4 Discussion

Since its discovery in 2001, HMPV has been considered an important cause of ARTIs. Some studies have shown that the prevalence of HMPV is different in different regions and years. The detection rate of HMPV in Chinese hospitalized children with ARTIs is about 2%-18.2%18,24,25, and the detection rate of HMPV in ARTIs inpatients in other countries is 1.2%-20.3%12,26,27. In this study, we mainly explored the molecular epidemiology and clinical characteristics of HMPV infection in hospitalized children with ARTIs in Beijing from April 2018 to March 2019 and September 2020 to August 2021, and the impact of the COVID-19 epidemic on HMPV infection. The detection rate of HMPV from April 2018 to March 2019 was 7.9%, and the detection rate of HMPV from September 2020 to August 2021 was 1.7%. A previous study in our laboratory showed that the detection rate of HMPV in hospitalized children from April 2017 to March 2018 was 4.1%25, which indicated that the detection rate of HMPV in hospitalized children with ARTIs in Beijing from April 2018 to March 2019 was higher than the previous year (p =0.000), while the detection rate of HMPV decreased significantly during the period of the COVID-19 epidemic. It shows that the detection rate of HMPV in hospitalized children with ARTIs has been significantly reduced under the strict prevention and control of the COVID-19 epidemic in China(p =0.001). Mandy Jongbloed et al. reported that despite a 324% increase in HMPV testing during the COVID-19 outbreak in Europe, there was no increase in HMPV incidence28. Since the outbreak of the COVID-19, various countries have adopted different prevention and control strategies. A series of measures such as wearing masks, frequent hand washing and disinfection, delaying the start of school, and maintaining social distance have cut off the transmission of respiratory viruses and reduced HMPV infection. During the COVID-19 outbreak, the number of hospitalized children with ARTIs decreased significantly, and the number of samples collected from September 2020 to August 2021 decreased accordingly. Due to the small number of HMPV-positive samples, the virus mixed infection and clinical characteristics have not been counted yet.
From April 2018 to March 2019, 78.2% of HMPV infections occurred in hospitalized children aged ≤5 years, and 9.1% (16/176) occurred in children aged 3-4 years; There was no significant difference in HMPV detection rate between male and female children(p =0.185), which was consistent with the studies in China and Argentina25,27 . HMPV infection had typical seasonal distribution characteristics, with the detection rate in winter and spring significantly higher than that in summer and autumn (P =0.000), and the prevalence period of HMPV was concentrated from December to May of the following year, which was consistent with the results of a study in Guangzhou, China23. Research shows that HMPV epidemics are typical in early spring, while small peaks in summer appear to be related to local weather conditions22,27. In ARTIs cases, HMPV is often co-infected with other respiratory viruses, such as ADV, RSV, HRV and HPIV25,29,30. In this study, from April 2018 to March 2019, the proportion of HMPV-positive samples mixed with other viruses was 37.1%, of which HPIV3 had the highest mixed infection rate of 32.6%, which was similar to the findings of Fathima S27, which may related to the prevalence of HPIV3 in that year. Some studies have concluded that co-infection of HMPV with other respiratory viruses can aggravate clinical symptoms and prolong hospitalization18,32. However, in this study, there were no statistically significant differences in viral load, length of hospital stay, cough, fever, nasal obstruction, rhinorrhoea and other clinical symptoms between children with HMPV single infection and mixed infection, which is consistent with the results of previous research in our laboratory25. In addition, the relationship between HMPV viral load and clinical symptoms remains unclear25,33,34, and no association between clinical symptoms and HMPV viral load was found in the study.
There are five gene subtypes in HMPV, so it is of great significance to explore the relationship between genetic diversity of HMPV and clinical characteristics. In this study, for HMPV samples from April 2018 to March 2019, 78 NPAs samples were successfully amplified by nested PCR amplification of the conserved fragment of the F gene. The phylogenetic analysis showed that 82.1% (64/78) of the prevalent strains belonged to A2b subtype, 11.5% belonged to B1 subtype and 6.4% belonged to B2 subtype, and no A1 and A2a subtypes were found. We found that the viral load of HMPV A2b genotype samples was significantly higher than that of B samples (p =0.009). In addition, there was a statistically significant difference between A2b subtype and B subtype in the number of children with hospitalization time > 7 days, and the hospitalization time of A2b subtype was longer than that of B1 subtype (p =0.030), which suggests that we should pay more attention to A2b subtype in pediatric clinic. From September 2020 to August 2021, only one HMPV case was identified by phylogenetic analysis as subtype B1 infection. From April 2017 to March 2018, the detection rate of B1 subtype (54.5%) were highest, followed by the A2b subtype (40.9%) in the previous study of our laboratory, and no HMPV A1 and A2a subtypes were found25. However, in this study, the A2b subtype was the predominant (82.1%), indicating that the main circulating strain of HMPV infection in Beijing in 2017-2019 switched from B1 subtype to A2b subtype. The study by Matsuzaki Y showed that laryngitis is more common in children with HMPV subtype B1 infection, while wheezing is more common in children infected with HMPV subtypes B1 and B2 than in children with subtype A2 infection35. Many data suggest that HMPV genotypes are associated with disease severity or clinical manifestations 36,37, but some studies have inconsistent conclusions 24,38, so it is still controversial whether HMPV genotypes are associated with clinical features.
This study was limited by the large decrease in hospitalized children with ARTIs during the COVID-19 outbreak. From September 2020 to August 2021, there were only 4 HMPV positive children of 232 hospitalized children with ARTIs, therefore a detailed epidemiological analysis of HMPV cases during this period was not possible. We will continue to collect respiratory samples from hospitalized children with ARTIs in the subsequent studies, and make a detailed analysis of the impact of SARS-CoV-2 on the prevalence of HMPV.