Nasopharyngeal Washes Research Articles

Development of a real-time multiplex RSV detection assay for difficult respiratory samples, using ultrasone waves and MNAzyme technology

Background Elderly infected with Human Respiratory Syncytial Virus (RSV) often bear low viral loads that stay below the detection limits of commercial assays. A more sensitive detection of RSV infections can improve patient management, guide containment strategies, and possibly prevent morbidity and mortality among populations most severely affected by RSV. Objective To test the sensitivity for RSV detection by using an alternative extraction method in combination with a new amplification procedure. Study design Nasopharyngeal washes and sputum samples ( n = 78) form clinical cases, and broncheo-alveolar lavages ( n = 27) from an experimental RSV rat model were obtained. An ultrasone-based RNA extraction method was combined with a multi-component Nucleic Acid enzymes (MNAzyme) amplification procedure for simultaneous detection of RSV-A, RSV-B, and an Internal Extraction control IEC. Results Compared to standard real-time PCR technology, this method resulted in an increased detection sensitivity, ranging from 0.9 to 4.93 log (average 2.05 ± 1.01) for RSV-A and 0.76 to 4.28 log (average 1.30 ± 0.92) for RSV-B. Conclusions An ultrasone-based extraction method with MNAzyme amplification resulted in improved detection of RSV in different respiratory samples, including sputum. This generic method for nucleic acid extraction should be readily applicable for any other respiratory pathogen.

Identification of Respiratory Viruses in Adults: Nasopharyngeal versus Oropharyngeal Sampling

The optimal method for identifying respiratory viruses in adults has not been established. The objective of the study was to compare the sensitivities of three sampling methods for this purpose. One thousand participants (mean age, 63.1 +/- 17.8 years) were included. Of these, 550 were patients hospitalized for acute febrile lower respiratory tract infections and 450 were controls. Oropharyngeal swabs (OPS), nasopharyngeal swabs (NPS), and nasopharyngeal washings (NPW) were obtained from each participant and were tested for 12 respiratory viruses by a multiplex hydrolysis probes-based quantitative real-time reverse transcription-PCR. Patients were defined as positive for a specific virus if the virus was identified by at least one sampling method. In all, 251 viruses were identified in 244 participants. For the detection of any virus, the sensitivity rates for OPS, NPS, and NPW were 54.2%, 73.3%, and 84.9%, respectively (for OPS versus NPS and NPW, P < 0.00001; for NPS versus NPW, P < 0.003). Maximal sensitivity was obtained only with sampling by all three methods. The same gradation of sensitivity for the three sampling methods was found when influenza viruses, coronaviruses, and rhinoviruses were analyzed separately. The three sampling methods yielded equal sensitivity rates for respiratory syncytial virus. We conclude that nasopharyngeal sampling has a higher rate of sensitivity than oropharyngeal sampling and that the use of NPW has a higher rate of sensitivity than the use of NPS with a rigid cotton swab for the identification of respiratory viruses in adults. Sampling by all three methods is required for the maximal detection of respiratory viruses.

Human Bocavirus Can Be Cultured in Differentiated Human Airway Epithelial Cells

In 2005, a human bocavirus was discovered in children with respiratory tract illnesses. Attempts to culture this virus on conventional cell lines has failed thus far. We investigated whether the virus can replicate on pseudostratified human airway epithelium. This cell culture system mimics the human airway environment and facilitates culturing of various respiratory agents. The cells were inoculated with human bocavirus-positive nasopharyngeal washes from children, and virus replication was monitored by measuring apical release of the virus via real-time PCR. Furthermore, we identified different viral mRNAs in the infected cells. All mRNAs were transcribed from a single promoter but varied due to alternative splicing and alternative polyadenylation, similar to what has been described for bovine parvovirus and minute virus of canines, the other two members of the Bocavirus genus. Thus, transcription of human bocavirus displays strong homology to the transcription of the other bocaviruses. In conclusion, we report here for the first time that human bocavirus can be propagated in an in vitro culture system and present a detailed map of the set of mRNAs that are produced by the virus.

