Optimized detection of respiratory viruses in nasopharyngeal secretions

Nasopharyngeal secretions (NPS) from 121 (110 pediatric) patients with acute respiratory infections were examined for respiratory virus detection by: i) conventional virus isolation in cell cultures (CC) using HEp-2, LLC-MK2, and MDCK cells; ii) rapid virus isolation using shell vial cultures (SVC) of a mixture (MIX) of mink lung epithelial cells (Mv1Lu) and human lung carcinoma (A549) cells in comparison to LLC-MK2 and MDCK cells; iii) direct fluorescent antibody (DFA) assay on NPS cells. A pool of monoclonal antibodies (MAbs) to influenzavirus A and B, parainfluenzavirus types 1 to 3, adenoviruses and respiratory syncytial virus (RSV), as well as single MAbs to the same viruses, were used for virus identification in all three procedures. Results on 101 NPS examined in parallel showed a sensitivity of 89.5%, 73.7%, and 81.6% for CC, SVC, and DFA, respectively, with the relevant negative predictive values of 94.0%, 86.3%, and 90.0%. Specificity and positive predictive values were 100%. However, the combination of DFA and SVC gave best results in terms of sensitivity (94.7%) and negative predictive value (95.5%). Use of the new MIX cell culture system in the SVC procedure enhanced virus detection, while use of the MAb pool allowed prompt identification of negative samples and saving of reagents and time for all three procedures. The combination of DFA and SVC allows diagnosis of the large majority of viral respiratory infections within 48h, while conventional virus isolation on CC may be limited to laboratories involved in research and epidemiological studies.     

GeXP多重PCR技术同时检测12种常见呼吸道病毒

本研究建立了一种基于GeXP多重基因表达遗传分析系统的多重RT-PCR检测方法,该方法可以同时检测12种呼吸道病毒,包括流感病毒A型和B型、季节性H1N1、副流感病毒1～3型、人鼻病毒、人偏肺病毒、腺病毒、呼吸道合胞病毒A型和B型、人博卡病毒。针对病原体保守区序列设计12种病毒的特异性引物,分别用已验证的阳性标本为模板检验多重体系的特异性。多重检测体系在10~3拷贝/μL水平可同时检测到12种病毒。另检测24份临床标本,以real-time RT-PCR为参考标准,进一步验证检测体系。结果表明,这种基于GeXP系统的新方法灵敏度高、特异性强,可以快速同时检测12种常见呼吸道病毒。     

Serodiagnosis of cilia-associated respiratory bacillus infection by the indirect immunofluorescence assay technique

Antibody to cilia-associated respiratory (CAR) bacillus was detected by the indirect immunofluorescence assay (IFA) technique using tracheal sections of infected mice as antigen in serum samples collected from rats infected naturally and experimentally. Nine of 23 cases of natural infection were positive in IFA antibody, with titres ranging from 1:10 to 1:80, and all these antibody-positive cases were also histologically positive. The remaining 14 cases were negative in both IFA antibody and histological diagnosis, even though some of them were infected with Sendai virus and Mycoplasma pulmonis. In the experimental infection, serum samples collected from 18 rats on days 4, 7, 14, 21, 28 and 56 post-inoculation (PI) (three rats for each point) and examined for IFA antibody revealed that seroconversion occurred in one rat on day 14 PI and in three rats on day 21 PI. Antibody titres of 1:80 to 1:160 remained to the termination of the experiment. The IFA technique was useful for the diagnosis of CAR bacillus infection except in the early stage of the infection.     

Development of a multiplex RT-RPA assay for simultaneous detection of three viruses in cucurbits

Begomoviruses and criniviruses, vectored by whiteflies (Bemisia tabaci), are important threats to crops worldwide. In recent years, the spread of cucurbit leaf crumple virus (CuLCrV), cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV) on cucurbit crops has been reported to cause devastating crop losses in many regions of the world. In this study, a multiplex recombinase polymerase amplification (RPA) assay, an isothermal technique for rapid and simultaneous detection of DNA and RNA viruses CuLCrV, CYSDV and CCYV was developed. Highly specific and sensitive multiplex RPA primers for the coat protein region of these viruses were created and evaluated. The sensitivity of the multiplex RPA assay was examined using serially diluted plasmid containing the target regions. The results demonstrated that multiplex RPA primers have high sensitivity with a detection limit of a single copy of the viruses. The multiplex RPA primers were specific to the target as indicated by testing against other begomoviruses, potyviruses and an ilarvirus, and no nonspecific amplifications were noted. The primers simultaneously detected mixed infection of CCYV, CYSDV and CuLCrV in watermelon and squash crude extracts. This study is the first report of a multiplex RPA assay for simultaneous detection of mixed infection of DNA and RNA plant viruses.     

