Detection Of Multiple Viruses Research Articles

Optimization of Amplicon-Based Sequencing for Large-Scale Diagnostics of Known Pome Viruses and Viroids

The implementation of reliable methods for the simultaneous detection of multiple viruses is challenging in plant viral diagnosis. In the present study, we report the development and effectiveness of an optimized version of a robust multiplexed PCR (HiPlex) followed by simultaneous targeted amplicon sequencing to detect 14 viruses and 5 viroids of pome fruit trees. We used amplicon sequencing data from 1,400 pome fruit tree samples previously diagnosed with the HiPlex v.1.0, which included 290 primer pairs for pome viruses. The analysis enabled the identification of highly efficient and specific primer pairs to be assembled in a new HiPlex (dubbed HiPlex v.2.0). Additionally, newly designed primers were implemented for 30% of targeted viruses and viroids. Overall, 116 primer pairs were assembled into the HiPlex v.2.0, resulting in a reduction of the number of primers and multiplex PCR reactions needed by 60 and 50%, respectively. The new HiPlex also includes two nepoviruses, tomato ringspot virus and tobacco ringspot virus, showing flexibility at augmenting and expanding the number of targeted diagnosed viruses using this approach. This optimized set of primers increased the cost-effectiveness and sensitivity while reducing background amplification. Comparable results for virus and viroid detection were obtained using the HiPlex v.2.0 and individual RT-qPCRs, even at a 10,000-fold dilution, demonstrating similar sensitivity to standard PCR-based diagnostics. This new HiPlex-based amplicon sequencing represents a powerful, sensitive, and low-cost tool for large-scale screening of pome fruit trees for all reported viruses and viroids infecting these crops. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Simultaneous Detection of Grapevine Viruses via Multiplex Reverse Transcription Polymerase Chain Reaction: An Academic Approach

Grapevine viruses pose a significant threat to the viticulture industry, resulting in reduced grape yield, poor fruit quality, and economic loss. Accurate and timely detection of multiple viruses are essential for effective disease management and the implementation of appropriate control measures. The aim of this study was to assess the presence of grapevine fanleaf virus (GFLV), grapevine fleck virus (GFkV), grapevine leafroll-associated virus‑1 (GLRaV-1), arabis mosaic virus (ArMV), and grapevine virus A (GVA) in 320 plant samples collected from the Aegean region of Turkey using the powerful technique of multiplex reverse transcription-polymerase chain reaction (RT-PCR). Through comprehensive analysis, 119 of the 320 infected samples were identified using PCR. The distribution of the detected viruses revealed the presence of 28 GFLV, 31 GLRaV‑1, 12 GFkV, 21 GVA, and 27 ArMV. By comprehensively analyzing the samples, the researchers were able to simultaneously detect and identify the target viruses using a single, cost-effective assay. These results reveal the prevalence and distribution of these viruses in the Aegean region, providing valuable insights into the viral landscape of grapevines in this important viticultural area. The high detection rates of these viruses highlight the urgent need for improved disease management strategies to safeguard vine health and productivity.

A novel surface-enhanced Raman based molecular identification platform for multiplexed and highly accurate clinical diagnosis of viral diseases.

During the infection process, the interactions among respiratory viruses impact the dynamics of transmission and clinical outcomes. Therefore, efficient molecular detection methods provide a basis for rational drug use and effective health management. Surface-enhanced Raman scattering (SERS) is an ultra-sensitive spectroscopic technique capable of generating extremely narrow spectra (∼1-2 cm-1), enabling simultaneous detection of multiple targets. By judiciously designing plasmonic nanostructures as SERS substrates, Raman signals can be amplified by several orders of magnitude (∼105-1015), facilitating the detection of trace biomolecules. In this highlight, we highlight the work about a novel SERS platform for the high-precision multi-virus molecular identification. This may offer a highly sensitive, specific, and accurate method for the detection of multiple viruses.

A Rapid and Novel Multiplex PCR Assay for Simultaneous Detection of Multiple Viruses Associated with Bovine Gastroenteritis.

