This study has been approved by the Health Research Ethics Committee, National Institute of Health Research and Development, Ministry of Health, Indonesia.

The pre-publication history for this paper can be accessed here:

http://www.biomedcentral.com/1471-2334/12/189/prepub

Nucleic acids were extracted from all samples using Microlab Nimbus IVD (Seegene Inc.), and RNAs were used for cDNA synthesis using cDNA Synthesis Premix (Seegene Inc.). The samples were tested by using Anyplex II RV16 detection kit (Seegene Inc.) according to the manufacturer's instructions. The assay was used to detect Flu-A, Flu-B, RSV A, RSVB, AdV, HMPV, HCoV-229E, HCoV-NL63, HCoV-OC43, PIV-1, PIV-2, PIV-3, PIV-4, HRV, HEV, and HBoV. Reaction mixtures for virus detection were divided into two panels: A and B. Each panel was used to detect 8 viruses with appropriate controls. Two types of DNA and 14 types of RNA viruses were amplified and detected by using CFX 96 Real-Time PCR Thermal cycler (Bio-Rad). Seegene Viewer software was used to analyze the amplification results. The study was approved by the Research and Ethical Committee of King Abdul-Aziz Medical City, Riyadh.

The pre-publication history for this paper can be accessed here:

http://www.biomedcentral.com/1471-2334/14/189/prepub

Of the 172 that tested positive for respiratory agents, there were 24 (14.0%) cases with mixed infections of two or three viruses. No patient was infected with more than three viruses. Twenty-one cases were infected with two viruses, with the most frequent mixture being adenovirus and influenza A virus (n = 5). Other frequent viral mixtures were adenovirus and rhinovirus A (n =3) and influenza A virus and coronavirus OC43 (n =3) (Table 3). Adenovirus was present in 62.5% (n = 15) of mixed infections which was almost a half (46.9%) of all detections for this virus. Respiratory syncytial virus B was the only aetiology that was not detected in a mixed infection.

Total viral nucleic acid was extracted from 190 μl of viral transport medium using RiboSpin v_RD GeneAll extraction Kit from Seegene (Seegene Inc., Seoul, South Korea). A 10 μl internal control which is inserted in the package was added to each of the 190 μl samples for internal amplification control to check the PCR process. 40 μl elution buffer was added according to the manufacturer instructions. Seeplex® RV16 ACE Multiplex detection (Seegene Inc., Seoul, South Korea), a multiplex real time PCR platform, which is able to detect 16 viruses including human adenovirus (ADV), influenza A and B viruses (Flu A, Flu B), human parainfluenza viruses 1/2/3/4 (PIV 1/2/3/4), human rhinoviruses A/B/C (RV A/B/C), human respiratory syncytial viruses A and B (RSV A, RSV B), human bocaviruses 1/2/3/4 (BoV1/2/3/4), human coronaviruses 229E, NL63 and OC43 (CoV-229E, CoV-NL63, and CoV-OC43), human metapneumovirus (hMPV), and human enterovirus (EV) was used. The protocol followed manufacturer's instruction as described before.

cDNA synthesis process was performed using cDNA synthesis premix (Seegene Inc., Seoul, South Korea) with 8 μl of RNA, 2 μl random hexamer primer, and 10 μl mix of transcriptase, MgCl2, dNTP, and buffer. The multiplex reaction was performed using Biorad CFX 96 Real Time Thermal Cycler. The reaction mixture was first denatured at 95°C for 15 min, followed by 50 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 60 s, extension at 72°C for 30 s, and a final extension step at 55°C for 30 s. The melting curve temperature from 55°C to 85°C (5 s/0, 5°C) was used to read the amplification. Any positive result was detected as a peak in electropherogram, compared to positive control.

JL and NM performed the operation of GeXP experiment. CZ was responsible for providing the clinical samples and leading the evaluation of GeXP result using RVP method with JL, MY and MW. Dr. WX offered great help in the supplement of different clinical samples as well as shared previous experimental data. Dr. XM designed and coordinated the study, analyzed data and drafted the manuscript with JL. All authors read and approved the final version of this manuscript.

