RAZOR: a database of PCR primers targeting human respiratory viruses

A molecular assay prognostic of survival in resected nonsquamous non-small cell lung cancer designed to meet the need for improved risk stratification in early-stage disease has recently been described. This assay measures the expression levels of 14 genes using RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. The assay underwent blinded clinical validation in 2 large international cohorts involving approximately 1500 patients; the analytical precision and reproducibility of this assay, however, have not yet been reported. For each of the 14 TaqMan quantitative polymerase chain reaction (PCR) primer and probe sets used in the molecular prognostic assay, the linear range, PCR efficiency, limits of blank, limits of quantitation, and quantitative bias were determined using serial dilutions of pooled RNA extracted from FFPE samples. The reproducibility of the entire molecular assay was determined by performing repeat testing of FFPE samples over multiple days. The linear range of individual quantitative TaqMan PCR primer and probe sets was between 2(10)- and 2(15)-fold input RNA. The median C(T) of the quantitative PCR primer and probe sets at 10 ng of input RNA was 24.3; the median efficiency was 91.2%. The median quantitative bias across all quantitative PCR primer and probe sets was 0.75% (range, 0.32% to 1.32%). In repeat testing, the mean SD of the risk score (scaled from 1 to 100) was 2.18, with a mean coefficient of variation of 0.08. The molecular prognostic assay presented in this study demonstrates high precision and reproducibility, validating its clinical utility as a reliable prognostic tool that can contribute to the management of patients with early-stage disease.

Acute otitis media (AOM) is a common ear infection caused by respiratory viruses and bacteria of the nasopharynx. The present study aimed to detect various respiratory viruses and bacteria in middle ear fluid (MEF) and nasopharyngeal aspirates (NPA) using polymerase chain reaction (PCR). We collected MEF and NPA samples from 122 pediatric patients with AOM. Real-time PCR detected 11 types of respiratory viruses (respiratory syncytial virus A/B, parainfluenza virus 1/2/3, human metapneumovirus, influenza virus A/B, adenovirus, human bocavirus and rhino virus) and 7 types of bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydia pneumoniae, Streptococcus pyogenes, Legionella pneumophila and Moraxella catarrhalis). MEF specimens were also examined using bacterial culture. At least 1 respiratory viral or bacterial pathogen was detected in MEF of 120 cases (98%) by viral and bacterial PCR and of 93 cases (76%) by viral PCR and bacterial culture. Respiratory viruses were detected in NPA of 84 cases (69%) and MEF of 67 cases (55%). The most common virus detected in MEF was respiratory syncytial virus (21%), followed by parainfluenza virus (15%). All the viruses present in MEF were also detected in NPA specimens. Bacteria were detected by PCR in MEF of 109 cases (89%); H. influenzae was the most frequently detected (65%). In many cases, pediatric AOM was found to constitute a respiratory polymicrobial infection. Multiplex PCR was useful to detect multiple respiratory viruses and bacteria in AOM. To understand intractable AOM, further studies regarding the clinical features of each viral and bacterial coinfection are required.

The measurement of vitellogenin (vtg) gene transcription has been shown to be a reliable indicator of exposure to estrogenic compounds. Unfortunately, the relatively poor molecular characterization of North American fish species has hindered its application to a larger number of ecologically important species. The current research aimed to demonstrate specific amplification of vtg gene transcripts in three model (zebrafish, rainbow trout, and medaka) and six nonmodel (emerald shiner, pearl dace, smallmouth bass, creek chub, white sucker, and golden redhorse) fish species. Quantitative polymerase chain reaction (QPCR) primers for model species were designed from publicly available vtg sequences. Successful amplification of vtg was demonstrated in fish exposed to 17alpha-ethinylestradiol (EE(2)) for all model species. Vitellogenin primers for selected nonmodel species were designed from published sequences of closely related species. Multiple primers were developed targeting different regions of the vtg gene. The successful amplification of vtg was confirmed through size and sequence analysis for all nonmodel species with the exception of the white sucker, in which amplifications failed. Furthermore, QPCR primers and conditions were quantitative over five orders of magnitude in at least one species (pearl dace) exposed to 5 ng/L of EE(2) for 24 h. The selected species are found in a wide array of ecological habitats that span the United States. Inclusion of vtg transcriptional analysis for wild, ecologically relevant fish in monitoring studies may aid in understanding the extent of estrogenic exposure in aquatic ecosystems across the United States.

