- Email: [email protected]
Detection of nine respiratory RNA viruses using three multiplex RT-PCR assays incorporating a novel RNA internal control transcript
Journal of Virological Methods 176 (2011) 9–13
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Detection of nine respiratory RNA viruses using three multiplex RT-PCR assays incorporating a novel RNA internal control transcript Helen Auburn a , Mark Zuckerman a , Simon Broughton b , Anne Greenough b , Melvyn Smith a,∗ a b
South London Specialist Virology Centre, King’s College Hospital NHS Foundation Trust, Bessemer Wing, Denmark Hill, London SE5 9RS, UK Department of Child Health, 4th Floor Golden Jubilee Wing, King’s College Hospital NHS Foundation Trust, Bessemer Road, London SE5 9RS, UK
a b s t r a c t Article history: Received 11 February 2011 Received in revised form 9 May 2011 Accepted 11 May 2011 Available online 19 May 2011 Keywords: Respiratory viruses Multiplex RT-PCR RNA internal control
Real-time PCR is a significant improvement over viral isolation and immunofluorescence for routinely detecting respiratory viruses. We developed three real-time internally controlled multiplex RT-PCR assays for detecting nine respiratory viruses. An internal control transcript consisting of a chimeric plasmid was synthesised and incorporated into each multiplex to monitor amplification efficiency, including inhibition. Each multiplex assay was developed on the Rotor-Gene 3000 and evaluated using RNA extracts from 126 nasopharyngeal aspirates from 112 pre-term infants. All 44/126 (35%) samples positive by immunofluorescence were confirmed by multiplex RT-PCR. Additionally, respiratory syncytial virus RNA was detected in 5 samples, influenza A virus RNA in 2 samples and thirteen (10%) dual infections by multiplex RT-PCR were noted. Inclusion of the RNA internal control did not affect the amplification efficiency of the target sequences and only 2 of 1256 (0.2%) samples tested over a 12 month period were inhibitory. Together with the improved sensitivity of the internally controlled multiplex RT-PCR assays over the older technology and the ability to detect co-infections, the internal control monitored the efficiency of both the RT and PCR steps and indicated inhibition, saving time and costs on running duplicate samples with a “spiked” inhibition control. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Respiratory RNA viruses are associated with increased morbidity and mortality among high-risk populations including the elderly, immunocompromised individuals, pre-term infants and neonates (Weinberg et al., 2002; Van Elden et al., 2003). Real-time PCR is now recognised as the most sensitive and rapid choice for respiratory virus diagnosis compared with viral isolation and immunofluorescence. Several multiplex reverse transcription (RT-PCR) assays have been developed and evaluated employing real-time technology to detect respiratory viruses (Templeton et al., 2004; Bellau-Pujol et al., 2005; Gunson et al., 2005; Scheltinga et al., 2005; Brittain-Long et al., 2008; Chidlow et al., 2009; Lassaunièrea et al., 2010). Previously, nested PCR was used to increase sensitivity and specificity (Templeton et al., 2004; Bellau-Pujol et al., 2005). However, this prolongs time to detection and is a potential source of carry-over contamination leading to false positive results. An internal control is an essential requirement for routine diagnostic PCR assays to monitor nucleic acid extraction, amplification
∗ Corresponding author. Tel.: +44 020 3299 6974; fax: +44 020 3299 6476. E-mail address: [email protected] (M. Smith). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.05.017
efficiency and inhibition in clinical samples, since inhibitory samples are known to suppress amplification, decreasing the sensitivity of the PCR, leading to false negative results (Rosenstraus et al., 1998). This paper describes the development of three internally controlled multiplex one-step RT-PCR assays for respiratory virus detection on the Rotor-Gene 3000. Each multiplex was designed to detect three respiratory viruses according to seasonality, covering an extended range of the key pathogens. For specific detection of each viral amplicon, TaqMan probes were used that carried three different fluorophores, 6-carboxy-4,5-dichloro-2,7dimethoxyfluorescein (JOE), 5-carboxyfluorescein (FAM) and the sulphoindocyanine dye (Cy5), the internal control probe was labelled with 5-carboxy-X-rhodamine (ROX). To monitor for inhibition of the RT-PCR, a novel RNA internal control was developed, adopting approaches described by Hodgson et al. (2007) and Tang et al. (2005). This choice of internal control allows for increased stability and a straightforward synthesis when stocks are low. For clinical evaluation, results were compared with those from an immunofluorescence assay. Overall, 126 nasopharyngeal aspirates collected from 112 premature infants who presented with signs of lower respiratory tract infections during the winter
10
H. Auburn et al. / Journal of Virological Methods 176 (2011) 9–13
seasons of 2002/3–2004/5 were tested. This was part of a longterm prospective study investigating the impact of viral lower respiratory tract infections on respiratory morbidity and healthcare utilisation during infancy (Broughton et al., 2005, 2006). 2. Materials and methods 2.1. Preparation of viral culture for stock generation and direct immunofluorescence assay Viral cultures were set up by inoculating 200 l of nasopharyngeal aspirate onto 75 cm3 flasks of continuous human laryngeal carcinoma (HEp-2) cells for respiratory syncytial viruses A and B (RSV A and B), and on rhesus monkey kidney (LLC-MK2) cells for influenza A and B viruses and parainfluenza viruses types 1, 2 and 3. Subsequently, cell cultures were incubated for 1 week at 33 ◦ C and 37 ◦ C, respectively, and examined daily for a cytopathic effect (CPE). A positive CPE was confirmed by direct immunofluorescence with specific monoclonal antibodies according to the manufacturer’s instructions (Imagen, DakoCytomation, Cambridge, UK). Virus stocks were subsequently prepared for the multiplex RT-PCR assays according to standard procedures. 2.2. Positive control material for human metapneumovirus and human rhinovirus Nasopharyngeal aspirates positive for human metapneumovirus and human rhinovirus were identified through routine evaluation by monoplex RT-PCR and subsequently used for the development of the multiplex RT-PCR assays. 2.3. Viral RNA extraction Viral RNA was extracted from virus stocks and nasopharyngeal aspirates using QIAamp viral RNA minikits (Qiagen, West Sussex, UK) according to the manufacturer’s protocol. RNA was either tested immediately or stored in aliquots of 20 l at −80 ◦ C for up to seven days until RT-PCR analysis. 