Acute respiratory infection (ARI) of viral aetiology is one of the main causes of morbidity and mortality worldwide [1Hall MJ, DeFrances CJ, Williams SN, Golosinskiy A, Schwartzman A. National Hospital Discharge Survey: 2007 summary Natl Health Stat Report 2010; 29: 1-20-24.-4Niska R, Bhuiya F, Xu J. National hospital ambulatory medical care survey: 2007 emergency department summary Natl Health Stat Report 2010; 26: 1-31.]. Besides classical respiratory viruses (RV), i.e. influenza viruses (Flu), respiratory syncytial virus (RSV), human rhinovirus (HRV), parainfluenza virus (PIV) 1-4, adenovirus (ADV), enterovirus (EV) and human coronavirus (HCoV) OC43 and 229E [5Henderson FW, Clyde WA Jr, Collier AM, et al. The etiologic and epidemiologic spectrum of bronchiolitis in pediatric practice J Pediatr 1979; 95: 183-90.-9Atmar RL, Piedra PA, Patel SM, Greenberg SB, Couch RB, Glezen WP. Picornavirus the most common respiratory virus causing infection among patients of all ages hospitalized with acute respiratory illness J Clin Microbiol 2012; 50: 506-8.], other “new” RV have currently been added to this list, such as human metapneumovirus (hMPV) [10Van den Hoogen BG, Osterhaus DM, Fouchier RA. Clinical impact and diagnosis of human metapneumovirus infection Pediatr Infect Dis J 2004; 23: S25-32.], SARS-CoV [11Ksiazek TG, Erdman D, Goldsmith CS, et al. A novel coronavirus associated with severe acute respiratory syndrome N Engl J Med 2003; 348: 1953-66.], HCoV NL63 and HKU1 [12Van der Hoek L, Pyrc K, Jebbink MF, et al. Identification of a new human coronavirus Nat Med 2004; 10: 368-73.,13Woo PC, Lau SK, Huang Y, Tsoi HW, Chan KH, Yuen KY. Phylogenetic and recombination analysis of coronavirus HKU1, a novel coronavirus from patients with pneumonia Arch Virol 2005; 150: 2299-311.], human bocavirus (hBoV) [14Allander T, Tammi MT, Eriksson M, Bjerkner A, Tiveljung-Lindell A, Andersson B. Cloning of a human parvovirus by molecular screening of respiratory tract samples Proc Natl Acad Sci USA 2005; 102: 12891-6.] and others. Most RV cause benign and self-limited infections in immunocompetent adults. However, clinical impact of ARI increases when they affect children, elderly, immunocompromised individuals and/or subjects with chronic underlying diseases, in whom RV constitute an important cause of hospitalisations, mainly during the cold months of the year [15Monto AS. Epidemiology of viral respiratory infections Am J Med 2002; 112(Suppl 6A): 4S-12S.,16Navarro-Marí JM, Perez-Ruiz M. Virus respiratorios: viejos y nuevos virus. Revisión de métodos diagnósticos Enferm Infecc Microbiol Clin 2007; 25(Suppl 3): 60-5.]. They involve important costs for health systems and constitute a frequent cause of school and work absenteeism. In certain circumstances, emerging RV cause a great impact in health systems worldwide, as occurred with SARS-CoV and Flu pandemics, the last one taking place in 2009.

Aetiological diagnosis of viral ARI is crucial in order to avoid unnecessary antibiotic use, to establish the appropriate use of antiviral drugs and a comprehensive cohort of hospitalised patients that minimizes the risk of nosocomial transmission, and to provide epidemiological information for an early release of recommendations for the prevention and treatment and, finally, to reduce the overall costs derived from patients´ management.