Bovine viral diarrheal virus (BVDV) and bovine coronavirus (BCoV) are prevalent viral infections in buffalo calves that result in significant economic losses globally. However, Bovine picobirnavirus (BPBV) Group I and II has been an emerging causes of gastrointestinal infection as has been detected with mixed of BVDV as well as BCV. To combat economic losses and viral infection, a rapid and innovative multiplex-PCR assay (M-PCR) was developed to simultaneously identify BVDV, BCV, and BPBV. The assay employed three primer pairs, each specific to a particular virus. Notably, the primers for BCV and BVDV, targeting the transmembrane (M) Mebus gene and 5'UTR genes, respectively, were self-designed. To validate the assay, 300 samples of buffalo calf feces were subjected to the standardized multiplex PCR. The results demonstrated that 54 (18%) samples tested positive for multiple viruses, with 16.67% samples infected by BVDV, 0.9% by BCoV, and 0.13% by BPBV, as detected by the M-PCR assay. In summary, this developed assay is characterized by high specificity, sensitivity, throughput, and speed, enabling the simultaneous detection of the three viruses in a single reaction tube. Consequently, it holds potential for epidemiological investigations. It is worth noting that, to the best of our knowledge, this is the first reported multiplex assay for the worldwide detection of BVDV, BCoV, and BPBV. This novel assay promises to aid in the detection of mixed infections in the gastrointestinal tract.

Implementing a multiplex-PCR test for the diagnosis of acute gastroenteritis in hospitalized children: Are all enteric viruses the same?

BackgroundMultiplex-PCR is a valuable tool for diagnosing viral acute gastroenteritis (AGE), enabling the detection of multiple pathogens. However, distinguishing between active disease and shedding poses challenges. This study aimed to evaluate viral AGE epidemiology and compare clinical characteristics among the five most common viruses. MethodsRotavirus vaccine was introduced in 2010, with 70% coverage achieved in southern Israel in two years. All rectal swabs for multiplex-PCR targeting rotavirus, norovirus, adenovirus, astrovirus and sapovirus from hospitalized diarrheic children <5 years were included, from December 2017 through March 2022. Detection of the same virus within two months was considered a single episode. Clinical analysis included episodes with single-virus detection and negative bacterial PCR. ResultsAmong 5,879 rectal swabs, 2,662 (45.3%) tested positive for at least one virus, with 245 (9.2%) showing multiple virus detection. Rotavirus was the most prevalent. While rotavirus exhibited typical winter-spring seasonality in 2018–19, an unusual off-season surge was observed during the second year of the COVID-19 pandemic. Among negative bacterial PCR episodes, 34.6% had mucus stool, 5.9% had bloody stool, and 29.3% received antibiotics. Astrovirus or sapovirus infections were associated with higher rates of hospital-acquired AGE and immunodeficiency (P<0.05), whereas rotavirus infections had higher rates of dehydration severity and acute kidney injury (P<0.05). DiscussionEnteric viruses were detected in 45.3% of rectal swabs from hospitalized children with diarrhea. Despite vaccination efforts, rotavirus remained prevalent and caused more severe disease. Continuous surveillance using multiplex-PCR is crucial for accurate management and future prevention strategies for viral AGE.

Exploring viral infections in honey bee colonies: insights from a metagenomic study in southern Brazil.

