DISCUSSION

The high detections of viral pathogens in this study are not surprising though such a broad- spectrum etiology research has not been done previously in the study location. Most upper respiratory infections are of viral etiology9_{. In a study of viruses and bacteria in the etiology}

of the common cold, different strains of rhinoviruses were found to be the primary cause of the infection28_{. Further, it has been indicated previously that viruses such as coronavirus,}

influenza virus, respiratory syncytial virus (RSV), parainfluenza virus, adenovirus, and Metapneumovirus are contributing agents to common cold.29

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It is important to understand detection of cytomegalovirus, which is one of the eight herpes viruses that affects humans, with a likelihood to infect immunocompromised patients30_{. The}

RPM also detected an Influenza A matrix gene that is generic and the strain of the Influenza A pathogen is unknown. Surprisingly, serotypes for influenza A can be detected using FilmArray. However, given that screening via FilmArray showed no presence of influenza serotypes, this affirms the broader detection scope and higher resolution of the RPM system. Influenza increases in clinical severity with increase in the age of the patient and persons with other health disorders, causing an intermittent seasonal morbidity and mortality worldwide31_{. The global burden of influenza varies every year with higher burdens in seasons}

when influenza A (H3N2) viruses predominate32_{, rather than influenza A (H1N1) and B}

viruses33,34_.

It was surprising that there was no detection of RSV though investigated at the three tiers from the nasal and throat swabs. This poor performance of the tests for detecting RSV could be due to the sample type used; nasal and throat swabs instead of nasal wash or nasopharyngeal aspirate. Nasopharyngeal aspirate (NPA) or nasal wash specimens have been reported35,36,37, _{to be sufficient for sampling respiratory viruses though the use of NPAs is}

not ideal for children. In a comparative specimen sampling, using NPAs, throat swabs, (TS) and nasal swabs (NS) in the viral etiologies of acute respiratory infections; Do et al38_,

reported that the overall diagnostic yield from NPAs was superior. In a related study, Sung et al39 _{compared NPA and NS specimen collection methods to results from three different}

assays: Immunofluorescence test (IF), viral culture (VC) and more importantly, PCR. Using the Cohen Kappa test (95% Confidence Interval, 0.6-0.8 meaning high agreement) there was a high concordance between NS and NPA for PCR tests involving Adenovirus, Influenza A, Influenza B, Parainfluenza and RSV. Do et al.38_{recommended the combination of NS and}

TS for generally sampling .

In the area of diagnosis, current methods employed to diagnose and detect respiratory pathogens include various polymerase chain reaction (PCR) assays40,41,42 _{and antigen tests}

using different fluorescence and enzyme-linked immunoassays 43_{. Though enzyme-linked}

immunoassays are easy to use and suited for use especially in the field and in cases where many samples are to be tested for pathogens, it was not available for this study.Rapid lateral flow immunoassays were used. They are however unreliable in the sense that: they

sometimes fail to detect pathogens and they only indicate the presence or absence of a pathogen but not the genetic sequence, concentration or other information that may be relevant to clinicians or researchers. PCR, on the other hand, is currently used not only as a confirmatory assay but also as a diagnostic test. It is economical in terms of time and resources, but the several methods for real-time PCR assay design have very low sensitivities for many clinical applications44_{, this is due mainly to the probe design and quality of primers.}

Also, in resource-poor settings PCR is not applicable in many areas due to resource limitations and low technical capabilities, but the rapid automated PCR system used in this study, is easy to use, runs for a shorter period and reduces turn-over time. However, unlike other PCR assays, the thermocycler processes one sample at a time and is not ideal for handling bulk samples during outbreak situations and is also expensive to run, as each pouch cost about 100 US dollars, making 120 pouches worth 12,000 US dollars, excluding value of operator time.

Generally, there were detections of bacterial pathogens on both molecular platforms used, with pathogens: Streptococcus pneumonia, Klebsiella pneumonia, Chlamydophila pneumonia and Mycoplasma pneumonia, four bacterial causative agents associated with atypical and community-acquired pneumonia45,46_{; Haemophilus influenzae, which exist both}

as a commensal in adults47,48 _{and as a serious source of morbidity in children, causing}

meningitis, pneumonia and bacteremia47,48_{; Moraxella catarrhalis which is responsible for}

certain bronchopulmonary infections in adults and otitis media and sinusitis in children49,50

. These results are consistent with other studies that have also indicated the role bacteria play in infections of the nasal sinuses and pharynx leading to rhinosinusitis and pharyngitis, epiglottitis and laryngotracheitis9_{. In addition pneumonia disease alone can kill over 1.6} million people each year, with vast a majority of its victims coming from the world’s poorest

countries such as Sierra Leone. 51_{. This study also indicates high bacterial activity in the}

causation of respiratory infections in Bo, Sierra Leone.

Though computational methods, including gene resequencing, microarray scan analysis, and comparisons against data found on GeneBank by alignment, do confirm the presence of certain pathogens; biological methods used in this study can be a cause for the contrast in detection and identification of emerging and infectious respiratory agents. The storage of the nasopharyngeal swab samples is a possible source of discrepancy. The FTA cards, protein

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saver types, may have been better in stabilizing DNA and protein-rich samples and less efficient in preserving RNA-based microorganisms, in this case respiratory viruses, that were stored on to these cards. Additionally, spotting preparations for these samples may have diluted them to a degree that lowered sensitivity. Sample storage may account for the absence of RNA-based viruses during detection using RPM-Flu v.3.1. Nonetheless, when comparing diagnostic capabilities, FilmArray is simpler and thus faster, but RPM-Flu carries greater detection resolution and is capable of detecting and identifying a broader range of pathogens while differentially separating apart notable strains and sequencing divergences for both bacteria and viruses.