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      The Source of Respiratory Syncytial Virus Infection In Infants: A Household Cohort Study In Rural Kenya

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          Abstract

          Background.  Respiratory syncytial virus (RSV) vaccine development for direct protection of young infants faces substantial obstacles. Assessing the potential of indirect protection using different strategies, such as targeting older children or mothers, requires knowledge of the source of infection to the infants.

          Methods.  We undertook a prospective study in rural Kenya. Households with a child born after the preceding RSV epidemic and ≥1 elder sibling were recruited. Nasopharyngeal swab samples were collected every 3–4 days irrespective of symptoms from all household members throughout the RSV season of 2009–2010 and tested for RSV using molecular techniques.

          Results.  From 451 participants in 44 households a total of 15 396 nasopharyngeal swab samples were samples were collected, representing 86% of planned sampling. RSV was detected in 37 households (84%) and 173 participants (38%) and 28 study infants (64%). The infants acquired infection from within (15 infants; 54%) or outside (9 infants; 32%) the household; in 4 households the source of infant infection was inconclusive. Older children were index case patients for 11 (73%) of the within-household infant infections, and 10 of these 11 children were attending school.

          Conclusion.  We demonstrate that school-going siblings frequently introduce RSV into households, leading to infection in infants.

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          Real-time RT-PCR detection of 12 respiratory viral infections in four triplex reactions

          1 Introduction Traditional viral culture, usually in combination with direct immunofluoresence (DIF), is the gold standard for the laboratory diagnosis of viral respiratory infection. However, these methods are insensitive, laborious, have prolonged turn-around times, and cannot detect all recognised viral respiratory pathogens. PCR is more sensitive and specific than traditional methods and can be used to detect fastidious viruses. Real-time PCR is at least as sensitive as nested gel-based PCR protocols and offers increased rapidity (results available within the working day). The use of specific labelled probes ensures easy interpretation when used in a multiplex format. We describe four triplex TaqMan™-based RT-PCR methods adapted from published methods and further developed in-house for the diagnosis of 12 viral respiratory pathogens. 2 Selection of primers and probes Primers and probes for each triplex are described in full (Table 1 ). Real-time RT-PCR assays for human metapneumovirus (hMPV) (Mackay et al., 2003), RSV A and B (van Elden et al., 2003), and coronavirus 229E and OC43 (van Elden et al., 2004) were published previously. Although the primers for the detection of influenza A and influenza B, rhinovirus, and parainfluenza 1 and 2 were from previously published methods (Bredius et al., 2004, Templeton et al., 2004), TaqMan™ probes for these pathogens were adapted from the original molecular beacons. In-house real-time RT-PCR methods were developed for coronavirus NL63 and parainfluenza 3 using Beacon designer 2.0 (Premier Biosoft International) and Primer Express (Applied Biosystems). Conserved target regions were identified using BLAST (www.ncbi.nlm.nih.gov/blast). Regions within the 1a and haemagluttinin genes were chosen for coronavirus NL63 and parainfluenza 3, respectively. The primers and probes were shown to detect all submitted NL63 and parainfluenza 3 sequences. No interfering secondary structures were observed using the mfold algorithm (www.bioinfo.rpi.edu). Table 1 Primers and probes used in triplex real-time RT-PCR assays Triplex Pathogen Primer sequences (concentration in nM) Probe sequence (concentration in nM) Target 1 Influenza A AAAGCGAATTTCAGTGTGAT (1000) 6FAM-CCC TCT TCG GTG AAA GCC CT-BHQ (300) NS1 gene GAAGGCAAT GTGAGATTT (500) 

 Influenza B GTCCATCAAGCTCCAGTTTT (1000) CY5-CCTCCGTCTCCACCTACT TCGTT-BHQ (300) Nucleoprotein gene TCTTCTTACAGCTTGCTTGC (500) 

 Human metapneumovirus AACCGTGTACTAAGTGATGCACTC (500) VIC-CTTTGCCATACTCAATGAACAAAC-TAMRA (300) Nucleocapsid protein gene CATTGTTTGACCGGCCCCATAA (500) 

 2 RSV A AGATCAACTTCTGTCATCCAGCAA (1000) 6FAM-CACCATCCAACGGAGCACAGGAGAT-BHQ (300) Nucleocapsid protein gene TTCTGCACATCATAATTAGGAG (250) 