Strong Association between Respiratory Viral Infection Early after Hematopoietic Stem Cell Transplantation and the Development of Life-Threatening Acute and Chronic Alloimmune Lung Syndromes

Purpose :Chlamydia trachomatis is one of the most common sexually transmitted diseases and is also a cause of pneumonia in infants. Respiratory infections by respiratory viruses are also common for infants. The objectives of this study were to identify the clinical manifestations and to determine the prevalence of C. trachomatis respiratory infections and coinfections by respiratory viruses in infants younger than 6 months of age. Methods : For this study, we enrolled 6 months or younger infants who were admitted to the Dankook University Hospital between January 2002 and July 2007, with respiratory symptoms. Nasopharyngeal aspirates or throat swabs were collected within s d of hospitalization and C. trachomatis was detected using polymerase chain reaction (PCR). Patients who tested positive underwent multiplex PCR for respiratory viruses. Results : A total of 690 patients underwent chlamydial PCR testing and 36 (5.2%) had positive results. Of the 36, 28 (78%) were male; 30 were vaginally delivered. From the 36 patients positive for C. trachomatis , 26 underwent multiplex respiratory viral PCR; 12 were coinfected with viruses. Respiratory syncytial virus (RSV) was the most frequent pathogen that was detected in 6 patients. Increased C-reactive protein and fever were significant in patients coinfected with respiratory viruses. Conclusion :C. trachomatis can infected in infants delivered by cesarean section as well as in 6 months old or younger infants. Infant with C. trachomatis respiratory infections can also be coinfected with respiratory infection also coinfected with respiratory viruses. Further studies are needed to better understand the prevalence rates of the this infection and its coinfection rate with respiratory viruses. (Korean J Pediatr 2008;51:729-735)