This study reported a high prevalence of respiratory viruses in children ≤ 5 years using a custom assay and an FTD assay. Good concordance was observed for all the viruses between both assays except for RSVA/B. However larger numbers of positive samples need to be tested for thorough evaluation of less prevalent viruses. The custom primer and probe mix was much more economical than the commercial FTD kit. Our study suggests that this custom multiplex real-time RT-PCR can be used for simultaneous and rapid detection of multiple viruses in resource limited settings. This will help prevent unnecessary use of antibiotics and permit timely initiation of supportive therapy/antiviral drugs if available.

In conclusion, nasal application of carrageenan spray in children as well as in adults suffering from virus-confirmed common cold reduced duration of disease, increased viral clearance and reduced relapses. Therefore, carrageenan nasal spray can be regarded as a safe and effective treatment with a high potential for reducing social and economic burden caused by common cold.

The principles of the 4S network were approved by the Ministry of Health in its guidelines for influenza surveillance policy, finalized with the support of Pasteur Institute in Dakar and the Strengthening Influenza Sentinel Surveillance in Africa (SISA) project funded by the WHO. The protocol and oral consent were determined as routine surveillance activity, and therefore non-research by the Senegalese National Ethics committee and the steering committee for 4S network, an entity representing MoH, IPD, WHO and Clinicians in compliance with all applicable National regulations governing the protection of human subjects. Data were collected in an objective of surveillance and are anonymous. The information provided to participants was an informal description of the study. Respiratory specimens were collected, only after informed consent was granted, verbally, to local health care workers by the patients or parents in the case of minors. Oral consent was documented in the patient form with two questions about received information and about oral consent. Patients could refuse to participate, no specimen will be taken. For the surveillance activities, written consent is judged not necessary by the Senegalese national ethics committee, which has also previously approved the work of the National Influenza Center. Collections of non-sensitive data or an observation from normal care in which participants remain anonymous do not require ethics committee review. The patients included in this study were of all ages and consulted the sentinel sites due to influenza-like symptoms; the patients, or parents in the case of minors, accept the tests for respiratory viruses largely because they are free and safe.

From October 2013 to June 2016 pilgrims and people returning from the Middle East presented with respiratory symptoms and having an epidemiological link were enrolled through active screening at the point of entry. After risk assessment by the medical team, suspected patients were admitted to the Kurmitola General Hospital isolation unit. In addition, travelers arriving with no clinical presentation received a health card mentioning the sign and symptoms of MERS-CoV infection. Self reported cases who developed symptoms within 14 days of arrival were included in sample collection along with admitted patients. The health care providers were instructed to use WHO standard Personal Protective Equipment (PPE) including N95 masks during handling of the patients.

Detection of respiratory viruses, including HPIV 1–4, HRSV, HMPV, HCoV-OC43, HCoV-229E, HCoVNL63, HCoV-HKU1, HRV, HAdV, and HBoV was performed using a RT-PCR Kit «AmpliSens ARVI-screen-FL» (Interlabservice, Russia), and IFVA and IFVB virus detection was performed using a RT-PCR Kit « AmpliSens Influenza virus A/B-FL» (Interlabservice, Russia) according to the manufacturer's instructions. Positive and negative controls were included in each run.

The need for informed consent was waived in view of the observational nature of the study with no interventions performed. The protocol and standardized clinical form, including the waiver of informed consent, were approved by the Asan Medical Center Institutional Review Board (IRB number: 2010-0079).

The tracheal aspirate specimens were first treated with an equal volume of Sputasol solution (Oxoid, Basingstok, UK) in a 37 °C water bath for liquefaction. The total RNA/DNA was then extracted from the liquefaction sample using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) per the manufacturer's instructions. The nucleic acids was stored in aliquots at −80 °C until use.