Several diagnostic methods have been described for RV. They can be classified in direct detection methods, which detect the virus or a part of it in the clinical sample, including viral culture, antigen detection methods and molecular techniques; and indirect methods, based on the detection of immune response to the virus by serologic assays. In clinical practice, serology is not useful for the diagnosis of viral infections, such as ARI, that cause frequent recurrences and are mainly restricted to the respiratory mucosa. Thus, in these cases, direct detection of the virus in the site of infection has to be a priority [16Navarro-Marí JM, Perez-Ruiz M. Virus respiratorios: viejos y nuevos virus. Revisión de métodos diagnósticos Enferm Infecc Microbiol Clin 2007; 25(Suppl 3): 60-5.,17Loeffelholz M, Chonmaitree T. Advances in diagnosis of respiratory virus infections Int J Microbiol 2010; 2010: 126049.].

Advances in diagnostic virology for detecting RV have been focussed on nucleic acids amplification techniques (NAAT), since they are more sensitive than traditional methods such as viral culture or antigen detection assays. Moreover, NAAT are the only available method for most new RV because they do not grow or grow poorly in cell culture and for which no efficient antigen detection methods have been commercialised. NAAT offer additional advantages with respect to other classical methods as less turnaround time and less effect of virus viability on the efficiency of the assay [18Mahony JB. Detection of respiratory viruses by molecular methods Clin Microbiol Rev 2008; 21: 716-47.].

On the contrary, some disadvantages have been attributed to NAAT: increased reagents’ costs, higher hands-on time, the need of knowing at least a part of the target genome to design appropriate primers and/or probes. Indeed, many RV are RNA viruses, subjected to a greater genetic variability that may affect the sensitivity and reliability of the molecular method.

Currently, many commercial molecular assays have been developed that allow partial or complete automation of the procedure. Semi-automated or automated assays have solved in part one disadvantage of NAAT, which is the hands-on time. Many commercial NAAT require minimal management of the specimen, taking no more than few minutes.

With the increasing availability of easy-to-perform and multiplex NAAT for detecting RV, cost-effective diagnostic algorithms should be used. Whether a more or less complete virological study should be carried out on a certain patient’s sample, depends not only on the resources and type of laboratory, but also on clinic-epidemiologic aspects such as patient’s age, severity of the disease, underlying conditions, etc.

This article describes the molecular methods used to detect respiratory viruses and the contribution that increasing automation, especially on molecular assays, has made to improve laboratory diagnosis. The possibilities of integration of these new assays on each laboratory portfolio are also discussed.

Traditional direct methods for RV are viral culture and antigen detection assays. Among indirect methods, many serologic assays, such as EIA, haemmagglutination inhibition, complement fixation, etc. have been used for RV. However, serology is not useful in clinical practice since diagnosis must be achieved by demonstration of seroconversion or seroincrease of IgG antibodies, between acute-phase and convalescent-phase serum samples [16Navarro-Marí JM, Perez-Ruiz M. Virus respiratorios: viejos y nuevos virus. Revisión de métodos diagnósticos Enferm Infecc Microbiol Clin 2007; 25(Suppl 3): 60-5.,18Mahony JB. Detection of respiratory viruses by molecular methods Clin Microbiol Rev 2008; 21: 716-47.].

In past decades, viral culture was the gold standard for the aetiological diagnosis of RV. No automation has been reported for this technology. Rather, advances in this field of clinical virology have been focused on shortening detection times and on increasing sensitivity. Currently, this procedure is limited to reference laboratories that are provided with the appropriate infrastructure, with the main objective of recovering the viral strain for further genetic and/or antigenic characterisation.

Traditional viral culture requires at least 7 days for yielding a result. To solve this problem, the next step in the viral culture methodology was the introduction of the shell-vial assay, based on centrifugation-enhanced adsorption of the sample onto the cells monolayer reproduced on cover slips. Detection is commonly achieved by fluorescent assay with monoclonal antibodies on the cell monolayer. This method reduces the time to obtain a result to 24-48 h without a deleterious effect on sensitivity and specificity.

