The Identification of Respiratory Viruses

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Table of Contents

Literature Review
Methods to Be Analyzed
Sensitivity Tests: CDS and CLSI
Manual Identification Methods
Emerging Technologies, Novel Testing Methods, and Latest Instrumentation
Fully Automated Methods
Conclusion
References

Literature Review

The identification of respiratory viruses is a critical task as it determines the ability of a specialist to target an effective intervention plan and to take the essential preventative measures. Therefore, the problem of selecting an appropriate identification method should be considered particularly attentively. Today, evaluation methods are abundant and varied in simplicity, rapidity, and qualifications requirements [1].

The present paper provides a review of relevant literature and summarizes the scientific findings related to different evaluation approaches and techniques. The key aim of the literature review resides in providing a guideline for the laboratory that seeks to improve its sample processing strategy within a three-month period.

Methods to Be Analyzed

Target Organisms

The table below illustrates the scope of these organisms and provides a brief overview of the available methods.

Organism	Associated Diseases	Available Identification Methods	Proposed Method	Rationale
Klebsiella species	Pneumonia, empyema, lung abscess, septicemia in lungs, community acquired pulmonary infection [2]	Phenotypic identification (biotyping, bxylosidase tests, etc.), molecular identification (PCR assay, MALDI-TOF, etc.), serotyping	Molecular identification	Fast, efficient, and reliable
Streptococcus pyogenes	Pharyngitis, Tonsillitis, Epiglottitis [3]	Gram staining, catalase test method, latex test agglutination test	Direct culture	Cheap and reliable
Corynebacterium diptheriae	Diphtheria, inflamed pseudomembrane, starts manifest on tonsils, larynx and trachea, sore throat, and fever [4]	Tellurite agar, Gram stain, biochemical tests, API, MALDI-TOF	MALDI-TOF	100% accuracy, rapid identification, relatively cheap
Aspergillus species	Invasive pulmonary aspergillosis, chronic necrotising aspergillosis, aspergilloma [5]	Subculture from primary culture, cyclohexamide responses test, Temperature tolerance test, PCR, MALDI-TOF	Subculture from primary culture (CZA)	Cheap, can metabolize inorganic nitrogen; might be likewise applied to identifying Candida Albicans
Pseudomonas species	Predominantly Paeruginosa, sinusitis, nosocomial pneumonia [6]	Gram staining, culture, biochemical tests, nucleic acid detection, APU, MALDI-TOF	Gram staining and culture	Low-cost and simple to apply
Haemophilus species	Epiglottitis, otitis media, sinusitis, tracheobronchitis, exacerbation of chronic bronchitis, and pneumonia [6]	API NH kit, VITEK, X, and V-Factor test, Porphyrin synthesis (ALA) test	Porphyrin synthesis (ALA) test	Results in 4 hours, easy to use, relatively cheap
Haemophilus Influezae	Infections in meninges, subcutaneous tissues, pleura, lungs, etc. [7]	Primary culture, gram staining, tests for growth factors X and V, Serotyping by slide agglutination	Serotyping by slide agglutination	Distinguishes encapsulated strains from unencapsulated strains
Staph species	Pneumonia, hospital-acquired, bronchitis, hospital acquired, lung abscess, empyema [8]	API STAPH, RAPIDEC Staph, saphaurex		Standardized, reliable, simple to use, identifies a wide scope of species
Moraxella Catarrhalis	Otitis media, sinusitis, laryngitis, bronchitis, bronchopneumonia, exacerbations in COPD [9]	Culture, gram staining, Cat screen	Cat screen	Simple to use, immediate results, does not require setting a susceptibility test
Candida species	Candida pulmonary disease, tracheobronchitis, laryngeal candidiasis, oropharyngeal candidiasis, oesophageal candidiasis, pneumonia [6]	CHROM Agar, BIGGY Agar, Germ Tube Test, API, MALDI-TOF, PCR, VITEK,	CHROM Agar	Cheap, short turnaround time, does not require further biochemical test
Samia Ibrahim	Tuberculosis [3]	PCR, antimicrobial susceptibility test, serology test, DNA fingerprint method, molecular method	Molecular method	Easy to use, ensures high accuracy and sensitivity
Streptococcus pneumoniae	Pneumonia [3]	Optochin test, commercial latex agglutination kits	Optochin test	Cheaper techniques with similar results
Legionella species	Pontiac fever, Legionnaires’ disease	Culture-based techniques and biochemical tests	Culture-based techniques	Detects a large scope of Legionella species

The table above illustrates the methods that can be currently used in the laboratory. In the meantime, it is likewise proposed to review the techniques that can be implemented in the nearest future. First and foremost, it is essential to point out the criteria that will be applied to the analysis of the manual identification kits. Hence, the key aspects that will be considered are the methods’ cost, exploitation simplicity, efficacy, accuracy, and turnaround time [3]. The target organisms can be viewed in the table above.

