key: cord-333026-9f6ecg30
authors: Kompanikova, J.; Zumdick, A.; Neuschlova, M.; Sadlonova, V.; Novakova, E.
title: Microbiologic Methods in the Diagnostics of Upper Respiratory Tract Pathogens
date: 2017-03-03
journal: Clinical Research and Practice
DOI: 10.1007/5584_2017_10
sha: 
doc_id: 333026
cord_uid: 9f6ecg30

Upper respiratory tract infection (URI) is a nonspecific term used to describe acute infections involving the nose, paranasal sinuses, pharynx, and larynx above the vocal cords. The aim of this study was to provide a summary of the most common pathogens of URI and to compare advantages and disadvantages of traditional and new rapid microbiological tests used to identify them. Blood samples were simultaneously examined by the enzyme-linked immunosorbent assay (ELISA) and by the FilmArray Respiratory Panel for eight different pathogens in a total of 15 tests performed in nasopharyngeal swabs. The ELISA method is unable to identify the pathologic agent until the host’s immune system elicits a response. The method is readily available in many laboratories at a low cost, which puts less strain on economic resources. The FilmArray(®) Panel, on the other hand, is more expensive, but it is fast and exact in the identification of a broad spectrum etiologic agents. Nonetheless, since most repiratory tract infections are viral in origin and there is no treatment available, the diagnosis provided by the FilmArray Panel does not provide any additional clinical benefit and thus should be used only whenever necessary on the individual basis.

Upper respiratory tract infections (URIs) involve the moist surface of the eyes and eyelids, the nasolacrimal ducts, the middle ear, paranasal sinuses, mastoid air cells, and the main respiratory passage of the nose and throat as far as the epiglottis and vocal cords. Acute URIs include the common cold, pharyngitis, epiglottitis, and laryngotracheitis (Nester et al. 1995) . A variety of viruses, bacteria, fungi, and parasites can infect the respiratory tract. Transmission of organisms occurs by aerosol droplet or direct hand-to-hand contact with infected secretions, with subsequent passage to the nares or eyes. Most URIs are of viral etiology (Dasaraju and Liu 1996) . Epiglottitis and laryngotracheitis are exceptions with severe cases likely caused by Haemophilus influenzae type B. Bacterial pharyngitis is often caused by Streptococcus pyogenes. Bacterial and viral upper respiratory infections produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment depends on the correct identification of a pathogen involved as antibiotics provide little or no benefit with viral infections (Nester et al. 1995) .

The URIs are in 69% of viral origin (Mäkelä et al. 1998) . Orthomyxoviruses (influenza A and B), paramyxoviruses (parainfluenza and respiratory syncytial viruses), coronaviruses, adenoviruses, and enteroviruses (coxsackie and ECHO viruses) cause common cold. However, most colds are caused by more than 89 types of rhinoviruses (Musher 2003; Cooper et al. 2001 ). More than 40 strains of adenoviruses cause pharyngitis resembling a strep throat. Rhinoviruses are unresponsive to antibiotics and other medications that control bacterial infections. Antibiotic treatment of adenovirus infections is of no value and sometimes can even be harmful, because it supresses normal bacterial flora and enables the resistant opportunistic pathogens to grow in an uncontrolled way (Nester et al. 1995 (Murray et al. 2005) . The most common URI of bacterial origin is pharyngitis caused by Group A beta-hemolytic streptococci, with Streptococcous pyogenes as a main representative, which accounts for 5-10% of pharyngitides (Poole and Portugal 2005 

The FilmArray Panel is a multiplexed nucleic acid test intended for the simultaneous qualitative detection and identification of multiple respiratory pathogen nucleic acids in nasopharyngeal swabs. This new platform combines automated sample preparation, nucleic acid extraction, and polymerase chain reaction (PCR)-based detection from a single unprocessed sample in 1 h (BioFire Diagnostics; Salt Lake City, UT). This method allows for identification of 21 different respiratory pathogens from a nasopharyngeal swab, 18 of viral etiology and three of bacterial origin (Idaho Technology 2007).

In the present study we seek to determine the individual advantages and disadvantages of different diagnostic tools for pathogens underlying the URIs, as well as under which circumstances a specific method would have an advantage over another one. Table 1 . These results corresponded with a number of respiratory infections, as diagnosed before hand, such as URIs, pneumonia, acute inflammation of nasopharynx, acute bronchitis, hypothermia unrelated to external temperature, and undefined fever.

For comparison, specimens obtained from two patients were tested simultaneously with traditional ELISA and FilmArray Panel methods. A venous blood sample and a nasopharyngeal swab were taken from both patients for either method, respectively. In one of these patients, all results were negative and no etiologic agent could be identied by the ELISA method. In total, 28 days were was required to obtain the full panel of results (Table 2 ; Patient 1). In addition, culture of the specimen obtained from a nasopharyngeal swab from the same patient also was negative for any pathogens. However, nasal swab specimen, examined by the FilmArray Panel, yielded a positive result for human rhinovirus or human enterovirus (Table 3 ; Patient 1), which was negative when with ELISA.