The analysis of RSV occurrence for the different studied populations and among symptomatics and asymptomatics was made using chi‐squared, odds ratio, Fisher's exact test, Student's t test for independent samples, and ANOVA. The programs used were OpenEpi version 2.3.1 and GraphPad. A P value of <.05 was considered statistically significant.

Nasopharyngeal aspirates were obtained upon admission by gently flushing the children's nostrils with 4 mL of sterile saline solution. All nasopharyngeal aspirates were tested within 24 hours of collection for respiratory syncytial viruses (A and B), human metapneumovirus, human rhinovirus, influenza A and B viruses, parainfluenza (A, B, and C) virus, coronavirus (A and B), and adenovirus. Total viral RNA was extracted from nasopharyngeal aspirates (300 µL) using Viral Gene-spin Viral DNA/RNA Extraction Kits (iNtRON, Seongnam, Korea) and stored at -80℃. First-strand cDNA was synthesized using Revert Aid First Strand cDNA Synthesis Kits (Fermentas Inc., Burlington, ON, Canada), followed by polymerase chain reactions using Seeplex Respiratory Viruses Detection Kits-1 (Seegene, Seoul, South Korea) and the GeneAmp PCR system 9700 (Applied Biosystems, Waltham, MA, USA). Each 20 µL reaction mixture included 3 µL of cDNA, 4 µL of 5× RV1A or 5× RV1B primer, and 10 µL of 2× Multiplex Master Mix. Briefly, 2.5 µL of extracted RNA was mixed with 5× buffer, 0.2mM of each dNTP, 0.5µM of each primer13141516), 1 µL of enzyme mix, and DEPC-treated ultrapure water to a final volume of 25 µL. After incubations at 50℃ for 30 minutes and at 94℃ for 15 minutes, the reactions were subjected to 40 cycles of denaturation for 30 seconds at 94℃, annealing for 90 seconds at 60℃, and extension for 90 seconds at 72℃, followed by a final extension at 72℃ for 10 minutes. The amplified products were analyzed on 2% agarose gels containing 0.5 g/mL ethidium bromide.

Viral nucleic acids were extracted from nasal and throat swabs using RNA/DNA extraction kit «RIBO-sorb» (Interlabservice, Russia) according to the manufacturer’s instructions. The extracted viral nucleic acid was immediately used to perform the reaction of reverse transcription using commercial kit "REVERTA-L" (Interlabservice, Russia).

Data and swabs result from a surveillance system that received regulatory approvals, including the CNIL (National Commission for Information Technology and Civil Liberties Number 1592205) approval in July 2012. All the patients have received oral information and gave their consent for swab and data collection. Data were collected for surveillance purpose and are totally anonymous.

NPA sample (200 μl) from the patient was subjected to total nucleic extraction after addition of internal control bacteriophage MS2 (20 μl) using the MagNA Pure LC Total Nucleic Acid Isolation kit (Roche Diagnostics, Germany) on Magna Pure LC 2.0 platform, following the manufacturer's instructions. The method is based on magnetic-bead technology. The procedure included cellular destruction, nucleic acid binding on beads, and washing steps to remove cellular and purified nucleic acid elution. Extracted nucleic acids were eluted in 50 µl of elution buffer and stored at −80°C for RVP FAST assay.

The study was performed at a medical ICU of the Asan Medical Center, a tertiary referral hospital in Seoul, Republic of Korea. This university-affiliated teaching hospital has 2700 beds and eight ICUs. During the study period, most of the adult patients with severe HAP requiring ICU care were referred to the medical ICU. The medical ICU is a closed 28-bed unit managed by five board-certified intensivists. All intensivists attend structured twice daily bedside rounds. Fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) was preferably performed on patients with bilateral interstitial pattern infiltration or non-resolving pneumonia, at the discretion of the physician’s judgment. The BAL protocol has been described in detail elsewhere.

