Clinical Nasopharyngeal Swab Research Articles

Pneumococcal carriage and serotype distribution among children with and without pneumonia in Mozambique, 2014-2016.

BackgroundPneumococcal colonization is a precursor to pneumonia, and pneumococcal conjugate vaccines (PCV) can decrease vaccine-type (VT) colonization. Pneumococcal colonization studies are traditionally done among healthy children in the community; however, VT colonization prevalence may differ between these children and those with pneumonia. We assessed overall and VT pneumococcal colonization and factors associated with colonization among children with and without pneumonia after Mozambique introduced 10-valent PCV (PCV10) in 2013.MethodsWe used data from ongoing pneumonia surveillance in children aged <5 years and from cross-sectional nasopharyngeal colonization surveys conducted in October 2014 –April 2015 and October 2015 –May 2016. Pneumonia was defined using WHO standard criteria for radiologically confirmed pneumonia. Children with pneumonia enrolled from January 2014 –April 2016 were compared to children without pneumonia enrolled from the cross-sectional surveys. Clinical data and nasopharyngeal (NP) swabs were collected from each child. NP specimens were cultured for pneumococci, and culture-negative specimens from children with pneumonia underwent polymerase chain reaction (PCR).ResultsOf 778 and 927 children with and without pneumonia, 97.4% and 27.0% were exposed to antibiotics before swab collection, respectively. Based on culture, pneumococcal colonization was 45.1% for children with and 84.5% for children without pneumonia (P<0.001); VT pneumococcal colonization was 18.6% for children with and 23.4% for children without pneumonia (P = 0.02). The addition of PCR in children with pneumonia increased overall and VT-pneumococcal colonization to 79.2% and 31.1%, respectively. In multivariable analysis including PCR results, pneumonia was associated with VT pneumococcal colonization (adjusted OR: 1.4, 95%CI: 1.10–1.78).ConclusionVaccine-type pneumococcal colonization remains common among children with and without pneumonia post-PCV10 introduction in Mozambique. In a population of children with high antibiotic exposure, the use of PCR for culture-negative NP swabs can improve assessment of pneumococcal colonization and circulating serotypes.

A four year seasonal survey of the relationship between outdoor climate and epidemiology of viral respiratory tract infections in a temperate climate

BackgroundThe relation between weather conditions, viral transmission and seasonal activity of respiratory viruses is not fully understood. ObjectivesTo investigate the impact of outdoor weather in a temperate climate setting on the seasonal epidemiology of viruses causing respiratory tract infections, particularly influenza A (IFA). Study designIn total, 20,062 clinical nasopharyngeal swab samples referred for detection of respiratory pathogens using a multiplex PCR panel, between October 2010 and July 2013, were included. Results of PCR detection were compared with local meteorological data for the same period. ResultsLow temperature and vapor pressure (VP) were associated with weekly incidence of IFA, respiratory syncytial virus, metapneumovirus, bocavirus and adenovirus but no association with relative humidity was found. The incidence of human rhinovirus and enterovirus was independent of temperature. During seasonal IFA outbreaks, the weekly drop of average temperature (compared with the week before) was strongly associated with the IFA incidence recorded the following week. ConclusionA sudden drop in outdoor temperature might activate the annual influenza epidemic in a temperate climate by facilitating aerosol spread in dry air. These conditions also seem to affect the incidence of other respiratory pathogens but not human rhino- or enterovirus, suggesting that routes of infection other than aerosol may be relevant for these agents.

Isolating Influenza RNA from Clinical Samples Using Microfluidic Oil-Water Interfaces.

The effective and robust separation of biomolecules of interest from patient samples is an essential step in diagnostic applications. We present a platform for the fast extraction of nucleic acids from clinical specimens utilizing paramagnetic PMPs, an oil-water interface, a small permanent magnet and a microfluidic channel to separate and purify captured nucleic acids from lysate in less than one minute, circumventing the need for multiple washing steps and greatly simplifying and expediting the purification procedure. Our device was able to isolate influenza RNA from clinical nasopharyngeal swab samples with high efficiency when compared to the Ambion® MagMAXTM Viral RNA Isolation Kit, sufficiently separating nucleic acid analytes from PCR-inhibiting contaminants within the lysate while also critically maintaining high integrity of the viral genome. We find that this design has great potential for rapid, efficient and sensitive nucleic acid separation from patient sample.