 RSV B AAGATGCAAATCATAAATTCACAGGA (1000) CY5-TTTCCCTTCCTAACCTGGACATA-BHQ (300) TGATATCCAGCATCTTTAAGTA (1000) 

 Rhinovirus TGGACAGGGTGTGAAGAGC (1000) VIC-TCCTCCGGCCCCTGAATG-TAMRA (300) Five untranslated region CAAAGTAGTCGGTCCCATCC (1000) 

 3 Parainfluenza 1 ACCTACAAGGCAACAACATC (1000) CY5-CAAACGATGGCTGAAAAAGGGA-BHQ (300) HN gene CTTCCTGCTGGTGTGTTAAT (500) 

 Parainfluenza 2 CCATTTACCTAAGTGATGGAA (1000) VIC-AATCGCAAAAGCTGTTCAGTCAC-TAMRA (300) HN gene CGTGGCATAATCTTCTTTTT (1000) 

 Parainlfuenza 3 CCAGGGATATAYTAYAAAGGCAAAA (1000) 6FAM-TGGRTGTTCAAGACCTCCATAYCCGAGAAA-BHQ (300) HN gene CCGGGRCACCCAGTTGTG (1000) 

 4 Coronvirus 229E CAGTCAAATGGGCTGATGCA (1000) 6FAM-CCCTGACGACCACGTTGTGGTTCA-BHQ (300) AAAGGGCTATAAAGAGAATAAGGTATTCT (1000) 

 Coronavirus OC43 CGATGAGGCTATTCCGACTAGGT (125) CY5-TCCGCCTGGCACGGTACTCCCT-BHQ (300) CCTTCCTGAGCCTTCAATATAGTAACC (1000) 