Full-term and preterm infants admitted to neonatal intensive care units (NICUs) face a high risk of infections, due to the immaturity of their innate and adoptive immune systems, inadequate protection through maternal immunity and the need for repeated invasive procedures (1). In recent years, our knowledge of viral infections in neonates has increased, due to multiplex polymerase chain reaction (PCR)-based techniques. It is now possible to screen for as many as 17 viruses from a single mucus sample (2). However, the role of viral respiratory tract infections in the symptoms of infants admitted to NICUs at birth is still poorly understood. In a recent review, 32 respiratory viral outbreaks in NICUs were reported (3). These were caused by several different respiratory viruses, including respiratory syncytial virus (RSV) (89 patients in 11 outbreaks), enteroviruses (101 patients in 10 outbreaks) and adenovirus (79 patients in six outbreaks). In addition, outbreaks of coronavirus, rhinovirus, influenza A virus and parainfluenza virus infections have been reported (4–7). An epidemic may result in the temporary closure of a NICU. Recently, a NICU was closed for 28 days after adenovirus type 19 caused an outbreak of keratoconjunctivitis, which affected 12 NICU infants, two NICU staff members, two relatives of patients and two members of the ophthalmologic team (8). We report on an observational study on the use of a multiplex PCR in a NICU in Finland. The study was carried out at Turku University Hospital, the only tertiary level NICU in south-west Finland, which serves a population of about 750 000. An average of 600–700 infants are admitted to the NICU annually, resulting in approximately 6000 patient care days per year. About one-third of the infants are preterm, and 50–85 very low birth weight infants are treated annually. Two to four patients are treated in one room. Parents are encouraged to stay with their infant and provided with unlimited access. Visitors with respiratory tract infection symptoms are not allowed into the NICU, and siblings under school age cannot visit if there is an RSV outbreak in the community. Otherwise, healthy siblings are allowed access. During the study period from 1 January 2009 to 30 June 2011, 1589 infants were admitted to the unit and 76 (5%) were evaluated for respiratory viruses. A nasopharyngeal aspirate was taken from infants if they presented with symptoms of respiratory infection, such as rhinorrhea, sneezing, increased bronchial secretions, episodes of bradycardia and/or desaturations, or if they had been exposed to someone with a respiratory infection. We collected and analysed 139 samples from 76 infants for this study. Nasopharyngeal aspirates were collected in sterile tubes and analysed on the same or following day. Nucleic acids were extracted from the aspirates using Nuclisense easyMag extractor (Biomerieux, Boxtel, the Netherlands). Respiratory virus genomes were detected using Seeplex RV12 multiplex PCR assay for adenovirus, influenza A and B viruses, parainfluenza types 1–3 viruses, RSVa, RSVb, rhinovirus, human metapneumovirus, and coronaviruses 229E/NL63 and OC43/HKU1 (Seegene, Seoul, Korea). In addition, specimens were tested in a real-time PCR assay for enteroviruses, rhinovirus and RSV (9). During the two-and-a-half-year study period, no outbreaks of viral respiratory tract infection occurred in the NICU. Of the 139 samples taken from 76 infants, 28 samples (20%) from 15 infants (20%) were positive for one or more viruses. Six babies were positive for rhinovirus, five for parainfluenza type 3 virus, one for parainfluenza type 2

Heparin is a well-known glycosaminoglycan extracted from porcine intestines. Increased vigilance for transmissible spongiform encephalopathy in animal-derived pharmaceuticals requires methods to prevent the introduction of heparin from ruminants into the supply chain. The sensitivity, specificity, and precision of the quantitative polymerase chain reaction (PCR) make it a superior analytical platform for screening heparin raw material for bovine-, ovine-, and caprine-derived material. A quantitative PCR probe and primer set homologous to the ruminant Bov-A2 short interspersed nuclear element (SINE) locus (Mendoza-Romero et al. J. Food Prot. 67:550–554, 2004) demonstrated nearly equivalent affinities for bovine, ovine, and caprine DNA targets, while exhibiting no cross-reactivity with porcine DNA in the quantitative PCR method. A second PCR primer and probe set, specific for the porcine PRE1 SINE sequence, was also developed to quantify the background porcine DNA level. DNA extraction and purification was not necessary for analysis of the raw heparin samples, although digestion of the sample with heparinase was employed. The method exhibits a quantitation range of 0.3–3,000 ppm ruminant DNA in heparin. Validation parameters of the method included accuracy, repeatability, precision, specificity, range, quantitation limit, and linearity.

With the reduction in access to polymerase chain reaction (PCR) testing and changes in testing guidelines in Australia, a reduced number of people are seeking testing for coronavirus disease (COVID-19), limiting the opportunity to monitor disease transmission. Knowledge of community transmission of COVID-19 and other respiratory viruses is essential to better predict subsequent surges in cases during the pandemic to alert health services, protect vulnerable populations and enhance public health measures. We describe a methodology for a testing-based sentinel surveillance program to monitor disease in the community for early signal detection of SARS-CoV-2 and other respiratory viruses. A longitudinal active testing-based sentinel surveillance program for respiratory viruses (including SARS-CoV-2, influenza A, influenza B and Respiratory Syncytial Virus) will be implemented in some regions of Queensland. Adults will be eligible for enrolment if they are part of specific community groups at increased risk of exposure and have not had a COVID-19 infection in the last 13 weeks. Recruitment via workplaces will occur in-person, via email and through online advertisement. Asymptomatic participants will be tested via PCR for SARS-CoV-2 infection by weekly self-collected nasal swabs. In addition, symptomatic participants will be asked to seek SARS-CoV-2 and additional respiratory virus PCR testing at nominated COVID-19 testing sites. SARS-CoV-2 and respiratory virus prevalence data will be analysed weekly and at the end of the study period. Once implemented, this surveillance program will determine the weekly prevalence of COVID-19 and other respiratory viruses in the broader community by testing a representative sample of adults, with an aim to detect early changes in the baseline positivity rate. This information is essential to define the epidemiology of SARS-CoV-2 in the community in near-real time to inform public health control measures and prepare health services and other stakeholders for a rise in service demand.

