Multiplex Real-time Reverse Transcription Polymerase Research Articles

Etiology of Acute Lower Respiratory Illness Hospitalizations Among Infants in 4 Countries.

Recent studies explored which pathogens drive the global burden of pneumonia hospitalizations among young children. However, the etiology of broader acute lower respiratory tract infections (ALRIs) remains unclear. Using a multicountry study (Albania, Jordan, Nicaragua, and the Philippines) of hospitalized infants and non-ill community controls between 2015 and 2017, we assessed the prevalence and severity of viral infections and coinfections. We also estimated the proportion of ALRI hospitalizations caused by 21 respiratory pathogens identified via multiplex real-time reverse transcription polymerase chain reaction with bayesian nested partially latent class models. An overall 3632 hospitalized infants and 1068 non-ill community controls participated in the study and had specimens tested. Among hospitalized infants, 1743 (48.0%) met the ALRI case definition for the etiology analysis. After accounting for the prevalence in non-ill controls, respiratory syncytial virus (RSV) was responsible for the largest proportion of ALRI hospitalizations, although the magnitude varied across sites-ranging from 65.2% (95% credible interval, 46.3%-79.6%) in Albania to 34.9% (95% credible interval, 20.0%-49.0%) in the Philippines. While the fraction of ALRI hospitalizations caused by RSV decreased as age increased, it remained the greatest driver. After RSV, rhinovirus/enterovirus (range, 13.4%-27.1%) and human metapneumovirus (range, 6.3%-12.0%) were the next-highest contributors to ALRI hospitalizations. We observed substantial numbers of ALRI hospitalizations, with RSV as the largest source, particularly in infants aged <3 months. This underscores the potential for vaccines and long-lasting monoclonal antibodies on the horizon to reduce the burden of ALRI in infants worldwide.

Molecular characterisation and tracking of severe acute respiratory syndrome coronavirus 2 in Thailand, 2020-2022.

The global COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first detected in China in December 2019. To date, there have been approximately 3.4 million reported cases of COVID-19 and over 24,000 deaths in Thailand. In this study, we investigated the molecular characteristics and evolution of SARS-CoV-2 in Thailand from 2020 to 2022. Two hundred sixty-eight SARS-CoV-2 isolates, collected mostly in Bangkok from COVID-19 patients, were characterised by partial genome sequencing. Moreover, the viruses in 5,627 positive SARS-CoV-2 samples were identified as viral variants - B.1.1.7 (Alpha), B.1.617.2 (Delta), B.1.1.529 (Omicron/BA.1), or B.1.1.529 (Omicron/BA.2) - by multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) assays. The results revealed that B.1.36.16 caused the predominant outbreak in the second wave (December 2020-January 2021), B.1.1.7 (Alpha) in the third wave (April-June 2021), B.1.617.2 (Delta) in the fourth wave (July-December 2021), and B.1.1.529 (Omicron) in the fifth wave (January-March 2022). The evolutionary rate of the viral genome was 2.60 × 10-3 (95% highest posterior density [HPD], 1.72 × 10-3 to 3.62 × 10-3) nucleotide substitutions per site per year. Continued molecular surveillance of SARS-CoV-2 is crucial for monitoring emerging variants with the potential to cause new COVID-19 outbreaks.

A multiplex real-time reverse transcription polymerase chain reaction assay for differentiation of classical and variant II strains of avian infectious bronchitis virus.