2.4. Primers and probes The primers and probes (Metabion, Martinsried, Germany) are described in Table 1. TaqMan probes and primers for human rhinovirus and (RSV A and B) were reported previously (Van Elden et al., 2003; Bredius et al., 2004; Gunson et al., 2005), although the VIC fluorophore was replaced with JOE. Primers for influenza A and B viruses, parainfluenza viruses types 1, 2 and 3 (Templeton et al., 2004) and human metapneumovirus (Mackay et al., 2003) were described previously. Additionally, primers were re-designed for parainfluenza virus type 2, and TaqMan probes were designed for influenza A and B viruses, human metapneumovirus and parainfluenza viruses types 1, 2 and 3. 2.5. Real-time multiplex one-step RT-PCR assay The cycling conditions and the concentrations of each primer and probe-set were evaluated to determine the optimal threshold cycle (Ct) for each monoplex (data not shown). Each multiplex was evaluated to ensure that no inhibition or cross-reactions occurred. Each multiplex was further tested with and without the internal control to ensure that no inhibition occurred and to determine its optimal concentration (Table 4). RT-PCR was carried out using Qiagen QuantiTect Probe RT-PCR kits (Qiagen, West Sussex, UK) with the internal control added to each extraction to monitor the complete RT-PCR. Each 30 l reaction mixture consisted of 1 l of RNA internal control (8 × 102 copies/l), 10 l of RNA eluate, forward
and reverse primers (10 pmoles each), TaqMan probes (5 pmoles each), 0.5 l of Qiagen RT enzyme, and 15 l of 2X QuantiTect Probe RT-PCR master mix in 200 l reaction vessels in a 72-well rotor (Corbett Research, Cambridge, UK). The RT-PCR consisted of 10 min reverse-transcription at 50 ◦ C, 15 min incubation at 95 ◦ C, followed by 50 cycles of: 15 s denaturation at 95 ◦ C and 30 s annealing and extension at 57 ◦ C. Fluorescence measurements were carried out at each annealing step and the total reaction time was approximately 1.5 h. Assay controls included PCR products for influenza A and B viruses, parainfluenza viruses types 1, 2 and 3, RSV A and B, human metapneumovirus and human rhinovirus, previously gel-purified using QIAquick gel extraction kits (Qiagen, West Sussex, UK). 2.6. Nasopharyngeal aspirates from pre-term infants Samples were placed on ice immediately after collection and delivered to the laboratory in viral transport medium. Viral culture and immunofluorescence were carried out on the day of collection by inoculating 200 l of each sample onto HEp-2 cells, human alveolar epithelial cell line A-549, human epithelial lung (HEL) cells and, LLC-MK2 cells. The remaining samples were stored at −80 ◦ C for up to seven days until RNA extraction. A positive cytopathic effect (CPE) was confirmed for influenza A and B viruses, parainfluenza viruses types 1, 2 and 3, and RSV by the immunofluorescence assay described previously. 2.7. Design of the RNA internal control The construction of the RNA internal control was based on the PCR methods reported by Tang et al. (2005) and Hodgson et al. (2007). The 188 bp amplicon for the control was produced using composite primers, incorporating the RNA internal control forward and reverse primer sequences and sequences complementary to the pGEM-T easy vector (Promega, Southampton, UK). The resulting internal control has identical primer-binding sites to the RNA internal control primers, but contains an internal sequence derived from the vector DNA and detected by a different probe and fluorophore combination (Table 1). The PCR programme consisted of an initial hold at 95 ◦ C for 10 min, followed by 50 cycles of: 15 s denaturation at 95 ◦ C and 60 s combined annealing/extension at 60 ◦ C. The amplicon was purified from a 2% agarose gel using a QIAquick gel extraction kit (Qiagen, West Crawley, UK) for cloning. 2.8. Cloning the chimeric insert into the pGEM-T vector system The purified insert was cloned into a pGEM-T easy vector according to the manufacturer’s instructions. The recombinant plasmids were extracted using QIAprep Miniprep kits (Qiagen, West Sussex, UK) following manufacturer’s instructions. 2.9. Synthesis of the RNA transcript for use as the internal control The RNA internal control was synthesised from linearised VZV/pGEM-T template using the MEGAshortscript T7 transcription kit (Ambion, Warrington, UK) according to the manufacturer’s instructions. RNA transcripts were purified with the MEGAclear kit (Ambion, Warrington, UK), treated with DNAse I (Ambion, Warrington, UK), and stored at −80 ◦ C. 2.9.1. Testing inhibition of the RNA internal control in the multiplex RT-PCR assays The detection limit of the RNA internal control was determined on a dilution series (8 × 1010 –8 × 10−1 copies/l). Amplification was initially performed using RNA internal control primers. Subsequently, to assess any inhibition of the multiplex RT-PCR assays by
H. Auburn et al. / Journal of Virological Methods 176 (2011) 9–13
11
Table 1 Primer and probe sequences used in the multiplex RT-PCR assays. Multiplex 1
Pathogen Influenza A virus Influenza B virus Human metapneumovirus
2
Respiratory syncytial virus A
Respiratory syncytial virus B
Rhinovirus 3
Parainfluenza virus type 1 Parainfluenza virus type 2 Parainfluenza virus type 3
1, 2, 3
RNA internal control Composite primers
Primer Sequences (5 , 3 ) c
(F) aaa gcg aat ttc agt gtg at (R) gaa ggc aat ggt gag att tc (F) gtc cat caa gct cca gtt ttc (R) tct tct tac agc ttg ctt gcc (F) cat gcc cac tat aaa agg tca ge (R) cac ccc agt ctt tct tga aae (F) aga tca act tct gtc atc cag caaa (R) ttc tgc aca tca taa tta gga gd (F) aag atg caa atc ata aat tca cag gaa (R) tga tat cca gca tct tta agt aa (F) tgg aca ggg tgt gaa gag cb (R) caa agt agt cgg tcc cat ccb (F) acc tac aag gca aca aca tcc (R) ctt cct gct ggt gtg tta atc (F) gga gat tgc ctc gat ttc acg acf (R) gtc tca gtt cag cta gat cagf (F) gga gca ttg tgt cat ctg tcc (F) tag tgt gta atg cag ctc gtc (F) acc tta aaa ctc act acc agtf (R) cta atc caa ggc ggg tgc atf (F) acc tta aaa ctc act acc agt GGC CGA GCG CAG AAG TGGf,g (R) cta atc caa ggc ggg tgc atG ACG AGC GTG ACA CCA CGf,g
Probe Sequences (5 , 3 )
Target Gene
Amplicon Size
JOE-cac cga aga ggg agc aat tgt tgg cg-BHQ1f Cy5-cct ccg tct cca cct act tcg ttc cc-BHQ2f FAM-tga gtc ttt gtc aag cag cgt tag cat g-BHQ1f FAM-cac cat cca acg gag cac agg aga t-BHQ1a
NS protein
103
NS protein
144
L gene
169
NP gene
83
Cy5-ttt ccc ttc cta acc tgg aca ta-BHQ2d
NP gene
102
JOE-tcc tcc ggc ccc tga atg-BHQ1d
5 UTR
143
FAM-ttg gtc tac aac ccg aaa tga taa ctc cac gg-BHQ1f JOE-tct gct gca ggg ttt cca att ttc agg act-BHQ1f Cy5-tgt ttc gga tgg cca gct cgt tta ctc-BHQ2f ROX-tcgccagttaatagtttgcgcaacgBHQ1f
HN gene
128
HN gene
249
HN gene
153
Vector
188
Abbreviations: F = forward; R = reverse. Primers and probe sequences published below: a Van Elden et al. (2003). b Bredius et al. (2004). c Templeton et al. (2004). d Gunson et al. (2005). e Scheltinga et al. (2005). f This study. g Sequences shown in upper case are derived from the pGEM-T easy vector.
the RNA internal control, respiratory viral targets (from nasopharyngeal aspirates) were assayed with and without the control (8 × 103 –8 × 101 copies/l), and the threshold cycles (Cts) compared (Table 4).
RT-PCR. Table 2 shows the results of the comparison of immunofluorescence and multiplex RT-PCR.