 Coronavirus NL63 ACGTACTTCTATTATGAAGCATGATATTAA (1000) VIC-ATTGCCAAGGCTCCTAAACGTACAGGTGTT-TAMRA (300) AGCAGATCTAATGTTATACTTAAAACTACG (1000) 3 Selection of flourophores All real-time RT-PCR assays were developed for use on the ABI 7500 real-time PCR system. For multiplex real-time PCR, ABI recommend that probes are labelled with the dyes FAM and VIC, as they are distinguishable from each other because they have different emission wavelength maxima (518 nm and 554 nm). We chose CY5 as the third dye as its emission wavelength maxima is far removed from both FAM and VIC (670 nm). No cross-talk was observed. 4 Protocol Respiratory controls and samples were extracted using the Qiagen blood minikit on the Biorobot 9604 using standard protocols. Amplification was carried out in a 25 μl reaction volume using the Invitrogen superscript III One step q-RT-PCR system containing 10 μl of extracted sample. Primers and probes were added to each PCR reaction at the concentration shown (Table 1). Reverse transcription was performed for 15 min at 50 °C. Platinum taq polymerase was activated 95 °C for 2 min and 40 cycles of PCR performed at 95 °C for 8 s and 60 °C for 34 s using an ABI 7500 SDS. Total reaction time per triplex reaction was approximately 75 min. 5 Analytical sensitivity All real-time PCR assays were previously assessed in their original single target or duplex formats using panels of known culture and DIF or nested PCR positive and negative samples. All of the real-time RT-PCR assays were more sensitive than traditional or nested RT-PCR methods (data not shown). All real-time RT-PCR assays (except coronavirus NL63 and parainfluenza 2) were also assessed using quality control molecular diagnostics (QCMD) respiratory panel (Forde et al., in press). Each real-time RT-PCR test detected the appropriate target at the appropriate end point dilution. 6 Sensitivity compared to current diagnostic methods Four multiplex real-time RT-PCR assays were developed for the simultaneous detection of 1. influenza A, influenza B, and hMPV; 2. RSV A, RSV B, and rhinovirus; 3. parainfluenza 1, 2, and 3; 4. coronavirus 229E, OC43, and NL63. Each triplex real-time RT-PCR assay was initially assessed using positive controls and compared to the previous duplicate or single target format. Triplex assays were then compared to panels of tissue culture and DIF and/or nested RT-PCR positive samples (where available). To determine whether mixed infections would reduce triplex sensitivity end point dilutions of each viral target were tested in both a single target and a pooled target (containing 10 different viral targets at the same endpoint dilution) format. 6.1 Multiplex real-time RT-PCR for influenza A, influenza B, and human metapneumovirus Positive control samples for influenza A, influenza B, and hMPV were tested in triplicate wells using the triplex assay in parallel with the previous duplex or single target format (Table 2 ). There was no significant loss of sensitivity (as observed by cycle threshold (Ct)) between methods. The triplex assay was then tested on 11 influenza A positive samples. The triplex assay detected all the previous positive samples. The endpoint dilution of both the single target controls and pooled format controls were detected by the triplex assay showing that “mixed infections” would not reduce the sensitivity of this assay. Table 2 Comparison of Ct of positive controls in triplicate for influenza A, influenza B, and HmPv in single, duplex, and triplex RT-PCR reactions Mean Ct (±S.E.M.) Flu A in duplex 29.91 (0.1) Flu A in triplex 30.46 (0.32) Flu B in duplex 25.90 (0.21) Flu B in triplex 26.22 (0.53) hMPV in single 29.83 (0.38) hMPV in triplex 28.93 (0.18) 6.2 Multiplex real-time RT-PCR for RSV A, RSV B, and rhinovirus Positive samples for RSV A, RSV B, and rhinovirus were tested in a triplex format in triplicate and compared to duplex or single target format (Table 3 ). No loss in sensitivity was observed for RSV A and RSV B. The sensitivity of the rhinovirus real-time RT-PCR improved when incorporated in the triplex assay (as shown by a reduction in the Ct). This increase in sensitivity was sample specific (only occurred with the positive control) as no reduction in Ct was observed when the testing the rhinovirus positive samples. The triplex assay was then assessed on 42 RSV positive samples (NPA samples positive by DIF and nested gel-based PCR) and 11 rhinovirus positive samples. The triplex assay detected all previously positive samples. The endpoint dilution of both the single target controls for RSV A, RSV B, and rhinovirus and pooled format controls (containing 10 viral targets) were detected by the triplex assay showing that “mixed infections” would not reduce the sensitivity of this assay. Table 3 Comparison of Ct for RSV A, RSV B, and rhinovirus in single, duplex, and triplex RT-PCR reactions Mean Ct (±S.M.E.) RSV A duplex 17.43 (0.28) RSV A triplex 17.19 (0.15) RSV B duplex 22.06 (0.22) RSV B triplex 21.17 (0.28) Rhino single 26.05 (0.13) Rhino triplex 23.85 (0.02) 6.3 Multiplex real-time RT-PCR for parainfluenza 1, 2, and 3 Positive control samples for parainflueza 1, 2, and 3 were tested in a triplex format, either with the new PF3 assay or the original test. The introduction of the new PF3 assay had no effect on the sensitivity of the PF1 and PF2 RT-PCR assays (Table 4 ). However, the new PF3 RT-PCR was more sensitive than the previous method. The new triplex assay was then compared to the published method on 19 DIF PF3 positive samples. The new method detected one additional sample (19 versus 18). The endpoint dilution of both the single target controls and pooled format controls were detected by the triplex assay showing that “mixed infections” would not reduce the sensitivity of this assay. Table 4 Comparison of published parainfluenza triplex assay with triplex parainfluenza assay incorporating new PF3 RT-PCR assay Ct of PF1 control Ct of PF2 control Ct of PF3 control Published parainfluenza triplex assay 28.34 24.17 28.37 Published parainfluenza triplex assay with new PF3 PCR 27.75 25.02 24.24 6.4 Multiplex real-time RT-PCR for coronavirus 229E, OC43, and NL63 Positive samples for coronavirus 229E, OC43, and NL63 were tested in a triplex format and in the duplex or single target format (Table 5 ). No significant changes in Ct were observed. The endpoint dilution of both the single target controls and pooled format controls were detected by the triplex assay showing that “mixed infections” would not reduce the sensitivity of this assay. Table 5 Comparison of Ct for single, duplex, and triplex RT-PCR assays for conrovirus 229E, OC43, and NL63 Ct of OC43 control Ct of 229E control Ct of NL63 control OC34/229E/NL63 assay 23.76 31.9 26.67 OC43/229E assay 23.63 31.3 NL63 assay 26.23 We have shown these triplex real-time RT-PCR assays to be at least as sensitive our previous RT-PCR assays. The rapidity, stability, and ease of use of these triplex real-time RT-PCR assays results in improved turn-around-times (12 pathogens within the working day), easier interpretation, and increased cost effectiveness. The implementation of these assays will no doubt improve patient management, infection control procedures, and the effectiveness of surveillance systems.
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            Identification of a recombinant live attenuated respiratory syncytial virus vaccine candidate that is highly attenuated in infants.