A packaged paper fluidic-based microdevice for detecting gene expression of influenza A virus

Pathotyping and subtyping of influenza A virus were performed with a packaged paper fluidic-based analytical microdevice (PFAM) after one-step reverse transcription-polymerase chain reaction (RT-PCR). The PFAM contains two test lines: one for detecting M gene to identify the influenza A virus and another for haemagglutinin subtyping to determine the viral strain among H1N1, H3N2, and H5N1. The M gene and the haemagglutinin gene (H1, H3, and H5 genes) were amplified by using the Digoxigenin and the Texas Red modified primers, respectively, in the multiplex RT-PCR. The amplicon products were loaded in the conjugate pad of the PFAM in which the streptavidin coated gold nanoparticles were linked with the biotin moieties that were incorporated in the middle of the DNA strands, and then captured by the anti-Digoxigenin and anti-Texas Red immobilized on the test lines. Influenza A H1N1, H3N2, and H5N1 could be identified with a limit of detection of 102 copies of RNA templates in 10min. Pathotyping and subtyping of the clinical nasopharyngeal swab samples were also analyzed whose results were confirmed by real-time RT-PCR.

Impact of 2009 pandemic influenza among Vietnamese children based on a population‐based prospective surveillance from 2007 to 2011

BackgroundInfluenza virus is one of the major viral pathogens causing pediatric acute respiratory infection (ARI). The spread of pandemic influenza A (A(H1N1)pdm09) in 2009 around the globe had a huge impact on global health.ObjectiveTo investigate the impact of A(H1N1)pdm09 on pediatric ARI in Vietnam.Study designAn ongoing population-based prospective surveillance in central Vietnam was used. All children aged <15 years residing in Nha Trang city, enrolled to the ARI surveillance in Khanh Hoa General Hospital, from February 2007 through March 2011 were studied. Clinical data and nasopharyngeal swab samples were collected. Influenza A was detected and genotyped by multiplex polymerase chain reaction assays and sequencing.ResultsAmong enrolled 2736 hospitalized ARI cases, 354 (13%) were positive for influenza A. Genotyping results revealed that seasonal H3N2 and H1N1 (sea-H1N1) viruses were cocirculating before A(H1N1)pdm09 appeared in July 2009. The A(H1N1)pdm09 replaced the sea-H1N1 after the pandemic. The majority of influenza A cases (90%) were aged <5 years with incidence rate of 537 (387–775) per 100 000 population. Annual incidence rates of hospitalized influenza cases for pre-, initial and post-pandemic periods among children aged <5 year were 474, 452, and 387 per 100 000, respectively. Children with A(H1N1)pdm09 were elder, visited the hospital earlier, less frequently had severe signs, and were less frequently associated with viral coinfection compared with seasonal influenza cases.ConclusionsThe A(H1N1)pdm09 did not increase the influenza annual hospitalization incidence or disease severity compared with seasonal influenza among pediatric ARI cases in central Vietnam.

Subtyping Clinical Specimens of Influenza A Virus by Use of a Simple Method To Amplify RNA Targets

This work presents the clinical application of a robust and unique approach for RNA amplification, called a simple method for amplifying RNA targets (SMART), for the detection and identification of subtypes of H1N1 pandemic, H1N1 seasonal, and H3N2 seasonal influenza virus. While all the existing amplification techniques rely on the diffusion of two molecules to complex RNA structures, the SMART achieves fast and efficient amplification via single-molecule diffusion. The SMART utilizes amplifiable single-stranded DNA (ssDNA) probes, which serve as reporter molecules for capturing specific viral RNA (vRNA) sequences and are subsequently separated on a microfluidic chip under zero-flow conditions. The probe amplification and detection are performed using an isothermal (41°C) amplification scheme via a modified version of nucleic acid sequence-based amplification (NASBA). In our study, 116 consecutive, deidentified, clinical nasopharyngeal swab samples were analyzed independently in a blinded fashion using the SMART, reverse transcription-PCR (RT-PCR), antigen (Ag) testing, and viral culture. The SMART was shown to have a limit of detection (LOD) of approximately 10(5) vRNA copies/ml, corresponding with a time-to-positivity (TTP) value of 70 min for real-time detection. The SMART correctly detected influenza virus in 98.3% of the samples with a subtyping accuracy of 95.7%. This work demonstrates that the SMART represents a highly accurate diagnostic platform for the detection and subtyping of influenza virus in clinical specimens and offers significant advantages over the current commercially available diagnostic tools.

