Prevalence of human respiratory viruses in adults with acute respiratory tract infections in Beijing, 2005–2007

To determine the aetiological role and epidemiological profile of common respiratory viruses in adults with acute respiratory tract infections (ARTIs), a 2-year study was conducted in Beijing, China, from May 2005 to July 2007. Nose and throat swab samples from 5808 ARTI patients were analysed by PCR methods for common respiratory viruses, including influenza viruses (IFVs) A, B, and C, parainfluenza viruses (PIVs) 1–4, enteroviruses (EVs), human rhinoviruses (HRVs), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), human coronaviruses (HCoVs) OC43, 229E, NL63, and HKU1, and adenoviruses (ADVs). Viral pathogens were detected in 34.6% of patient samples, and 1.6% of the patients tested positive for more than one virus. IFVs (19.3%) were the dominant agents detected, followed by HRVs (6.5%), PIVs (4.3%), EVs (3.2%), and HCoVs (1.1%). ADVs, RSV and HMPV were also detected (<1%). The viral detection rates differed significantly between infections of the lower and upper respiratory tracts in the sample population: PIVs, the second most commonly detected viral agents in lower acute respiratory tract infections (LRTIs), were more prevalent than in upper acute respiratory tract infections, indicating that the pathogenic role of PIVs in LRTIs should be investigated. Currently, this study is the largest-scale investigation of respiratory virus infections in China with multiple agent detection, providing baseline data for further studies of respiratory virus infections in adults with ARTIs.     

Polymerase chain reaction for detection of herpesvirus simiae (B virus) in clinical specimens

Summary A polymerase chain reaction (PCR) was designed which is specific toMacaca fascicularis (cynomolgus monkey) isolates of B virus. The PCR primers produced the expected 188 basepair product from the Cyno 2 strain and seven other cynomolgus monkey isolates of B virus. Oligomer hybridization with a 31-mer oligonucleotide was used to confirm the origin of this product. The PCR failed to amplify DNA of Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, and other alphaherpesviruses (herpes simplex virus types 1 and 2, four SA 8 isolates and three rhesus isolates of B virus). PCR testing of swabs obtained from four orally-infected cynomolgus monkeys confirmed the presence of B virus DNA in samples previously shown to be positive by culture. In addition, PCR detected B virus in several swabs from infected monkeys that were culture negative. Total DNA extracts from the trigeminal and sacral ganglia of these animals were tested by nested PCR and B virus DNA was detected in the trigeminal ganglia of 3 of the 4 orally-infected cynomolgus monkeys. Nested PCR did not detect B virus DNA in total DNA extracts obtained from the brains of the four monkeys.     

Investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations

Summary. Two training centres for working dogs were monitored for one year to determine the presence of viruses and viral antibodies and their association with canine infectious respiratory disease (CIRD). Tonsillar swabs and serum were obtained from dogs on entry into the kennels and in regular intervals thereafter. Additional samples were collected during outbreaks of CIRD. The swabs were examined by virus culture and PCR for canine parainfluenza virus, canine adenovirus, canine herpesvirus (CHV) and canine respiratory coronavirus (CRCoV). Furthermore the prevalence of antibodies to CHV and CRCoV was determined. During this study CIRD was reported mainly in one of the two kennels investigated. In that kennel antibody responses to CRCoV indicated a seasonal occurrence of the virus, which coincided with two outbreaks of respiratory disease. CHV antibody responses were detected throughout the year. In the other kennel, which reported few cases of CIRD a high prevalence of antibodies to CRCoV was detected on entry but only sporadic seroconversions to CRCoV or CHV. By PCR three dogs were found positive for CRCoV in one kennel whereas all PCR tests for other viruses were negative for both kennels. Virus culture failed to detect any viruses in either kennel.     

Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections

Viruses are the major cause of pediatric acute respiratory tract infection (ARTI) and yet many suspected cases of infection remain uncharacterized. We employed 17 PCR assays and retrospectively screened 315 specimens selected by season from a predominantly pediatric hospital-based population. Before the Brisbane respiratory virus research study commenced, one or more predominantly viral pathogens had been detected in 15.2% (n = 48) of all specimens. The Brisbane study made an additional 206 viral detections, resulting in the identification of a microbe in 67.0% of specimens. After our study, the majority of microbes detected were RNA viruses (89.9%). Overall, human rhinoviruses (HRVs) were the most frequently identified target (n = 140) followed by human adenoviruses (HAdVs; n = 25), human metapneumovirus (HMPV; n = 18), human bocavirus (HBoV; n = 15), human respiratory syncytial virus (HRSV; n = 12), human coronaviruses (HCoVs; n = 11), and human herpesvirus-6 (n = 11). HRVs were the sole microbe detected in 37.8% (n = 31) of patients with suspected lower respiratory tract infection (LRTI). Genotyping of the HRV VP4/VP2 region resulted in a proposed subdivision of HRV type A into sublineages A1 and A2. Most of the genotyped HAdV strains were found to be type C. This study describes the high microbial burden imposed by HRVs, HMPV, HRSV, HCoVs, and the newly identified virus, HBoV on a predominantly paediatric hospital population with suspected acute respiratory tract infections and proposes a new formulation of viral targets for future diagnostic research studies.     

Human metapneumovirus and human bocavirus associated with respiratory infection in Apulian population

We have studied the occurrence of hBoV, hMPV and InfA-B in an Apulian population with respiratory tract infections. During influenza season 2008-2009, 116 oropharingeal swabs were collected from patients affected by Influenza-Like Illness (ILI). The PCR products of hMPV M and HBoV NP-1 genes were sequenced. 78 out of 116 samples were positive for at least one respiratory virus; hBoV was detected in 53, hMPV in 22 and InfA-B in 41 out of 116 swabs. A high rate of hBoV infection in adult (18.9%) and elderly (26.4%) subjects was found. The co-infection rate was higher for hMPV (18/22 cases, 81.8%) compared to hBoV (26/53 cases, 49.1%), and InfA-B (25/41 cases, 61.0%). Co-infections were common in children. hBoV positive samples shared a high level of genetic similarity with the hBoV1 genotype, and hMPV positive samples clustered with A2 subgroup. Our results suggest that hBoV and hMPV play a role in ILI.     

Newly identified respiratory viruses in children with asthma exacerbation not requiring admission to hospital

There are few data describing the comprehensive identification in and influence of newly identified respiratory viruses on asthma exacerbations. Most studies focus on inpatients. In this preliminary study, the point prevalence and the associations of picornavirus species described recently and human bocavirus (HBoV) with the recovery from exacerbations in non‐hospitalized asthmatic children (median age 5.1 years) were examined. Human rhinoviruses (HRVs) were present in 52.6% of specimens, HBoV‐1 was in 7.7%. Viral co‐detections occurred in 25.6% of children and were associated (P = 0.04) with lower asthma quality of life scores upon presentation than were single viral detections. The undifferentiated presence or absence of virus did not influence the severity of asthma or recovery however when virus species were examined individually, specific clinical associations emerged. HRV species C (HRV‐Cs) were the viruses most frequently detected as single virus detections. Among 41 genotyped HRVs, more HRV‐Cs (n = 23) were identified than HRV‐As (n = 16) however HRV‐A detection was associated (P = 0.01) with worse asthma symptoms and cough for longer than was HRV‐C detection. Larger, PCR‐based studies are required to elucidate further the true impact of HRV species in childhood asthma exacerbations of both hospitalized and non‐hospitalized cohorts. J. Med. Virol. 82:1458–1461, 2010. © 2010 Wiley‐Liss, Inc.     

Rapid detection of adenovirus in throat swab specimens by PCR during respiratory disease outbreaks among military recruits

We evaluated the performance of a generic PCR test to detect adenoviruses (AdV) in throat swab specimens collected from asymptomatic and ill military recruits with acute respiratory disease. Samples (n = 210) were collected at entry to basic training and at the time of large outbreaks of AdV-associated acute respiratory disease among military recruits at Fort Jackson, South Carolina, from 1997 to 1998. Compared to cell culture, a sensitivity of 99% and a specificity of 98% were noted for the PCR method to detect AdV in throat swabs. Similar results were obtained with or without DNA extraction, suggesting the absence of significant inhibitors for the PCR method in throat swab samples. No AdV was detected by culture or PCR in throat swabs from healthy recruits, suggesting the absence of latency or asymptomatic shedding. Throat swab specimens proved to be adequate, noninvasive samples to rapidly diagnose respiratory disease in young adults. This generic direct PCR proved to be a useful test for the rapid diagnosis of AdV-associated respiratory disease, detecting all serotypes tested to date and furnishing results within 6 h of specimen arrival. The use of this direct, rapid, sensitive, and specific assay would assist health care providers and public health practitioners in the early diagnosis, management, and control of AdV-associated respiratory disease.     