In the group of asymptomatic patients, RSV was detected in 3% (3/100) of the samples. The average age of these patients was 33.7 years and the mean CD4 T‐cell count was 499 cells/mm3, without difference between those infected or not infected by RSV (P = .94). Two RSV cases reported previous contact with a symptomatic person. We quantified RSV viral load for 2 samples and obtained 1.4 and 5.3 log copies/mL.In the group of symptomatic HIV individuals, the positivity of RSV was 7% (3/43) and the average age of these positive patients was 46 years. The mean CD4 of patients with positive samples was 442.6 cells/mm3. There was no statistically significant difference between the CD4 T‐cell count of RSV‐positive and the RSV‐negative ones. (P = .72). The average time from onset of symptoms until the date of sample collection was 11 days for patients with positive samples.

Only 1 of the individuals reported close contact with symptomatic children at home.

The viral load found for each positive sample of symptomatic patients was 5.3, 6.5, and 3.2 log copies/mL, with an average value of 4.8 log copies/mL.

Wide range of viruses is known to be associated with respiratory disease in humans. Adenoviruses, coronaviruses, human enteroviruses (HEV), human rhinoviruses (HRV), influenza viruses, parainfluenza viruses (PIV), and respiratory syncytial viruses (RSV) are well-known causes of acute respiratory tract infections (ARTI) in both industrialized and developing countries. Over the last decade, modern molecular techniques have led to the discovery of several previously unknown respiratory tract viruses, including human metapneumovirus (hMPV), two new human coronavirus types [2, 3], human bocavirus (HBoV), and two new human polyomaviruses [5, 6]. The significance of these novel viruses has been reviewed recently [7, 8].

It is widely accepted that common cold is almost always caused by viruses, most frequently by HRV, and viral infections are considered to contribute to the generation of complications of common cold, such as acute otitis media and sinusitis. Moreover, different viruses, including influenza viruses and RSV, are also frequently detected in samples obtained from patients with lower respiratory tract infection (LRTI), either alone or together with pathogenic bacteria. Several recent reports, including some from Africa, suggest viruses as potential etiologic agents in pneumonia in children [10–13], or exacerbations of asthma [14–16].

Several studies underscore the importance of respiratory tract viruses in Nigerian patients, but these studies were carried out before the introduction of modern molecular diagnostic techniques [14, 17–19]. The present study was designed to identify viral agents associated with respiratory infections among young children in Nigeria using modern, validated molecular techniques. We wanted to explore the presence of different virus groups, including some of the newer ones detected by only molecular techniques.

2017.

The study was approved by the Ethical Committee of the Oyo State Ministry of Health. Participation of children in the study was voluntary and required informed consent from the parents. Inclusion criteria were recent onset of symptoms suggestive for respiratory tract infection, such as cough, coryza, repeated sneezing, and/or difficulty in breathing. Patients were recruited between February and May, 2009, and included hospitalized patients, children seen at emergency departments, and outpatient clinics at 3 different children's hospitals in Ibadan, Oyo state. Demographic and clinical information, including age, sex and clinical symptoms, was recorded during the medical visit by means of a structured questionnaire.

A nasal swab sample was obtained from children by inserting a sterile nylon swab (Regular Flocked swab, Cat. No. 520CS01, Copan Diagnostics Inc., Murrieta, Calif USA) into the nostril to a depth of 2–4 cm, and retracting it with a rotating motion, in order to trap epithelial cells in the swab. With a second swab, a throat specimen was collected by rubbing the tonsils and the posterior wall of the pharynx. The 2 swabs were then placed in a vial containing 2 mL of RNAlater solution (RNAlater Tissue Collection, Applied Biosystems, Espoo, Finland). The specimens were transported to the laboratory on the same day in an ice pack and stored at −70°C until further processing.

A total of 356 nasopharyngeal aspirate and throat swab samples were collected from patients with SARI by a trained technician using a sterile nylon flocked swab and placed in viral transport medium (VTM), labelled and transported on ice at the earliest to Advanced research lab (ICMR Grade-1 Virology Lab) of SMS Medical college Jaipur for further processing and storage of the samples. The study was approved by the institutional ethics committee.