A Disposable Microfluidic Virus Concentration Device Based on Evaporation and Interfacial Tension

We report a disposable and highly effective polymeric microfluidic viral sample concentration device capable of increasing the concentration of virus in a human nasopharyngeal specimen more than one order of magnitude in less than 30 min without the use of a centrifuge. The device is fabricated using 3D maskless xurography method using commercially available polymeric materials, which require no cleanroom operations. The disposable components can be fabricated and assembled in five minutes. The device can concentrate a few milliliters (mL) of influenza virus in solution from tissue culture or clinical nasopharyngeal swab specimens, via reduction of the fluid volume, to tens of microliters μL). The performance of the device was evaluated by nucleic acid extraction from the concentrated samples, followed by a real-time quantitative polymerase chain reaction (qRT-PCR). The viral RNA concentration in each sample was increased on average over 10-fold for both cultured and patient specimens compared to the starting samples, with recovery efficiencies above 60% for all input concentrations. Highly concentrated samples in small fluid volumes can increase the downstream process speed of on-chip nucleic acid extraction, and result in improvements in the sensitivity of many diagnostic platforms that interrogate small sample volumes.

Does a 10-valent pneumococcal-Haemophilus influenzae protein D conjugate vaccine prevent respiratory exacerbations in children with recurrent protracted bacterial bronchitis, chronic suppurative lung disease and bronchiectasis: protocol for a randomised controlled trial.

BackgroundRecurrent protracted bacterial bronchitis (PBB), chronic suppurative lung disease (CSLD) and bronchiectasis are characterised by a chronic wet cough and are important causes of childhood respiratory morbidity globally. Haemophilus influenzae and Streptococcus pneumoniae are the most commonly associated pathogens. As respiratory exacerbations impair quality of life and may be associated with disease progression, we will determine if the novel 10-valent pneumococcal-Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) reduces exacerbations in these children.MethodsA multi-centre, parallel group, double-blind, randomised controlled trial in tertiary paediatric centres from three Australian cities is planned. Two hundred six children aged 18 months to 14 years with recurrent PBB, CSLD or bronchiectasis will be randomised to receive either two doses of PHiD-CV or control meningococcal (ACYW135) conjugate vaccine 2 months apart and followed for 12 months after the second vaccine dose. Randomisation will be stratified by site, age (<6 years and ≥6 years) and aetiology (recurrent PBB or CSLD/bronchiectasis). Clinical histories, respiratory status (including spirometry in children aged ≥6 years), nasopharyngeal and saliva swabs, and serum will be collected at baseline and at 2, 3, 8 and 14 months post-enrolment. Local and systemic reactions will be recorded on daily diaries for 7 and 30 days, respectively, following each vaccine dose and serious adverse events monitored throughout the trial. Fortnightly, parental contact will help record respiratory exacerbations. The primary outcome is the incidence of respiratory exacerbations in the 12 months following the second vaccine dose. Secondary outcomes include: nasopharyngeal carriage of H. influenzae and S. pneumoniae vaccine and vaccine- related serotypes; systemic and mucosal immune responses to H. influenzae proteins and S. pneumoniae vaccine and vaccine-related serotypes; impact upon lung function in children aged ≥6 years; and vaccine safety.DiscussionAs H. influenzae is the most common bacterial pathogen associated with these chronic respiratory diseases in children, a novel pneumococcal conjugate vaccine that also impacts upon H. influenzae and helps prevent respiratory exacerbations would assist clinical management with potential short- and long-term health benefits. Our study will be the first to assess vaccine efficacy targeting H. influenzae in children with recurrent PBB, CSLD and bronchiectasis.Trial registrationAustralia and New Zealand Clinical Trials Registry (ANZCTR) number: ACTRN12612000034831.