Gene Expression of Nucleic Acid-Sensing Pattern Recognition Receptors in Children Hospitalized for Respiratory Syncytial Virus-Associated Acute Bronchiolitis

Given the critical role of pattern recognition receptors (PRRs) in acid nucleic recognition in the initiation of innate immunity and the orchestration of adaptive immunity, the aim of this study was to determine whether any heterogeneity of PRR expression in the airway tracts of infants with respiratory syncytial virus (RSV) infection might explain the broad clinical spectrum of RSV-associated bronchiolitis in infants. For this purpose, the levels of melanoma differentiation-associated protein-5 (MDA-5), retinoic acid inducible gene-1 (RIG-1), and Toll-like receptor 3 (TLR-3), TLR-7, TLR-8, and TLR-9 mRNAs were evaluated, using TaqMan quantitative reverse transcription-PCR, in cells from nasopharyngeal washes collected from 157 infants suffering from acute bronchiolitis whether or not they were associated with respiratory viruses. High interindividual variability was observed in both virus-positive and -negative infants; however, the relative gene expression levels of MDA-5, RIG-1, TLR-7, and TLR-8 were significantly higher in the virus-infected group, whereas the expression levels of TLR-3 and TLR-9 were not significantly different. The differences in the gene expression of MDA-5, RIG-1, TLR-7, and TLR-8 were more evident in infants with RSV infection than in those with bocavirus or rhinovirus infection. In RSV-infected infants, PRR-mRNA levels also were analyzed in relation to interferon protein levels, viral load, clinical severity, days of hospitalization, age, and body weight. A significant positive correlation was observed only between RSV viral load and RIG-1 mRNA levels. These findings provide the first direct evidence that, in infants with respiratory virus-associated bronchiolitis, especially RSV, there are substantial changes in PRR gene expression; this likely is an important determinant of the clinical outcome of bronchiolitis.

Rhinoviruses Cause Severe Respiratory Infections

In recent years, sensitive molecular testing has revealed that rhinoviruses may play a role in respiratory infections beyond the common cold. Now, investigators have linked rhinoviruses to severe lower respiratory tract infections (LRTIs) in children. Upper respiratory tract samples (nasopharyngeal washes or swabs) were collected from 43 patients aged <18 years consecutively admitted …

Infektionen mit dem humanen Bocavirus (hBoV): Eine wichtige Ursache schwerer viraler Atemwegsinfektionen im frühen Kindesalter

Apart from established pathogens of lower respiratory tract infections, such as respiratory syncytial virus (RSV), an increasing number of additional agents has been identified in recent years. In 2005 the human bocavirus (hBoV) has been isolated from respiratory tract samples and has been reported worldwide with frequencies ranging from 1.5 to 18.3% in respiratory samples from children with airway infections. We investigated 173 specimens of a total number of 162 children who were inpatients with severe respiratory tract infections most of whom required oxygen therapy. We analyzed respiratory tract samples (83% nasopharyngeal washes, 15% tracheal secretions, 2% bronchoalveolar lavages) for adenoviruses, influenza A und B viruses, parainfluenzaviruses types 1 to 3 and RSV using antigen-specific immunofluorescence assays. Additionally we tested human metapneumovirus (hMPV) and hBoV using a PCR assay. 35.8% specimens were negative in all assays, 54.3% were positive for RSV and 9.8% were positive for adeno-, influenza-, parainfluenzaviruses or hMPV. HBoV could be detected in 17 specimens (9.8%), defining HBoV to be the second most frequent pathogen. Nine of these patients showed a coinfection with RSV, one with parainfluenza virus. Viral loads did range from 2x10 (2) to 5.6x10 (10) genome equivalents/ml with higher viral loads being observed in the first days after disease onset. Most children were infected in the months between December and April. Half of the patients with isolated HBoV infection showed rhinopharyngitis, a third suffered from pulmonary obstruction and nearly every second required oxygen therapy. However, no HBoV-specific symptoms were found. HBoV is a common pathogen causing viral respiratory tract infection in infants and young children. Among the here reported patients HBoV was the second most frequent identified pathogen. X-ray studies frequently revealed peribronchial and pneumonic infiltrates with only moderately elevated laboratory inflammatory markers. So far, no HBoV-specific clinical symptoms are known. Additional questions for example related to the way of transmission and optimal treatment remain to be investigated in prospective studies.