Although viral culture may represent an open system for detecting multiple viral pathogens, there is no single cell line that allows the growth of all RV, and there is no RV that may grow in any cell line. Thus, to detect the most prevalent RV, multiple cell lines must be used. The combination of shell-vial assay with the inclusion of different cell lines in the same vial has represented a subsequent step that has facilitated the recovery of RV isolates from cell cultures [19Navarro-Marí JM, Sanbonmatsu-Gámez S, Pérez-Ruiz M, De La Rosa-Fraile M. Rapid detection of respiratory viruses by shell vial assay using simultaneous cultura of HEp-2, LLC-MK2, and MDCK cells in a single vial J Clin Microbiol 1999; 37: 2346-7.-21Weinberg A, Brewster L, Clark J, Simoes E. ARIVAC consortium. Evaluation of R-Mix shell vials for the diagnosis of viral respiratory tract infections J Clin Virol 2004; 30: 100-5.]. NAAT have recently contributed to increase the sensitivity of viral culture. PCR assays can be carried out on the cell culture supernatant to detect the isolates. This approach has demonstrated the highest sensitive reading procedure, and has allowed identifying viral isolates in cell monolayers with no cytopathic effect [22Pérez-Ruiz M, Yeste R, Ruiz-Pérez MJ, Ruiz-Bravo A, de la Rosa-Fraile M, Navarro-Marí JM. Testing of diagnostic methods for detection of influenza virus for optimal performance in the context of an influenza surveillance network J Clin Microbiol 2007; 45: 3109-10.]. However, viral culture is not useful for detection of new RV, since they do not grow or grow poorly in cell culture. For this purpose, the best alternative is NAAT.

The availability of monoclonal antibodies has allowed the development of different technologies for detecting viral antigens from clinical samples. Fluorescent and chromatographic immunoassays are rapid, easy to perform and have proved their utility, mainly for RSV and Flu. The minimum (hands-on and hands-off) turnaround time that these techniques require for obtaining results have converted them as referral point-of-care diagnostic assays. However, sensitivity widely depends on the commercial method and on the type of patient and sample, ranging from 10% to 70% in the case of pandemic Flu [23Centers for Disease Control and Prevention. Evaluation of rapid Influenza diagnostic tests for detection of Novel Influenza A (H1N1) Virus — United States, 2009 MMWR 2009; 58: 826-9.]. Higher viral loads and thus, higher sensitivity of antigen detection methods for RSV and Flu have been observed in the paediatric population versus adults and in nasopharyngeal aspirates versus nasopharyngeal exudates [24Utokaparch S, Marchant D, Gosselink JV, et al. The relationship between respiratory viral loads and diagnosis in children presenting to a pediatric hospital emergency department Pediatr Infect Dis J 2011; 30: e18-23.,25Lieberman D, Lieberman D, Shimoni A, Keren-Naus A, Steinberg R, Shemer-Avni Y. Identification of respiratory viruses in adults: nasopharyngeal versus oropharyngeal sampling J Clin Microbiol 2009; 47: 3439-43.].

Recently, the goal of automation in these type of diagnostic tools has been achieved by a new commercial assay, the mariPOC^® respi test (ArcDia, Turku, Finland). This allows rapid detection and differentiation between viral and bacterial respiratory infections (Flu A, Flu B, RSV, hMPV, PIV 1, PIV2, PIV3, ADV and Streptococcus pneumoniae). The test is very simple to operate and hands-on-time is less than a minute per sample. Results for highly positive samples are obtained in 20 minutes whereas low positive and negative samples are reported in 2 hours.

