In the study, we tested the performance of the 4-Plex multiplex respiratory panel assay on the Alinity m automated molecular system for the simultaneous detection of Flu-A, Flu-B, hRSV and SARS-CoV-2 to determine the rate of overlooked Flu-A/B and hRSV infections in early 2022 and to investigate the epidemiological pattern of Flu-A/B and hRSV infections in the first “post COVID-19” season. The Alinity m 4-plex was selected because it has good performance, high throughput, random access, semi-batch functionality, STAT (urgent) prioritization and the ability to load and run multiple different assays simultaneously, making it suitable for both inpatient and outpatient diagnostics [9–13]. Full integration with the LIS enables automatic sample recognition, ordering of tests/markers per sample, and release of results, minimizing the possibility of errors.
We started the study already in February 2022, when SARS-CoV-2 was still the most frequently detected virus (66.1%). Although the dominance of SARS-CoV-2 infections was undeniable, Flu-A (1.6%), hRSV (0.7%), and Flu-B (0.1%) were also detected, which would otherwise have been overlooked if the focus had been solely on the detection of SARS-CoV-2. These results suggest that the emergence of SARS-CoV-2 and the implementation of nonpharmaceutical preventive measures has clearly impacted the prevalence of other respiratory viruses, as the detected infections with Flu-A, Flu-B and hRSV were lower than in previous seasons, although the number of tests was higher [14]. Before the emergence of SARSCoV-2, the epidemiology of respiratory viral infections in Slovenia followed an established and fairly uniform pattern. Starting with a peak of hRSV infections in December to February, followed by a peak of Flu-A infections in late January and Flu-B infections in March [14]. The disruption of the usual respiratory virus season in Slovenia due to the emergence of SARS-CoV-2 was observed before the time of this study. Preventive measures were already in place and strictly followed between March 2020 and May 2021. No hRSV infection was detected during this time. After a one- year gap, hRSV cases reappeared in late spring and summer, with a peak in September 2021, suggesting that hRSV must have survived in the population, although it was clearly hampered by the pandemic and preventive measures [15]. Similarly, Flu-A and Flu-B infections in Slovenia were not detected during the 2020/21 season and only reappeared in March 2022 [14]. SARS-CoV-2 appears to be superior to other respiratory viruses in terms of resistance and infectivity [16]. Varela et al. (2021) reported that SARSCoV-2 was detected in 52.2% of samples, and only 0.4% of samples were positive for influenza in late spring and summer 2020. Human RSV also showed a significant decrease in the number of hospitalized cases with acute viral bronchiolitis compared to the previous respiratory season [17]. Infections with other respiratory viruses were also less common in the study by Uhteg et al. (2022), which was conducted in a period from October 2019 to December 2021 and ended just before the first period of this study. They found that the most common nonSARSCoV-2 infections were caused by FluA (10.1%), Flu-B (8.5%), and hRSV (5.4%). All available data suggest that COVID-19 prevention measures resulted in reduced transmission of influenza and hRSV, but were not sufficient to prevent the spread of the highly infectious SARS-CoV-2 virus [18].
In the second part of the present study, laboratory diagnostics of respiratory viruses in our institute slowly returned to a similar framework as before COVID-19. The origin of the samples also changed from screening of the general population back to targeted testing of inpatients (Fig. 4). Compared to the previous part of the study, we detected a lower proportion of SARS-CoV-2 positive samples (9.6%) and an increase in cases of Flu-A (3.5%), hRSV (2.8%), and Flu-B (0.6%). We hypothesize that the number of SARS-CoV-2 infections has decreased due to the evolution of the virus (the Omicron variant replicates less efficiently than earlier variants and causes less cellular damage in infected cells [19]), global vaccination efforts, the substantial proportion of the population already exposed to the virus, and the lifting of non-pharmaceutical preventive measures. The latter in particular, is leading to increased humantohuman contact, allowing other respiratory viruses to thrive again. A relative increase in influenza virus activity has already been reported in Australia compared to previous influenza seasons (2020/2021). Early surveillance data during the 2021/2022 influenza season in the Northern Hemisphere suggests that sporadic cases of influenza virus infectionsare re-emerging, but not at the same level of activity as before the COVID-19 pandemic. During the 2021/2022 influenza season, Flu-B virus predominated in China, while other Asian countries reported cases of both Flu-A and FluB [20]. During the 2022–2023 winter season, several countries in the Northern Hemisphere experienced an increase in influenza and hRSV infections. In Egypt, after two years of decline, a resurgence of influenza and hRSV was reported in children under 16 years of age. Higher infection rates were observed compared to pre-pandemic period [21].
Already in early 2022, when we detected SARS-CoV-2 in the majority of samples tested, codetection with other respiratory viruses were present in 1.2% of the samples analysed. Interestingly, a similar rate of co-detection was observed in the entire winter season 22/23 (1.7%). However, in the second part of study, co-detections other than SARS-CoV-2 were observed in 0.04% of the samples; hRSV/Flu-A co-detection occurred in 7 samples and Flu-A/Flu-B co-detection was observed in 1 sample. The estimates of co-detection in various cohort studies are also consistent with these results, ranging up to 3%[17, 22–23]. On the other hand, there are studies that report higher rates of co-detection [22]. Kim et al. (2020) found that 20% of SARS-CoV-2 positive patients were also positive for another respiratory viral pathogen, with hRSV being the most common at 5.2% [24]. Swets et al (2022) found co-detection with other respiratory viruses in 8.4% of patients, most commonly influenza and hRSV [25]. As indicated by the available literature, the extent of infections and co-detection with other respiratory viruses besides SARS- CoV-2 was low to moderate.
One limitation of the study should be mentioned: since the included respiratory samples were selected sequentially as soon as they newly arrived at the laboratory and only SARS-CoV-2 follow-up tests were excluded, the selection of the study population might be biased. However, this effect is mitigated by a larger number of samples tested, which has not yet been done [3, 11–13].
In conclusion, this study shows that infections with Flu-A/B and hRSV were relatively rare during the pandemic. The re-emergence of Flu-A/B and hRSV infections in the wake of the pandemic suggests that differential diagnosis will play a very important role in detecting and distinguishing SARS-CoV-2, Flu-A/B, or hRSV patients in the coming season. The ability to rapidly test for SARS-CoV-2, Flu-A, Flu-B, and hRSV on the Alinity m or similar analyzers will expand the ability to further increase testing capacity with the goal of containing the spread of viral respiratory infections other than just SARS-CoV-2. With its urgent sample priority processing feature, the Alinity m can deliver rapid results (a mean sample-to-response TAT of 176 minutes) to select appropriate therapy or diagnose patients in hospital prior to surgery. At the same time, it enables the processing of a large number of samples, which will be particularly important in the future when the respiratory virus season starts.