Multiplex MassTag-PCR for respiratory pathogens in pediatric nasopharyngeal washes negative by conventional diagnostic testing shows a high prevalence of viruses belonging to a newly recognized rhinovirus clade

Background Respiratory infections are the most common infectious diseases in humans worldwide and are a leading cause of death in children less than 5 years of age. Objectives Identify candidate pathogens in pediatric patients with unexplained respiratory disease. Study design Forty-four nasopharyngeal washes collected during the 2004–2005 winter season from pediatric patients with respiratory illnesses that tested negative for 7 common respiratory pathogens by culture and direct immunofluorescence assays were analyzed by MassTag-PCR. To distinguish human enteroviruses (HEV) and rhinoviruses (HRV), samples positive for picornaviruses were further characterized by sequence analysis. Results Candidate pathogens were detected by MassTag PCR in 27 of the 44 (61%) specimens that previously were rated negative. Sixteen of these 27 specimens (59%) contained picornaviruses; of these 9 (57%) contained RNA of a recently discovered clade of rhinoviruses. Bocaviruses were detected in three patients by RT-PCR. Conclusions Our study confirms that multiplex MassTag-PCR enhances the detection of pathogens in clinical specimens, and shows that previously unrecognized rhinoviruses, that potentially form a species HRV-C, may cause a significant amount of pediatric respiratory disease.

Intranasal vaccination of infant mice induces protective immunity in the absence of nasal-associated lymphoid tissue

Intranasal (i.n.) immunization is an effective regimen for the prophylaxis of respiratory diseases in early life. The aim of this study was to assess the need for nasal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLN) in induction of protective immunity following mucosal vaccination of infant mice. We developed surgical techniques to eliminate NALT and CLN in young (8 days old) mice. i.n. vaccination of NALT- or CLN-deficient mice with pneumococcal polysaccharide conjugate vaccine plus interleukin-12 as a mucosal adjuvant (days 10 and 17) was followed by i.n. pneumococcal challenge (days 24-28). Mice were sacrificed on day 31 and nasal mucosal and systemic immune responses as well as pneumococcal colonization in the middle ear and nasopharynx were assessed. Elimination of NALT did not impair the ability of infant (3 weeks old) mice to produce nasal or serum antibody responses following i.n. immunization. In contrast, surgical removal of CLN significantly impaired the ability to express IgA antibody in nasopharyngeal washes and total antibody in serum. Similarly, protection against pneumococcal colonization in the nasopharynx and middle ears of immunized mice was decreased in the absence of CLN but not in the absence of NALT. These findings suggest that surgical removal of NALT tissue, at least in a mouse model, does not affect the ability to respond to subsequent i.n. vaccination. In addition, in the young mice CLN play a more important role than NALT for induction of protective mucosal and systemic antibody responses following i.n. immunization.

Human Metapneumovirus and Human Coronavirus NL63

To the Editor.—We read with great interest the article by Lambert et al that studied the role of 2 new respiratory viruses (human metapneumovirus [hMPV] and human coronavirus NL63 [hCoV-NL63]) in healthy preschool-aged children using parent-collected specimens with molecular techniques.1 The study showed that these viruses circulated in Melbourne, Australia, during 2003, and an association between child care and acute respiratory illness was proposed.1 We believe that some methodologic aspects of this study may have impaired the accuracy of the assessment of the role of these 2 viruses in such a population. Current literature shows that there are differences between respiratory samples collected by nose/throat swabs and nasopharyngeal aspirates regarding their potential to detect and identify respiratory pathogens.2 Tracheal secretion is less suitable for detection of respiratory viruses than nasopharyngeal washes and bronchoalveolar lavage.2 Another important point is the classification of symptoms, based entirely on parental experience. There are many subjective signs that, for an inexperienced person, would be difficult to recognize. All conclusions of an association between acute respiratory illness and virus incidence are based on symptom classifications (A and B), which may be incorrect.Finally, different methods were used to determine the incidence of several respiratory viruses. It has been shown that the sensitivity and specificity of real-time polymerase chain reaction (PCR), conventional PCR, and nested PCR may be completely different. In this case, hMPV and hCoV-NL63 were identified by using the most sensitive techniques (real-time and nested PCR), which might lead to an overestimation of the role of hMPV and hCoV-NL63 in community-acquired infections. However, there are no gold standards for detection of respiratory viruses to which both conventional tests and real-time PCR can be compared.3

