Background
Human metapneumovirus (HMPV) is a negative-stranded RNA virus classified in the
Pneumoviridae family [
1]. HMPV infections are commonly associated with mild respiratory symptoms, but severe cough, bronchiolitis and pneumonia have also been reported, sometimes accompanied by high fever, myalgia and vomiting [
2]. Several risk factors associated with more severe disease due to HMPV infection in adults have been identified, which include patients with pulmonary disease or congestive heart disease, healthy elderly patients with age over 65 years old, long term stay in hospital care facilities and immunocompromised patients [
3].
Previous reports had attempted to associate the genetic diversity of HMPV, which are classified as genotypes A and B (with further classification into sub-lineages - A1, A2a, A2b, B1 and B2) [
4] with disease severity [
5‐
7]. For instances, it was found that HMPV genotype A infection in children caused more severe illnesses (e.g. higher risk of pneumonia and oxygen saturation < 90%, need for hospitalization, and longer stay of intensive care unit) compared with genotype B infection [
7]. In contrast, some studies reported that genotype B infection was one of the risk factor for severe disease [
5] with more pathological signs on chest X-ray compared with genotype A infection [
6]. Furthermore, some associations remain debatable as several studies found no direct correlation between HMPV genotypes and severity of illness [
8‐
10]. On the other hand, apart from viral genetic diversity, HMPV viral load was recognized as a risk factor associated with more severe disease outcome leading to hospitalization [
11,
12].
Although HMPV genetic diversity continues to be described and linked with disease severity in hospitalized children [
6], pediatrics [
13], elderly adults [
10] and immunocompromised patients [
3], reports that address this association have been limited in the adult outpatient settings. A recent study which showed that HMPV can also cause respiratory outbreaks in adults [
14] highlights the fact that adults may play a role in the transmission and evolutionary dynamics of the virus, and more severe disease could occur in adults during an outbreak.
Hence, in this study, we sought to investigate the possible linkage of genetic diversity on symptom severity in adult outpatients with HMPV infection presenting acute upper respiratory tract infections (URTI) [
15]. Using an improved molecular assay for viral load quantification, we also assessed the correlation of HMPV viral load in nasopharyngeal specimens on symptom severity.
Discussion
Studies assessing the risk factors such as genetic diversity and viral load that contribute to the burden of illness caused by HMPV infection have generally focused on high-risk populations such as hospitalized children [
27] or adult patients [
28], immunocompromised elderly [
29], or hematopoietic cell transplant recipients [
30], with limited reports from outpatients presenting with acute respiratory tract symptoms. While individuals seeking and receiving outpatient care may not experience severe respiratory complications, their illnesses contribute significantly to the overall disease burden and transmission of the virus to susceptible individuals. The present study investigated the severity of acute respiratory symptoms caused by HMPV infection in a generally adult outpatient population, and assessed the virological and demographical factors that may be associated with the degree of symptom severity.
A standardized four-category scoring of the severity of common cold symptoms has been used as a complementary measure for the impact of respiratory illness in outpatients [
16‐
19]. By summing up the scores of all individual symptoms [
20], we could not observe any significant symptom severity differences (or TSSS) between HMPV genotypes/sub-lineages in the adult outpatients (Table
2), suggesting that the genetic diversity of HMPV as shown in this study played limited role as a predictor for the severity of symptoms in the outpatient settings. Moreover, patients infected with a recently described sub-lineage A2, which was identified based on the estimated genetic distances between sub-lineages in the
F and
G genes and was also found in other countries such as China, Vietnam and Cambodia [
15], did not show more severe symptoms compared to other sub-lineages. Several previous studies have reported the association of HMPV genetic diversity with disease severity in children. In one report, children infected by genotype A were found to experience more severe acute respiratory illness (based on a scoring method that measures the need for hospitalization, oxygen saturation < 90% at hospital admission, and intensive care unit stay) as compared with genotype B infection [
7]. In contrast, a separate study on young children suggested that genotype B infection may result in greater hospitalization rate and higher clinical score (using two scoring methods [
7,
31] compared to genotype A [
6]. Yet, no significant differences in the severity of illness or clinical manifestation have been reported between HMPV genotypes in children in several other studies [
8,
9,
32,
33], which used other scoring methods. The contrasting findings in these studies highlighted that the relationship between HMPV genetic diversity and disease severity remains undetectable, probably due in part to the lack of a consensus and standardized severity scoring method for outpatients or hospitalized patients. Inclusion of other objective assessment including body temperature, chest X-ray and also respiration rate may help to improve the analysis and reduce discordant findings between studies. In addition, the inconclusive association between HMPV genetic diversity and symptom severity could also be due to the low number of HMPV-positive samples in this study as well as several other studies [
7,
9], which may reduce the statistical power for associations [
34].