The mariPOC^® system uses polystyrene microparticles as solid phase reaction carriers for the immunocomplex formation. Viral antigens are captured by specific antibodies fixed onto the microsphere and bind to a fluorescently-labelled conjugate. The amount of immunocomplexes formed on the microsphere is proportional to the analyte concentration. Detection of the fluorescence signal is carried out by separation-free two-photon excitation fluorescence detection assay, the ArcDia^TM TPX (TPX). In contrast to other antigen detection methods, TPX assay avoids the need for multiple washes, and readout is achieved by on-line monitoring of reaction kinetics [26Koskinen JO, Vainionpää R, Meltola NJ, Soukka J, Hänninen PE, Soini AE. Rapid method for detection of influenza a and B virus antigens by use of a two-photon excitation assay technique and dry-chemistry reagents J Clin Microbiol 2007; 45: 3581-8.]. Preliminary studies have reported a sensitivity and specificity of this method comparable to ultrasensitive fluorescent immunoassays (DELPHIA^® method) [27Koskinen JO, Vainionpää R, Soini AE. Novel multianalyte point-of-care test for pathogen-speci?c diagnosing of respiratory infections Clin Microbiol Infect 2009; 15(Suppl S4): S556.,28Toivola H, Koskinen JM, Lappalainen M, Ruonamo R, Antikainen P, Koskinen JO. Performance evaluation of multianalyte mariPOC® respiratory infection test for adenovirus types 1–8, 14, 19 and 21 Clin Microbiol Infect 2012; 18(Suppl S3): 787-8.] and offers the advantage of automated result readout, allowing connections to laboratory information systems.

NAAT, particularly the polymerase chain reaction (PCR), have expanded the range of pathogens that can be identified in clinical laboratories. They have shown their utility in clinical diagnosis of viral respiratory infections. Molecular assays have greater sensitivity than conventional methods as viral culture or antigen detection and indeed, they are able to detect new viral agents. Another advantage of NAAT compared with viral culture is that they do not require viable viruses. Thus, they have converted in the gold standard for detection of RV. Thus, most advances in clinical diagnostic virology have been made on NAAT.

NAAT are especially useful for detecting “low-positive” patients. Yearly, during periods of RSV and Flu epidemics, the availability of NAAT eases the management of hospitalised patients and the control of outbreaks, since antigen detection tests are not sensitive enough for certain population groups, such as immunosuppressed individuals.

Flu, HRV, PIV 1, PIV 3, PIV 4, ADV and HCoV (except SARS-CoV) usually cause upper respiratory tract infections (URTI), whereas RSV, hMPV and PIV 2 have been frequently associated to lower respiratory tract infections (LRTI) [18Mahony JB. Detection of respiratory viruses by molecular methods Clin Microbiol Rev 2008; 21: 716-47.].

Most RV are RNA viruses except ADV. Two alternatives are feasible for reversetranscription (RT) of viral RNA into its appropriate cDNA PCR target. One approach is the use of random and/or oligo(dT) primers in the RT step. Subsequently, PCR can be directed against any RV with specific primers. The main disadvantage of this procedure is the need for opening the RT tube to carry out the PCR. One-step RT-PCR protocols have been reported elsewhere for most RNA viruses, and solve the problem the former protocol has. These are based on the inclusion in the reaction mix of reversetranscriptases and DNA polymerases for both the RT and the PCR steps, or of enzymes with reversetranscriptase and DNA polymerase activities.

Monoplex PCRs have been published and commercial kits have been developed for detecting the most prevalent RV [18Mahony JB. Detection of respiratory viruses by molecular methods Clin Microbiol Rev 2008; 21: 716-47.]. Whether a simpler or a more complete virological study must be carried out depends on many factors: severity of the disease, type of patient, capacity of the laboratory, availability of validated and optimized trade-marked commercial kits, number of samples that the laboratory can process, etc.

Nevertheless, in many cases, clinical signs and symptoms of ARI are not specific of a single RV, and mild and severe infections have been described for all viruses mentioned above. Currently, monoplex PCR is not useful in a clinical laboratory equipped enough to conduct an extended aetiological diagnosis. However, the advent of the 2009 pandemic allowed many laboratories for equipping with the infrastructure necessary to carry out PCR assays for detection of pandemic Flu. In these circumstances, when a rapid answer is encouraging, monoplex PCRs provide an efficient response.