The decline in honey bee colonies in different parts of the world in recent years is due to different reasons, such as agricultural practices, climate changes, the use of chemical insecticides, and pests and diseases. Viral infections are one of the main causes leading to honey bee population declines, which have a major economic impact due to honey production and pollination. To investigate the presence of viruses in bees in southern Brazil, we used a metagenomic approach to sequence adults' samples of concentrated extracts from Apis mellifera collected in fifteen apiaries of six municipalities in the Rio Grande do Sul state, Brazil, between 2016 and 2017. High-throughput sequencing (HTS) of these samples resulted in the identification of eight previously known viruses (Apis rhabdovirus 1 (ARV-1), Acute bee paralysis virus (ABPV), Aphid lethal paralysis virus (ALPV), Black queen cell virus (BQCV), Bee Macula-like virus (BeeMLV), Deformed wing virus (DWV), Lake Sinai Virus NE (LSV), and Varroa destructor virus 3 (VDV-3)) and a thogotovirus isolate. This thogotovirus shares high amino acid identities in five of the six segments with Varroa orthomyxovirus 1, VOV-1 (98.36 to 99.34% identity). In contrast, segment 4, which codes for the main glycoprotein (GP), has no identity with VOV-1, as observed for the other segments, but shares an amino acid identity of 34-38% with other glycoproteins of viruses from the Orthomyxoviridae family. In addition, the putative thogotovirus GP also shows amino acid identities ranging from 33 to 41% with the major glycoprotein (GP64) of insect viruses of the Baculoviridae family. To our knowledge, this is the second report of a thogotovirus found in bees and given this information, this thogotovirus isolate was tentatively named Apis thogotovirus 1 (ATHOV-1). The detection of multiple viruses in bees is important to better understand the complex interactions between viruses and their hosts. By understanding these interactions, better strategies for managing viral infections in bees and protecting their populations can be developed.

Detection of multiple viruses and viroid in apple trees in Brazil and their possible association with decline

In an attempt to provide additional information on the virome in apples in Southern Brazil in face of recurrent events of tree decline, plant samples of regional apple orchards have been submitted to HTS (high-throughput sequencing) analysis. Besides common latent viruses, results showed additionally the presence of apple rubbery wood virus 2 (ARWV 2) and citrus concave gum-associated virus (CCGaV) in orchards with decline history.

High-throughput detection of a large set of viruses and viroids of pome and stone fruit trees by multiplex PCR-based amplicon sequencing.

A comprehensive diagnostic method of known plant viruses and viroids is necessary to provide an accurate phytosanitary status of fruit trees. However, most widely used detection methods have a small limit on either the number of targeted viruses/viroids or the number of samples to be evaluated at a time, hampering the ability to rapidly scale up the test capacity. Here we report that by combining the power of high multiplexing PCR (499 primer pairs) of small amplicons (120-135bp), targeting 27 viruses and 7 viroids of fruit trees, followed by a single high-throughput sequencing (HTS) run, we accurately diagnosed the viruses and viroids on as many as 123 pome and stone fruit tree samples. We compared the accuracy, sensitivity, and reproducibility of this approach and contrast it with other detection methods including HTS of total RNA (RNA-Seq) and individual RT-qPCR for every fruit tree virus or viroid under the study. We argue that this robust and high-throughput cost-effective diagnostic tool will enhance the viral/viroid knowledge of fruit trees while increasing the capacity for large scale diagnostics. This approach can also be adopted for the detection of multiple viruses and viroids in other crops.

#19 Viral DNAemia and Viral Seropositivity are Associated with Mortality in Pediatric Patients with Severe Sepsis