Identification of avian infectious bronchitis virus (IBV) genotypes is essential for controlling infectious bronchitis (IB) disease, because vaccines that differ from the circulating strains might not provide efficient cross-protection. In Egypt, IBV strain typing is a difficult process, due to the widespread distribution of four genotype lineages (GI-13, GI-23, GI-1, and GI-16), which may contribute to IBV vaccination failure. In this study, we developed a multiplex real-time quantitative reverse transcription polymerase chain reaction (mRT-qPCR) assay that targets highly conserved areas of the S1 gene in order to detect classical (G1) and Egyptian variant II (G23) strains in allantoic fluids and clinical samples. The viral genotyping technique was assessed usingcommercially available vaccines as well as local strains, and 16 field isolates were tested to investigate its clinical applicability. The assay was found to be specific for the detection of classical and VAR II strains and did not detect the VAR I strain or other avian pathogens such as Newcastle disease virus, avian influenza virus (H9N2 and H5N8), or infectious bursal disease virus. The results also showed that 28 out of 41 samples tested positive for IBV utilizing rt-qRT-PCR targeting the N gene and that 26 out of the 28 positive samples were genotyped by mRT-qPCR targeting the S1 gene, whereas the remaining two samples that were not genotyped were VAR 1 (4/91) and VAR I (793/B). Interestingly, the testing could identify combined infections in one sample, indicating a mixed infection with both genotypes. The real-time RT-PCR assay could detect viral RNA at concentrations as low as 102 EID50 /ml for both classical and variant II. This assay is rapid, specific, and sensitive. It appears to be a valuable tool for regular disease monitoring that can be used to differentiate as well as identify viruses.

High genetic diversity of human rhinovirus among pilgrims with acute respiratory tract infections during the 2019 Hajj pilgrimage season

ObjectivesAcute respiratory tract infections (ARIs) due to human rhinoviruses (HRVs) are common in pilgrims during the annual Hajj pilgrimage. The objective of this study was to investigate the genetic diversity of HRV among pilgrims with respiratory symptoms during Hajj 2019. MethodsHRV infection was detected using multiplex real-time reverse transcription polymerase chain reaction. Cycle sequencing was performed on positive samples and the sequences were subjected to phylogenetic analysis. ResultsA total of 19 HRV-positive respiratory samples were sequenced. All three serotypes of HRV were identified: HRV-A (13; 68.42%) was more common than HRV-B (2; 10.53%) and HRV-C (4; 21.05%). HRV-A species were found to be of genotypes A101, A21, A30, A57, A23, A60, and A11. HRV-B species belonged to genotypes B4 and B84, and HRV-C species were of genotypes C15, C3, and C56. ConclusionSequencing studies of respiratory tract viruses in pilgrims are important. We provide preliminary evidence of high diversity of HRV genotypes circulating in pilgrims in a restricted area during Hajj. This requires further clinical and sequencing studies of viral pathogens in larger cohorts of overseas and local pilgrims.

A Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay With Enhanced Capacity to Detect Vesicular Stomatitis Viral Lineages of Central American Origin.

Vesicular stomatitis virus (VSV) causes a disease in susceptible livestock that is clinically indistinguishable from foot-and-mouth disease. Rapid testing is therefore critical to identify VSV and rule out FMD. We previously developed and validated a multiplex real-time reverse transcription polymerase chain reaction assay (mRRT-PCR) for detection of both VS New Jersey virus (VSNJV) and VS Indiana virus (VSIV). However, it was subsequently apparent that this assay failed to detect some VSNJV isolates in Mexico, especially in genetic group II, lineage 2.1. In order to enhance the sensitivity of the mRRT-PCR for VSNJV, parts of the assay were redesigned and revalidated using new and improved PCR chemistries. The redesign markedly improved the assay by increasing the VSNJV detection sensitivity of lineage 2.1 and thereby allowing detection of all VSNJV clades. The new assay showed an increased capability to detect VSNJV. Specifically, the new mRRT-PCR detected VSNJV in 100% (87/87) of samples from Mexico in 2006-2007 compared to 74% for the previous mRRT-PCR. Furthermore, the analytical sensitivity of the new mRRT-PCR was enhanced for VSNJV. Importantly, the modified assay had the same sensitivity and specificity for VSIV as the previously published assay. Our results highlight the challenges the large genetic variability of VSV pose for virus detection by mRRT-PCR and show the importance of frequent re-evaluation and validation of diagnostic assays for VSV to ensure high sensitivity and specificity.