Detection of anti-Candida antibodies by the indirect immunofluorescence assay in patients with cancer in the orofacial region

An indirect immunofluorescence assay was performed to detect antibodies toCandida albicans blastospores and germ tubes. Serum specimens were obtained from 82 patients with neoplastic diseases in the orofacial region and thrush of the oral mucosa.C. albicans was identified in the oral cavity of 63 patients investigated but serum anti-Candida antibodies were detected in only 23 of them. Serological examination showed that titers of antibodies toC. albicans blastospores ranged from 1∶20 to 1∶1280. High titers from 1∶640 to 1∶1280 were detected in patients without antibiotic, cytostatic, or radiotherapeutic treatment. The titers of antibodies toC. albicans germ tubes ranged from 1∶20 to 1∶640. Our results indicate that titers of antibodies to theC. albicans germ tubes were lower and were detected in a smaller number of patients.     

136例急性呼吸道感染患儿病原菌特点及鼻咽分泌物中呼吸道合胞病毒和人偏肺病毒基因分析

目的 分析136例急性呼吸道感染患儿的病原菌特点,并对患儿鼻咽分泌物中呼吸道合胞病毒(RSV)、人偏肺病毒(hMPV)进行检测,为后续研究提供参考。 方法 选择2017年1月至2019年4月我院收治的136例急性呼吸道感染患儿为研究对象,分析患儿呼吸道感染病原菌分布、耐药性,并对不同性别、年龄和感染部位患儿鼻咽分泌物中hMPV和RSV基因进行检测。 结果 136例急性呼吸道感染患儿共检出病原菌164株,其中革兰阴性菌占70.73%(116/164),革兰阳性菌占25.00%(41/164),真菌占4.27%(7/164);以铜绿假单胞菌(26.22%)、肺炎克雷伯菌(17.07%)、大肠埃希菌(15.24%)、金黄色葡萄球菌(12.20%)为主。药敏试验显示,铜绿假单胞菌、肺炎克雷伯菌、大肠埃希菌均对哌拉西林/他唑巴坦、头孢哌哃/舒巴坦、亚胺培南、左氧氟沙星、阿米卡星的耐药性较低(耐药率0.05)。1~2岁患儿hMPV检出率最高,0~5岁患儿RSV检出率较高。喘息性支气管炎、毛细支气管炎患儿hMPV检出率均较高,支气管肺炎、毛细支气管炎患儿RSV检出率均较高。 结论 急性呼吸道感染患儿病原菌以革兰阴性菌为主,应结合病原菌的药敏试验结果为患儿选择更有针对性的药物进行治疗。此外,应重视患儿RSV、hMPV感染情况,采取科学有效的方式对急性呼吸道感染进行预防和治疗。     

Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica)

The major viruses infecting apple cultivars throughout the world including India are apple mosaic virus (ApMV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and recently, a new virus, apple necrotic mosaic virus (ApNMV), was reported from mosaic-infected apple cultivars in India. The aim of this study was to detect the ApNMV virus along with the other three viruses (ApMV, ASPV and ASGV) simultaneously by multiplex RT-PCR. Four primer-pair-produced amplicons of 670, 550, 350 and 210 bp corresponding to ApNMV, ApMV, ASPV and ASGV, respectively, were found to be specific for these viruses when tested individually. The annealing temperature (55°C), primer concentration (0·8 µl) and other components of the master mix were standardized for the development of one-step m-RT-PCR assay. The m-RT-PCR protocol developed was further validated with 30 samples from seven symptomatic or asymptomatic apple cultivars, which revealed the presence of more than one virus in these cultivars. Most of the viruses were found to be present either alone or in mixed infection; however, ASPV was more common in tested cultivars. An easy, cost-effective and rapid multiplex RT-RCR protocol was developed to detect the four viruses, which infect apple plants either in individually or together in the field. This assay will help in the surveying and indexing of apple germplasm and the distribution of all four viruses in the apple growing regions of India.     

Simultaneous detection of eight immunosuppressive chicken viruses using a GeXP analyser-based multiplex PCR assay

Background

Immunosuppressive viruses are frequently found as co-infections in the chicken industry, potentially causing serious economic losses. Because traditional molecular biology methods have limited detection ability, a rapid, high-throughput method for the differential diagnosis of these viruses is needed. The objective of this study is to develop a GenomeLab Gene Expression Profiler Analyser-based multiplex PCR method (GeXP-multiplex PCR) for simultaneous detection of eight immunosuppressive chicken viruses.