3.3. Detection of dual infections 3. Results 3.1. Evaluation of multiplex RT-PCR inhibition The initial concentration of the internal control transcript was determined using a nanodrop spectrophotometer (NanoDrop Technologies, UK) to be 300 ng/l and adjusted to 8 × 1010 RNA copies/l. The detection limit of the internal control was determined by testing it at ten-fold serial dilutions and found to be 8 × 100 copies/l. Respiratory virus RNA targets were amplified with and without the RNA internal control at dilutions ranging from 8 × 103 to 8 × 101 copies/l in the multiplex RT-PCR assays and the Ct values compared, the optimal concentration was found to be 8 × 102 copies/l. Table 4 shows that inclusion of the internal control resulted in similar Ct values of respiratory targets in comparison to amplification without the RNA internal control.
Thirteen (10.3%) dual infections were identified from nasopharyngeal aspirates from 12 infants by the multiplex RT-PCR assay (Table 3). These included: RSV B and human rhinovirus infections (n = 6), human metapneumovirus and RSV A (n = 2), human metapneumovirus and human rhinovirus (n = 1), human metapneumovirus and parainfluenza virus type 3 (n = 1), RSV A and RSV B (n = 1), influenza A virus and RSV A (n = 1) and influenza A virus and human rhinovirus (n = 1).
Table 2 Comparison of the numbers of respiratory RNA viruses detected in nasopharyngeal aspirates using immunofluorescence assays and real-time multiplex RT-PCR assays. Respiratory virus
Samples positive by immunofluorescence assay
Samples positive by multiplex RT-PCR assays
Influenza A virus Influenza B virus Human metapneumovirus Parainfluenza virus type 1 Parainfluenza virus type 2 Parainfluenza virus type 3 RSV A RSV B RSV (Total A + B) Human rhinovirus
3 0 Not tested 0 1 4 Not tested Not tested 36 Not tested
5 0 7 0 1 4 28 13 41 36
3.2. Clinical evaluation of the real-time multiplex RT-PCR assays A total of 126 nasopharyngeal aspirates collected from 112 preterm infants were used. Viral culture with immunofluorescence and multiplex RT-PCR were carried out on each sample. Forty-four samples (35%) were positive for at least one of the nine respiratory viruses in the multiplex assay, confirming all immunofluorescence positive results. Additionally, RSV and influenza A virus RNA was detected in five and two immunofluorescence-negative samples respectively. In total, 94 positives were detected (75%) by multiplex
12
H. Auburn et al. / Journal of Virological Methods 176 (2011) 9–13
Table 3 Dual infections detected in nasopharyngeal aspirates collected from 112 pre-term infants using the real-time multiplex RT-PCR assays. Respiratory virus dual infections
Number of dual infections
Human rhinovirus and RSV B Human metapneumovirus and RSV A Human metapneumovirus and human rhinovirus Human metapneumovirus and parainfluenza type 3 RSV A and RSV B Influenza A virus and RSV A Influenza A virus and human rhinovirus
6 2 1 1 1 1 1
Table 4 Ct values for multiplex RT-PCR with and without dilution series of the RNA internal control. (Each measurement represents the mean of three replicates.). Respiratory virus (RNA IC expressed as copies/l) RSV A + RNA ic 103 RSV A + RNA ic 102 RSV A + RNA ic 101 RSV A only RSV B + RNA ic 103 RSV B + RNA ic 102 RSV B +RNA ic 101 RSV B only Human rhinovirus + RNA ic 103 Human rhinovirus + RNA ic 102 Human rhinovirus + RNA ic 101 Human rhinovirus only Influenza A + RNA ic 103 Influenza A + RNA ic 102 Influenza A + RNA ic 101 Influenza A only Influenza B + RNA ic 103 Influenza B + RNA ic 102 Influenza B + RNA ic 101 Influenza B only Human metapneumovirus + RNA ic 103 Human metapneumovirus + RN0A ic 102 Human metapneumovirus + RNA ic 101 Human metapneumovirus only Parainfluenza virus type 1 + RNA ic 103 Parainfluenza virus type 1 + RNA ic 102 Parainfluenza virus type 1 + RNA ic 101 Parainfluenza type 1 virus only Parainfluenza virus type 2 + RNA ic 103 Parainfluenza virus type 2 + RNA ic 102 Parainfluenza virus type 2 + RNA ic 101 Parainfluenza type 2 virus only Parainfluenza virus type 3 + RNA ic 103 Parainfluenza virus type 3 + RNA ic 102 Parainfluenza virus type 3 + RNA ic 101 Parainfluenza type 3 virus only RNA ic 103 only RNA ic 102 only RNA ic 101 only
Ct
Ct of RNA IC
23.06 23.17 23.96 23.52 27.44 28.00 28.35 28.53 21.11 21.10 22.15 21.95 19.88 20.99 20.89 21.21 17.25 19.39 17.51 18.07 22.50 22.36 22.48 22.21 24.74 24.81 25.03 25.12 19.36 19.21 19.59 19.39 17.51 17.57 17.64 18.48 – – –
22.66 35.79 Undetectable – 22.28 25.53 31.37 – 22.14 25.97 42.99 – 21.88 25.02 30.66 – 22.14 29.94 Undetectable – 21.92 26.81 38.72 – 21.78 24.92 33.45 – 21.67 26.28 45.17 – 28.46 40.50 Undetectable – 23.68 27.46 33.02
Abbreviations: IC = internal control.
4. Discussion Inhibitory substances are found frequently in clinical samples, which may carry-over during nucleic acid extraction and can result in a significant reduction in the sensitivity of a PCR leading to inaccurate quantitative results, or worse, false-negative results (Hoorfar et al., 2004; Nolan et al., 2006). The inclusion of an internal control is an essential requirement for all real-time PCR assays for monitoring for the presence of inhibitory substances. The use of a plasmid-based DNA internal control in other real-time PCR assays in our laboratory has demonstrated satisfactory and reproducible results. In this study, a novel chimeric RNA internal control was developed to monitor inhibition of the multiplex RT-PCR assays. Although this choice of RNA internal control was not used to mon-
itor the quality of the RNA extraction in our assays, it can be included in the sample prior to extraction. Therefore, it has the added benefits of being multi-purpose, suitable for both in-house DNA and real-time RT-PCR assays and monitoring both extraction and amplification steps. Once the plasmid vector has been generated, a life-long supply of the RNA internal control can be transcribed in a quick and straightforward procedure. Over twelve months, only 2 (0.2%) out of 1265 nasopharyngeal samples were inhibitory by the RNA internal control for the multiplex RT-PCR assays. Real-time PCR is the preferred choice for respiratory virus diagnosis due to its increased sensitivity, a major limitation of viral isolation that depends on viable tissue culture cells for detection. From the clinical evaluation, the multiplex RT-PCR assays detected all samples positive by immunofluorescence (27.8%), with an additional five (5.6%) respiratory syncytial viruses and three (2.4%) influenza A viruses. Furthermore, of the 41 (32.5%) respiratory syncytial virus infections, 28 (22.2%) were type A and 13 (10.32%) type B. The other advantage of the multiplex RT-PCR is its ability to detect mixed infections, which are often undetected in viral culture with immunofluorescence due to the masking of cytopathic effects between one or more viruses. In this study, thirteen dual infections were detected in samples from twelve patients. The majority were RSV B in combination with human rhinovirus, followed by human metapneumovirus and RSV A. There is conflicting evidence that dual infections are known to enhance the clinical severity of lower respiratory infections (Aberle et al., 2005; Semple et al., 2005; Wolf et al., 2006; Pierangeli et al., 2007; Manoha et al., 2007) therefore, the detection of co-infections may be extremely useful in clinical management as well as providing epidemiological information. However, since some reports (Wolf et al., 2006; Van Woensel et al., 2006; Garcia-Garcia et al., 2006) suggest no correlation between dual infections and clinical severity, larger studies using multiplex assays are required to clarify the situation. Compared with immunofluorescence, the one-step internally controlled multiplex RT-PCR assays were more sensitive for detecting respiratory viruses in this unique group of patients. Furthermore, the range of respiratory virus types detected can be increased by using the real-time assays compared with traditional methods. Further, the novel RNA internal control is simple and easy to produce, having no effect on the efficiency of the multiplex RTPCR reactions, including inhibition of the target sequences. This reliable system will improve surveillance of respiratory infections, as well as the management of patients in specific clinical settings in the advent of more antiviral agents being available.