Utility of Direct Fluorescent Antibody Testing of Nasopharyngeal Washes in Children With and Without Respiratory Tract Illness

Utility of Direct Fluorescent Antibody Testing of Nasopharyngeal Washes in Children With and Without Respiratory Tract Illness

Neonatal Respiratory Tract Involvement by Trichomonas vaginalis: A Case Report and Review of the Literature

Only occasional cases of Trichomonas spp. infection have been reported in neonates, and these usually represent vaginal infections with Trichomonas vaginalis acquired during passage down the birth canal. We report the case of a 2-week-old girl who was brought by her mother to the Children's Emergency Clinic at our institution for symptoms of lethargy and poor appetite. The neonate was subsequently diagnosed with herpetic encephalitis and developed increasing respiratory difficulty, requiring intubation. Routine viral culture of a nasopharyngeal wash showed no viral organisms, but trichomonads were abundant microscopically on the viral culture medium. Molecular studies identified the organism as T. vaginalis. The significance of this organism as a neonatal respiratory pathogen and its contribution to neonatal respiratory distress are discussed.

Sensitive Commercial NASBA Assay for the Detection of Respiratory Syncytial Virus in Clinical Specimen

The aim of the study was to evaluate the usability of three diagnostic procedures for the detection of respiratory syncytial virus in clinical samples. Therefore, the FDA cleared CE marked NOW® RSV ELISA, the NucliSENS® EasyQ RSV A+B NASBA, and a literature based inhouse RT-PCR protocol were compared for their relative sensitivities. Thereby, NASBA turned out to be the most sensitive method with a total number of 80 RSV positive samples out of a cohort of 251 nasopharyngeal washings from patients suffering from clinical symptoms, followed by the inhouse RT-PCR (62/251) and ELISA (52/251). Thus, NASBA may serve as a rapid and highly sensitive alternative for RSV diagnostics.

Rapid molecular detection of influenza outbreaks in nursing homes

Background Nursing home influenza outbreaks occur in spite of established vaccination programs, and require rapid and sensitive laboratory confirmation for timely intervention. Objectives To evaluate diagnostic approaches for rapid confirmation of nursing home influenza outbreaks. Study design Influenza virus real-time PCR and Directigen Flu A+B enzyme immunoassay were performed on nasopharyngeal swabs, nasopharyngeal washes and throat swabs collected from residents with clinical suspicion of influenza during seven probable nursing home outbreaks in 2004–2005 and 2005–2006. The efficacy of specimen sampling and transport management by Public Health Service outbreak team was evaluated. Results PCR detected influenza RNA in 80% (68/85) of specimens from 81% (38/47) residents, confirming six suspected outbreaks. Immunoassay sensitivity was highest on nasopharyngeal swabs (38%; 11/29) with a positive predictive value of 100% compared to PCR. Nasopharyngeal swabs were equally sensitive to nasopharyngeal washes by PCR. Nasopharyngeal wash sampling appeared unpractical due to common underlying disability of residents. Outbreak team support was associated with a shorter time to PCR diagnosis compared to outbreaks with no logistical support (mean, 28.2 h vs. 84 h; P = 0.05). Conclusions Influenza real-time PCR on nasopharyngeal swabs, obtained by Public Health Service outbreak teams, enabled rapid and sensitive confirmation of nursing home influenza outbreaks.

A BRIEF COMMUNICATION

To determine whether there is an airway IFN response in infants with acute bronchiolitis and to establish whether the rate of such a response is related to the severity of illness, the expression of some IFN-induced genes was measured in nasopharyngeal washes from 39 infants with acute bronchiolitis. The results indicate that in infants with a virus-associated acute bronchiolitis there is a strong activation of IFN system and that the severity of illness is inversely related to the level of expression of IFN-induced genes. This suggests that the IFN response plays an important role in determining virus–associated respiratory disease in early life.

Mixing of nasopharyngeal and oropharyngeal samples to identify potential respiratory pathogens in adults

A mixed culture of oropharyngeal swabs, nasopharyngeal swabs and nasopharyngeal washings, taken from 400 patients, was compared to separate cultures of the same samples. The mixed culture identified Streptococcus pneumoniae in 37 of 40 (93%) patients with positive samples, Hemophilus influenzae in 28 of 29 (97%), and Moraxella catarrhalis in 94 of 94 (100%). These sensitivity rates clearly justify the use of mixed cultures instead of separate cultures for clinical and epidemiological purposes. The reduction in costs stemming from the use of mixed cultures may have a decisive influence when considering this test for extensive clinical and epidemiological purposes.

