Comparison of three techniques for detection of respiratory viruses in nasopharyngeal aspirates from children with lower acute respiratory infections

A comparison of immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), and isolation in tissue culture (TC) for detection of respiratory viruses was performed on 496 nasopharyngeal aspirates from children under 5 years of age with lower acute respiratory infections who were receiving attention at three hospitals in Buenos Aires, Argentina. All samples were tested by the three methods for respiratory syncytial virus (RSV), influenza A and B, adenovirus, and parainfluenza 1 and 3. Viral diagnosis was made in 167 samples (33.7%); of these, 124 (74.3%) were isolated in TC, whereas 120 (71.8%) were detected by ELISA and 127 (76%) by IF. RSV was detected in 121 samples, mainly by ELISA and IF. The sensitivity and specificity of each rapid technique as compared with isolation in TC were similar, reaching 98% and 92%, respectively. When ELISA was compared with IF, the sensitivity was 95%, and the specificity was 98%. Adenovirus was detected in 18 patients by TC. For this virus, rapid techniques sensitivity as compared with TC was low (almost 22%). Parainfluenza 3 was readily detected by IF and TC; influenza A, B and parainfluenza 1 were detected in few samples; and tissue culture proved more efficient than rapid techniques. The results indicate that both rapid techniques are good tools for the detection of most respiratory viruses except for adenovirus, for which TC cannot be omitted.     

Comparison of the Luminex xTAG respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections

Detection of respiratory viruses using sensitive real-time nucleic acid amplification tests (NATs) is invaluable for patient and outbreak management. However, the wide range of potential respiratory virus pathogens makes testing using individual real-time NATs expensive and laborious. The objective of this study was to compare the detection of respiratory virus targets using the Luminex xTAG respiratory viral panel (RVP) assay with individual real-time NATs used at the Provincial Laboratory of Public Health, Calgary, Alberta, Canada. The study included 1,530 specimens submitted for diagnosis of respiratory infections from December 2006 to May 2007. Direct-fluorescent-antigen-positive nasopharyngeal samples were excluded from this study. A total of 690 and 643 positives were detected by RVP and in-house NATs, respectively. Kappa correlation between in-house NATs and RVP for all targets ranged from 0.721 to 1.000. The majority of specimens missed by in-house NATs (96.7%) were positive for picornaviruses. Samples missed by RVP were mainly positive for adenovirus (51.7%) or respiratory syncytial virus (27.5%) by in-house NATs and in general had low viral loads. RVP allows for multiplex detection of 20 (and differentiation between 19) respiratory virus targets with considerable time and cost savings compared with alternative NATs. Although this first version of the RVP assay has lower sensitivity than in-house NATs for detection of adenovirus, it has good sensitivity for other targets. The identification of picornaviruses and coronaviruses and concurrent typing of influenza A virus by RVP, which are not currently included in our diagnostic testing algorithm, will improve our diagnosis of respiratory tract infections.     

常见呼吸道病毒分子鉴别诊断技术的建立

目的 建立一种常见呼吸道感染病毒的快速检测方法,为尽早诊断、减少疾病的传播以及为临床提供良好的治疗依据.方法 采用液体芯片检测技术结合靶向多重RT-PCR技术建立可以同时检测13种呼吸道病原的检测技术.结果 该方法特异性方面检测13种常见呼吸道病毒没有交叉,标本的检出率为100%;灵敏度方面达到10e2-10e1（pfu/ml）.结论 该分子鉴别诊断可应用常见呼吸道病毒的检测,协助诊断病毒引起的病毒性呼吸道感染.     