References Aberle, J.H., Aberle, S.W., Pracher, E., Hutter, H.P., Kundi, M., Popow-Kraupp, T., 2005. Single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response. Pediatr. Infect. Dis. J. 24, 605–610. Bellau-Pujol, S., Vabret, A., Legrand, L., Dina, J., Gouarin, S., Petitjean-Lecherbonnier, J., Pozzeto, B., Ginerva, C., Freymuth, F., 2005. Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses. J. Virol. Methods 126, 53–63. Bredius, R.G., Templeton, K.E., Scheltinga, S.A., Claas, E.C., Kroes, A.C., Vossen, J.M., 2004. Prospective study of respiratory viral infections in pediatric hemopoietic stem cell transplantation patients. Pediatr. Infect. Dis. J. 23, 518–522. Brittain-Long, R., Nord, S., Olofsson, S., Westin, J., Anderson, L.-M., Lindh, M., 2008. Multiplex real-time PCR for detection of respiratory tract infections. J. Clin. Virol. 41, 53–56. Broughton, S., Roberts, A., Fox, G., Pollina, E., Zuckerman, M., Chaudhry, S., Greenough, A., 2005. Prospective study of healthcare utilisation and respiratory morbidity due to RSV infection in prematurely born infants. Thorax 60, 1–11. Broughton, S., Zuckerman, M., Auburn, H., Smith, M., Fox, G., Greenough, A., 2006. Chronic respiratory morbidity following viral lower respiratory tract infections in prematurely born infants. Paediatr. Infect. Dis. J. 1, 205–211.
H. Auburn et al. / Journal of Virological Methods 176 (2011) 9–13 Chidlow, G.R., Harnett, G.B., Shellam, G.R., David, W., Smith, D.W., 2009. An economical tandem multiplex real-time pcr technique for the detection of a comprehensive range of respiratory pathogens. Viruses 1, 42–56. Garcia-Garcia, M.L., Calvo, C., Martin, F., Perez-Brena, P., Acosta, B., Casas, I., 2006. Human metapneumovirus infections in hospitalised infants in Spain. Arch. Dis. Child. 91, 290–295. Gunson, R., Collins, N., Carman, T.C W.F., 2005. Real-time RT-PCR detection of 12 respiratory viral infections in four triplex reactions. J. Clin. Virol. 33, 341–344. Hodgson, J., Zuckerman, M., Smith, M., 2007. Development of a novel internal control for a real-time PCR for HSV DNA types 1 and 2. J. Clin. Virol. 38, 217–220. Hoorfar, J., Malorny, B., Abdulmawjood, A., Cook, N., Wagner, M., Fach, P., 2004. Practical considerations in design of internal amplification controls for diagnostic PCR assays. J. Clin. Microbiol. 42, 1863–1868. Lassaunièrea, R., Kresfeldera, T., Ventera, M., 2010. A novel multiplex real-time RTPCR assay with FRET hybridization probes for the detection and quantitation of 13 respiratory viruses. J. Virol. Methods 165, 254–260. Mackay, I.M., Jacob, K.C., Woolhouse, D., Waller, K., Syrmis, M.W., Whiley, D.M., 2003. Molecular assays for detection of human metapneumovirus. J. Clin. Microbiol. 41, 100–105. Manoha, C., Espinosa, S., Aho, S.L., Huet, F., Pothier, P., 2007. Epidemiological and clinical features of hMPV RSV and RVs infections in young children. J. Clin. Virol. 38, 221–226. Nolan, T., Hands, R.E., Bustin, S.A., 2006. Quantification of mRNA using real-time RT-PCR. Nat. Protocols 1, 1559–1582. Pierangeli, A., Gentile, M., Di Marco, P., Pagnotti, P., Scagnolari, C., Trombetti, S., Lo Russo, L., Tromba, V., Moretti, C., Midulla, F., Antonelli, G., 2007. Detection and typing molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in Rome, Italy. J. Med. Virol. 79, 463–468. Rosenstraus, M., Wang, Z., Chang, S.Y., Debonville, D., Spadoro, J.P., 1998. An internal control for routine diagnostic PCR: design, properties, and effect on clinical performance. J. Clin. Microbiol. 36, 191–197.
13
Semple, M.G., Cowell, A., Dove, W., 2005. Dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis. J. Infect. Dis. 191, 382–386. Scheltinga, S.A., Templeton, K.E., Beersma, M.F., Claas, E.C., 2005. Diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time RNA PCR. J. Clin. Virol. 33, 306–311. Tang, Y., Wang, Q., Saif, Y.M., 2005. Development of ssRNA internal control template reagent for a multiplex RT-PCR to detect turkey astroviruses. J. Virol. Methods 126, 81–86. Templeton, K., Scheltinga, S.A., Beersma, M.F., Kroes, A.C., Claas, E.C., 2004. Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4. J. Clin. Microbiol. 42, 1564– 2156. Van Elden, L.J., Van Loon, A.M., Van der Beek, A., Hendriksen, K.A., Hoepelman, A.I., Van Kraai, M.J., 2003. Applicability of a real-time quantitative PCR assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults. J. Clin. Microbiol. 41, 4378–4381. Van Woensel, J.B.M., Bos, A.P., Lutter, R., Rossen, J.W.A., Schuurman, R., 2006. Absence of human metapneumovirus co-infection in cases of severe respiratory syncytial virus infection. Pediatr. Pulmonol. 41, 872–874. Weinberg, W., Zamora, M.R., Li, S., Torres, F., Hodges, T.N., 2002. The value of polymerase chain reaction for the diagnosis of viral respiratory tract infections in lung transplant recipients. J. Clin. Microbiol. 25, 171– 175. Wolf, D., Greenberg, D., Kalkstein, D., Shemer-Avni, Y., Givon-Lavi, N., Saleh, N., Goldberg, M.D., Dagan, R., 2006. Comparison of human metapneumovirus, respiratory syncytial virus and influenza A virus lower respiratory tract infections in hospitalized young children. Paediatr. Infect. Dis. J. 25, 320– 324.