A microbead-incorporated centrifugal sample pretreatment microdevice

In this study, we present a novel bead-incorporated centrifugal sample pretreatment microdevice to purify influenza A H3N2 viral RNA. Simple revolution per minute (RPM) control can lead to RNA capture on a bead-bed, and the sequential loading of a washing solution and an elution solution. Tetraethoxy orthosilicate (TEOS)-treated glass microbeads were utilized as a capture matrix. The sample pretreatment microdevice consists of four reservoirs for storing an RNA sample, a washing solution, an elution solution, and a collected sample, and they were merged at the microbead-bed microchannel. The washing solution reservoir and the elution solution reservoir were connected to the bead-bed microchannel through a capillary valve and a siphon channel, respectively. An RNA sample (a lysed influenza A H3N2 virus), a washing solution (70% ethanol) and an elution solution (water or a reverse transcription-polymerase chain reaction (RT-PCR) cocktail) were loaded into the designated reservoirs, and they were successively transported to the bead-bed by RPM control owing to the optimized channel design. Purified RNAs could be obtained in 440 s. Then, a target H3 gene was amplified by an off-chip based real-time RT-PCR to evaluate the capture efficiency of RNA on our proposed microdevice. 81% of RNAs were successfully captured and purified. Interestingly, the use of the RT-PCR cocktail itself as an elution solution resulted in a 76% capture yield. Furthermore, we successfully performed RNA purification from the clinical nasopharyngeal swabs to identify the subtype of the influenza A virus. This platform provides high potential for the direct integration of the sample pretreatment microdevice into the downstream micro-PCR unit to create a total genetic analysis microsystem.

Real-Time Detection of Influenza A, Influenza B, and Respiratory Syncytial Virus A and B in Respiratory Specimens by Use of Nanoparticle Probes

Seasonal epidemics of influenza and respiratory syncytial virus are responsible for significant morbidity and mortality worldwide. Infrequently, novel or reemergent strains of influenza A virus have caused rapid, severe global pandemics resulting in millions of fatalities. The ability to efficiently and accurately detect and differentiate respiratory viruses is paramount for effective treatment, infection control, and epidemiological surveillance. We evaluated the ability of two FDA-cleared nucleic acid-based tests, the semiautomated respiratory virus nucleic acid test (VRNAT) and the fully automated respiratory virus nucleic acid test SP (RVNAT(SP)) (Nanosphere Inc., Northbrook, IL) to detect influenza A virus, influenza B virus, and respiratory syncytial virus A and B (RSV A/B) from clinical nasopharyngeal swab specimens. Detection of viral RNA in both tests is based on nucleic acid amplification followed by hybridization to capture probes immobilized on a glass slide. A novel technology utilizing gold nanoparticle-conjugated probes is utilized to detect the presence of captured target DNA. This microarray-based approach to detection has proven to be more sensitive than the traditional culture/direct fluorescent-antibody assay (DFA) method for detecting RSV and influenza viruses in clinical specimens, including the novel 2009 H1N1 strain. Specifically, we report 98.0% sensitivity and 96.5% specificity for the VRNAT compared to culture/DFA. Further, the VRNAT detected virus in an additional 58% of specimens that were culture negative. These data were confirmed using bidirectional sequencing. Evaluation of the fully automated RVNAT(SP), which is built on the same detection technology as the VRNAT but contains an updated processor enabling complete automation, revealed the two tests to be functionally equivalent. Thus, the RVNAT(SP) is a fully automated sample-to-result test capable of reliable detection of select respiratory viruses directly from clinical specimens in 3.5 h.