High detection rates of nucleic acids of a wide range of respiratory viruses in the nasopharynx and the middle ear of children with a history of recurrent acute otitis media

Both bacteria and viruses play a role in the development of acute otitis media, however, the importance of specific viruses is unclear. In this study molecular methods were used to determine the presence of nucleic acids of human rhinoviruses (HRV; types A, B, and C), respiratory syncytial viruses (RSV; types A and B), bocavirus (HBoV), adenovirus, enterovirus, coronaviruses (229E, HKU1, NL63, and OC43), influenza viruses (types A, B, and C), parainfluenza viruses (types 1, 2, 3, 4A, and 4B), human metapneumovirus, and polyomaviruses (KI and WU) in the nasopharynx of children between 6 and 36 months of age either with (n = 180) or without (n = 66) a history of recurrent acute otitis media and in 238 middle ear effusion samples collected from 143 children with recurrent acute otitis media. The co-detection of these viruses with Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis was analyzed. HRV (58.3% vs. 42.4%), HBoV (52.2% vs. 19.7%), polyomaviruses (36.1% vs. 15.2%), parainfluenza viruses (29.4% vs. 9.1%), adenovirus (25.0% vs. 6.1%), and RSV (27.8% vs. 9.1%) were detected significantly more often in the nasopharynx of children with a history of recurrent acute otitis media compared to healthy children. HRV was predominant in the middle ear and detected in middle ear effusion of 46% of children. Since respiratory viruses were detected frequently in the nasopharynx of both children with and without a history of recurrent acute otitis media, the etiological role of specific viruses in recurrent acute otitis media remains uncertain, however, anti-viral therapies may be beneficial in future treatment and prevention strategies for acute otitis media.     

Determination of a universal nucleic acid extraction procedure for PCR detection of gastroenteritis viruses in faecal specimens

Four nucleic acid extraction protocols were examined for their suitability for extraction of the ssRNA, dsRNA and dsDNA genomes of gastroenteritis viruses, for PCR detection. Protocol (A), employed specimen lysis with guanidinium thiocyanate, extraction with phenol–chloroform–isoamyl alcohol and nucleic acid purification by size-fractionated silica particles. Protocol (B), utilised specimen lysis with guanidinium thiocyanate and nucleic acid purification by silica, followed by phenol–chloroform–isoamyl alcohol extraction. Protocol (C), employed specimen lysis with guanidinium thiocyanate and nucleic acid purification by RNAID glass powder. Protocol (D), employed specimen lysis with sodium dodecyl sulphate, proteinase K digestion and extraction with phenol–chloroform–isoamyl alcohol. Of the four protocols, (B) appeared to be a suitable candidate ‘universal’ nucleic acid extraction procedure for PCR detection of different viral agents of gastroenteritis in a single nucleic acid extract of a faecal specimen, irrespective of genome composition. Omission of the phenol–chloroform extraction step did not affect negatively the ability of protocol (B) to allow PCR detection of gastroenteritis viruses in faecal specimens. PCR detection of NLVs, astroviruses, rotaviruses and adenoviruses, in single nucleic acid extracts of faecal specimens obtained from the field, confirmed the universality of the modified protocol (B). We propose the modified protocol (B) as a ‘universal’ nucleic acid extraction procedure, for monoplex PCR detection of gastroenteritis viruses in single nucleic acid extracts of faecal specimens and for development of multiplex PCR for their simultaneous detection.     

A prospective study of prevalence of respiratory viruses causing acute respiratory infection in pediatric in-patients during pre- COVID times

PurposeTo find out the prevalence of respiratory viruses causing Acute Respiratory Infection in pediatric in-patients during Pre-COVID times.MethodsNasal swabs were collected from children in the age group of 1 month–16 years who were admitted at our hospital with Acute Respiratory Infection. Samples were subjected to nucleic acid extraction and Real time polymerase chain reaction to detect 16 RNA viruses and 2 DNA viruses. The results were interpreted in context of most prevalent viruses detected, their seasonal distribution, co-infecting viruses, co-morbidities in patients with effect thereof and use and effect of antibiotics in those positive for viral etiology.ResultsOf the 250 children recruited in the study, viral pathogen was detected in 74% cases. RSV was the most common virus detected with 36.2% positivity (92/254) followed by rhino/entero (19.2%, 49/254), PIV 1,2,3,4 (9.4%, 24/254), Influenza A,B,C (8.2%, 21/254), adenovirus & HBoV (6.2%, 16/254), coronavirus HKU1, NL63, OC43, 229E (4.3%, 11/254), H1N1 (4.7%, 12/254) and hMPV (0.7%, 2/254). Co-infection with 2 or more viruses was seen in 34% cases. Among the cases on whom antibiotics were started, they were withdrawn following test results in 42.3% of the cases.ConclusionThe prevalence of viral etiology is high amongst children especially ≤2 years. RSV, rhino/enterovirus, PIV 1,2,3,4 and Influenza virus were more prevalent than others. Rapid, early detection of virus with multiplex PCR will help in early cohorting of the patients thus reducing nosocomial spread of these viruses and prevent injudicious use of antibiotics.     