Detection of multiple respiratory pathogens during primary respiratory infection: nasal swab versus nasopharyngeal aspirate using real-time polymerase chain reaction

In this study, we present the multiple detection of respiratory viruses in infants during primary respiratory illness, investigate the sensitivity of nasal swabs and nasopharyngeal aspirates, and assess whether patient characteristics and viral load played a role in the sensitivity. Healthy infants were included at signs of first respiratory tract infection. Paired nasopharyngeal aspirates and nasal swabs were collected. Real-time polymerase chain reaction (PCR) was carried out for 11 respiratory pathogens. Paired nasopharyngeal aspirates and nasal swabs were collected in 98 infants. Rhinovirus (n = 67) and respiratory syncytial virus (n = 39) were the most frequently detected. Co-infection occurred in 48% (n = 45) of the infants. The sensitivity of the nasal swab was lower than the nasopharyngeal aspirate, in particular, for respiratory syncytial virus (51% vs. 100%) and rhinovirus (75% vs. 97%). The sensitivity of the nasal swab was strongly determined by the cycle threshold (CT) value (p < 0.001). The sensitivity of the swab for respiratory syncytial virus, but not rhinovirus, was 100% in children with severe symptoms (score ≥11). It is concluded that, for community-based studies and surveillance purposes, the nasal swab can be used, though the sensitivity is lower than the aspirate, in particular, for the detection of mild cases of respiratory syncytial virus (RSV) infection.     

Comparison of four clinical specimen types for detection of influenza A and B viruses by optical immunoassay (FLU OIA test) and cell culture methods

Although laboratory diagnosis of respiratory viruses has been widely studied, there is a relative insufficiency of literature examining the impact of specimen type on the laboratory diagnosis of influenza A and B. In a clinical study comparing the FLU OIA test with 14-day cell culture, clinical specimens from nasopharyngeal swabs, throat swabs, nasal aspirates, and sputum were obtained from patients experiencing influenza-like symptoms. A total of 404 clinical specimens were collected from 184 patients. Patients were defined as influenza positive if the viral culture of a specimen from any sample site was positive. Patients were defined as influenza negative if the viral cultures of specimens from all sample sites were negative. By this gold standard, culture and FLU OIA test results for each sample type were compared. For each of the four specimen types, the viral culture and FLU OIA test demonstrated equal abilities to detect the presence of influenza A or B virus or viral antigen. Sputum and nasal aspirate samples were the most predictive of influenza virus infection. Throat swabs were the least predictive of influenza virus infection, with both tests failing to detect influenza virus in nearly 50% of the throat samples studied.     

Absence of oropharyngeal vaccinia virus after vaccinia (smallpox) vaccination.

BACKGROUND: With the resumption of the vaccinia (smallpox) vaccination, questions regarding transmission risk prompted this study to determine whether vaccinia virus could be detected in the oropharynx of adults recently vaccinated with vaccinia (smallpox) vaccine. German, Russian, and American studies on the oropharyngeal presence of vaccinia virus revealed conflicting results in different age groups. OBJECTIVE: To measure vaccinia viral particle or antigen presence in the oropharynx of adult health care workers after vaccination with vaccinia (smallpox) vaccine using viral culture and high-sensitivity assays (polymerase chain reaction [PCR] and electrochemiluminescence) and to determine whether there is an association between the presence of vaccinia virus and adverse reactions. METHODS: A total of 155 adults (primary vaccinees and revaccinees) were enrolled for 1 baseline and 5 subsequent throat swabs. The swabs were evaluated using viral culture, PCR, and electrochemiluminescence. RESULTS: Of the 155 participants, 144 had more than 2 throat swabs in the 2 weeks after vaccination. Of the 801 specimens evaluated, there were no positive results by culture, PCR, or electrochemiluminescence except in the control samples (n = 6), which were positive by all 3 methods. CONCLUSIONS: Based on the absence of detectable vaccinia virus in this study population, one can be 95% certain that the true rate of vaccinia virus in the oropharynx of adults during the 2 weeks after vaccination with vaccinia (smallpox) vaccine is 0% to 3.3%. These data should be reassuring to the medical community and support the Advisory Committee on Immunization Practice guidelines that respiratory precautions are not necessary after vaccinia (smallpox) vaccination in healthy adults.     