Multiplex PCR assays have been the most widely used molecular methods for RV detection in laboratories of clinical virology capable of developing in-house and/or robust published protocols. The advantage with respect to monoplex PCR is the cost savings compared to multiple monoplex PCRs and an easier processing of runs. Indeed, multiplex PCR extends the portfolio of virological diagnostic methods. Although advantages are strong enough to compensate for any disadvantage, multiplex PCR have intrinsic inconveniences: as the number of targets increase, the possibility of losing sensitivity and specificity increases as well, due to competition among primers and viral targets that consequently yields unspecific PCR products. Indeed, multiplex PCR has to be optimized to ensure the adequate amplification of each viral genome. Multiplex PCR assays have demonstrated that co-detections of RV in a sample are a common event [29Cilla G, Oñate E, Perez-Yarza EG, Montes M, Vicente D, Perez-Trallero E. Viruses in community-acquired pneumonia in children aged less than 3 years old: High rate of viral coinfection J Med Virol 2008; 80: 1843-9.]. Whether these co-detections express true infections by all viruses and/or prolonged shedding of certain agents is difficult to demonstrate, although some authors have pointed out the role of co-detections of RV in the severity of the disease [30Mansbach JM, Piedra PA, Teach SJ, et al. Prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis Arch Pediatr Adolesc Med 2012; 166(8 ): 700-6.]. Table 1 shows the most important advantages and disadvantages of different methods for detection of RV.

Table 1.

Methods for Detection of Respiratory Viruses from Clinical Samples. Advantages and Disadvantages

Table 2.

Fully-Automated Assays for Detection and Identification of Respiratory Viruses

Automation in molecular assays, aiming at a continuous reduction of hands-on time, yields higher reproducibility and offers possibility of detecting several pathogens with a minimum processing.

The availability of automated procedures for detecting RV is increasing day by day. This obliges the virologists to choose the best alternative taking into account laboratory throughput and epidemiologic, clinical and cost-effectiveness parameters since, in many instances, each commercial assay requires its own apparatus. Indeed, commercial diagnostic kits are less versatile and often, more expensive than well-optimized in-house PCR.

Molecular procedures for achieving automation goals for RV detection could be divided in two main straightforward phases: procedures and protocols for a stepwise automation (semi-automation) and fully-automated NAAT.

The optimal use of the diagnostic tools for RV detection depends on the correct choice of the method in combination with other clinic-epidemiologic criteria. The origin of the sample, patient’s age, predisposing factors for severe disease and the prevalence of a specific RV in a certain area and within a study period must be considered for an appropriate diagnostic algorithm. The overwhelming demand for RV detection in clinical samples has been evident and continuous since the advent of the 2009 influenza pandemic, when clinicians and health institutions have realised that, in many circumstances, an aetiological diagnosis of ARI is desirable. Thus, clinical laboratories must take into account all these points and the availability of infrastructure and resources, to organise their workflow and portfolio related to the diagnosis of viral ARI.

A rational algorithm for viral diagnosis of ARI could be divided in four levels of increasing use of diagnostic tools depending on the type of patient [53Navarro-Marí JM, Pérez-Ruiz M, Galán JC, et al. Circulation of other respiratory viruses and viral co-infection during the 2009 pandemic influenza Enferm Infecc Microbiol Clin 2012; 30(Supl 4): 25-31.].

Overall, viral diagnosis in outpatients with mild ARI is not probably cost-effective, and should not be routinely performed, except for studies from surveillance programs or of epidemiological interest.

Antigen detection methods for RSV and/or Flu may be enough for patients attended at emergency units that do not fulfil severity clinical criteria for hospitalisation. Some individuals should constitute an exception of this protocol as pregnant women, patients with severe underlying conditions and other special situations (immunosupression, obesity, residents in chronic-care facilities, etc) for which a more extended aetiological study and/or the use of NAAT for the most prevalent RV is encouraging [54Fiore AE, Fry A, Shay D, Gubareva L, Bresee JS, Uyeki TM. Centers for Disease Control and Prevention (CDC). Antiviral agents for the treatment and chemoprophylaxis of influenza. Recommendations of the Advisory Committee on Immunization Practices (ACIP) MMWR Recomm Rep 2011; 60: 1-24.-57Van Leeuwen JC, Goossens LK, Hendrix RM, Van Der Palen J, Lusthusz A, Thio BJ. Equal virulence of rhinovirus and respiratory syncytial virus in infants hospitalized for lower respiratory tract infection Pediatr Infect Dis J 2012; 31: 84-6.].