E型肝炎ウイルスの遺伝子型3および4型株迅速鑑別のためのマルチプレックスreal-time RT-PCR法における検出試薬の比較検討

E型肝炎ウイルスの遺伝子型3および4型株迅速鑑別のためのマルチプレックスreal-time RT-PCR法における検出試薬の比較検討

English

Zika, yellow fever and dengue viruses are three important flaviviruses that are epidemic and endemic in Central Africa, Middle and South America, Southeast Asia and Pacific Islands. Since these three viral diseases present with similar symptoms and there is a possibility of multiple flaviviruses co-infection, it is important to develop an effective and quick diagnostic tool. Here, we designed and validated a one-step triplex real time reverse transcription polymerase chain reaction (RT-PCR) using TaqMan probes targeting NS5 region for detecting zika virus (ZIKV), dengue virus-4 (DENV-4) and yellow fever virus (YFV). The assay was highly specific for the target viral RNA corresponding to ZIKV, DENV-4 and YFV strains. The sensitivity of the assay was compared to one-step, RT-PCR, it showed a 100-fold higher sensitivity than RT-PCR with ZIKV and DENV and 10-fold higher sensitivity with YFV. In conclusion, the one-step, triplex, TaqMan, real time RT-PCR assay identified and distinguished ZIKV, DENV-4 and YFV by targeting single gene. Key words: Zika, yellow fever, dengue, multiplex real time reverse transcription-polymerase chain reaction (RT-PCR).

Internally Controlled, Multiplex Real-Time Reverse Transcription PCR for Dengue Virus and Yellow Fever Virus Detection.

The differential diagnosis of dengue virus (DENV) and yellow fever virus (YFV) infections in endemic areas is complicated by nonspecific early clinical manifestations. In this study, we describe an internally controlled, multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) for the detection of DENV and YFV. The DENV-YFV assay demonstrated specific detection and had a dynamic range of 2.0-8.0 log10 copies/μL of eluate for each DENV serotype and YFV. Clinical performance was similar to a published pan-DENV assay: 48/48 acute-phase samples from dengue cases were detected in both assays. For YFV detection, mock samples were prepared with nine geographically diverse YFV isolates over a range of concentrations. The DENV-YFV assay detected 62/65 replicates, whereas 54/65 were detected using a reference YFV rRT-PCR. Given the reemergence of DENV and YFV in areas around the world, the DENV-YFV assay should be a useful tool to narrow the differential diagnosis and provide early case detection.

Respiratory viral infection`s frequency and clinical outcome in symptomatic children with cancer: A single center experience from a middle-income country.

Büyükkapu-Bay S, Kebudi R, Görgün Ö, Meşe S, Zülfikar B, Badur S. Respiratory viral infection`s frequency and clinical outcome in symptomatic children with cancer: A single center experience from a middle-income country. Turk J Pediatr 2018; 60: 653-659. In developing countries, acute respiratory tract infections are a significant cause of morbidity and mortality in children, particularly in pediatric cancer patients. A majority of these illnesses are precipitated by viral infections. In our country, studies were conducted on the single respiratory viral infection in a pediatric hematology-oncology unit; however, the analysis of respiratory viral infections in children with cancer is lacking. The present study aimed to provide analysis of multiple respiratory viral infections and clinical outcome in children with cancer who receive chemotherapy and show signs and symptoms of respiratory tract infections. During January, 2014 and January, 2015 children with cancer under treatment who presented with respiratory tract infections were assessed for viruses by using multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR). Specimens were collected by nasal swabbing at in-patient and out-patient clinics. Overall, 72 samples of respiratory tract infection episodes, collected from children with cancer were evaluated with the simultaneous detection of 20 respiratory viruses. A respiratory viral pathogen was obtained in 56.9% samples. Rhinovirus (24.3%) and co-infection with two viruses (19.5%) were the most frequently isolated pathogens. There were four (9.6%) samples of severe pneumonia. Patients with febrile neutropenic episodes and pneumonia were hospitalized and treated with broad-spectrum antibiotics. Other non-neutropenic and mild respiratory tract infections were treated with supportive care as outpatient procedures. There were no deaths. Because there are no effective antiviral agents for certain respiratory viruses, infection control and early diagnosis are crucial in preventing the spread of infection. Clinical findings and serological results of viral respiratory tract infections help us to accurately determine the treatment approach and avoid the unnecessary use of antibiotics.

Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR.

A one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan probes was developed for the simultaneous detection of Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) in total RNA of pome trees extracted with a CTAB method. The sensitivity of the method was established using in vitro synthesized viral transcripts serially diluted in RNA from healthy, virus-tested (negative) pome trees. The three viruses were simultaneously detected up to a 10−4 dilution of total RNA from a naturally triple-infected apple tree prepared in total RNA of healthy apple tissue. The newly developed RT-qPCR assay was at least one hundred times more sensitive than conventional single RT-PCRs. The assay was validated with 36 field samples for which nine triple and 11 double infections were detected. All viruses were detected simultaneously in composite samples at least up to the ratio of 1:150 triple-infected to healthy pear tissue, suggesting the assay has the capacity to examine rapidly a large number of samples in pome tree certification programs and surveys for virus presence.

Multiplex real-time reverse transcription polymerase chain reaction for differential detection of H5, N1, and N8 genes of highly pathogenic avian influenza viruses

Rapid and differential diagnosis of highly pathogenic avian influenza virus (HPAIV) subtype H5 is essential for the effective prevention and control of outbreaks caused by this pathogen. In this study, we describe a one-step multiplex real-time reverse transcription polymerase chain reaction (mRRT-PCR), using H5-, N1-, and N8-specific primers and probes, for differential detection of two HPAIVs (H5N1 and H5N8) and other H5-subtype AIVs. Using the mRRT-PCR assay, we were able to detect H5N1, H5N8, and other H5-subtype AIVs in a one-tube reaction, with high specificity; furthermore, using an in silico PCR program, we confirmed that this assay can detect nearly all H5, N1, and N8 genes of AIVs currently available in the Influenza Sequence Database. The limit of detection of the assay was determined to be as low as 100 copies/reaction for each target gene, and was comparable to limits of detection of previously reported mRRT-PCR assays. Thus, the mRRT-PCR assay described here can serve as a rapid and reliable differential diagnostic tool for the monitoring and surveillance of H5N1, H5N8, and other H5-subtype AIVs in countries where these pathogens are problematic.

Viruses causing severe acute respiratory infections (SARI) in children ≤5 years of age at a tertiary care hospital in Rajasthan, India.

Background & objectives:Severe acute respiratory infection (SARI) is one of the leading causes of death among children worldwide. As different respiratory viruses exhibit similar symptoms, simultaneous detection of these viruses in a single reaction mixture can save time and cost. The present study was done in a tertiary care children's hospital for rapid identification of viruses causing SARI among children less than or equal to five years of age using multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) kit.Methods:A total of 155 throat swabs were collected from equal number of children suspected to have SARI and processed for extraction of nucleic acids using automated extraction system. Multiplex real-time RT-PCR was done to identify the viruses in the samples.Results:The overall positivity for viruses in the study was found to be 72.9 per cent with a co-infection rate of 19.5 per cent. Human metapneumovirus (HMPV) was the predominant virus detected in 25.7 per cent children followed by influenza A (H1N1)pdm09, human rhinovirus (HRV) and human adenovirus (HAdV) in 19.9, 11.0 and 8.8 per cent children, respectively. The HMPV was at its peak in February 2013, HAdV showed two peaks in March-April, 2012 and November 2012-March 2013 while HRV was detected throughout the year.Interpretation & conclusions:Multiplex real-time PCR helped in rapid identification of viruses. Seventeen viruses were detected in SARI cases with overall positivity of 72.9 per cent. HMPV was the most predominant virus. However, for better clinico-virological correlation, studies are required with complete work up of all the aetiological agents, clinical profile of patients and treatment outcome.