Assessment of laboratory performance of nucleic acid amplification tests for detection of Mycobacterium tuberculosis

During implementation of the Centers for Disease Control and Prevention's Mycobacterium tuberculosis nucleic acid amplification (NAA) evaluation program, 27.1% of participants used the same biological safety cabinet for NAA and specimen processing; 28.8% reported not using unidirectional workflow. An association between false positives and adverse responses to quality assurance questions (P = 0.04) illustrated the need for following NCCLS recommendations.     

Comparison of three nucleic acid amplification tests for detection of Chlamydia trachomatis in urine specimens

Traditionally, culture and immunoassays have been performed for the detection of sexually transmitted diseases, including Chlamydia trachomatis. However, these assays may often require invasive specimen collection methods, such as female cervical and male urethral swabs. Recently, nucleic acid amplification tests (NAATs) have been approved for testing for the presence of C. trachomatis in urine samples. Our objective was to compare the sensitivities and specificities of C. trachomatis detection in urine samples with three NAATs: the Abbott LCx (LCx), BD ProbeTec ET (ProbeTec), and Gen-Probe APTIMA Combo 2 (AC2). Urine specimens (n = 506) were collected from both symptomatic and asymptomatic males and females from various high school health clinics. Specimens were tested for C. trachomatis with the three NAATs, and a true-positive result was defined as any two positive NAATs. The C. trachomatis prevalence was 14.8% (75 of 506 samples). Of the 75 urine samples defined as true positives, LCx detected 72, ProbeTec 72, and AC2 detected 75. The sensitivities of LCx, ProbeTec, and AC2 for C. trachomatis detection were 96.0, 96.0, and 100%, and the specificities were 99.1, 100, and 98.8%, respectively. Four of five samples that were positive with AC2 and negative with LCx and ProbeTec were found to be positive with an alternative target TMA-based NAAT, APTIMA C. trachomatis, suggesting that they may have been true positives. Two of four uniquely positive LCx samples available for subsequent testing were both found to be positive by Roche PCR. We found that the LCx, ProbeTec, and AC2 NAATs are highly sensitive and specific methods for the detection of C. trachomatis in urine specimens and can be recommended for noninvasive screening of C. trachomatis in urine.     

Chlamydia trachomatis infections in Greece: first prevalence study using nucleic acid amplification tests

The present retrospective study was initiated to determine the prevalence of Chlamydia trachomatis and to assess the risk factors for infection in adult women and men presenting to general practitioners, gynecologists, dermatologists, and family-planning centers in Greece. The study was carried out in four different Greek hospital centers using highly sensitive nucleic acid amplification techniques. Altogether, 16,834 women and 1,035 men were enrolled from October 1998 to April 2004. Two types of specimens were collected from each patient: cervical swabs from women, urethral swabs from men, and first-catch urine from women and men. All specimens were examined with the Cobas Amplicor C. trachomatis polymerase chain reaction assay (Roche Molecular Systems, Branchburg, NJ, USA) or the LC × C. trachomatis ligase chain reaction assay (Abbott Laboratories, Abbott Park, IL, USA). Demographic and behavioral data were collected by clinicians using a standardized questionnaire. A total of 704 (3.9%) patients were infected with C. trachomatis. The prevalence among female patients was 3.5% and that among male patients 11.2%. Among infected patients, 88% were under 30 years of age, 71% reported more than one sexual partner, and 91% reported a new sexual partner within the last year. In conclusion, the prevalence of C. trachomatis infection in Greece is low. Young age and new and multiple sexual partners within the last year were factors consistently associated with an increased risk of chlamydial infection.     

Simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-PCR assays

A 4-tube multiplex RT-PCR (mRT-PCR), which showed higher sensitivity over conventional methods, was previously developed for the diagnosis of 14 viral pathogens of the respiratory tract. Herein the mRT-PCR was compared to the commercial Luminex mPCR-microsphere flow cytometry assay (Resplex II) which allows the detection of 12 different viruses. Eleven different viruses were identified in 91 nasopharyngeal swabs of children with acute respiratory infection, influenza A (IAV) and B, respiratory syncytial virus (RSV), human rhinovirus (hRhV), human echovirus, parainfluenza viruses (PIV) 1, 2, 3 and 4, human metapneumovirus (hMPV), and human coronavirus NL63. The results of the two techniques showed 53 and 40 positive patients by the Resplex II assay and mRT-PCR, respectively, with a concordance in 35 positive and 33 negative patients (74.7%). Individual RT-PCR tests were performed to control viruses not simultaneously detected by the two multiplex assays. The major virus misdiagnosed by mRT-PCR was IAV whereas the major viruses misdiagnosed by Resplex II were PIV1, 3 and 4. The mRT-PCR remains a simple, rapid, and specific assay for the specific detection of respiratory viruses, and can be easily implemented with standards in clinical laboratories at a low cost.     

Two-year prospective study of single infections and co-infections by respiratory syncytial virus and viruses identified recently in infants with acute respiratory disease

A prospective 2-year analysis including 322 infant patients with acute respiratory disease (ARD) hospitalized in a pediatric department in northern Italy was carried out to evaluate the role as respiratory pathogens or co-pathogens of recently identified viruses. The presence of respiratory syncitial virus (RSV), human Metapneumoviruses (hMPVs), human Bocaviruses (hBoVs), and human Coronaviruses (hCoVs) was assayed by molecular detection and clinical symptoms evaluated. Nasopharyngeal aspirates from 150 of the 322 infants (46.6%) tested positive for at least one pathogen. Ninety samples (28.0%) tested positive for RSV RNA (61.5% genotype A and 38.5% genotype B), 46 (14.3%) for hMPV RNA (71.7% subtype A and 28.3% subtype B), 28 (8.7%) for hCoV RNA (39.3% hCoV-OC43, 35.7% hCoV-NL63, 21.4% hCoV-HKU1, and 3.6% hCoV-229E), and 7 (2.2%) for hBoV DNA (of the 6 typed, 50% subtype 1 and 50% subtype 2); 21/150 samples revealed the presence of 2 or more viruses. Co-infection rates were higher for hMPVs, hCoVs, and hBoV (38.3%, 46.4%, and 57.1%,) and lower for RSV (23.3%). RSV was associated with the presence of complications (P < 0.001) and hypoxia (P < 0.015). When the presence of RSV alone and the RSV-hMPV co-infections were considered, RSV mono-infected patients resulted to have longer hospitalization and higher hypoxia (P < 0.001). The data highlight that (i) RSV has a central role as a respiratory pathogen of infants, (ii) the wide circulation of recently identified viruses does not reduce the clinical and epidemiological importance of RSV, and that (iii) recently identified agents (hMPVs, hBoVs, and hCoVs) act as primary pathogens or co-pathogens.     

Burden of respiratory viruses in patients with acute respiratory failure

Respiratory viruses (RVs) are ubiquitous pathogens that represent a major cause of community‐acquired pneumonia and chronic pulmonary diseases exacerbations. However, their contribution to acute respiratory failure events requiring intensive care unit admission in the era of rapid multiplex molecular assay deserves further evaluation. This study investigated the burden of viral infections in non immunocompromised patients admitted to the intensive care unit for acute respiratory failure using a multiplex molecular assay. Patients were investigated for RVs using immunofluoresence testing and a commercial multiplex molecular assay, and for bacteria using conventional culture. Half the patients (34/70, 49%) had a documented RVs infection. No other pathogen was found in 24 (71%) patients. Viral infection was detected more frequently in patients with obstructive respiratory diseases (64% vs. 29%; P = 0.0075). Multiplex molecular assay should be considered as an usefull diagnostic tool in patients admitted to the intensive care unit with acute respiratory failure, especially those with acute exacerbations of chronic obstructive pulmonary disease and asthma. J. Med. Virol. 86:1198–1202, 2014. © 2013 Wiley Periodicals, Inc.