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Detection of nine respiratory RNA viruses using three multiplex RT-PCR assays incorporating a novel RNA internal control transcript Helen Auburn a , Mark Zuckerman a , Simon Broughton b , Anne Greenough b , Melvyn Smith a,∗ a b
South London Specialist Virology Centre, King’s College Hospital NHS Foundation Trust, Bessemer Wing, Denmark Hill, London SE5 9RS, UK Department of Child Health, 4th Floor Golden Jubilee Wing, King’s College Hospital NHS Foundation Trust, Bessemer Road, London SE5 9RS, UK
a b s t r a c t Article history: Received 11 February 2011 Received in revised form 9 May 2011 Accepted 11 May 2011 Available online 19 May 2011 Keywords: Respiratory viruses Multiplex RT-PCR RNA internal control
Real-time PCR is a significant improvement over viral isolation and immunofluorescence for routinely detecting respiratory viruses. We developed three real-time internally controlled multiplex RT-PCR assays for detecting nine respiratory viruses. An internal control transcript consisting of a chimeric plasmid was synthesised and incorporated into each multiplex to monitor amplification efficiency, including inhibition. Each multiplex assay was developed on the Rotor-Gene 3000 and evaluated using RNA extracts from 126 nasopharyngeal aspirates from 112 pre-term infants. All 44/126 (35%) samples positive by immunofluorescence were confirmed by multiplex RT-PCR. Additionally, respiratory syncytial virus RNA was detected in 5 samples, influenza A virus RNA in 2 samples and thirteen (10%) dual infections by multiplex RT-PCR were noted. Inclusion of the RNA internal control did not affect the amplification efficiency of the target sequences and only 2 of 1256 (0.2%) samples tested over a 12 month period were inhibitory. Together with the improved sensitivity of the internally controlled multiplex RT-PCR assays over the older technology and the ability to detect co-infections, the internal control monitored the efficiency of both the RT and PCR steps and indicated inhibition, saving time and costs on running duplicate samples with a “spiked” inhibition control. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Respiratory RNA viruses are associated with increased morbidity and mortality among high-risk populations including the elderly, immunocompromised individuals, pre-term infants and neonates (Weinberg et al., 2002; Van Elden et al., 2003). Real-time PCR is now recognised as the most sensitive and rapid choice for respiratory virus diagnosis compared with viral isolation and immunofluorescence. Several multiplex reverse transcription (RT-PCR) assays have been developed and evaluated employing real-time technology to detect respiratory viruses (Templeton et al., 2004; Bellau-Pujol et al., 2005; Gunson et al., 2005; Scheltinga et al., 2005; Brittain-Long et al., 2008; Chidlow et al., 2009; Lassaunièrea et al., 2010). Previously, nested PCR was used to increase sensitivity and specificity (Templeton et al., 2004; Bellau-Pujol et al., 2005). However, this prolongs time to detection and is a potential source of carry-over contamination leading to false positive results. An internal control is an essential requirement for routine diagnostic PCR assays to monitor nucleic acid extraction, amplification
∗ Corresponding author. Tel.: +44 020 3299 6974; fax: +44 020 3299 6476. E-mail address: [email protected] (M. Smith). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.05.017
efficiency and inhibition in clinical samples, since inhibitory samples are known to suppress amplification, decreasing the sensitivity of the PCR, leading to false negative results (Rosenstraus et al., 1998). This paper describes the development of three internally controlled multiplex one-step RT-PCR assays for respiratory virus detection on the Rotor-Gene 3000. Each multiplex was designed to detect three respiratory viruses according to seasonality, covering an extended range of the key pathogens. For specific detection of each viral amplicon, TaqMan probes were used that carried three different fluorophores, 6-carboxy-4,5-dichloro-2,7dimethoxyfluorescein (JOE), 5-carboxyfluorescein (FAM) and the sulphoindocyanine dye (Cy5), the internal control probe was labelled with 5-carboxy-X-rhodamine (ROX). To monitor for inhibition of the RT-PCR, a novel RNA internal control was developed, adopting approaches described by Hodgson et al. (2007) and Tang et al. (2005). This choice of internal control allows for increased stability and a straightforward synthesis when stocks are low. For clinical evaluation, results were compared with those from an immunofluorescence assay. Overall, 126 nasopharyngeal aspirates collected from 112 premature infants who presented with signs of lower respiratory tract infections during the winter
10
H. Auburn et al. / Journal of Virological Methods 176 (2011) 9–13
seasons of 2002/3–2004/5 were tested. This was part of a longterm prospective study investigating the impact of viral lower respiratory tract infections on respiratory morbidity and healthcare utilisation during infancy (Broughton et al., 2005, 2006). 2. Materials and methods 2.1. Preparation of viral culture for stock generation and direct immunofluorescence assay Viral cultures were set up by inoculating 200 l of nasopharyngeal aspirate onto 75 cm3 flasks of continuous human laryngeal carcinoma (HEp-2) cells for respiratory syncytial viruses A and B (RSV A and B), and on rhesus monkey kidney (LLC-MK2) cells for influenza A and B viruses and parainfluenza viruses types 1, 2 and 3. Subsequently, cell cultures were incubated for 1 week at 33 ◦ C and 37 ◦ C, respectively, and examined daily for a cytopathic effect (CPE). A positive CPE was confirmed by direct immunofluorescence with specific monoclonal antibodies according to the manufacturer’s instructions (Imagen, DakoCytomation, Cambridge, UK). Virus stocks were subsequently prepared for the multiplex RT-PCR assays according to standard procedures. 2.2. Positive control material for human metapneumovirus and human rhinovirus Nasopharyngeal aspirates positive for human metapneumovirus and human rhinovirus were identified through routine evaluation by monoplex RT-PCR and subsequently used for the development of the multiplex RT-PCR assays. 2.3. Viral RNA extraction Viral RNA was extracted from virus stocks and nasopharyngeal aspirates using QIAamp viral RNA minikits (Qiagen, West Sussex, UK) according to the manufacturer’s protocol. RNA was either tested immediately or stored in aliquots of 20 l at −80 ◦ C for up to seven days until RT-PCR analysis. 2.4. Primers and probes The primers and probes (Metabion, Martinsried, Germany) are described in Table 1. TaqMan probes and primers for human rhinovirus and (RSV A and B) were reported previously (Van Elden et al., 2003; Bredius et al., 2004; Gunson et al., 2005), although the VIC fluorophore was replaced with JOE. Primers for influenza A and B viruses, parainfluenza viruses types 1, 2 and 3 (Templeton et al., 2004) and human metapneumovirus (Mackay et al., 2003) were described previously. Additionally, primers were re-designed for parainfluenza virus type 2, and TaqMan probes were designed for influenza A and B viruses, human metapneumovirus and parainfluenza viruses types 1, 2 and 3. 