Molecular epidemiology of enteric viruses and genotyping of rotavirus A, adenovirus and astrovirus among children under 5 years old in Gabon

This study aimed to determine the prevalence of enteric viruses causing gastroenteritis, and the circulating stains, in Gabonese children under five years old who visited health centers between March 2010 and June 2011. Stool specimens were collected and sent for analysis to CIRMF (Centre International de Recherches Médicales de Franceville). Stools were screened for six enteric viruses (rotavirus, adenovirus, norovirus I and II, sapovirus, human astrovirus) by means of a multiplex real-time reverse transcription polymerase chain reaction, and Rotavirus A, Adenovirus and Astrovirus were genotyped. Among the 317 specimens analyzed, 193 (60.9%) were positive for at least one enteric virus. Rotavirus A (RVA) (27.1%) was the most frequently detected virus, followed by human Adenovirus (HAdV) (19.6%), Norovirus II (NoVs-II) (13.9%), Norovirus I (NoVs-I) (9.1%), Sapovirus (SaV) (9.5%) and human Astrovirus (HAstV) (6.3%). One-third of the 193 positive samples contained more than one virus. The most common Rotavirus A genotype was G6P[6]. Various HAdV serotypes were found. HAstV-1 was identified. These findings improve our knowledge of circulating enteric viruses in Gabon. The emergence of unusual G6P[6] strain of rotavirus A, predominant, suggested a particular epidemiological surveillance of circulating rotavirus strains during the introduction of vaccination in Gabon.

Impacto del virus de la gripe A (H1N1) 2009 en un servicio de urgencias de un hospital de tercer nivel

Background The 2009 H1N1 influenza A virus infection pandemic was an important challenge for public health systems worldwide. Material and methods A prospective study including all patients with an influenza-like illness, with microbiological criteria for 2009 H1N1 virus, from July to December 2009 seen in the paediatric emergency department. Viral testing was performed using multiplex real-time reverse transcription polymerase chain reaction (RT-PCR). We analysed the number of visits to, and hospital admissions from, our emergency department. We compared patients with laboratory-confirmed 2009 H1N1 virus with non-confirmed ones. Results A total of 1144 patients with microbiological criteria were identified. Of these, 513 (44.8%) were admitted to hospital, 12 of them (1%) to the PICU and 3 (0.3%) died. The majority of the patients (824; 72%) had some underlying medical condition. Of the 1144 patients, 412 (36%) had RT-PCR confirmed 2009 H1N1 infection, and 732 (64%) were not confirmed. The mean age of patients with 2009 H1N1 infection was higher than those not infected (median age: 7.4 vs 4.1 years; p < .001). Laboratory-confirmed 2009 pandemic H1N1 influenza patients had more underlying high-risk conditions (OR: 2.21 [1.65-2.96]), suffered from pneumonia in less cases (OR: 0.33 [0.23-0.49]) and were admitted to hospital in less cases (OR: 0.19 [0.14-0.24]). In our emergency department, we identified an important increase in the number of visits (12%) and admissions (5.7%) compared to the previous year, 2008. Conclusions The 2009 pandemic H1N1 influenza caused significant morbidity but the mortality was not significant. The majority of children with laboratory-confirmed 2009 H1N1 virus had uncomplicated illnesses despite the increased presence of high-risk conditions.