Viral Infection in Adults Hospitalized With Community-Acquired Pneumonia: Prevalence, Pathogens, and Presentation

BackgroundThe potential role of respiratory viruses in the natural history of community-acquired pneumonia (CAP) in adults has not been well described since the advent of nucleic amplification tests (NATs).MethodsFrom 2004 to 2006, adults with CAP who were admitted to five hospitals were prospectively enrolled in the study, and clinical data, cultures, serology, and nasopharyngeal swabs were obtained. NATs from swabs were tested for influenza, human metapneumovirus (hMPV), respiratory syncytial virus (RSV), rhinovirus, parainfluenza virus 1–4, coronaviruses (OC43, 229E, and NL63), and adenovirus.ResultsA total of 193 patients were included; the median age was 71 years, 51% of patients were male, and 47% of patients had severe CAP. Overall, 75 patients (39%) had a pathogen identified. Of these pathogens, 29 were viruses (15%), 38 were bacteria (20%), 8 were mixed (4%), and the rest were “unknown.” Influenza (n = 7), hMPV (n = 7), and RSV (n = 5) accounted for most viral infections; other infections included rhinovirus (n = 4), parainfluenza (n = 3), coronavirus (n = 4), and adenovirus (n = 2). Streptococcus pneumoniae was the most common bacterial infection (37%). Compared with bacterial infection, patients with viral infection were older (76 vs 64 years, respectively; p = 0.01), were more likely to have cardiac disease (66% vs 32%, respectively; p = 0.006), and were more frail (eg, 48% with limited ambulation vs 21% of bacterial infections; p = 0.02). There were few clinically meaningful differences in presentation and no differences in outcomes according to the presence or absence of viral infection.ConclusionsViral infections are common in adults with pneumonia. Easily transmissible viruses such as influenza, hMPV, and RSV were the most common, raising concerns about infection control. Routine testing for respiratory viruses may be warranted for adults who have been hospitalized with pneumonia.

Multiplex real-time PCR for detection of respiratory tract infections

Background Broad diagnostics of respiratory infection by molecular assays has not yet won acceptance due to technical difficulties and high costs. Objectives To evaluate clinical applicability of multiplex real-time PCR. Study design An assay targeting influenza virus A (IfA) and B (IfB), parainfluenza 1-3 (PIV), human metapneumovirus (MPV), respiratory syncytial virus (RSV), rhinovirus (RV), enterovirus (EV), adenovirus (AdV), human coronaviruses (229E, OC43, NL63), M. pneumoniae and Ch. pneumoniae was developed and run daily on consecutive clinical nasopharyngeal swab samples. Results An etiology was identified in 48% of the 954 samples, with IfA in 25%, RV in 20%, MPV in 10% and M. pneumoniae in 10% of the positive. By a rational procedure costs could be reduced and the customer price set relatively low (€33 per sample). Conclusion Streamlined testing and cost limitation is achievable and probably critical for implementation of a broad molecular diagnostics of respiratory infections.

Simultaneous Detection ofChlamydophila pneumoniaeandMycoplasma pneumoniaeby Use of Molecular Beacons in a Duplex Real-Time PCR

A real-time PCR was designed for detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae such that each pathogen could be detected in a single tube and differentiated using molecular beacons marked with different fluorochromes. This duplex PCR, targeting the P1 adhesion gene for M. pneumoniae and the ompA gene for C. pneumoniae, was compared with two conventional PCR assays targeting the 16S rRNA gene and the ompA gene. A total of 120 clinical throat and nasopharyngeal swab samples were tested. DNA extraction was performed using an alkali denaturation/neutralization method, and real-time amplification, detection, and data analysis were performed using a Rotor-Gene 2000 real-time rotary analyzer (Corbett Life Science, Sydney, Australia). Using conventional PCR as a reference in an analysis of 120 samples, 13 of 14 samples positive for C. pneumoniae were detected by the novel real-time PCR. In an analysis of M. pneumoniae, 22 samples were positive in the conventional PCR and the novel assay detected 24 positive samples. When using the conventional PCR as a reference, sensitivity and specificity were 93% and 100%, respectively, for C. pneumoniae and 100% and 98%, respectively, for M. pneumoniae. With an overall agreement of 98.8%, this suggests that performance of the new duplex real-time PCR is comparable to that of conventional PCR.

Prevalence of serotypes and molecular epidemiology of Streptococcus pneumoniae strains isolated from children in Beijing, China: identification of two novel multiply-resistant clones.