Bronchiolitis due to respiratory syncytial virus in hospitalized children: a study of seasonal rhythm

The objective of this study was to describe the rhythm of respiratory syncytial virus (RSV) bronchiolitis seasonal outbreaks in hospitalized children. Data was collected from 1324 patients, who were admitted to our hospital with bronchiolitis, over an 11-year period, from 1994 to 2004. The epidemic onset was established according to the epidemic index. Virological diagnosis was made with immunofluorescent assay from nasopharyngeal washings. Rhythm study was carried-out by spectral analysis with the fast-Fourier transformed and cosinor method. Epidemics begin in September (45%) and October (55%); the highest peak was observed in January, the minimum in August and the end in February (73%), March (18%) and April (9%). When the epidemic outbreak begins sooner, the end is sooner as well. Epidemic onset varies but not its length and the onset was less variable than its conclusion. Spectral analysis showed a 12-months cyclic period along the study years and cosinor analysis demonstrated significant circannual rhythm. When data was segregated by long and short hospital stay, no significant differences were found between the rhythms. Comorbid association among bronchiolitis, otitis and gastroenteritis was very common. Bronchiolitis epidemics onset and conclusion varies along time years in hospitalized infants and showed circannual rhythmicity with a 12-months period.

Naso- and oropharyngeal potential respiratory pathogens in adults with nonpneumonic lower respiratory tract infection

The objective of this prospective study was to determine positive isolation rates for potential respiratory pathogens (PRPs) in the naso- and oropharynx of adults hospitalized for nonpneumonic lower respiratory tract infection (NPLRTI), compared with patients with community-acquired pneumonia (CAP) and healthy controls. The study population was 315 non–chronic obstructive pulmonary disease adults hospitalized with febrile lower respiratory tract infection (158 NPLRTI and 157 CAP) and 450 control subjects. Each participant was sampled by oropharyngeal swab, nasopharyngeal swab, and nasopharyngeal washings that were tested by conventional bacteriologic methods to identify PRP. At least 1 of the samples was positive for at least 1 of the 3 PRP bacteria in 55 NPLRTI patients (35%) compared with 51 CAP patients (33%) (NS) and 100 controls (22%) ( P = 0.003 compared with NPLRTI and P = 0.02 compared with CAP). Samples were positive for Streptococcus pneumoniae in 14 NPLRTI patients (9%) compared with 29 CAP patients (19%) ( P = 0.02) and 16 controls (4%) (NPLRTI P = 0.015, CAP P < 0.0001). The corresponding rates for Haemophilus influenzae were 23 (15%), 16 (10%), and 60 (13%) (NS for all 3 comparisons), and for Moraxella catarrhalis, 28 (18%), 25 (16%), and 48 (11%), respectively (NPLRTI versus controls, P = 0.03, NS other comparisons). We conclude that the rate of positive naso/oropharyngeal isolates for at least 1 of the 3 PRP bacteria in NPLRTI patients is similar to the corresponding rates for CAP patients and is higher in both groups than in controls.

Simkania Negevensis in Bronchoalveolar Lavage of Lung Transplant Recipients: A Possible Association with Acute Rejection

Simkania negevensis is a novel organism closely related to chlamydiae. The organism has been associated with community acquired pneumonia and acute exacerbation of chronic obstructive pulmonary disease. The prevalence and pathogenic potential of S. negevensis is not known in lung transplant recipients. In this multicenter study comparative analysis of bronchoalveolar lavage (BAL) in lung transplants (Tx) and kidney Tx, immunocompromised and nasopharyngeal (NP) washes of immunocompetent patients was done. The BAL specimens were tested by nested polymerase chain reaction (PCR) for C. pneumoniae and S. negevensis. Selected S. negevensis positive PCR cases were confirmed by culture. In the initial 41 BAL samples S. negevensis was detected in 97.5% (40/41) of lung transplant recipients as compared to 14.1% (1/7) in other organ transplant recipients (P<0.0001). In the sequential samples of 19 lung transplant recipients, 59% (24/41) had concomitant positive PCR and rejection as compared to 30% (3/10) who had negative PCR but had rejection (P=0.16). S. negevensis infection had hazard ratio of 3.29 (95% CI: 0.73-14.76; P=0.11) for developing acute rejection. S. negevensis is highly prevalent in liver Tx recipients and may be associated with acute rejection.

