussis-like syndrome in children under 5 years old hospitalized.

BACKGROUND: Acute respiratory infections (ARIs) represent an important cause of morbidity and mortality in children, remaining a major public health concern, especially affecting children under 5 years old from low-income countries. Unfortunately, information regarding their epidemiology is still limited in Peru. METHODS: A secondary data analysis was performed from a previous cross-sectional study conducted in children with a probable diagnosis of Pertussis from January 2010 to July 2012. All samples were analyzed via Polymerase Chain Reaction (PCR) for the following etiologies: Influenza-A, Influenza-B, RSV-A, RSV-B, Adenovirus, Parainfluenza 1 virus, Parainfluenza 2 virus, Parainfluenza 3 virus, Mycoplasma pneumoniae and Chlamydia pneumoniae. RESULTS: A total of 288 patients were included. The most common pathogen isolated was Adenovirus (49%), followed by Bordetella pertussis (41%) from our previous investigation, the most prevelant microorganisms were Mycoplasma pneumonia (26%) and Influenza-B (19.8%). Coinfections were reported in 58% of samples and the most common association was found between B. pertussis and Adenovirus (12.2%). CONCLUSIONS: There was a high prevalence of Adenovirus, Mycoplasma pneumoniae and other etiologies in patients with a probable diagnosis of pertussis. Despite the presence of persistent cough lasting at least two weeks and other clinical characteristics highly suspicious of pertussis, secondary etiologies should be considered in children under 5 years-old in order to give a proper treatment.

Characterization of human parainfluenza virus-3 circulating in Israel, 2012-2015.

BACKGROUND: Human parainfluenza virus 3 (hPIV-3) causes respiratory tract infection. OBJECTIVES: The objective of this study was to describe the epidemiology of hPIV-3 infection among hospitalized patients and characterize the circulating strains. STUDY DESIGN: A cross-sectional study was conducted using respiratory samples of 15,946 hospitalized patients with respiratory symptoms in 2012-2015 in Israel. All samples were subjected to q-PCR and q-RT-PCR to determine the presence of hPIV-3 and other respiratory viruses. Samples positive for hPIV-3 were subjected to molecular typing and phylogenetic analysis. RESULTS: Overall, 547 samples 3.4% (95% CI 3.2-3.7) were positive for hPIV-3. Of these 87 (15.9%) were mixed infections; 41.4% with adenovirus, 40.2% with RSV (40.2%) and 19.5% influenza A viruses. The prevalence of hPIV-3 was highest (5.1%) in children aged 0-4 years. Hospitalization in oncology department was associated with increased likelihood of hPIV-3 infection: adjusted odds ratio [aOR] 2.29 (95% confidence intervals [CI] 1.78-2.96), as well as hospitalization in organ transplantation department: aOR 3.65 (95% CI 2.80-4.76). The predominant lineages were C3c (62.3%) and C1b (24.6%), followed by sub-lineages C5 (8.7%) and C3b (2.9%). A new sub-lineage emerged in our analysis, named C1d, which was 17 (1.5%) nucleotide different from C1a, 25 (2.2%) nucleotide different from C1b and 24 (2.1%) nucleotide different from C1c. DISCUSSION: Young children and immunocompromised patients are likely the risk groups for severe respiratory infections with hPIV-3. Strains belonging to lineages C3c and C1b, which are present worldwide, should be targeted in vaccine development. The emergence of new lineage might have public health implications and on vaccine development.

Viral Entry Properties Required for Fitness in Humans Are Lost through Rapid Genomic Change during Viral Isolation.

Human parainfluenza viruses cause a large burden of human respiratory illness. While much research relies upon viruses grown in cultured immortalized cells, human parainfluenza virus 3 (HPIV-3) evolves in culture. Cultured viruses differ in their properties compared to clinical strains. We present a genome-wide survey of HPIV-3 adaptations to culture using metagenomic next-generation sequencing of matched pairs of clinical samples and primary culture isolates (zero passage virus). Nonsynonymous changes arose during primary viral isolation, almost entirely in the genes encoding the two surface glycoproteins-the receptor binding protein hemagglutinin-neuraminidase (HN) or the fusion protein (F). We recovered genomes from 95 HPIV-3 primary culture isolates and 23 HPIV-3 strains directly from clinical samples. HN mutations arising during primary viral isolation resulted in substitutions at HN's dimerization/F-interaction site, a site critical for activation of viral fusion. Alterations in HN dimer interface residues known to favor infection in culture occurred within 4 days (H552 and N556). A novel cluster of residues at a different face of the HN dimer interface emerged (P241 and R242) and imply a role in HPIV-3-mediated fusion. Functional characterization of these culture-associated HN mutations in a clinical isolate background revealed acquisition of the fusogenic phenotype associated with cultured HPIV-3; the HN-F complex showed enhanced fusion and decreased receptor-cleaving activity. These results utilize a method for identifying genome-wide changes associated with brief adaptation to culture to highlight the notion that even brief exposure to immortalized cells may affect key viral properties and underscore the balance of features of the HN-F complex required for fitness by circulating viruses.IMPORTANCE Human parainfluenza virus 3 is an important cause of morbidity and mortality among infants, the immunocompromised, and the elderly. Using deep genomic sequencing of HPIV-3-positive clinical material and its subsequent viral isolate, we discover a number of known and novel coding mutations in the main HPIV-3 attachment protein HN during brief exposure to immortalized cells. These mutations significantly alter function of the fusion complex, increasing fusion promotion by HN as well as generally decreasing neuraminidase activity and increasing HN-receptor engagement. These results show that viruses may evolve rapidly in culture even during primary isolation of the virus and before the first passage and reveal features of fitness for humans that are obscured by rapid adaptation to laboratory conditions.