            Recombination technology can be used to create live attenuated respiratory syncytial virus (RSV) vaccines that contain combinations of known attenuating mutations. Two live attenuated, recombinantly derived RSV vaccine candidates, rA2cp248/404 Delta SH and rA2cp248/404/1030 Delta SH, were evaluated in 31 adults and in 95 children >/=6 months old. rA2cp248/404/1030 Delta SH was subsequently evaluated in 44 infants 1-2 months old. These vaccine candidates share 4 attenuating genetic elements and differ only in a missense mutation (1030) in the polymerase gene. Both vaccines were highly attenuated in adults and RSV-seropositive children and were well tolerated and immunogenic in RSV-seronegative children. Compared with that of rA2cp248/404 Delta SH, replication of rA2cp248/404/1030 Delta SH was restricted in RSV-seronegative children (mean peak titer, 10(4.3) vs. 10(2.5) plaque-forming units [pfu]/mL), indicating that the 1030 mutation had a potent attenuating effect. Although rA2cp248/404/1030 Delta SH was well tolerated in infants, only 44% of infants who received two 10(5.3)-pfu doses of vaccine had detectable antibody responses. However, replication after administration of the second dose was highly restricted, indicating that protective immunity was induced. At least 4 of 5 attenuating genetic elements were retained in recovered vaccine viruses. rA2cp248/404/1030 Delta SH is the first RSV vaccine candidate to be sufficiently attenuated in young infants. Additional studies are needed to determine whether rA2cp248/404/1030 Delta SH can induce protective immunity against wild-type RSV.
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              Comparison of nasopharyngeal flocked swabs and aspirates for rapid diagnosis of respiratory viruses in children.

              The quality of clinical specimens is a crucial determinant for virological diagnosis. We compared the viral diagnostic yield for influenza A and respiratory syncytial virus (RSV) from the recently developed nasopharyngeal flocked swabs (NPFS) with nasopharyngeal aspirates (NPA) collected in parallel from 196 hospitalized children with acute respiratory infection during the peak period of influenza A and RSV activity in Hong Kong. Specimens were tested by RT-PCR for influenza A and RSV and viral load determined. They were also tested by direct immunofluorescence (DIF) for influenza A and B, RSV, parainfluenza types 1-3 and adenovirus. Both NPA and NPFS had excellent sensitivity (100%) for detecting influenza A by RT-PCR but NPA was slightly more sensitive than NPFS for detecting RSV by both RT-PCR (100% vs. 92.3%) and DIF (87.2% vs. 84.6%) and for detecting influenza A by DIF (90.2% vs. 82.9%). Viral load for influenza A in NPA and NPFS was not significantly different but that for RSV was higher in NPA. NPA remains the optimal specimen for diagnosis of respiratory infections by RT-PCR and DIF. However, collection of NPFS is easier to perform in an out-patient setting, was more acceptable to parents and less likely to generate aerosols than NPA engendering potentially less infection control hazard.
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                Author and article information

                Journal
                J Infect Dis
                J. Infect. Dis
                jid
                jinfdis
                The Journal of Infectious Diseases
                Oxford University Press
                0022-1899
                1537-6613
                01 June 2014
                23 December 2013
                23 December 2013
                : 209
                : 11
                : 1685-1692
                Affiliations
                [1 ]KEMRI –Wellcome Trust Research Programme , Kilifi, Kenya
                [2 ]School of Life Sciences and WIDER, University of Warwick , Coventry, United Kingdom
                Author notes
                Correspondence: Patrick K. Munywoki, PhD, KEMRI–Wellcome Trust Research Programme, Hospital Road, PO Box 230, Kilifi, Kenya ( pmunywoki@ 123456kemri-wellcome.org ).
                Article
                jit828
                10.1093/infdis/jit828
                4017365
                24367040
                91464f26-b2f9-4a22-8861-bf8d4cb38ccb
                © The Author 2013. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 02 October 2013
                : 02 December 2013
                Categories
                Major Articles and Brief Reports
                Viruses
                Editor's choice

                Infectious disease & Microbiology
                rsv,infants,siblings,transmission,households
                Infectious disease & Microbiology
                rsv, infants, siblings, transmission, households

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