Molecular diagnostics for invasive lung infections in children.

Rationale for the studyReal‐time polymerase chain reaction (PCR) for respiratory viruses is more sensitive, yet more expensive, than conventionally used direct immunofluorescence (DIF). We determined the impact of real‐time PCR, additional to DIF, on antibiotic prescription in ventilated children with lower respiratory tract infection (LRTI) at admission to the pediatric intensive care unit (PICU).MethodsFirst, a multicenter survey study was performed. Subsequently, in a prospective study, children (≤5 years) with LRTI were tested at admission by DIF and PCR. Positive DIF results were reported at the end of the first working day. PICU physicians reported antibiotic treatment on the second working day. After informing them of the PCR result antibiotic treatment was reevaluated.ResultsThe multicenter survey study (94 respondents) showed that PCR decreased antibiotic use (P < 0.001). In the prospective study 38 children were included, of which 19 (50%) were DIF positive. Of the 19 DIF negative patients 12 (63%) were treated with antibiotics before revealing the PCR result; the PCR test was positive in 9 out of 12. Revealing PCR results did not alter antibiotic treatment. In 7 DIF negative patients antibiotics not given, the PCR test was positive.ConclusionIn contrast to their responses to the survey study, in real‐life PICU physicians did not let their antibiotic prescription be influenced by respiratory real‐time PCR in children ventilated for LRTI. Pediatr. Pulmonol. 2011; 46:428–434. © 2010 Wiley‐Liss, Inc.

Introduction and ObjectiveThe emergence of the COVID‐19 pandemic raised questions about the interaction between severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and other respiratory viruses. The objective of this study is to validate the impact of the SARS‐CoV‐2 pandemic and its interventional measures on the respiratory viruses' transmission/infection rates.MethodsA retrospective chart review was conducted for cancer patients who underwent laboratory‐confirmed respiratory virus polymerase chain reaction (PCR) testing from January 2018 to June 2022. COVID‐19 PCR tests from March 2020 to June 2022 were also included. Joinpoint regression analysis was applied to evaluate trends in respiratory virus rates. Statistical analysis was performed using Statistical Package for Social Science software.ResultsA total of 6298 respiratory virus PCRs and 40,000 COVID‐19 PCRs were performed. Data showed a significant decrease in respiratory viruses' positive cases, total respiratory tests, and respiratory viruses' activity during the pandemic period compared with the pre‐pandemic period (p = .0209, .026, and .028, respectively). The joinpoint regression analysis showed a significant decrease of 13.85% in the tested positive cases of respiratory viruses between the years 2018 and 2022. Monthly, the analysis indicated a significant decrease in the positive cases by 13.46% from December 2019 to May 2021. Weekly analysis following lockdown initiation showed a reduction in respiratory virus cases.ConclusionThis study provides valuable insights into the interplay between COVID‐19 and other respiratory viruses, suggesting that the measures taken for COVID‐19 were effective in reducing the spread of viral respiratory infections, aiding future infection control strategies to protect vulnerable populations, including cancer patients, from seasonal respiratory infections.