Abstract Background Sepsis remains the leading cause of pediatric mortality worldwide. While sepsis recognition campaigns have improved early mortality, current contributors to mortality are unclear. Little attention has been paid to the incidence of latent viral infection in septic pediatric patients, but resultant immune remodeling may affect future host response to acute illness. We report a large multi-center study of viral DNAemia and viral seropositivity in pediatric patients with severe sepsis. Method 401 patients from 9 Pediatric Intensive Care Units (PICUs) in the Eunice Kennedy Shriver National Institutes of Child Health and Human Development Collaborative Pediatric Critical Care Research Network were enrolled. Enrollment criteria included severe sepsis, indwelling central venous or arterial catheter, and age between 44 weeks gestation and 18 years. Blood samples were collected twice per week up to 28 days of admission or until death or PICU discharge. Viral DNAemia was defined by qPCR detection of EBV, CMV, HSV, adenovirus, TTV, HHV6, and/or parvovirus B19. CMV, EBV, HSV and HHV6 serologies (IgG) were performed on one sample from each subject. Infection status was defined as uninfected (serologically negative without detectable DNAemia), acute infection (serologically negative with DNAemia), reactivated infection (serologically positive with DNAemia), or latent infection (serologically positive without DNAemia). Results Of 401 pediatric patients with severe sepsis, 55% were male, 39% previously healthy, and 27% immunocompromised. 44 subjects (11%) died in the PICU. 56% of subjects had documented infection(s) on enrollment (63% bacterial, 50% viral, and 2% fungal). Viral DNAemia, excluding TTV, was detected in 57% of immunocompromised patients and 44% of non-immunocompromised patients. Among patients with viral DNAemia, detection was due to reactivation in 91% of subjects with EBV DNAemia, 63% of those with CMV, and 100% of subjects with HSV and HHV-6. After adjusting for age, PRISM, previously healthy and immunosuppressed status, higher mortality was observed in patients with viral DNAemia due to CMV (OR 3.29 [1.46,7.21], p=0.005), adenovirus (3.55 [1.45,8.38] p=0.006), and HHV-6 (2.29 [1.11,4.63] p=0.025). DNAemia with multiple viruses, excluding TTV, during ICU admission was also independently associated with increased mortality in both immunocompromised (OR 5.51 [1.61,22.6] p=0.001) and non-immunocompromised subjects (OR 3.93 [1.36,11.5] p=0.015). Seropositivity was common (HSV 33%, CMV 42%, EBV 61%, HHV6 98%). Mortality was significantly higher in subjects who were seropositive for EBV (OR 11.5 [2.24, 211.2], p&amp;lt;0.001) and HSV (OR 2.65, [1.03, 7.21], p=0.004). Mortality was increased if more than one virus antibody was detected (OR 6.89 [1.98, 43.5], p&amp;lt;0.001) and highest when all four virus antibodies were detected. With HHV6, DNAemia increased mortality associated with seropositivity (18% vs. 7%, p=0.024). Conclusion In this study, viral DNAemia was common in pediatric patients with severe sepsis and was associated with mortality. Detection of multiple viruses during ICU admission increased risk of mortality, even in patients without preexisting immunosuppression. CMV and adenovirus reactivation were most strongly associated with mortality. Viral seropositivity was also strongly associated with ICU mortality independent of the presence of viral DNAemia. Future analyses will investigate mechanisms by which viral DNAemia and seropositivity contribute to pediatric sepsis mortality.

Changes in virus detection in hospitalized children before and after the severe acute respiratory syndrome coronavirus 2 pandemic.

The impact of strengthening preventive measures against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection on the prevalence of respiratory viruses in children was examined. After the SARS‐CoV‐2 pandemic, the rate of multiple virus detection among hospitalized children decreased. Immediately after the SARS‐CoV‐2 pandemic, respiratory syncytial and parainfluenza viruses were rarely detected and subsequently reemerged. Human metapneumovirus and influenza virus were not consistently detected. Non‐enveloped viruses (bocavirus, rhinovirus, and adenovirus) were detected to some extent even after the pandemic. Epidemic‐suppressed infectious diseases may reemerge as susceptibility accumulates in the population and should continue to be monitored.

Development of a simple one-step multiplex RT-PCR system for simultaneous detection of DNA and RNA viruses of cucurbit leaf crumple virus, cucurbit yellow stunting disorder virus, squash vein yellowing virus, and cucurbit chlorotic yellows virus

A highly sensitive one-step multiplex RT-PCR (OMRT-PCR) system with an internal plant gene control was developed to simultaneously detect single and mixed RNA and DNA viral pathogens affecting watermelon and squash in the Southeastern United States. Current methods rely on a singleplex detection system for several viruses which is time-consuming for large-scale field monitoring. The gene-specific primers for one DNA virus, cucurbit leaf crumple virus (CuLCrV), and three RNA viruses, cucurbit yellow stunting disorder virus (CYSDV), squash vein yellowing virus (SqVYV), and cucurbit chlorotic yellows virus (CCYV), were used for the development of OMRT-PCR. A plant gene, rubisco from cucurbits was used as an internal control in the standardization of the assay and to monitor false amplification. In addition, we examined the use of total nucleic acid as a template in OMRT-PCR for simultaneously detection of mixed viral infection. The OMRT-PCR demonstrated high sensitivity and detected all four virus infections from 1 ng of total plant nucleic acid in a single reaction. The multiple virus detection approach was successful in the diagnosis of 16 watermelon and 18 squash samples collected from commercial fields in Florida. The OMRT-PCR system is specific, rapid, sensitive, and cost-efficient for the detection of multiple viruses. This technique is expected to improve the ease in the detection of CuLCrV, CYSDV, SqVYV and CCYV and assist in the monitoring of the viral pathogens in commercial cucurbit production fields.