Sensitivity Tests: CDS and CLSI

The two methods that should be analyzed in this context are CLSI sensitivity testing and CDS sensitivity testing. The methods offer similar frameworks for carrying out control diffusion methods: the examined strains are defined as resistant, susceptible, and less susceptible [3]. The CDS method is preferable considering the specificity of the context described above. A recent study revealed a series of CDS’ competitive advantages over the CLSI. Firstly, the CDS method proves to be more accurate as it offers complete agreement with the Etest minimal concentration opposite to the CLSI technique. Secondly, the CDS method is better suited for use in laboratories that work with small specimen numbers. Third, the techniques showed higher cost-effectiveness and simplicity of use. Finally, the output generated under the CBS method can be easily processed and directly compared between local and international laboratories [10]. Upon considering the advantages described above, it is recommended to choose the CDS sensitivity test.

Manual Identification Methods

Oxoid Strep Grouping and Prolex Strep Grouping

Oxoid strep grouping helps identify streptococcal groups demonstrating high accuracy and short waiting time. However, the method is rather costly as it requires purchasing a set of the relevant kits [11]. This technique is commonly compared to the Prolex strep grouping method aimed at rapid evaluation of a wide range of organisms. Proflex Streptococcal Grouping Latex Kit is a specially designed platform that helps identify a wide scope of streptococcus organisms at a minimal waiting time. Researchers point out that this technique might require carrying out further biochemical tests [12, 13]. At that, the method is low-cost and effective, which makes it all the more commendable for inclusion in the manual. The cost of the relevant set is less considerable [14]. Practice reveals that this test shows the shortest agglutination time in contrast to other tests of similar character [15]. Therefore, the Proflex strep grouping method is considered to be more appropriate for the improvement of the sample processing standards in the laboratory.

Gram Stain

This technique might be the least costly of all the methods described above. Amid the health market technology turnabout, this method is still widely used due to its simplicity and high accuracy [13]. The technique can be applied to the identification of A. haemolyticum, Serratia species, Legionella species, and other organisms. The reliability of Gram Stain is proved by a vast body of empirical evidence.

Staph Latex Testing

This technique is aimed at identifying the presence of staphylococci colonies. The use of the kit does not require many instruments or special skills. The essential kit can be purchased from many vendors at a reasonable cost. Practice shows that the test ensures accurate results and can be carried out in small laboratories, which speak of it as commendable [14].

On the whole, four methods can be recommended on the basis of the literature review: the Prolex strep grouping, Staph latex testing, gram staining, and the CDS sensitivity test.

Emerging Technologies, Novel Testing Methods, and Latest Instrumentation

Semi-Automated Methods

API

The efficacy of the API method seems to be one of the most ambiguous questions based on the literature analysis. API is a well-established technique of identifying microorganisms to the levels of species. The identification kits and other products manufactured by BioMérieux (API’s producer) can be used to determine Gram-positive and Gram-negative bacteria. One of the unique benefits of API is the durability of the test strips, making it possible to always have an API test at hand. However, some researchers state that the test results are not as accurate as they could have been with the application of other identification techniques, such as Vitek [16]. Other studies show that the difference is not significant and that the accuracy of the method’s results is still higher than that retrieved through conventional techniques. From the standpoint of the time required to receive the result, the method is likewise similar to those described above. From this perspective, it allows for receiving the necessary data rapidly [17]. The cost-effectiveness of the method is determined not only by the expenses that the laboratory will bear due to the purchase of the necessary equipment but the fees spent on the personnel training as well. As a result, taking into account that the implementation of this technique is rather costly, while its efficiency is ambiguous, this method cannot be recommended in the framework of the sample processing improvement strategy.

Cat Screen

The Cat Screen test allows detecting the enzyme butyrate esterase in order to identify Moraxella catarrhalis. Although there is little information on the applicability, efficiency, simplicity, and precision of the test, it is known to be commonly used along with such techniques as Gram stain and oxidase test. The significance of this particular method consists primarily in its rapidity. Similarly to a number of other rapid tests capable of confirming the presence of Moraxella catarrhalis, the Cat Screen is reliant on the species’ capability to hydrolyze tributyrin, which enables the test to identify it immediately and distinguish between Moraxella and other bacteria, such as the Neisseria, which does not take part in tributyrin hydrolysis. The technique is reportedly simple to apply and ensures fast results. Another advantage of the method is that it implies no need for purchasing additional equipment. The kit can be purchased from a wide range of vendors [18]. It might be recommended that this method is implemented as a supplementary tool to provide the confirmation of the Moraxella catarrhalis identification.