2.5. Real-time multiplex one-step RT-PCR assay The cycling conditions and the concentrations of each primer and probe-set were evaluated to determine the optimal threshold cycle (Ct) for each monoplex (data not shown). Each multiplex was evaluated to ensure that no inhibition or cross-reactions occurred. Each multiplex was further tested with and without the internal control to ensure that no inhibition occurred and to determine its optimal concentration (Table 4). RT-PCR was carried out using Qiagen QuantiTect Probe RT-PCR kits (Qiagen, West Sussex, UK) with the internal control added to each extraction to monitor the complete RT-PCR. Each 30 l reaction mixture consisted of 1 l of RNA internal control (8 × 102 copies/l), 10 l of RNA eluate, forward
and reverse primers (10 pmoles each), TaqMan probes (5 pmoles each), 0.5 l of Qiagen RT enzyme, and 15 l of 2X QuantiTect Probe RT-PCR master mix in 200 l reaction vessels in a 72-well rotor (Corbett Research, Cambridge, UK). The RT-PCR consisted of 10 min reverse-transcription at 50 ◦ C, 15 min incubation at 95 ◦ C, followed by 50 cycles of: 15 s denaturation at 95 ◦ C and 30 s annealing and extension at 57 ◦ C. Fluorescence measurements were carried out at each annealing step and the total reaction time was approximately 1.5 h. Assay controls included PCR products for influenza A and B viruses, parainfluenza viruses types 1, 2 and 3, RSV A and B, human metapneumovirus and human rhinovirus, previously gel-purified using QIAquick gel extraction kits (Qiagen, West Sussex, UK). 2.6. Nasopharyngeal aspirates from pre-term infants Samples were placed on ice immediately after collection and delivered to the laboratory in viral transport medium. Viral culture and immunofluorescence were carried out on the day of collection by inoculating 200 l of each sample onto HEp-2 cells, human alveolar epithelial cell line A-549, human epithelial lung (HEL) cells and, LLC-MK2 cells. The remaining samples were stored at −80 ◦ C for up to seven days until RNA extraction. A positive cytopathic effect (CPE) was confirmed for influenza A and B viruses, parainfluenza viruses types 1, 2 and 3, and RSV by the immunofluorescence assay described previously. 2.7. Design of the RNA internal control The construction of the RNA internal control was based on the PCR methods reported by Tang et al. (2005) and Hodgson et al. (2007). The 188 bp amplicon for the control was produced using composite primers, incorporating the RNA internal control forward and reverse primer sequences and sequences complementary to the pGEM-T easy vector (Promega, Southampton, UK). The resulting internal control has identical primer-binding sites to the RNA internal control primers, but contains an internal sequence derived from the vector DNA and detected by a different probe and fluorophore combination (Table 1). The PCR programme consisted of an initial hold at 95 ◦ C for 10 min, followed by 50 cycles of: 15 s denaturation at 95 ◦ C and 60 s combined annealing/extension at 60 ◦ C. The amplicon was purified from a 2% agarose gel using a QIAquick gel extraction kit (Qiagen, West Crawley, UK) for cloning. 2.8. Cloning the chimeric insert into the pGEM-T vector system The purified insert was cloned into a pGEM-T easy vector according to the manufacturer’s instructions. The recombinant plasmids were extracted using QIAprep Miniprep kits (Qiagen, West Sussex, UK) following manufacturer’s instructions. 2.9. Synthesis of the RNA transcript for use as the internal control The RNA internal control was synthesised from linearised VZV/pGEM-T template using the MEGAshortscript T7 transcription kit (Ambion, Warrington, UK) according to the manufacturer’s instructions. RNA transcripts were purified with the MEGAclear kit (Ambion, Warrington, UK), treated with DNAse I (Ambion, Warrington, UK), and stored at −80 ◦ C. 2.9.1. Testing inhibition of the RNA internal control in the multiplex RT-PCR assays The detection limit of the RNA internal control was determined on a dilution series (8 × 1010 –8 × 10−1 copies/l). Amplification was initially performed using RNA internal control primers. Subsequently, to assess any inhibition of the multiplex RT-PCR assays by
H. Auburn et al. / Journal of Virological Methods 176 (2011) 9–13
11
Table 1 Primer and probe sequences used in the multiplex RT-PCR assays. Multiplex 1
Pathogen Influenza A virus Influenza B virus Human metapneumovirus
2
Respiratory syncytial virus A
Respiratory syncytial virus B
Rhinovirus 3
Parainfluenza virus type 1 Parainfluenza virus type 2 Parainfluenza virus type 3
1, 2, 3
RNA internal control Composite primers
Primer Sequences (5 , 3 ) c
(F) aaa gcg aat ttc agt gtg at (R) gaa ggc aat ggt gag att tc (F) gtc cat caa gct cca gtt ttc (R) tct tct tac agc ttg ctt gcc (F) cat gcc cac tat aaa agg tca ge (R) cac ccc agt ctt tct tga aae (F) aga tca act tct gtc atc cag caaa (R) ttc tgc aca tca taa tta gga gd (F) aag atg caa atc ata aat tca cag gaa (R) tga tat cca gca tct tta agt aa (F) tgg aca ggg tgt gaa gag cb (R) caa agt agt cgg tcc cat ccb (F) acc tac aag gca aca aca tcc (R) ctt cct gct ggt gtg tta atc (F) gga gat tgc ctc gat ttc acg acf (R) gtc tca gtt cag cta gat cagf (F) gga gca ttg tgt cat ctg tcc (F) tag tgt gta atg cag ctc gtc (F) acc tta aaa ctc act acc agtf (R) cta atc caa ggc ggg tgc atf (F) acc tta aaa ctc act acc agt GGC CGA GCG CAG AAG TGGf,g (R) cta atc caa ggc ggg tgc atG ACG AGC GTG ACA CCA CGf,g
Probe Sequences (5 , 3 )
Target Gene
Amplicon Size
JOE-cac cga aga ggg agc aat tgt tgg cg-BHQ1f Cy5-cct ccg tct cca cct act tcg ttc cc-BHQ2f FAM-tga gtc ttt gtc aag cag cgt tag cat g-BHQ1f FAM-cac cat cca acg gag cac agg aga t-BHQ1a
NS protein
103
NS protein
144
L gene
169
NP gene
83
Cy5-ttt ccc ttc cta acc tgg aca ta-BHQ2d
NP gene
102
JOE-tcc tcc ggc ccc tga atg-BHQ1d
5 UTR
143
FAM-ttg gtc tac aac ccg aaa tga taa ctc cac gg-BHQ1f JOE-tct gct gca ggg ttt cca att ttc agg act-BHQ1f Cy5-tgt ttc gga tgg cca gct cgt tta ctc-BHQ2f ROX-tcgccagttaatagtttgcgcaacgBHQ1f
HN gene
128
HN gene
249
HN gene
153
Vector
188
Abbreviations: F = forward; R = reverse. Primers and probe sequences published below: a Van Elden et al. (2003). b Bredius et al. (2004). c Templeton et al. (2004). d Gunson et al. (2005). e Scheltinga et al. (2005). f This study. g Sequences shown in upper case are derived from the pGEM-T easy vector.
the RNA internal control, respiratory viral targets (from nasopharyngeal aspirates) were assayed with and without the control (8 × 103 –8 × 101 copies/l), and the threshold cycles (Cts) compared (Table 4).
RT-PCR. Table 2 shows the results of the comparison of immunofluorescence and multiplex RT-PCR.