It has been described in several studies that HMPV reinfection can occur during an adult’s life due to transient immunity or incomplete protective immune responses for the newly evolved genotype [
29,
35], indicating that the elderly individuals are equally susceptible to reinfection. Furthermore, the possible immune dysregulation which lead to a decreased viral clearance in the elderly will further increase their risk of severe symptomatic infection [
28] and lead to higher rates of hospitalization [
36,
37]. However, unlike previous studies, we found that elderly patients who were ≥ 65 years old had less severe symptoms compared to young and adult patients who were < 65 years old. As our study was conducted in the outpatient clinic, it is possible that elderly patients with severe respiratory symptoms may have opted to seek immediate treatment at inpatient settings, leading to sampling bias. Hence, such contrasting observation may be due to the limited sampling on elderly patients who experience more severe symptoms compared to the young and adult patients. Similarly, even though the Indian ethnic group may appear to be experiencing more severe symptoms (based on higher TSSS score) compared to other ethnic groups in this study (Table
2), such observation needs to be interpreted with caution given the limited number of patients in our cohort. Overall, it remains unclear if host demographic factors could be contributing to a more severe symptom outcome in HMPV infection.
The availability of an up-to-date RT-qPCR assay is important for sensitive, specific and rapid detection and quantification of HMPV. Previously, assays for the detection and quantification of HMPV were developed based on limited genome sequences that belonged to the five known genetic lineages/sub-lineages of HMPV: A1, A2 (A2a and A2b sub-lineage), B1 and B2 [
37,
38]. Besides, most published assays were designed to target the conserved regions of the nucleoprotein (
N) gene [
39‐
41] as it is the highly transcribed and conserved gene [
24,
42], even though nucleotide mismatches between primer/probe sets with reference sequences have been reported [
28,
41,
43,
44]. Thus, in this study, using an updated alignment of complete reference genomes (
n = 135), primers/probes were designed to target the conserved region of the
M2 gene with a minimum coverage of 99.3% of the global HMPV sequences (Additional file
1). The viral load in all but three HMPV-positive specimens was successfully captured with the lowest quantifiable concentration estimated at approximately 13 RNA copies/μl, which was more sensitive than previously published methods [
38,
43].
High nasopharyngeal HMPV viral load has been implicated as an important risk factor for severe symptoms in children who were hospitalized [
12,
45] or admitted for emergency care [
46]. However, in this study, no correlation was observed between viral load and symptom severity in adult outpatients (Table
2, Fig.
2 and Additional file
5). Interestingly, it was found that patients infected with genotype A had a significantly higher peak viral load compared to genotype B-infected patients around 3–5 days of symptom onset, suggesting that genotype A may have a better replication fitness (or replication capacity) and higher transmissibility than genotype B during this period. However, the viral load of genotype A-infected patients who enrolled ≥6 days were observed to be significantly lower compared those who enrolled 3–5 days after symptom onset, due to the fact they enrolled much later during the course of infection, in which most viruses would have been cleared by immunity. Interestingly, our findings corroborates with a previous report that demonstrated the differences in replication fitness between HMPV genotypes in vitro and in vivo [
47]. The study by Aerts et al. showed that HMPV genotype A replicates to a significantly higher titers than genotype B in LLC-MK2 cells and in the lungs of BALB/c mice on day 4 post-infection, but the viral titers of genotype A decreased more rapidly than genotype B after day 4 [
47]. As observed in other viral genotypes/serotypes [
48,
49], the differences in replication capacity may contribute to the competitive, transmission and epidemiological fitness differences between HMPV genotypes [
50], which in turn may dictate the spread and evolution of both genotypes in the human population.