Diagnóstico etiológico de las infecciones respiratorias agudas de origen vírico en un hospital pediátrico de Gran Canaria

ObjetivoLas infecciones respiratorias agudas (IRA) de origen vírico son una causa frecuente de consulta y hospitalización pediátrica. El objetivo de este estudio fue conocer la etiología de dichas infecciones en la isla de Gran Canaria.MétodosDurante 3 años (de mayo de 2002 a mayo de 2005) se recogieron 1957 lavados nasofaríngeos de 1.729 niños atendidos en Urgencias con síntomas compatibles con IRA. En todas las muestras se realizó una técnica rápida de detección de antígeno de virus respiratorio sincitial (VRS) y, en las que se obtuvo resultado negativo, inmunofluorescencia (IF) y cultivo celular (CC).ResultadosLa mediana de edad fue de 2 meses (intervalo: 0,03-119). Se identificó el agente causal del cuadro respiratorio en 1.032 niños (59,7%). El VRS se detectó en 769 niños (74,5%). Los demás virus identificados, por orden de frecuencia, fueron: virus parainfluenza, rinovirus, adenovirus, virus de la gripe, enterovirus y coronavirus. Se encontraron diferencias estadísticamente significativas al comparar la edad y el tipo de virus detectado: los adenovirus fueron responsables de cuadros en niños de mayor edad (mediana: 6 meses; intervalo: 1-74). Hubo 6 casos de infección mixta. La sensibilidad de la IF en relación con el CC fue del 55,8%, y la especificidad del 99,2%.ConclusionesLos virus respiratorios son responsables de un alto número de casos de IRA, principalmente el VRS. Su identificación es determinante en el tratamiento clínico de los pacientes y en el empleo adecuado de antibacterianos y antivirales.

Papel del rinovirus en las infecciones respiratorias en niños hospitalizados

AntecedentesEl rinovirus se considera un agente causal de cuadros catarrales banales, sin embargo se ha descrito como un agente inductor de exacerbaciones asmáticas en adultos y niños mayores. En nuestro medio no se ha descrito el papel del rinovirus en infecciones respiratorias de niños hospitalizados.ObjetivosDescribir las infecciones confirmadas por rinovirus en niños hospitalizados por infección respiratoria en un hospital de segundo nivel.Pacientes y métodosEstudio descriptivo prospectivo de las infecciones confirmadas por rinovirus en niños hospitalizados por fiebre o infección respiratoria en la temporada 2004-2005. Para el diagnóstico virológico se realizó inmunofluorescencia y reacción en cadena de la polimerasa (PCR) en aspirado nasofaríngeo. Se describen las características clínicas de los pacientes.ResultadosSe describen un total de 76 niños hospitalizados con infección por rinovirus, lo que supuso el 25 % de los pacientes hospitalizados por procesos respiratorios o fiebre. El rinovirus fue el segundo agente viral identificado tras el virus respiratorio sincitial (29,9% de los hospitalizados). El 71,1 % de los pacientes fueron menores de 2 años. Los diagnósticos más frecuentes fueron sibilancias recurrentes en el 60,5 %, bronquiolitis en 23,7 %, neumonía en el 7,9 % e infección respiratoria de vías altas en el 5,3%. Presentaron fiebre de más de 38 °C el 57,9 % de los niños e infiltrado radiológico el 23,7%. Presentaron hipoxia el 43,4% de los niños. En niños mayores de 2 años el diagnóstico fue crisis asmática en 21 de los 22 casos.ConclusionesLos rinovirus se detectaron en un importante porcentaje de los niños hospitalizados a consecuencia de infección respiratoria, siendo precedidos en frecuencia sólo por el virus respiratorio sincitial. En nuestra serie es el agente viral más frecuentemente asociado con episodios de sibilancias recurrentes en niños mayores de 2 años, y el segundo en los más pequeños.