Comparison of Luminex xTAG® RVP fast assay and real time RT‐PCR for the detection of respiratory viruses in adults with community‐acquired pneumonia

Community‐acquired pneumonia (CAP) is the third cause of death worldwide. Viruses are frequently detected in adult CAP. Highly sensitive diagnostic techniques should be used due to poor viral shedding. Different sampling methods can affect viral detection, being necessary to establish the optimal type of sample for identifying respiratory viruses in adults. The detection rates of respiratory viruses by Luminex xTAG® RVP fast assay, real time RT‐PCR (rtRT‐PCR) (Sacace®), and immunofluorescence assay (IFA) in adult CAP were performed in nasopharyngeal swabs (NPS) and aspirates (NPA) from 179 hospitalized adults. Positivity was 47.5% for Luminex®, 42.5% for rtRT‐PCR (P = 0.3), and 2.7% for IFA (2.7%) (P < 0.0). The sensitivity, specificity, and kappa coefficient of xTAG® RVP compared with rtRT‐PCR were 84.2%, 79.6%, and 0.62%, respectively. Luminex® and rtRT‐PCR detected 65 (58.0%) and 57 (50.9%) viruses in 112 NPA and 35 (34.3%) and 31 (30.4%) in 102 NPS, respectively (P < 0.01). xTAG® RVP is appropriate for detecting respiratory viruses in CAP adults. Both molecular techniques yielded better results with nasopharyngeal aspirate than swabs. J. Med. Virol. 88:1173–1179, 2016 . © 2016 Wiley Periodicals, Inc.

     

Non-invasive sample collection for respiratory virus testing by multiplex PCR

Background

Identifying respiratory pathogens within populations is difficult because invasive sample collection, such as with nasopharyngeal aspirate (NPA), is generally required. PCR technology could allow for non-invasive sampling methods.

Objective

Evaluate the utility of non-invasive sample collection using anterior nare swabs and facial tissues for respiratory virus detection by multiplex PCR.

Study design

Children aged 1 month–17 years evaluated in a pediatric emergency department for respiratory symptoms had a swab, facial tissue, and NPA sample collected. All samples were tested for respiratory viruses by multiplex PCR. Viral detection rates were calculated for each collection method. Sensitivity and specificity of swabs and facial tissues were calculated using NPA as the gold standard.

Results

285 samples from 95 children were evaluated (92 swab-NPA pairs, 91 facial tissue-NPA pairs). 91% of NPA, 82% of swab, and 77% of tissue samples were positive for ≥ 1 virus. Respiratory syncytial virus (RSV) and human rhinovirus (HRV) were most common. Overall, swabs were positive for 74% of virus infections, and facial tissues were positive for 58%. Sensitivity ranged from 17 to 94% for swabs and 33 to 84% for tissues. Sensitivity was highest for RSV (94% swabs and 84% tissues). Specificity was ≥95% for all viruses except HRV for both collection methods.

Conclusions

Sensitivity of anterior nare swabs and facial tissues in the detection of respiratory viruses by multiplex PCR varied by virus type. Given its simplicity and specificity, non-invasive sampling for PCR testing may be useful for conducting epidemiologic or surveillance studies in settings where invasive testing is impractical or not feasible.     

Use of oligonucleotide microarrays for rapid detection and serotyping of acute respiratory disease-associated adenoviruses

The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a long oligonucleotide microarray capable of identifying the most common adenovirus serotypes associated with adult respiratory tract infections (serotypes 3, 4, 7, 16, and 21) and a representative member of adenovirus subgroup C (serotype 6) that is a common cause of childhood ARD and that often persists into adulthood. Analyses with prototype strains demonstrated unique hybridization patterns for representative members of adenovirus subgroups B(1), B(2), C, and E, thus allowing serotype determination. Microarray-based sensitivity assessments revealed lower detection limits (between 1 and 100 genomic copies) for adenovirus serotype 4 (Ad4) and Ad7 cell culture lysates, clinical nasal washes, and throat swabs and purified DNA from clinical samples. When adenovirus was detected from coded clinical samples, the results obtained by this approach demonstrated an excellent concordance with those obtained by the more established method of adenovirus identification as well as by cell culture with fluorescent-antibody staining. Finally, the utility of this method was further supported by its ability to detect adenoviral coinfections, contamination, and, potentially, recombination events. Taken together, the results demonstrate the usefulness of the simple and rapid diagnostic method developed for the unequivocal identification of ARD-associated adenoviral serotypes from laboratory or clinical samples that can be completed in 1.5 to 4.0 h.