Simultaneous detection of influenza virus type B and influenza A virus subtypes H1N1, H3N2, and H5N1 using multiplex real-time RT-PCR

Use of multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) for the simultaneous detection of influenza type B virus and influenza A virus subtypes H5N1, H3N2, and H1N1 has been described. The method exhibited a high specificity and sensitivity of approximately 10(1)-10(2) copies per microliter or 10(-3)-10(-2) TCID50/L for each subtype, as well as a high reproducibility with coefficient of variation (CV) ranging from 0.27% to 4.20%. The assays can be performed commendably on various models of real-time PCR instruments; including ABI7500, ROCH 2.0, and Mx3005p. In an analysis of 436 clinical samples from patients during the year 2009, this detection method has successfully identified 261 positive samples, as compared to only 189 positive samples using the conventional cell culture systems, and at the same time further differentiated them as 35 type B, 21 subtype H1N1, and 205 subtype H3N2. The results indicate that the multiplex real-time RT-PCR method is a potential tool for rapid screening of influenza virus from a large pool of clinical samples during flu pandemics and facilitates early influenza virus identification in most public health laboratories around the world.

Norovirus gastroenteritis in young children receiving human rotavirus vaccine

We explored whether human rotavirus vaccine had any efficacy against norovirus (NV)-associated gastroenteritis in young children. In an efficacy trial of rotavirus vaccine, 405 infants were immunized with a human rotavirus vaccine or placebo at a ratio of 2:1, and prospectively followed for acute gastroenteritis (AGE) from approximately 2 months to 2 y of age. Multiplex real-time reverse transcription polymerase chain reaction (Mrt RT-PCR) assays were used for detection and quantitation of NVs of genogroup I (GI) and genogroup II (GII) in stool specimens. NVs were detected in 155 (32%) of 485 episodes of AGE. Of these, NV was the only gastroenteritis virus detected in the stools in 142 (29%) episodes. GI and GII NVs were found in 12% and 88% of the cases, respectively. NV as the only gastroenteritis virus was detected in 36% of the infants in the rotavirus vaccine group and 27% in the placebo group. The clinical severity of NV-associated AGE in the vaccine and placebo recipients was not different. NVs were the most common etiologic agents of AGE in children under 2 y of age. Human rotavirus vaccine did not protect against NV gastroenteritis.

Isolation and characterization of avian metapneumovirus from chickens in Korea

Avian metapneumovirus (aMPV) causes upper respiratory tract infections in chickens and turkeys. Although the swollen head syndrome (SHS) associated with aMPV in chickens has been reported in Korea since 1992, this is the study isolating aMPV from chickens in this country. We examined 780 oropharyngeal swab or nasal turbinate samples collected from 130 chicken flocks to investigate the prevalence of aMPV and to isolate aMPV from chickens from 2004-2008. Twelve aMPV subtype A and 13 subtype B strains were detected from clinical samples by the aMPV subtype A and B multiplex real-time reverse transcription polymerase chain reaction (RRT-PCR). Partial sequence analysis of the G glycoprotein gene confirmed that the detected aMPVs belonged to subtypes A and B. Two aMPVs subtype A out of the 25 detected aMPVs were isolated by Vero cell passage. In animal experiments with an aMPV isolate, viral RNA was detected in nasal discharge, although no clinical signs of SHS were observed in chickens. In contrast to chickens, turkeys showed severe nasal discharge and a relatively higher titer of viral excretion than chickens. Here, we reveal the co-circulation of aMPV subtypes A and B, and isolate aMPVs from chicken flocks in Korea.

A low density oligonucleotide microarray for the detection of viral and atypical bacterial respiratory pathogens

Acute respiratory tract infections are a major cause of morbidity and mortality worldwide and exert a considerable economic burden on healthcare systems. Acute respiratory tract infections of the upper and lower respiratory tract are caused by a wide variety of viral and bacterial pathogens, which require comprehensive laboratory investigations. Conventional serological and immunofluorescence-based diagnostic methods for acute respiratory tract infections lack sensitivity when compared to polymerase chain reaction (PCR)-based approaches and the development of new diagnostic methodologies is required, to provide accurate, sensitive and rapid diagnoses. In the present study, a PCR-based low density oligonucleotide microarray was developed for the detection of 16 viral and two atypical bacterial pathogens. The performance of this DNA microarray-based analysis exhibited comparable sensitivities and specificities to multiplex real-time reverse transcription polymerase chain reactions (rtPCRs) confirming the potential diagnostic utility of the method. In contrast to routine multiplex PCR, the microarray incorporates an intrinsic redundancy as multiple and non-identical probes per target on the array allow direct intra-assay confirmation of positives. This study demonstrates that microarray technology provides a viable alternative to conventional serological-based approaches and multiplex PCR for pathogen identification in acute respiratory tract infections.

