Development and verification of multiplex real-time RT-PCR assay for classing and identification of HPIV1,HPIV2 and HPIV3 / 中国生物制品学杂志

@#ObjectiveTo develop and verify a multiplex real-time RT-PCR assay for simultaneous identification of human parainfluenza virus type 1(HPIV1),type 2(HPIV2)and type 3(HPIV3).MethodsThe whole genome sequences of HPIV1,HPIV2 and HPIV3 were downloaded from the database for alignment analysis,and the conserved regions were selected. Specific primers and probes were designed for the three viruses respectively to develop a multiplex real-time RTPCR assay with human ribonuclease P(RNase P)as theinternal quality control. The method was verified for the sensitivity,specificity and precision and used to detect 192 clinical samples.ResultsAfter optimization,the multiplex real-time RTPCR reaction system was determined to be 30 μL,including 10 × NeoscriptRTase/UNG Multi mix 3 μL,5 × Neoscript RT Premix Multi Buffer 6 μL,upstream and downstream primers of HPIV1,HPIV2 and HPIV3(100 μmol/L)0. 1 μL respectively,HPIV1,HPIV2,HPIV3 probes(100 μmol/L)0. 05 μL respectively,RNase P upstream and downstream primers(50 μmol/L)0. 06 μL respectively,RNase P probe(50 μmol/L)0. 03 μL respectively,template 15 μL,and ddH2O supplemented to 30 μL. The reaction conditions were 50 ℃ 20 min,95 ℃ 3 min and 45 cycles of 95 ℃ 15 s and54 ℃ 30 s. Fluorescence signals were collected during annealing in each cycle. The minimum detection limits of HPIV1,HPIV2 and HPIV3 were all 500 copies/mL by the multiplex real-time RT-PCR assay;The method showed no cross-reaction with influenza A virus,influenza B virus,respiratory syncytial virus andnovel coronaviruses. The coefficients of variation(CVs)in intra-and inter-groups of the recombinant plasmid standard mixture with three different concentrations were all less than 3%. HPIV1,HPIV2 and HPIV3 were detected in 192 clinical samples,and the positive rates were7. 81%,0. 05% and 3. 1%,respectively.ConclusionThe multiplex real-time RT-PCR assay for detection of HPIV1,HPIV2 and HPIV3 developed in this study has good sensitivity,specificity and precision,which has a high clinical application prospect in the field of rapid diagnosis and identification of HPIV.

Characteristics of hemagglutinin-neuraminidase gene of human parainfluenza virus type 3 among patients with acute respiratory tract infection in Henan Province / 中华微生物学和免疫学杂志

Objective:To analyze the molecular characteristics of hemagglutinin-neuraminidase (HN) gene of human parainfluenza virus type 3 (HPIV3) among the cases with acute respiratory tract infection (ARI) in Henan Province.Methods:Nasal/throat swab samples collected from patients with severe acute respiratory tract infection (SARI) in Luohe and patients with influenza-like illness (ILI) in Zhengzhou were used in this study. HPIV nucleic acids in the samples were detected using real-time fluorescent PCR. HPIV3-positive samples were subjected to RT-PCR for the amplification of HN genes and the sequences were analyzed with Sanger method. CExpress and MEGA7.0 software were used for sequences editing, evolution tree construction and gene sequence analysis.Results:A total of 374 throat swab samples collected form ARI cases in Luohe and Zhengzhou were tested and 20 (5.3%) of them were positive for HPIV3. Eighteen HPIV3 HN gene sequences were successfully amplified and all belonged to C3 subgroups, including 16 sequences of C3f genotype and two sequences of C3a genotype. The 18 HN gene sequences shared the homology of 97.6%-100.0% in nucleotide and 99.3%-100.0% in amino acid, but the differences between them and the prototype strain Wash/47885/57 were significant. There were 12 amino acid mutations shared by them, including four function-related mutations (H295Y, I391V, D556N and I53T). There were no significant differences in the nucleotide or amino acid sequences as compared with the epidemic strain of China/BCH4210A/2014.Conclusions:The C3f and C3a branches of HPIV3 were the epidemic genotypes in Henan Province in recent years and a local circulating prevalence might be established. Continuous and in-depth monitoring of HPIV3 C3 subtype would be of great significance for the prevention and control of HPIV3-associated diseases.