Simultaneous detection of 10 viruses in globe artichoke by a synthetic oligonucleotide-based DNA polyprobe

Most of the 26 viruses that infect globe artichoke are detrimental to the crop’s production, quality, and nutritional value, hamper the development of a nursery activity in the respect of current EU legislation and pose risks for the new plantings. Worldwide movement of virus-controlled plant propagative material require fast, accurate and feasible methods to guarantee reliable results and detection of multiple viruses in a single assay to reduce time and costs in routine tests. Here we describe the use of a digoxigenin-labelled polyprobe assembled from synthetic 50 nt single-stranded DNA sequences derived from genomes of the most commonly occurring viruses in globe artichoke. These included artichoke Italian latent virus (AILV), artichoke latent virus (ArLV), artichoke mottled crinkle virus (AMCV), turnip mosaic virus (TuMV), tomato infectious chlorosis virus (TICV), bean yellow mosaic virus (BYMV), cucumber mosaic virus (CMV), pelargonium zonate spot virus (PZSV), tomato spotted wilt virus (TSWV) and tobacco mosaic virus (TMV). The polyprobe described herein represent the first cheap and reliable example of a technology for broad-spectrum surveys in globe artichoke.

Simultaneous detection of four lily-infecting viruses by a multiplex RT-PCR assay

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) system was modified and evaluated for the simultaneous detection of multiple viruses in coinfected lily plants. Four major lily viruses, namely, lily symptomless, cucumber mosaic, lily mottle, and plantago asiatica mosaic viruses, were targeted by simultaneous detection. Analysis of PCR products confirmed the amplification of virus-specific DNA fragments of the expected sizes and nucleotide sequences from the target viruses. The cDNAs from singly infected plant samples were used as positive controls, and multiple infected samples were used to develop the multiplex RT-PCR system, which was then applied to leaf samples from diverse lily species and hybrids. The modified multiplex RT-PCR was successfully validated on the simultaneous detection of these viruses.

Detection of multiple viruses in oropharyngeal samples from Brazilian free-tailed bats (Tadarida brasiliensis) using viral metagenomics.

In this study, we analyzed the viral population in oropharyngeal samples from T. brasiliensis using a viral metagenomic approach. Genomes corresponding to members of the families Circoviridae, Genomoviridae, Herpesviridae, Paramyxoviridae, Coronaviridae, and Astroviridae were detected. This study provides the first preliminary understanding of the oropharyngeal virome of T. brasiliensis, which may guide the discovery and isolation of novel viruses in the future and highlights the need for continuing investigations in this regard.Graphic abstract Electronic supplementary materialThe online version of this article (10.1007/s00705-020-04825-x) contains supplementary material, which is available to authorized users.

Digital Pathology Platform for Respiratory Tract Infection Diagnosis via Multiplex Single-Particle Detections.

The variability of bioparticles remains a key barrier to realizing the competent potential of nanoscale detection into a digital diagnosis of an extraneous object that causes an infectious disease. Here, we report label-free virus identification based on machine-learning classification. Single virus particles were detected using nanopores, and resistive-pulse waveforms were analyzed multilaterally using artificial intelligence. In the discrimination, over 99% accuracy for five different virus species was demonstrated. This advance is accessed through the classification of virus-derived ionic current signal patterns reflecting their intrinsic physical properties in a high-dimensional feature space. Moreover, consideration of viral similarity based on the accuracies indicates the contributing factors in the recognitions. The present findings offer the prospect of a novel surveillance system applicable to detection of multiple viruses including new strains.