Accurate and timely diagnosis of viral ARI has additional benefits as preventing unnecessary use of antibiotics, thus, decreasing the overall costs of treatments and minimizing the selection of resistant organisms. Moreover, specific antiviral therapy is recommended for treating certain risk groups with Flu and RSV infection. Neuraminidase inhibitors are recommended in patients with suspected or confirmed Flu who require hospitalisation and/or in high-risk groups. Early treatment with neuraminidase inhibitors has demonstrated to decrease the risk of complications, to reduce the duration of the disease and to improve outcome [54Fiore AE, Fry A, Shay D, Gubareva L, Bresee JS, Uyeki TM. Centers for Disease Control and Prevention (CDC). Antiviral agents for the treatment and chemoprophylaxis of influenza. Recommendations of the Advisory Committee on Immunization Practices (ACIP) MMWR Recomm Rep 2011; 60: 1-24.]. Ribavirin is approved for the treatment of hospitalised infants, young children and immunocompromised adults with severe lower respiratory tract infections due to RSV, despite the limited evidence of benefit [58Ventre K, Randolph A. WITHDRAWN: Ribavirin for respiratory syncytial virus infection of the lower respiratory tract in infants and young children Cochrane Database Syst Rev 2010; 5CD000181,59Chemaly RF, Torres HA, Munsell MF, et al. An adaptive randomized trial of intermittent dosing schedule of aerosolized ribavirin in cancer patients with respiratory syncytial virus infections J Infect Dis 2012; 206(9 ): 1367-91.]. New automated assays may improve point-of-care diagnosis of RV since they include a wider range of pathogens within the same kit. In future, automated NAAT may be the alternative of antigen detection methods if costs become comparable and a reduction of turnaround time is achieved.

Samples from patients requiring hospitalisation with negative RSV or Flu antigen results should be subjected to extended virological study that includes viral culture assays and/or NAAT targeted at more or less RV depending on the severity of the disease and the need for a rapid result. With this premise, NAAT for detection of RSV and Flu (at the type and subtype level) in patients with less severe ARI, that do not require admission at intensive care units (ICU), should be carried out first of all. Otherwise, other molecular multiplexed methods of low complexity could be performed in certain patients that included hMPV, HRV and PIV detection, depending on patient’s age and/or the existence of underlying diseases. More complex NAAT that detect other less frequent RV should be reserved to certain situations such as more severe cases (ICU patients) in which previous NAAT for the most prevalent viruses have yielded negative results [60Kehl SC, Kumar S. Utilization of nucleic acid amplification assays for the detection of respiratory viruses Clin Lab Med 2009; 29: 661-71.-62Dundas NE, Ziadie MS, Revell PA, et al. A lean laboratory: operational simplicity and cost effectiveness of the LuminexTAG™ respiratory viral panel J Mol Diagn 2011; 13: 175-9.]. At this level, automated and semi-automated NAAT may provide higher throughput than in-house protocols.

In the future, the feasibility of introducing fully-automated NAAT in a wider range of health institutions can constitute an improvement in viral diagnosis of ARI. Basic and first-level hospitals may provide, as well, with an efficient point-of-care assay to ensure the appropriate management of their in-patients.

Automation in the field of clinical virology for diagnosis of ARI relies on molecular assays. New approaches have yielded point-of-care methods, optimal for a rapid detection of several RV.

Cost-effective laboratory methods must take into account the benefits of aetiological diagnosis depending on technical characteristics of the assays, on the availability of the appropriate resources and on clinic-epidemiologic criteria. Automated NAAT are expensive but probably the most cost-effective tools for severely ill hospitalised patients. Many automated commercial assays require their own instrument; thus, each laboratory should make its forecast for future to provide with the most feasible automation strategies. Since no expertise is required to carry out automated molecular assays, extended aetiological diagnosis of ARI can be achieved if first-level hospitals implement these techniques in their portfolio.