Clinical and epidemiological characteristics of human parainfluenza virus infections of children in southern Taiwan.

BACKGROUND: Human parainfluenza viruses (HPIV) 1-4 had been analyzed as being one of the most frequent causes of hospitalizations for young children with respiratory tract illnesses. METHODS: This retrospective study was performed from children virologically confirmed as HPIV infection through throat swab or nasopharyngeal aspirates at a tertiary care university hospital, between January 2012 and December 2014. HPIV4 was not checked and analyzed, due to not include in the commercial kit. The demographic, epidemiological, clinical presentations, diagnosis, treatment, outcomes, and laboratory data were analyzed. RESULTS: Totally 398 cases were enrolled, including 39 (9.8%) of HPIV1, 67 (16.8%) of HPIV2, and 292 (73.4%) of HPIV3. The mean age of HPIV-infected children was 2.9 year-old, and 50.5% were among one to three year-old. A total of 56.8% HPIV3-infected children were among one to three years old, however, no HPIV2-infected children was younger than one year-old. The HPIV1-infected patients were more common to develop wheezing and diagnose as acute bronchiolitis. HPIV2-infected children were more likely to have hoarseness (23.9%), and were associated with croup (25.4%). HPIV3 was isolated from two fatal cases, with neurological underlying diseases. CONCLUSION: The impact caused by HPIVs infections is significant in hospitalized children. In the current study, our results contribute to the epidemiologic, clinical and laboratory information of HPIV infection in children in the important areas of respiratory tract infection that could support the development of optimization management.

Prevalence of Human Parainfluenza Viruses and Noroviruses Genomes on Office Fomites.

The aim of this study was to evaluate the potential role of office fomites in respiratory (human parainfluenza virus 1-HPIV1, human parainfluenza virus 3-HPIV3) and enteric (norovirus GI-NoV GI, norovirus GII-NoV GII) viruses transmission by assessing the occurrence of these viruses on surfaces in office buildings. Between 2016 and 2017, a total of 130 surfaces from open-space and non-open-space rooms in office buildings located in one city were evaluated for HPIV1, HPIV3, NoV GI, and NoV GII viral RNA presence. Detection of viruses was performed by RT-qPCR method. Study revealed 27 positive samples, among them 59.3% were HPIV3-positive, 25.9% HPIV1-positive, and 14.8% NoV GII-positive. All tested surfaces were NoV GI-negative. Statistical analysis of obtained data showed that the surfaces of office equipment including computer keyboards and mice, telephones, and desktops were significantly more contaminated with respiratory viruses than the surfaces of building equipment elements such as door handles, light switches, or ventilation tracts (χ 2 p = 0.006; Fisher's Exact p = 0.004). All examined surfaces were significantly more contaminated with HPIVs than NoVs (χ 2 p = 0.002; Fisher's Exact p = 0.003). Office fomites in open-space rooms were more often contaminated with HPIVs than with NoVs (χ 2 p = 0.016; Fisher's Exact p = 0.013). The highest average concentration of HPIVs RNA copies was observed on telephones (1.66 × 102 copies/100 cm2), while NoVs on the light switches (1.40 × 102 copies/100 cm2). However, the Kruskal-Wallis test did not show statistically significant differences in concentration levels of viral RNA copies on surfaces between the all tested samples. This study unequivocally showed that individuals in office environment may have contact with both respiratory and enteric viral particles present on frequently touched surfaces.

[Detection and Analysis of Human Parainfluenza Virus Infection in Hospitalized Adults with Acute Respiratory Tract Infections].

OBJECTIVE: To investigate the prevalence and gene characteristics of different groups of human parainfluenza virus (HPIV) infection in hospitalized adults with acute respiratory tract infections (ARI). METHODS: RT-PCR was used to detect HPIV hemagglutinin (HA) DNA,which was extracted from sputum samples of 1 039 adult patients with ARI from March,2014 to June,2016. The HA gene amplified from randomly selected positive samples were sequenced to analyze the homology and variation. RESULTS: 10.6% (110/1 039) of these samples were positive for HPIV,including 8 cases of HPIV-1,22 cases of HPIV-2,46 cases of HPIV-3 and 34 cases of HPIV-4. Detectable rate varied among different groups of HPIV according to seasons of the year and ages of patients. No significant differences were found between the positive samples and the reference sequences. Compared with different reference strains of different regions,the genetic distance of nucleotide is the smallest between the strains tested in this study and the reference strains of other provinces and cities in China. CONCLUSION: In Chengdu region,HPIV virus is highly detected in ARI,all subtypes were detected with HPIV-3 being the main subtype.