Detection of multiple viruses potentially infecting humans in sewage water from Xinjiang Uygur Autonomous Region, China

The progress of sequencing technologies has facilitated metagenomics projects on environmental samples like sewage water. The present study concerned the analysis of sewage samples collected from 3 locations in Xinjiang Uygur Autonomous Region in China. The analysis focused on RNA viruses known to infect humans and identified viruses from 10 families. The proportion of human virus species in the sewage samples was relatively stable with an average of 17%. Thirty virus species known to infect humans were identified and they belonged to 6 families: Picornaviridae (12), Astroviridae (11), Reoviridae (3), Caliciviridae (2), Papillomaviridae (1) and Picobirnaviridae (1). A total of 16 full-length genomes were generated from Astroviridae, Picornaviridae (Salivirus and Kobuvirus) and Picobirnaviridae. Astroviruses appeared to be the most present viruses and were detected in all sewage samples. Analyzing the virome of sewage samples should help to monitor any potential risks to public health.

The Role of Viral Coinfection in Bronchiolitis Treated With High-Flow Nasal Cannula at Pediatric Emergency Department During 2 Consecutive Seasons: An Observational Study.

The role of multiple respiratory viruses in bronchiolitis treated with high-flow nasal cannula (HFNC) has not been thoroughly investigated. We evaluated the contribution of coinfection on clinical course of bronchiolitis treated with HFNC and on response to this treatment. We selected 120 children with bronchiolitis, younger than 12 months, admitted to Emergency Department between 2016 and 2018 and treated with HFNC. We compared single and multiple virus infections in relation to specific outcomes such as the clinical response to HFNC and the HFNC failure. The multiple virus infection was defined by the detection of 2 or more viruses in nasopharyngeal aspirates. The HFNC failure was defined as escalation to higher level of care, including Helmet-Continuous Positive Airway Pressure, invasive ventilation or transfer to pediatric intensive care unit within 48 hours from the time of HFNC initiation. We also performed a comparison between HFNC failure and HFNC not-failure groups according to the number of virus and the type of virus. The severity score post-HFNC initiation was significantly associated with coinfection [odds ratio (OR): 1.361; 95% confidence interval (CI): 1.036-1.786; P = 0.027]. The likelihood of coinfection decreased by 23.1% for each increase of saturation O2 after HFNC initiation (OR: 0.769; 95% CI: 0.609-0.972; P = 0.028). Atelectasis was more likely to occur in coinfection (OR: 2.923; 95% CI: 1.049-8.148; P = 0.04). The duration of HFNC treatment increased significantly in coinfection (OR: 1.018; 95% CI: 1.006-1.029; P = 0.002). No significant differences were described between HFNC failure and the number and the type of detected viruses. The detection of multiple viruses and the type of virus did not influence the HFNC failure, although the coinfection was associated with a deterioration of severity score, a longer HFNC treatment and a major presence of atelectasis. The role of coinfection on HFNC treatment might subtend a complex interplay between multiple viruses and host susceptibility.

High Detection Rates of Human Bocavirus in Infants and Small Children with Lower Respiratory Tract Infection from Croatia.

Human bocavirus (HBoV) is known to cause lower respiratory tract infections (LRTI) in children and may result in substantial morbidity and mortality. The aim of this study was to determine HBoV prevalence among hospitalized infants and small children with acute LRTI in Zagreb, Croatia, as well as to evaluate HBoV DNA quantity in samples in relation to the patients' age and co-infection with other respiratory viruses. During winter season 2016/2017, a total of 295 children younger than three years of age who were admitted to hospitals with LRTI were tested for the presence of HBoV, respiratory syncytial virus (RSV), adenovirus (ADV), parainfluenza virus (PIV) types 1 to 3, and human metapneumovirus (HMPV). HBoV was detected with a real-time PCR method, and the other viruses were diagnosed using monoclonal antibodies in direct fluorescence assay. Viral etiology was proven in 225/295 (76.3%) of patients. The most commonly diagnosed virus was RSV (59.3%), followed by HBoV (23.1%), PIVs (4.4%), ADV (3.1%), and HMPV (1.4%). HBoV-infected children were older than RSV-infected children; likewise, detection rates of HBoV infection increased with age, while RSV infection rates decreased with age. In 51% of HBoV-positive samples an additional respiratory virus was also detected. There was no difference in HBoV DNA quantity between samples with single virus detection and those with multiple virus detection (p = 0.056), although samples positive only for HBoV showed higher cycle threshold values. There was no difference in HBoV DNA quantity in samples of different age groups (p > 0.05). Frequent detection of HBoV in small children with LRTI, even in combination with other viruses, highlights its role in the pathogenesis of respiratory disease.