Editor, Herpes simplex keratitis (HSK) is a major cause of unilateral blindness in developed countries (Guess et al. 2007). A fast and highly sensitive test could assist in the diagnosis to guide adequate antiviral therapy. We aimed to report the use of herpes simplex virus (HSV) polymerase chain reaction (PCR) test for the diagnosis of HSK and its relationship to the outcomes in these patients over a 2-year period at the Sydney Eye Hospital, Australia. A retrospective case review of all patients who received antiviral medications for HSK was conducted. Cases were identified from HSV PCR swab results, pharmacy records and hospital coding data from 2012 to 2013. Clinical details were collated from the medical records. Herpes simplex virus type 1 and type 2 were detected using the HSV-1 HSV-2 VZV R-gene® kit, a real-time PCR on DNA extracted from human clinical samples (Argene, bioMérieux SA, Marcy-l'Etoile, France) according to manufacturer’s instructions. The tests were performed using a Roche LC480 thermal cycler for real-time amplification. The R-gene kit’s sensitivity ranged between 91% and 100% and specificity 95% and 100% depending on the study (Biomerieux 2016). Patients were sub-grouped depending on the clinical features: epithelial, stromal with ulceration (SHSK+U), stromal without ulceration (SHSK−U), endothelial, keratouveitis and active HSK with prior corneal graft. Outcome was determined when the initial antiviral therapy was stopped or changed and classified as clinically improved or worsened. Two hundred and fifty-two patients were included with HSV PCR performed in 166 (66%) at presentation. The HSV-1 PCR test was positive in 32/94 (34%) patients with epithelial HSK, 4/14 (29%) patients with SHSK+U, 3/11 (27%) patients with active HSK and corneal graft, and 6/33 (18%) patients with keratouveitis. Overall, the positivity rate was 27% (45/166). Of the patients with prior corneal graft, two were diagnosed with epithelial HSK and one with SHSK+U. A clinically improved or worsened outcome was determined for 112 of 166 patients (67%) tested with HSV PCR (Table 1). There was no statistical association between the type of outcome and HSV PCR result (p = 0.5). Polymerase chain reaction test has been reported as a highly sensitive diagnostic test for HSK (Subhan et al. 2004; Azher et al. 2017). Its diagnostic power varies depending on the type of HSK, previous acyclovir therapy, use of topical anaesthetics or dyes, and viral load in the specimen (Shoji et al. 2016). The PCR test most likely identifies patients with a dendritic ulcer as it is a manifestation of active viral infection (Azher et al. 2017). Consequently, clinicians requested the test to patients with a presence of a epithelial defect and provisional diagnosis of epithelial HSK [94/123, (76%)], SHSK+U [14/19, (74%)], keratouveitis [33/49, (67%)] or active HSK with corneal graft [11/25, (44%)]. Stromal HSK is thought to occur due to an immune response to the virus rather than via viral infection (Azher et al. 2017). It is also assumed that stromal and endothelial HSK are recurrent episodes (Guess et al. 2007) and the patients may have a previous positive PCR result. Due to the absence of an epithelial defect, it is not possible to access the stroma and therefore to obtain a sample of the area affected. This may explain the negative PCR results for this type of HSK (Azher et al. 2017). This may also be the reason why clinicians did not request as many PCR tests on patients with SHSK−U [11/22, (50%)] and endothelial HSK [3/14, (21%)] compared with the epithelial HSK (76%) and SHSK+U (74%) groups. The positivity rate for both, SHSK−U and endothelial HSK, was 0% supporting the above hypothesis. In conclusion, the HSV PCR test had an overall low positive rate. Regardless of the PCR result, most patients improved with the initial antiviral therapy, especially for epithelial HSK. A clinical diagnosis of HSK may therefore be sufficient to guide treatment.

The clinical impact of pneumocystis and viral PCR testing on bronchoalveolar lavage in immunosuppressed patients.

New Aspects on Human Rhinovirus Infections

A community-based, time-matched, case-control study of respiratory viruses and exacerbations of COPD