Human parainfluenza virus surveillance in pediatric patients with lower respiratory tract infections: a special view of parainfluenza type 4 / Vigilância dos vírus parainfluenza humanos em pacientes pediátricos com infecções do trato respiratório inferior: uma visão especial do parainfluenza tipo 4

Abstract Objective: Characterize the role of human parainfluenza virus and its clinical features in Brazilian children under 2 years of age presenting with acute lower respiratory tract infections. Methods: Real-time assays were used to identify strains of human parainfluenza virus and other common respiratory viruses in nasopharyngeal aspirates. One thousand and two children presenting with acute lower respiratory tract illnesses were enrolled from February 2008 to August 2010. Results: One hundred and four (10.4%) patients were human parainfluenza virus positive, of whom 60 (57.7%) were positive for human parainfluenza virus-3, 30 (28.8%) for human parainfluenza virus-4, 12 (11.5%) for human parainfluenza virus-1, and two (1.9%) for human parainfluenza virus-2. Seven (6.7%) patients had more than one strain of human parainfluenza virus detected. The most frequent symptoms were tachypnea and cough, similar to other viral respiratory infections. Clinical manifestations did not differ significantly between human parainfluenza virus-1, -2, -3, and -4 infections. Human parainfluenza virus-1, -3, and -4 were present in the population studied throughout the three years of surveillance, with human parainfluenza virus-3 being the predominant type identified in the first two years. Conclusion: Human parainfluenza viruses contribute substantially to pediatric acute respiratory illness (ARI) in Brazil, with nearly 30% of this contribution attributable to human parainfluenza virus-4.

Resumo Objetivo: Caracterizar o papel do VPH-4 e suas características clínicas em crianças brasileiras com menos de dois anos de idade com infecções agudas do trato respiratório inferior. Métodos: Ensaios em tempo real foram utilizados para identificar tipos de VPH e outros vírus respiratórios comuns em aspirados nasofaríngeos. Mil e duas crianças com doença aguda do trato respiratório inferior foram inscritas para participar de fevereiro de 2008 a agosto de 2010. Resultados: 104 (10,4%) pacientes eram VPH positivos, dos quais 60 (57,7%) eram positivos para VPH-3, 30 (28,8%) para VPH-4, 12 (11,5%) para VPH-1 e dois (1,9%) para VPH-2. Sete (6,7%) pacientes apresentaram mais de um tipo de VPH detectado. Os sintomas mais frequentes foram tosse e taquipneia, semelhantes a outras infecções respiratórias virais. As manifestações clínicas não diferiram de forma significativa entre as infecções por VPH-1, -2, -3 e -4. Os VPH-1, -3 e -4 estavam presentes na população estudada ao longo dos três anos de vigilância, e o VPH-3 foi o tipo predominante identificado nos primeiros dois anos. Conclusão: Os VPHs contribuem substancialmente para a DRA pediátrica no Brasil com quase 30% dessa contribuição atribuível ao VPH-4.

Epidemiological and clinical characteristics of respiratory human parainfluenza virus infection in children / 中华实用儿科临床杂志