3.3. Detection of dual infections 3. Results 3.1. Evaluation of multiplex RT-PCR inhibition The initial concentration of the internal control transcript was determined using a nanodrop spectrophotometer (NanoDrop Technologies, UK) to be 300 ng/l and adjusted to 8 × 1010 RNA copies/l. The detection limit of the internal control was determined by testing it at ten-fold serial dilutions and found to be 8 × 100 copies/l. Respiratory virus RNA targets were amplified with and without the RNA internal control at dilutions ranging from 8 × 103 to 8 × 101 copies/l in the multiplex RT-PCR assays and the Ct values compared, the optimal concentration was found to be 8 × 102 copies/l. Table 4 shows that inclusion of the internal control resulted in similar Ct values of respiratory targets in comparison to amplification without the RNA internal control.
Thirteen (10.3%) dual infections were identified from nasopharyngeal aspirates from 12 infants by the multiplex RT-PCR assay (Table 3). These included: RSV B and human rhinovirus infections (n = 6), human metapneumovirus and RSV A (n = 2), human metapneumovirus and human rhinovirus (n = 1), human metapneumovirus and parainfluenza virus type 3 (n = 1), RSV A and RSV B (n = 1), influenza A virus and RSV A (n = 1) and influenza A virus and human rhinovirus (n = 1).
Table 2 Comparison of the numbers of respiratory RNA viruses detected in nasopharyngeal aspirates using immunofluorescence assays and real-time multiplex RT-PCR assays. Respiratory virus
Samples positive by immunofluorescence assay
Samples positive by multiplex RT-PCR assays
Influenza A virus Influenza B virus Human metapneumovirus Parainfluenza virus type 1 Parainfluenza virus type 2 Parainfluenza virus type 3 RSV A RSV B RSV (Total A + B) Human rhinovirus
3 0 Not tested 0 1 4 Not tested Not tested 36 Not tested
5 0 7 0 1 4 28 13 41 36
3.2. Clinical evaluation of the real-time multiplex RT-PCR assays A total of 126 nasopharyngeal aspirates collected from 112 preterm infants were used. Viral culture with immunofluorescence and multiplex RT-PCR were carried out on each sample. Forty-four samples (35%) were positive for at least one of the nine respiratory viruses in the multiplex assay, confirming all immunofluorescence positive results. Additionally, RSV and influenza A virus RNA was detected in five and two immunofluorescence-negative samples respectively. In total, 94 positives were detected (75%) by multiplex
12
H. Auburn et al. / Journal of Virological Methods 176 (2011) 9–13
Table 3 Dual infections detected in nasopharyngeal aspirates collected from 112 pre-term infants using the real-time multiplex RT-PCR assays. Respiratory virus dual infections
Number of dual infections
Human rhinovirus and RSV B Human metapneumovirus and RSV A Human metapneumovirus and human rhinovirus Human metapneumovirus and parainfluenza type 3 RSV A and RSV B Influenza A virus and RSV A Influenza A virus and human rhinovirus
6 2 1 1 1 1 1
Table 4 Ct values for multiplex RT-PCR with and without dilution series of the RNA internal control. (Each measurement represents the mean of three replicates.). Respiratory virus (RNA IC expressed as copies/l) RSV A + RNA ic 103 RSV A + RNA ic 102 RSV A + RNA ic 101 RSV A only RSV B + RNA ic 103 RSV B + RNA ic 102 RSV B +RNA ic 101 RSV B only Human rhinovirus + RNA ic 103 Human rhinovirus + RNA ic 102 Human rhinovirus + RNA ic 101 Human rhinovirus only Influenza A + RNA ic 103 Influenza A + RNA ic 102 Influenza A + RNA ic 101 Influenza A only Influenza B + RNA ic 103 Influenza B + RNA ic 102 Influenza B + RNA ic 101 Influenza B only Human metapneumovirus + RNA ic 103 Human metapneumovirus + RN0A ic 102 Human metapneumovirus + RNA ic 101 Human metapneumovirus only Parainfluenza virus type 1 + RNA ic 103 Parainfluenza virus type 1 + RNA ic 102 Parainfluenza virus type 1 + RNA ic 101 Parainfluenza type 1 virus only Parainfluenza virus type 2 + RNA ic 103 Parainfluenza virus type 2 + RNA ic 102 Parainfluenza virus type 2 + RNA ic 101 Parainfluenza type 2 virus only Parainfluenza virus type 3 + RNA ic 103 Parainfluenza virus type 3 + RNA ic 102 Parainfluenza virus type 3 + RNA ic 101 Parainfluenza type 3 virus only RNA ic 103 only RNA ic 102 only RNA ic 101 only
Ct
Ct of RNA IC
23.06 23.17 23.96 23.52 27.44 28.00 28.35 28.53 21.11 21.10 22.15 21.95 19.88 20.99 20.89 21.21 17.25 19.39 17.51 18.07 22.50 22.36 22.48 22.21 24.74 24.81 25.03 25.12 19.36 19.21 19.59 19.39 17.51 17.57 17.64 18.48 – – –
22.66 35.79 Undetectable – 22.28 25.53 31.37 – 22.14 25.97 42.99 – 21.88 25.02 30.66 – 22.14 29.94 Undetectable – 21.92 26.81 38.72 – 21.78 24.92 33.45 – 21.67 26.28 45.17 – 28.46 40.50 Undetectable – 23.68 27.46 33.02
Abbreviations: IC = internal control.
4. Discussion Inhibitory substances are found frequently in clinical samples, which may carry-over during nucleic acid extraction and can result in a significant reduction in the sensitivity of a PCR leading to inaccurate quantitative results, or worse, false-negative results (Hoorfar et al., 2004; Nolan et al., 2006). The inclusion of an internal control is an essential requirement for all real-time PCR assays for monitoring for the presence of inhibitory substances. The use of a plasmid-based DNA internal control in other real-time PCR assays in our laboratory has demonstrated satisfactory and reproducible results. In this study, a novel chimeric RNA internal control was developed to monitor inhibition of the multiplex RT-PCR assays. Although this choice of RNA internal control was not used to mon-
itor the quality of the RNA extraction in our assays, it can be included in the sample prior to extraction. Therefore, it has the added benefits of being multi-purpose, suitable for both in-house DNA and real-time RT-PCR assays and monitoring both extraction and amplification steps. Once the plasmid vector has been generated, a life-long supply of the RNA internal control can be transcribed in a quick and straightforward procedure. Over twelve months, only 2 (0.2%) out of 1265 nasopharyngeal samples were inhibitory by the RNA internal control for the multiplex RT-PCR assays. Real-time PCR is the preferred choice for respiratory virus diagnosis due to its increased sensitivity, a major limitation of viral isolation that depends on viable tissue culture cells for detection. From the clinical evaluation, the multiplex RT-PCR assays detected all samples positive by immunofluorescence (27.8%), with an additional five (5.6%) respiratory syncytial viruses and three (2.4%) influenza A viruses. Furthermore, of the 41 (32.5%) respiratory syncytial virus infections, 28 (22.2%) were type A and 13 (10.32%) type B. The other advantage of the multiplex RT-PCR is its ability to detect mixed infections, which are often undetected in viral culture with immunofluorescence due to the masking of cytopathic effects between one or more viruses. In this study, thirteen dual infections were detected in samples from twelve patients. The majority were RSV B in combination with human rhinovirus, followed by human metapneumovirus and RSV A. There is conflicting evidence that dual infections are known to enhance the clinical severity of lower respiratory infections (Aberle et al., 2005; Semple et al., 2005; Wolf et al., 2006; Pierangeli et al., 2007; Manoha et al., 2007) therefore, the detection of co-infections may be extremely useful in clinical management as well as providing epidemiological information. However, since some reports (Wolf et al., 2006; Van Woensel et al., 2006; Garcia-Garcia et al., 2006) suggest no correlation between dual infections and clinical severity, larger studies using multiplex assays are required to clarify the situation. Compared with immunofluorescence, the one-step internally controlled multiplex RT-PCR assays were more sensitive for detecting respiratory viruses in this unique group of patients. Furthermore, the range of respiratory virus types detected can be increased by using the real-time assays compared with traditional methods. Further, the novel RNA internal control is simple and easy to produce, having no effect on the efficiency of the multiplex RTPCR reactions, including inhibition of the target sequences. This reliable system will improve surveillance of respiratory infections, as well as the management of patients in specific clinical settings in the advent of more antiviral agents being available.