Molecular epidemiology and environmental contamination during an outbreak of parainfluenza virus 3 in a haematology ward.

BACKGROUND: Although fomites or contaminated surfaces have been considered as transmission routes, the role of environmental contamination by human parainfluenza virus type 3 (hPIV-3) in healthcare settings is not established. AIM: To describe an hPIV-3 nosocomial outbreak and the results of environmental sampling to elucidate the source of nosocomial transmission and the role of environmental contamination. METHODS: During an hPIV-3 outbreak between May and June 2016, environmental surfaces in contact with clustered patients were swabbed and respiratory specimens used from infected patients and epidemiologically unlinked controls. The epidemiologic relatedness of hPIV-3 strains was investigated by sequencing of the haemagglutinin-neuraminidase and fusion protein genes. FINDINGS: Of 19 hPIV-3-infected patients, eight were haematopoietic stem cell recipients and one was a healthcare worker. In addition, four had upper and 12 had lower respiratory tract infections. Of the 19 patients, six (32%) were community-onset infections (symptom onset within <7 days of hospitalization) and 13 (68%) were hospital-onset infections (≥7 days of hospitalization). Phylogenetic analysis identified two major clusters: five patients, and three patients plus one healthcare worker. Therefore, seven (37%) were classified as nosocomial transmissions. hPIV-3 was detected in 21 (43%) of 49 environmental swabs up to 12 days after negative respiratory polymerase chain reaction conversion. CONCLUSION: At least one-third of a peak season nosocomial hPIV-3 outbreak originated from nosocomial transmission, with multiple importations of hPIV-3 from the community, providing experimental evidence for extensive environmental hPIV-3 contamination. Direct contact with the contaminated surfaces and fomites or indirect transmission from infected healthcare workers could be responsible for nosocomial transmission.

Characterization of the nasopharyngeal viral microbiome from children with community-acquired pneumonia but negative for Luminex xTAG respiratory viral panel assay detection.

In the present study, 50 nasopharyngeal swabs from children with community-acquired pneumonia (CAP) but negative for 18 common respiratory viruses, as measured by the Luminex xTAG Respiratory Viral Panel Assay, were subjected to multiplex metagenomic analyses using a next-generation sequencing platform. Taxonomic analysis showed that all sequence reads could be assigned to a specific species. An average of 95.13% were assigned to the Bacteria kingdom, whereas, only 0.72% were potentially virus derived. This snapshot of the respiratory tract virome revealed most viral reads to be respiratory tract related, classified into four known virus families: Paramyxoviridae, Herpesviridae, Anelloviridae, and Polyomaviridae. Importantly, we detected a novel human parainfluenza virus 3 (HPIV 3) strain with a 32-bp insertion in the haemagglutinin-neuraminidase (HN) gene that produced a negative result in the Luminex assay, highlighting the strength of virome metagenomic analysis to identify not only novel viruses but also viruses likely to be missed by ordinary clinical tests. Thus, virome metagenomic analysis could become a viable clinical diagnostic method.

Comparison of the Luminex xTAG Respiratory Viral Panel Fast v2 Assay With Anyplex II RV16 Detection Kit and AdvanSure RV Real-Time RT-PCR Assay for the Detection of Respiratory Viruses.

BACKGROUND: The accurate and rapid identification of the causative viruses is important for the timely diagnosis and management of respiratory infections. Multiplex molecular diagnostic techniques have been widely adopted to detect respiratory viruses. We compared the results of a newly upgraded, multiplex, molecular bead-based respiratory viral panel (RVP) assay with the results of Anyplex II RV16 detection kit and AdvanSure RV real-time RT-PCR assay. METHODS: We tested 254 respiratory specimens and cultured viral strains using the Luminex xTAG RVP Fast v2 assay (Luminex Molecular Diagnostics, Canada) and Anyplex II RV16 detection kit and compared the results. Specimens showing discordant results between the two assays were tested with a AdvanSure RV real-time RT-PCR assay. RESULTS: Of the 254 respiratory specimens, there was total agreement in the results between the xTAG RVP Fast v2 assay and the other real-time PCR assay in 94.1-100% of the specimens. The agreement levels were relatively low (94.1-97.6%) for specimens of adenovirus, coronavirus NL63, and parainfluenza type 3. In comparison to the other assay, the xTAG RVP Fast v2 assay detected a higher number of parainfluenza type 3 (4 cases) and metapneumovirus (9 cases). CONCLUSIONS: The xTAG RVP Fast v2 assay showed comparable capabilities compared with the other assays; it will be useful for identifying respiratory viral infections in patients with respiratory symptoms. Clinicians should be aware of the characteristics of the assays they use, since different assays show different detectability for each virus.