Results

Using chimeric primers, eight such viruses, including Marek's disease virus (MDV), three subgroups of avian leucosis virus (ALV-A/B/J), reticuloendotheliosis virus (REV), infectious bursal disease virus (IBDV), chicken infectious anaemia virus (CIAV) and avian reovirus (ARV), were amplified and identified by their respective amplicon sizes. The specificity and sensitivity of the optimised GeXP-multiplex PCR assay were evaluated, and the data demonstrated that this technique could selectively amplify these eight viruses at a sensitivity of 100 copies/20 μl when all eight viruses were present. Among 300 examined clinical specimens, 190 were found to be positive for immunosuppressive viruses according to this novel assay.

Conclusion

The GeXP-multiplex PCR assay is a high-throughput, sensitive and specific method for the detection of eight immunosuppressive viruses and can be used for differential diagnosis and molecular epidemiological surveys.

     

Detection of human bocavirus mRNA in respiratory secretions correlates with high viral load and concurrent diarrhea

Human bocavirus (HBoV) is a parvovirus recently identified in association with acute respiratory infections (ARI). Despite its worldwide occurrence, little is known on the pathogenesis of HBoV infections. In addition, few systematic studies of HBoV in ARI have been conducted in Latin America. Therefore, in order to test whether active viral replication of human bocavirus is associated with respiratory diseases and to understand the clinical impact of this virus in patients with these diseases, we performed a 3-year retrospective hospital-based study of HBoV in outpatients and inpatients with symptoms of Acute Respiratory Infections (ARI) in Brazil. Nasopharyngeal aspirates (NPAs) from 1015 patients with respiratory symptoms were tested for HBoV DNA by PCR. All samples positive for HBoV were tested by PCR for all other respiratory viruses, had HBoV viral loads determined by quantitative real time PCR and, when possible, were tested by RT-PCR for HBoV VP1 mRNA, as evidence of active viral replication. HBoV was detected in 4.8% of patients, with annual rates of 10.0%, 3.0% and 3.0% in 2005, 2006 and 2007, respectively. The range of respiratory symptoms was similar between HBoV-positive and HBoV-negative ARI patients. However, a higher rate of diarrhea was observed in HBoV-positive patients. High HBoV viral loads (>10⁸ copies/mL) and diarrhea were significantly more frequent in patients with exclusive infection by HBoV and in patients with detection of HBoV VP1 mRNA than in patients with viral co-infection, detected in 72.9% of patients with HBoV. In summary, our data demonstrated that active HBoV replication was detected in a small percentage of patients with ARI and was correlated with concurrent diarrhea and lack of other viral co-infections.     

Self-collected mid-turbinate swabs for the detection of respiratory viruses in adults with acute respiratory illnesses

Background

The gold standard for respiratory virus testing is a nasopharyngeal (NP) swab, which is collected by a healthcare worker. Midturbinate (MT) swabs are an alternative due to their ease of collection and possible self-collection by patients. The objective of this study was to compare the respiratory virus isolation of flocked MT swabs compared to flocked NP swabs.

Methods

Beginning in October 2008, healthy adults aged 18 to 69 years were recruited into a cohort and followed up for symptoms of influenza. They were asked to have NP and MT swabs taken as soon as possible after the onset of a fever or two or more respiratory symptoms with an acute onset. The swabs were tested for viral respiratory infections using Seeplex® RV12 multiplex PCR detection kit. Seventy six pairs of simultaneous NP and MT swabs were collected from 38 symptomatic subjects. Twenty nine (38%) of these pairs were positive by either NP or MT swabs or both. Sixty nine (91%) of the pair results were concordant. Two samples (3%) for hCV OC43/HKU1 and 1 sample (1%) for rhinovirus A/B were positive by NP but negative by MT. One sample each for hCV 229E/NL63, hCV OC43/HKU1, respiratory syncytial virus A, and influenza B were positive by MT but negative by NP.

Conclusions

Flocked MT swabs are sensitive for the diagnosis of multiple respiratory viruses. Given the ease of MT collection and similar results between the two swabs, it is likely that MT swabs should be the preferred method of respiratory cell collection for outpatient studies. In light of this data, larger studies should be performed to ensure that this still holds true and data should also be collected on the patient preference of collection methods.