References Aberle, J.H., Aberle, S.W., Pracher, E., Hutter, H.P., Kundi, M., Popow-Kraupp, T., 2005. Single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response. Pediatr. Infect. Dis. J. 24, 605–610. Bellau-Pujol, S., Vabret, A., Legrand, L., Dina, J., Gouarin, S., Petitjean-Lecherbonnier, J., Pozzeto, B., Ginerva, C., Freymuth, F., 2005. Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses. J. Virol. Methods 126, 53–63. Bredius, R.G., Templeton, K.E., Scheltinga, S.A., Claas, E.C., Kroes, A.C., Vossen, J.M., 2004. Prospective study of respiratory viral infections in pediatric hemopoietic stem cell transplantation patients. Pediatr. Infect. Dis. J. 23, 518–522. Brittain-Long, R., Nord, S., Olofsson, S., Westin, J., Anderson, L.-M., Lindh, M., 2008. Multiplex real-time PCR for detection of respiratory tract infections. J. Clin. Virol. 41, 53–56. Broughton, S., Roberts, A., Fox, G., Pollina, E., Zuckerman, M., Chaudhry, S., Greenough, A., 2005. Prospective study of healthcare utilisation and respiratory morbidity due to RSV infection in prematurely born infants. Thorax 60, 1–11. Broughton, S., Zuckerman, M., Auburn, H., Smith, M., Fox, G., Greenough, A., 2006. Chronic respiratory morbidity following viral lower respiratory tract infections in prematurely born infants. Paediatr. Infect. Dis. J. 1, 205–211.
H. Auburn et al. / Journal of Virological Methods 176 (2011) 9–13 Chidlow, G.R., Harnett, G.B., Shellam, G.R., David, W., Smith, D.W., 2009. An economical tandem multiplex real-time pcr technique for the detection of a comprehensive range of respiratory pathogens. Viruses 1, 42–56. Garcia-Garcia, M.L., Calvo, C., Martin, F., Perez-Brena, P., Acosta, B., Casas, I., 2006. Human metapneumovirus infections in hospitalised infants in Spain. Arch. Dis. Child. 91, 290–295. Gunson, R., Collins, N., Carman, T.C W.F., 2005. Real-time RT-PCR detection of 12 respiratory viral infections in four triplex reactions. J. Clin. Virol. 33, 341–344. Hodgson, J., Zuckerman, M., Smith, M., 2007. Development of a novel internal control for a real-time PCR for HSV DNA types 1 and 2. J. Clin. Virol. 38, 217–220. Hoorfar, J., Malorny, B., Abdulmawjood, A., Cook, N., Wagner, M., Fach, P., 2004. Practical considerations in design of internal amplification controls for diagnostic PCR assays. J. Clin. Microbiol. 42, 1863–1868. Lassaunièrea, R., Kresfeldera, T., Ventera, M., 2010. A novel multiplex real-time RTPCR assay with FRET hybridization probes for the detection and quantitation of 13 respiratory viruses. J. Virol. Methods 165, 254–260. Mackay, I.M., Jacob, K.C., Woolhouse, D., Waller, K., Syrmis, M.W., Whiley, D.M., 2003. Molecular assays for detection of human metapneumovirus. J. Clin. Microbiol. 41, 100–105. Manoha, C., Espinosa, S., Aho, S.L., Huet, F., Pothier, P., 2007. Epidemiological and clinical features of hMPV RSV and RVs infections in young children. J. Clin. Virol. 38, 221–226. Nolan, T., Hands, R.E., Bustin, S.A., 2006. Quantification of mRNA using real-time RT-PCR. Nat. Protocols 1, 1559–1582. Pierangeli, A., Gentile, M., Di Marco, P., Pagnotti, P., Scagnolari, C., Trombetti, S., Lo Russo, L., Tromba, V., Moretti, C., Midulla, F., Antonelli, G., 2007. Detection and typing molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in Rome, Italy. J. Med. Virol. 79, 463–468. Rosenstraus, M., Wang, Z., Chang, S.Y., Debonville, D., Spadoro, J.P., 1998. An internal control for routine diagnostic PCR: design, properties, and effect on clinical performance. J. Clin. Microbiol. 36, 191–197.
13
Semple, M.G., Cowell, A., Dove, W., 2005. Dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis. J. Infect. Dis. 191, 382–386. Scheltinga, S.A., Templeton, K.E., Beersma, M.F., Claas, E.C., 2005. Diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time RNA PCR. J. Clin. Virol. 33, 306–311. Tang, Y., Wang, Q., Saif, Y.M., 2005. Development of ssRNA internal control template reagent for a multiplex RT-PCR to detect turkey astroviruses. J. Virol. Methods 126, 81–86. Templeton, K., Scheltinga, S.A., Beersma, M.F., Kroes, A.C., Claas, E.C., 2004. Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4. J. Clin. Microbiol. 42, 1564– 2156. Van Elden, L.J., Van Loon, A.M., Van der Beek, A., Hendriksen, K.A., Hoepelman, A.I., Van Kraai, M.J., 2003. Applicability of a real-time quantitative PCR assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults. J. Clin. Microbiol. 41, 4378–4381. Van Woensel, J.B.M., Bos, A.P., Lutter, R., Rossen, J.W.A., Schuurman, R., 2006. Absence of human metapneumovirus co-infection in cases of severe respiratory syncytial virus infection. Pediatr. Pulmonol. 41, 872–874. Weinberg, W., Zamora, M.R., Li, S., Torres, F., Hodges, T.N., 2002. The value of polymerase chain reaction for the diagnosis of viral respiratory tract infections in lung transplant recipients. J. Clin. Microbiol. 25, 171– 175. Wolf, D., Greenberg, D., Kalkstein, D., Shemer-Avni, Y., Givon-Lavi, N., Saleh, N., Goldberg, M.D., Dagan, R., 2006. Comparison of human metapneumovirus, respiratory syncytial virus and influenza A virus lower respiratory tract infections in hospitalized young children. Paediatr. Infect. Dis. J. 25, 320– 324.