Three-hundred and seventy-six strains of Streptococcus pneumoniae isolated from clinical specimens and nasopharyngeal swabs from children at daycare centers and hospitals in Beijing China, between January 1997 and March 1998, were serotyped. Twenty-seven different serotypes were identified. The most prevalent serotypes in the carriage isolates were 6A, 19F, 23F, and 15 and were found in 66.8% of cases. Serotype data indicate that 51.8% of carrier strains would be included in the 11-valent conjugate vaccine formulation, while inclusion of vaccine-related serotypes, increased the potential vaccine coverage to 79.4%. Serotypes 7, 6B, 23F, 19F, 15, and 3 accounted for 62% of clinical strains, with 70% vaccine-related serotypes. DNA fingerprinting of 47 penicillin resistant and 71 penicillin-susceptible/macrolide-resistant strains by BOX polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE), and penicillin binding protein (PBP)-fingerprinting identified two novel clones: one a serotype 23F multiresistant clone resistant to penicillin, tetracycline, erythromycin, clindamycin, and variably resistant to chloramphenicol and trimethoprim-sulphamethoxazole; and the second a multiresistant penicillin-susceptible, macrolide-resistant serotype 6A clone, highly resistant also to tetracycline, clindamycin, and trimethoprim-sulphamethoxazole. The macrolide resistance determinant in 89% of erythromycin-resistant strains tested (penicillin-susceptible and penicillin-resistant) was the erm gene, both the erm and mef genes were simultaneously found in 6%, and mef alone in 3.4%. The data demonstrates that macrolide resistant strains in China include clonal strains and strains with dual mef and erm resistance determinants.

Brief report. Persistent rhinorrhoea in rural Indian children: prevalence and consequences

To determine whether persistent rhinorrhoea constitutes a significant problem requiring intervention, 17 rural day care centres (Balwadis) in Tamilnadu, India, were visited. Among 414 children in the Balwadis 92 (22 per cent) children with persistent rhinorrhoea (15 days duration or longer) were identified. Demographic and clinical data and nasopharyngeal swabs for bacterial culture were obtained from 56 such children and 91 age-matched controls from the same Balwadi. Type of housing or nutritional status did not appear to be significant risk factors. There was a significantly higher number of children aged 5-15 years in the household of cases as compared to controls (1.23 +/- 1.08 vs. 0.83 +/- 0.95, p = 0.02). Other illnesses were noted in 25 (44.6 per cent) cases and seven (7.7 per cent) controls (OR 11.5; CI, 4.13-33.4; p < 0.00001). Notably, chronic ear discharge was noted in 6 (11.7 per cent) cases but in none of the controls (p = 0.007). Streptococcus pneumoniae was isolated from nasopharyngeal swabs in 42/49 (85.7 per cent) cases and 44/80 (55 per cent) controls (p < 0.001) and H. influenzae from seven cases and five controls; S. pneumoniae was isolated in all children with chronic ear discharge and H. influenzae from one child. Serotypes of pneumococci commonly associated with otitis media, i.e., types 6, 14, 19, and 23 were isolated from 25 (51 per cent) cases and 16 (20 per cent) controls (OR 4.17; 95% CI, 1.78-9.85; p < 0.001). Persistent rhinorrhoea, presumably due to pneumococcus, is a common condition among rural Indian children and appears to be associated with chronic otitis media.

A Pertussis Outbreak in a Wisconsin Nursing Home

The epidemiologic features and clinical spectrum of pertussis in the elderly are poorly understood. In October 1985, the Wisconsin Division of Health investigated an outbreak of pertussis in residents of a nursing home in rural Wisconsin. Clinical information and nasopharyngeal swab and acute- and convalescent-phase serum specimens were obtained from all consenting residents and employees. Of 105 residents, 38 (36.2%) were seropositive, including four who were culture-positive for Bordetella pertussis. Culture-positive residents (age range, 52-81 years) had cough lasting 43-54 days. Three of these residents had paroxysmal cough, and all four had cough that interrupted sleep; none of the residents had cough with apnea or vomiting, and all recovered without sequelae. Of six seropositive residents with clinical pertussis, five lived on the south wing of the facility. Of 104 employees, 8 (7.7%) were seropositive, but none were culture-positive for B. pertussis. The higher attack rate for residents and the clustering of clinical cases were consistent with ongoing transmission within the nursing home.