     

Optimizing screening for acute human immunodeficiency virus infection with pooled nucleic acid amplification tests

Recent studies have shown the public health importance of identifying individuals with acute human immunodeficiency virus infection (AHI); however, the cost of nucleic acid amplification testing (NAAT) makes individual testing of at-risk individuals prohibitively expensive in many settings. Pooled NAAT (or group testing) can improve efficiency and test performance of testing for AHI, but optimizing the pooling algorithm can be difficult. We developed simple, flexible biostatistical models of specimen pooling with NAAT for the identification of AHI cases; these models incorporate group testing theory, operating characteristics of biological assays, and a model of viral dynamics during AHI. Pooling algorithm sensitivity, efficiency (test kits used per individual specimen evaluated), and positive predictive value (PPV) were modeled and compared for three simple pooling algorithms: two-stage minipools (D2), three-stage hierarchical pools (D3), and square arrays with master pools (A2m). We confirmed the results by stochastic simulation and produced reference tables and a Web calculator to facilitate pooling by investigators without specific biostatistical expertise. All three pooling strategies demonstrated improved efficiency and PPV for AHI case detection compared to individual NAAT. D3 and A2m algorithms generally provided better efficiency and PPV than D2; additionally, A2m generally exhibited better PPV than D3. Used selectively and carefully, the simple models developed here can guide the selection of a pooling algorithm for the detection of AHI cases in a wide variety of settings.     

Detection of respiratory viruses in gargle specimens of healthy children

BackgroundRespiratory tract viral infection is one of the most common and important diseases in children. Polymerase chain reaction (PCR) tests are often used to detect viruses in samples, it is difficult to interpret the clinical significance of PCR positivity, which may reflect a past, imminent or active asymptomatic infection due to their high sensitivity. Although single respiratory viruses have been detected in samples from children with symptoms, other respiratory viruses can also be detected simultaneously. However, the clinical importance of these findings for the symptoms is not known.ObjectivesTo investigate the prevalence of respiratory viruses among children without any symptoms such as acute respiratory illness and/or fever.Study designFrom week twenty-five 2013 to week twenty-six 2014, gargle samples were collected from children once a week and these samples were subjected to real-time PCR to detect respiratory viruses. On each sampling day, we asked the parents about their children’s health condition.ResultsAmong the 286 samples collected, 200 were from asymptomatic children. In the asymptomatic condition, human parechovirus, adenovirus, enterovirus, rhinovirus, coronavirus 229E and HKU1 were observed in 45 episodes. In samples from symptomatic children, parainfluenza viruses, respiratory syncytial virus and coronavirus OC43 were detected in addition to those mentioned above.ConclusionsVarious viruses of different species were detected in the specimens from the children regardless of their health status. It might be speculated that host factors such as the function of the immune system influence the clinical outcome of the infection. However, this needs to be studied further.     

Impact of patient characteristics on performance of nucleic acid amplification tests and DNA probe for detection of Chlamydia trachomatis in women with genital infections

The performance of nucleic acid amplified tests (NAAT) for Chlamydia trachomatis at the cervix and in urine was examined in 3,551 women, and the impacts of clinical findings (age, endocervical and urethral inflammation, menses, and gonococcal coinfection) were assessed. Ligase chain reaction (LCR) and first-generation uniplex PCR were studied relative to an unamplified DNA probe (PACE2) and to an expanded, independent diagnostic reference standard. Relative to the expanded standard, cervical or urine LCR was generally the most sensitive test in most subgroups. Increased detection by NAAT of cervical C. trachomatis over PACE2 was highest among women without mucopurulent endocervical discharge versus those with (relative increase in positivity with cervical LCR, 46%) and among women > or =20 years old versus younger women (relative increase in positivity with cervical LCR, 45%). The sensitivity of cervical PCR was highest when mucopurulent endocervical discharge was present (84%) and highest for cervical LCR when cervical gonococcal coinfection was detected (91%). Urethral inflammation was associated with higher sensitivities of urine LCR (86 compared to 70% when inflammation was absent) and PCR (82 compared to 62% when inflammation was absent). Menses had no effect on test performance. The effects of patient characteristics on test specificities were less pronounced and were closely related to observed sensitivities. These findings support expanded use of NAAT for screening and diagnosis of C. trachomatis in diverse clinical populations of women.