ELISA performed in a second patient showed an elevation of IgG against parainfluenza virus 1 (IgG positive -1.53, cut off -0.709, index -2.1). This result, however, is inconclusive regarding an acute current infection as it rather confirms a previous encounter with the virus. The result regarding influenza A infection was also negative (IgM -0.355, cut off -0.832, index -0.4; and IgG -0.083, cut off -0.381, index -0.2). The testing time for all eight pathogens amounted to 22 days (Table 2 ; Patient 2). The examination with the FilmArray Panel in this patient identified the etiologic agent as influenza A/H1-2009 (Table 3 ; Patient 2). The identification had to be confirmed with a serum hemagglutination inhibition test (HIT), which was done at the Department of Virology of the Regional Institute of Public Health, in Banská Bystrica, Slovakia. The HIT was performed twice 2 weeks apart to assess a possible change in antibody titers. While in the first sample influenza A and B titers were negative, there was a significant rise in the antibody titer against Table 4 . Health insurance gave 600 points for each of the 15 tests, which makes a total 9000 points, plus 150 points for sample culture from upper respiratory tract and 320 points for a culture form lower respiratory tract. Each insurance point has a value of 0.0066 €. The laboratory therefore received 59.40 € for running the ELISA and additionally 3.10 € for the cultures, which sums up to a total of 62.50 € or 224% of the real material costs. On the other hand, health insurance gave 2500 points for each of the 21 pathogen targets in the FilmArray Panel, which makes a total of 52,500 points and comes to 346.50 € or 210% of the real material costs amounting to 165.00 € per sample. The insurance reimbursed 11.70 € for influenza A and B testing each. Each type of influenza was tested twice giving the cost of 46.80 € in total. The actual laboratory costs for all HIT tests were 8.00 €, which is about 6 times less than the insurance reimbursement.

Laboratory testing is generally not recommended in the evaluation of upper respiratory infections. Tests for specific pathogens are helpful when therapy depends on the results. Targeted therapy is not available for most viruses that cause URI. Therefore, viral testing is rarely indicated for uncomplicated URIs in the outpatient setting. However, confirmation of a viral condition such as influenza may reduce inappropriate use of antibiotics (Balentine and Siamak 2015) . Considering the benfits for patient, the speed to identify the etiologic agent clearly favors the FilmArray System. This method readily identified the 

etiologic agent in both patients in whom it was applied in the present study, whereas the HIT identified the virus only in the repeat sample of the second patient. However, identification of the etiologic agent did not have any benefit for the first patient with mild symptomatology of coryza because no targeted treatment for human rhinovirus/enterovirus infection was needed. The symptomatology in the second patient was severe and the speed of pathogen identification is crucial for the commencement of appropriate treatment, especially in suspected cases of influenza where early administration of neuraminidase inhibitors significantly reduces mortality rates. In such cases, accuratelly targeted therapy has an enormous benefit for the patient. The laboratory costs to run one examination with different methods showed that the FilmArray multiplex PCR respiratory panel is more expensive than the ELISA, HIT, and the cultivation. One examination with the FilmArray panel brought a 181.50 € per patient profit for the laboratory, whereas the profit from running ELISA together with cultivation was 34.62 €, and that from HIT was 38.80 € per patient. Therefore, FilmArray respiratory panel is best in terms of profit margin for the laboratory and least favorable for health insurance.

Serologic diagnostic methods, such as ELISA and HIT, cannot identify the pathologic agent until the host's immune system elicits a response. However, advantages of those methods are that they are readily available in many laboratories and are least pricey for health insurance. The disadvantage is that the spectrum of pathogens detected is small. A clinical benefit of the FilmArray respiratory panel is that it is quick and exact and may identify a broader spectrum of possible pathogens. Since most URIs are viral in origin and there is no treatment available, the diagnosis provided by the FilmArray panel is not always necessary and the method should be used on an individual basis when clinically justified. From the economic standpoint, FilmArray respiratory panel is the most profitable for the laboratory. In critically ill patients, a spectrum of diagnostic methods should be used to obtain diagnosis as fast as possible. In patients with minor respiratory tract infections, a more rational approach should be undertaken since the speed and accuracy of diagnosis are less crucial. The decision to choose a specific diagnostic method rests with the medical caregiving staff.

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Acknowledgments This study was supported by Department of Microbiology and Immunology. We are thankful to our colleagues who provided expertise that greatly assisted the research.

The authors declare no conflicts of interest in relation to this article.