Objective To study the epidemiological and clinical features of human parainfluenza virus (HPIV) infection in children in Suzhou,and to provide the evidence-based foundation for early warning,diagnosis and treatment of respiratory infection in children.Methods The sputum specimens and medical history were obtained from children with acute respiratory tract infection hospitalized at the Childen's Hospital Affiliated to Soochow University from January 2006 to December 2015.Seven kinds of common respiratory viruses including respiratory syncytial virus,influenza virus A,influenza virus B,HPIV Ⅰ,HPIV Ⅱ,HPⅣV Ⅲ and adenovirus were detected by using the direct im-munofluorescence.Mycoplasma pneumoniae(MP),chlamydia pneumoniae,human bocavirns (hBoV) were detected by using fluorescence quantitative PCR.Rhinovirus and human metapneumovirus were detected by using reverse transcription-PCR.Sputum was cultured for bacteria.Results In 21 769 cases,the detection rate of HPIV positive was 3.21％ (829 cases),among which,HPIV Ⅰ,HPIV Ⅱ,HPIV Ⅲ were respectively detected in 113 cases (0.52％),16 cases (0.07 ％) and 700 cases (3.21％),respectively.There were 378 cases of simple infection and 428 cases of mixed infection,and the mixed infection was very common in Streptococcus pneumoniae,Haemophilus influenza,MP and hBoV.There was a difference in HPIV infection among genders,and the detection rate of the boys was higher than that of girls[4.14％ (563/13 591 cases) vs.3.25％ (266/8 178 cases),x2 =11.036,P =0.001].In the 28 d-1 year old and ＞ 1-3 year old group,the detection rate of HPIV was higher[4.71％ (494/10 476 cases) and 4.21％ (244/5 793 cases),respectively].In spring and summer,there was a higher detection rate of HPIV infection.The clinical manifestations with simple infection of HPIV Ⅰ and HPIV Ⅲ were cough,fever and wheezing.The rate of fever and shortness of breath in those of HPIV Ⅰ was 71.74％ (33/46 cases),10.87％ (5/46 cases),and that in HPIV Ⅲ was 40.12％ (134/334 cases),2.10％ (7/334 cases),HPIV Ⅰ infection was more likely to cause fever and shortness of breath than those of HPIV Ⅲ,there were significant differences (x2 =16.410,P ＜ 0.001;x2 =10.177,P =0.001).Pneumonia had the highest detection rate of viral infection.Conclusions HPIV Ⅲ is the leading pathogen among the types of HPIV in the hospitalized children in Suzhou area.Among the subtypes of HPIV,the peak of HPIV infection occurs in spring and summer.The children less than 3 years old are the most susceptible to parainfluenza virus,and the HPIV detection rate is gradually declines with age.

Lower Respiratory Tract Diseases Caused by Common Respiratory Viruses among Stem Cell Transplantation Recipients: A Single Center Experience in Korea

PURPOSE: To describe the incidence, clinical courses, and risk factors for mortality of lower respiratory tract diseases (LRDs) caused by common respiratory viruses (CRVs) in stem cell transplantation (SCT) recipients. MATERIALS AND METHODS: We retrospectively reviewed the medical records of 1038 patients who received SCT between January 2007 and August 2011 at a single center in Korea. RESULTS: Seventy-one CRV-LRDs were identified in 67 (6.5%) patients. The human parainfluenza virus (HPIV) was the most common causative pathogen of CRV-LRDs at 100 days [cumulative incidence estimate, 23.5%; 95% confidence interval (CI), 3.3–43.7] and 1 year (cumulative incidence estimate, 69.2%; 95% CI, 45.9–92.5) following SCT. The 30-day overall mortality rates due to influenza-LRDs, respiratory syncytial virus-LRDs, HPIV-LRDs, and human rhinovirus-LRDs were 35.7, 25.8, 31.6, and 42.8%, respectively. Co-pathogens in respiratory specimens were detected in 23 (33.8%) patients. The overall mortality at day 30 after CRV-LRD diagnosis was 32.8% (22/67). High-dose steroid usage (p=0.025), a severe state of immunodeficiency (p=0.033), and lymphopenia (p=0.006) were significantly associated with death within 30 days following CRV-LRD diagnosis in a univariate analysis. Multivariate logistic regression analysis revealed that high-dose steroid usage [odds ratio (OR), 4.05; 95% CI, 1.12–14.61; p=0.033] and lymphopenia (OR, 6.57; 95% CI, 1.80–24.03; p=0.004) were independent risk factors for mortality within 30 days of CRV-LRDs. CONCLUSION: CRV-LRDs among SCT recipients showed substantially high morbidity and mortality rates. Therefore, the implement of an active diagnostic approaches for CRV infections is required for SCT recipients with respiratory symptoms, especially those receiving high-dose steroids or with lymphopenia.

The epidemiology, clinical features, phylogenetics analysis of human parainfluenza virus / 国际儿科学杂志

Human parainfluenza virus (HPIV)is one of the most common pathogens of community acquired respiratory infection.The epidemiology and clinical features vary in different subtypes.HPIV also affect other systems except respiratory system.The sequences of nucleotide and amino acid showed high identity among the strains in the same type,while remaining some variation.Most research shows the variation associated with time and district.Hemagglutinin-neuraminidase(HN) protein shows most polymorphic among all the proteins of HPIV.Some amino acid variation among the active site of HN protein will affect its activity.The influence of many gene mutations,which lead to amino acid variation,and affect the activity of HN protein remains uncertain.Further study on the epidemiology,clinical features,phylogenetics analysis of HPIV plays an important role in the prevention and control of its prevalence.