A novel multiplex PCR for virus detection by melting curve analysis

BackgroundRapid and accurate laboratory diagnoses of viral infections are crucial for the management and treatment of patients with viral infections. Conventional methods for virus detection are labourious, time consuming, and only a single virus can be analysed in one assay. ObjectivesThe objective of this study was to develop a novel real-time PCR method for multiple virus detection by melting curve analysis using Taqman probes in a single reaction. Study designAs a model, six respiratory viruses were detected in one tube using three fluorophores. The specificity was assessed by cross-reaction tests with other common respiratory pathogens. The analytical sensitivity was assessed by testing the limit of detection of the assay using artificial plasmids as the positive template. The clinical evaluation of the established assay was evaluated for the detection of respiratory viruses in clinical samples, and the results were compared with direct fluorescent antibody testing (DFA). ResultsThe six respiratory viruses were clearly distinguished by their respective melting temperature values in the corresponding fluorescence detection channels. No cross reactions were observed by cross reaction tests. The detection limits of this assay were 2 to 2 × 103 copies per reaction for each virus. The clinical evaluation of this assay was demonstrated by analysing 352 clinical samples, and 67(19.0%) samples were positive for at least one virus. The accordance rate between the established PCR and DFA testing was high, and ranged from 94.57% to 100%. ConclusionsTaqman probe-based melting curve analysis is well suited for detection of multiple viruses in clinical and research laboratories because of its high throughput, reliability, and cost savings.

Respiratory viruses among children with non-severe community-acquired pneumonia: A prospective cohort study

BackgroundCommunity-acquired pneumonia (CAP) causes a major burden to the health care system among children under-5 years worldwide. Information on respiratory viruses in non-severe CAP cases is scarce. ObjectivesTo estimate the frequency of respiratory viruses among non-severe CAP cases. Study designProspective study conducted in Salvador, Brazil. Out of 820 children aged 2–59 months with non-severe CAP diagnosed by pediatricians (respiratory complaints and radiographic pulmonary infiltrate/consolidation), recruited in a clinical trial (ClinicalTrials.gov Identifier NCT01200706), nasopharyngeal aspirate samples were obtained from 774 (94.4%) patients and tested for 16 respiratory viruses by PCRs. ResultsViruses were detected in 708 (91.5%; 95%CI: 89.3–93.3) cases, out of which 491 (69.4%; 95%CI: 65.9–72.7) harbored multiple viruses. Rhinovirus (46.1%; 95%CI: 42.6–49.6), adenovirus (38.4%; 95%CI: 35.0–41.8), and enterovirus (26.5%; 95%CI: 23.5–29.7) were the most commonly found viruses. The most frequent combination comprised rhinovirus plus adenovirus. No difference was found in the frequency of RSVA (16.1% vs. 14.6%; P = 0.6), RSVB (10.9% vs. 13.2%; P = 0.4) influenza (Flu) A (6.3% vs. 5.1%; P = 0.5), FluB (4.5% vs. 1.8%; P = 0.09), parainfluenza virus (PIV) 1 (5.1% vs. 2.8%; P = 0.2), or PIV4 (7.7% vs. 4.1%; P = 0.08), when children with multiple or sole virus detection were compared. Conversely, rhinovirus, adenovirus, enterovirus, bocavirus, PIV2, PIV3, metapneumovirus, coronavirus OC43, NL63, 229E were significantly more frequent among cases with multiple virus detection. ConclusionsRespiratory viruses were detected in over 90% of the cases, out of which 70% had multiple viruses. Several viruses are more commonly found in multiple virus detection whereas other viruses are similarly found in sole and in multiple virus detection.