Immunomodulatory Effect of Cytosine-Phosphate-Guanosine (CPG)-Oligonucleotides in Nonasthmatic Chronic Rhinosinusitis: An Explant Model

The use of cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODNs) or immunostimulatory sequences (ISSs) in the treatment of airway diseases is gaining interest. Binding of the CpG-ODN ligand to Toll-like receptor 9 (TLR9) triggers a shift from a Th2- to a Th1-type response in the target tissue. In this study, we explored the potential use of CpG-ODN to dampen the predominantly Th2-driven chronic inflammatory state in our cohort of patients. An in vitro explant model comprising of sinonasal tissue from patients with asthma (n = 12) and without asthma (n = 11) were stimulated with CpG-ODN or Staphylococcus aureus enterotoxin B (SEB) or CpG-ODN in combination with SEB for 48 hours. Ten of the 12 asthma patients had nasal polyps. RNA was extracted for multiplex real-time reverse transcription polymerase chain reaction analysis and the 2(-delta deltaC(T)) method used to determine interleukin (IL)-5, p35 IL-12, interferon (IFN) gamma, and TLR9 expression levels. CpG-ODN significantly reduced IL-5 mRNA expression in patients without asthma (p = 0.0379) but not in the asthma-associated group. SEB alone caused an increase in IL-5 levels that could be dampened when CpG-ODN was added in combination with SEB. Significant differences in mean IL-5 expression levels between the asthmatic and nonasthmatic categories were detected (Welch t-test; **p = 0.0041). Asthmatic and nonasthmatic patients present as two distinct categories as reflected by significant differences in their IL-5 response to CpG-ODN (F = 11.93; ***p = 0.0008), SEB (F = 41.34; *p = 0.0476) and CpG-ODN with SEB (F = 13.2; *p = 0.0114). In contrast, no significant differences were observed in the expression levels of IL-12, IFN-gamma, and TLR9. Localized application of CpG-ODN on its own or in combination with SEB may potentially reduce the expression of the proinflammatory cytokine IL-5 in nonasthmatic patients and may be further developed as an immunotherapeutic agent.

Multiplex Real-Time Reverse Transcription–Polymerase Chain Reaction for the Detection of Three Viruses Associated with Poult Enteritis Complex: Turkey Astrovirus, Turkey Coronavirus, and Turkey Reovirus

Poult enteritis complex (PEC) is an economically important disease of young turkeys characterized by diarrhea, poor weight gain, and, in some cases, high mortality. Although PEC is considered to be a polymicrobial disease, numerous viruses, including turkey coronavirus (TCV), turkey astrovirus type 2 (TAstV-2), and avian reoviruses (ARVs), have been associated with PEC-like disease. Real-time reverse transcription-polymerase chain reaction (RRT-PCR), a highly sensitive and specific detection method for viral RNA, was developed in a multiplex format for the simultaneous detection of TAstV-2 and TCV and for the detection of two genetic types of ARV. Assay sensitivity was determined using in vitro transcribed RNA and varied by target between 150 gene copies for TAstV-2 alone and 2200 gene copies for TCV when multiplexed. Virus detection was evaluated with samples collected from poults inoculated at 1 day of age with each of the viruses. Cloacal swabs and intestinal samples were obtained at 1, 2, 3, 4, 6, 9, 14, 17, and 21 days after inoculation, processed, and tested for virus detection by RRT-PCR Cloacal swabs from TAstV-2- and TCV-infected poults were shown to have sensitivity for virus detection similar to that of intestinal samples when compared directly. ARV detection by RRT-PCR was compared with virus isolation and had similar sensitivity.