Rapid culture and identification of human parainfluenza viruses / 医学研究生学报

Objective Parainfluenza virus is an important pathogen of lower respiratory tract infections in infants and young children.This study was to search for a method for rapid culture and identification of human parainfluenza viruses from nasal swabs. Methods Nasal swab specimens were collected from 0-5 years old children with acute respiratory tract infection.The specimens were inoculated onto 96 plates with prefabricated LLC-MK2 cells and then centrifuged for 1 hour at 3000 r/min and also inoculated using the traditional culture method, followed by addition of virus mainte-nance medium containing 4 μg/mL TPCK trypsin.The cytopathic effect was observed daily, and hemagglutination and blood absorption tests were done at 2, 5, and 8 days after inoculation.In case of posi-tive result of either test, the specimen was subjected to immunofluo-rescence staining. Results Six strains of parainfluenza virus were isolated from the 83 nasal swab specimens, with a positive rate of 7.2%.There was a significant difference in the rate of separation be-tween the rapid and traditional culture methods after 2 days of culturing (7.2%vs 0%, P<0.05).The infected cells produced a cy-topathic effect that characterized by syncytium and crush formation.Hemagglutination and blood adsorption tests were positive at 4℃and negative at the room temperature.Immunofluorescence staining exhibited specific apple green fluorescence. Conclusion The method for rapid culture and identification of human parainfluenza viruses in nasal swab specimens was successfully established, which can be used to obtain and identify parainfluenza viruses with virulence and biological activity in 2 days.

Isolation & molecular characterization of human parainfluenza virus in Chennai, India.

Background & objectives: Human parainfluenza virus (HPIV) accounts for a significant proportion of lower respiratory tract infections in children as well as adults. This study was done to detect the presence of different subtypes of HPIV from patients having influenza like illness (ILI). Methods: Throat and nasal swabs from 232 patients with ILI who were negative for influenza viruses were tested by multiplex reverse transcription polymerase chain reaction(mRT-PCR) for the detection of human parainfluenza virus. All samples were inoculated in rhesus monkey kidney (LLC-MK2) cell line. Results: Of the 232 samples, 26(11.2%) were positive by mRT-PCR and nine (34.6%) showed cytopathic effect with syncytium formation for HPIV and all were HPIV-3 serotype, other serotypes like 1,2,4 were negative. The HPIV-3 strains (HN gene) were sequenced and analysed. Two novel mutations were identified at amino acid residues 295 and 297. Interpretation & conclusions: The mRT-PCR assay offers a rapid, sensitive and accurate diagnostic method for detection of HPIV which enables early detection and control. In our study there was a predominance of HPIV among 1-5 yr age group and the school going age group was less affected. Further studies need to be done to characterize HPIV isolated from different parts of the country.

Encefalomielitis aguda diseminada grave: Comunicación de un caso asociado a infección por virus parainfluenza 3 / Severe acute disseminated encephalomyelitis associated with parainfluenza 3 infection: Case report

Disseminated encephalomyelitis (ADEM) is an infrequent condition with considerable morbidity and mortality in adult patients. It requires a high level of suspicion and diagnosis emerges by gathering clinical information, laboratory exams and images studies. ADEM is related to an immunological phenomena occurring after a bacterial/viral infection or recent vaccination. Glucocorticoids are the first line treatment, reserving immunoglobulins and plasmapheresis to refractory cases. We report a male patient aged 25, with ADEM associated to parainfluenza 3 virus respiratory infection that required mechanical ventilation and that had a complete recovery only after plasmapheresis.

La encefalomielitis aguda diseminada es una enfermedad infrecuente pero de elevada morbi-mortalidad en pacientes adultos. Demanda una sospecha y diagnóstico precoz que requiere el concurso de información clínica, pruebas de laboratorio y estudio de imágenes. De sustrato inmunológico, se puede relacionar a una infección viral, bacteriana o inmunización reciente. Los glucocorticoides son el tratamiento de elección, mientras que la inmunoglobulina intravenosa y la plasmaféresis se reservan para casos refractarios. Se presenta el caso de una encefalomielitis aguda diseminada grave, en un paciente de sexo masculino de 25 años, asociado a una infección respiratoria por virus parainfluenza 3. Requirió conexión a ventilación mecánica y tuvo una respuesta completa con plasmaféresis.