Fully Automated Methods

PCR

PCR tests are capable of identifying a wide scope of influenza organisms, respiratory syncytial viruses, parainfluenza, etc. The test ensures rapid diagnostics and high results accuracy. Generally, there are eight PCR tubes available that serve to determine different types of organisms. The test is applied to the RNA and DNA retrieved from respiratory samples. It is currently considered to be a convenient alternative to the direct fluorescent-antibody assay (DFA). Developed PCR panels can identify up to 20 types of viruses [19]. Still, PCR is characterized by its amenability to inhibitors and contaminations, as well as its uneven stability in lab conditions, which constitute one of the major drawbacks of PCR application to the clinical specimen. Additionally, and not least because of the high sensitivity of the test, its results can be affected by a number of variables (what the target genes are, by what means the DNA is extracted, what methods are used to detect PCR products, etc.). In order to achieve maximum precision and account for all the variables, each application requires a procedure of calibration, which can be lengthy. At the same time, the method’s cost-effectiveness is disputed actively. Recent research examined the correlation between the test cost and its efficacy; according to the findings, and despite the fact that the method can be characterized as costly, it still proves to have a competitive advantage over the DFA technique [20]. Thus, the introduction of the new method will naturally require additional expenses, which are nevertheless likely to be compensated for in about a one-year period [21]. As a result, this method might be recommended for implementation.

Maldi-Tof

The matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) is used to perform rapid identification of organisms and ensure the discovery of biomarkers relevant to respiratory diseases. The key advantage of this technique resides in the fact that it allows to carry out faster identification and target the immediate treatment course [22]. This technique is considered to be a convenient alternative for such conventional methods as gram staining or sample cultures. The favorability of this method is further explained when one considers that polymers and dendrimers have a tendency to destabilize and fall to fragments when subjected to conventional techniques. The three steps necessary to complete the process include the application of the sample to a metal plate, its irradiation with a pulsating laser, and the very ionization of the molecules that ablate upon the irradiation. Although in theory, the process may seem lengthy, the main aim of implementing this technology is to reduce the detection time to the minimum [23]. The accuracy of the method has been empirically proved: recent research has revealed that the MALDI-TOF agreement level is significantly higher than that of phenotypic methods [24]. The MALDI-TOF spectra are oftentimes used together with other tests to locate diseases such as NEC. Devastating as it might be, the disease is more easily (and cost-effectively) located through MALDI-TOF feces analysis to differentiate mutated and functioning proteins. It might be suggested that the adoption of this method will be rather consuming from a financial perspective. However, the efficacy prospects are considered to be worth the contribution.

Vitek

Vitek is another product manufactured by BioMérieux: it is a fully automated method that allows identifying a wide range of species. The technology helps to carry out rapid and rational decision-making. One of the key advantages that it offers is the diversity of species that Vitek can process [25]. Hence, it is capable of identifying geographically diverse isolates, samples of different origins, and isolates that have a varied incubation period. The sample variance is not the only benefit of this method; some other advantages include comprehensibility achieved through user-friendly Windows-based software and a well-organized database, test cards coming in several generations, high discrimination, and extensive base of species – the features allowing for accuracy [26]. With these benefits in mind, research results evidencing the precision of the data retrieved with the help of Vitek being significantly higher than that collected through conventional phenotypical methods are not surprising [27]. Despite the self-proclaimed comprehensibility of the software, operating the technology requires special skills. The personnel needs to receive the training to learn to manage the Vitek databases and ensure their compliance with the corporate software – but then, the benefits of it compensate for the effort required. As long as the technology is integrated and the employees get used to the new program, the speed of their performance is likely to increase considerably [28].

Summarizing the analysis presented above, it must be pointed out that the Vitek technology seems to be the most reliable method for the automatic identification of organisms. This method does not receive an ambiguous assessment in the research overviews but is always singled out as effective and accurate. Other tests, such as the Cat Screen, lack reliable data and can only be implemented in tandem with other techniques. Vitek, on the other hand, can be successfully used separately and although it requires preparatory training before usage, the effort is justified. As a result, it is assumed rational that the laboratory should consider adopting the Vitek method.

Conclusion

The analysis presented above considers the efficacy of particular methods from different perspectives: cost, turnabout period, simplicity, etc., while considering the feasibility of implementing a certain method; it also evaluates whether this change will require additional training for the personnel. The evidence for the method’s efficacy and inefficacy was retrieved from peer-reviewed sources only and was considered valid in those cases only when the researchers provided some empirical evidence of the method’s success.

Thus, based upon a detailed review of the relevant literature, a series of evaluation techniques can be recommended. First, it is proposed to choose the CDS sensitivity test instead of the CLSI test. Second, the following manual identification methods should be recommended on the basis of the researchers’ feedback: the Prolex strep grouping (opposite to Oxford step grouping), Staph latex testing, and gram staining. As for the implementation of a semi-automated method, the laboratory can use the Cat Screen technique which implies the simplest and cheapest exploitation options. However, it is proposed that the laboratory prefers to implement a fully automated method to enhance the standards of the sample processing approach. In this case, it can pay attention to the Vitek method that represents a fine balance of cost and result accuracy. Alternately, it might consider the including Maldi-Tof in the manual as well.

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