Respiratory viruses can be detected in >90% of children with lower respiratory tract infections, such as bronchiolitis, due to the use of sensitive nucleic acid amplification tests (NAATs) for virus detection.1 NAATs also detect common respiratory viral coinfections, defined as the detection of >1 viral pathogen in the same sample. Even 4–5 different respiratory viruses have been detected in children with acute respiratory infection (ARI).2 In this review, we will discuss the clinical significance of multiple respiratory virus detection (MRVD) that should be the preferred term for this phenomenon3 because it has not been shown that all viruses detected are causing infection. It is possible that some of them are just innocent bystanders inducing no inflammatory response. A large number of studies have shown that MRVD occurs in 20%–40% of children with lower respiratory tract infections, but this finding is less common in adults.2–5 The occurrence of different respiratory viruses in MRVD varies widely in different studies. In a recent study on community-acquired pneumonia among 2222 US children, 38%, 34% and 35%, respectively, of respiratory syncytial virus (RSV), human rhinovirus and human metapneumovirus detections were associated with other viruses compared with 72% of adenovirus and 76% of coronavirus detections.6 In that study, human bocavirus was not tested for. Human bocavirus occurs most commonly associated with other viruses, which has raised the question of its potential pathogenicity and significance.7 However, enteroviruses are seldom associated with other viruses. In many studies, human bocavirus plus human rhinovirus is the most common MRVD. Prolonged asymptomatic excretion of a virus detectable by NAATs plus a new infection with a second positive NAATs result might imply MRVD at the molecular level without being a dual clinical infection.
With the use of virus culture or virus antigen detection, dual respiratory virus infections have seldomly been detected. In 1997, Drews et al8 reviewed 8 studies with a total of 1341 cases of respiratory viral infection detected by virus culture, serologic tests or polymerase chain reaction (PCR). Dual viral infection was noted in 67 (5%) cases. By considering the results of cell culture alone, 1200 infections were analyzed and 2.3% of them were MRVD. The age range was broad in patients either with MRVD (<1–79 years) or with single respiratory virus detection (SRVD) (<1–84.6 years). The authors estimated the hospitalization rate and found a significantly higher rate in patients with MRVD (46.3%) in comparison with patients with SRVD (21.7%; P < 0.01). Based on these findings, increased severity was inferred for patients with MRVD. It is important to note that this result was provided in a bivariate analysis in a heterogeneous group of patients. It is also necessary to highlight that 58% of patients with MRVD had underlying lung disease, and this information was not available for all enrolled patients.
A recent meta-analysis assessed the association between MRVD detected by NAATs and the severity of clinical disease in children and adults hospitalized with ARI.9 The protocol was developed a priori in accordance with Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRIMSA) guidelines. The exclusion criteria comprise studies reporting data only on outpatients, studies using multiple diagnostic techniques for viral detection and studies including viral-bacterial coinfections. The primary outcome was the length of hospital stay, and several secondary outcomes were defined: admission to intensive care unit (ICU), need for mechanical ventilation, oxygen requirements and mortality. From 1017 screened titles and abstracts, 21 studies were included, all of them cohort studies: 90% evaluated children, 80% exclusively enrolled children and 76% provided data for quantitative analysis. No difference was found in any of the previously defined outcomes. Higher mortality was found in preschool children with viral coinfections, but this result was based on one single study, which was graded as being at high risk of bias. Heterogeneity was considered to be high in all outcomes.
Another recent systematic review investigated the relation between MRVD and outcome.4 Only studies that measured MRVD as a risk factor for disease outcome and included the outcome measures (hospitalization to general ward, admission to ICU, bronchiolitis or pneumonia) were included. Nineteen studies from all over the world were analyzed. In 5 of the 8 studies, MRVD significantly increased the risk of admission to general ward, 1 found it did not and the other 2 had insignificant results. Similarly, on the risk of admission to ICU, some studies found that MRVD significantly increased the risk of admission to ICU whereas others did not. No evidence for or against MRVD and risk of bronchiolitis or pneumonia was found.
In a prospective observational cohort study conducted in 16 US hospitals, children <2 years of age hospitalized with bronchiolitis were enrolled.1 The authors assessed the presence of respiratory viruses detected by using PCR in nasopharyngeal aspirates and length of hospital stay. Of note, 72.0% of the participants had RSV and 25.6% had human rhinovirus. MRVD was present in 29.8% of the 2207 studied children. The length of hospital stay of 3 or more days was less likely among children with human rhinovirus alone and those with human rhinovirus plus non-RSV pathogens but more likely among children with RSV plus human rhinovirus. These results were provided in a multivariate analysis controlled for 15 demographic and clinical factors.
Another prospective study enrolled 592 children <15 years of age seen in 1 emergency room in Italy with radiographically confirmed community-acquired pneumonia.10 Seventeen respiratory viruses were searched for in respiratory secretion samples by using multiplex PCR. At least 1 respiratory virus was found in 435 (73.5%) patients, among which MRVD was present in 26.9%. Patients with MRVD showed radiographic evidence of alveolar pneumonia significantly more frequently than patients with SRVD.
One study in adults compared the clinical characteristics of hospitalized patients with ARI and MRVD with those of patients with SRVD.3 The study was carried out in a tertiary care teaching hospital. All patients aged 16 years and older with a positive multiplex reverse transcriptase (RT) PCR results were included. Patients’ demographics, presence of pathogens other than respiratory virus, presence of underlying diseases or conditions, presence of respiratory symptoms, history of mechanical ventilation, use of vasoconstrictive agents, history of supplemental oxygen therapy, admission to ICU and clinical outcomes were investigated. Overall, 593 adult patients were evaluated of which 190 had 1 positive respiratory virus multiplex RT-PCR test. MRVD was detected in 13.7% of them. The mean age was 62.7 years; 17.9% of the patients were admitted to the ICU and 4.7% died in the hospital. When the patients with MRVD were compared with those with SRVD, the authors found no difference in the initial clinical severity assessments. Likewise, the clinical outcomes of the patients with MRVD did not differ from those of the patients with SRVD. In a multivariate analysis, only chronic lung disease was associated with MRVD. Therefore, the authors concluded that MRVD was associated with chronic lung disease rather than the severity of the ARI.
It would be logical to think that 2–4 respiratory viruses with different inflammatory mechanisms induce greater inflammatory response and more severe clinical illness than 1 respiratory virus alone. However, this seems not to be the case, and controversial findings are being published. Since the discovery of interferon by Isaacs and Lindenmann11 in 1957, it has been clear that viruses can affect each other. Viral infection of an organism results in prevention or partial inhibition of another viral infection within the same host, a phenomenon called viral interference.12 Viruses may interact indirectly or directly, resulting in complementation or inhibition of each other. Recently, it was shown that influenza A and B viruses may affect each other.13 Human herpesvirus 6 and human herpesvirus 7 both suppress the replication CC chemokine receptor 5-tropic HIV-1.14 Human bocavirus may interfere with human rhinovirus-induced immune response.15 However, viral-viral interactions in vivo are still poorly understood and likely to be complex.
Most published reports on MRVD are subject to several limitations. Most studies have used only multiplex RT-PCR test kits capable of detecting many respiratory viruses. However, their sensitivity with regard to monoplex PCR tests may not be sufficient and only 40%–70% recovery rates are being reported in patients with clinical viral infection. In a study that performed multiplex and monoplex PCR tests, the detection rate was 45.9% and 62.6%, respectively, the sensitivity of multiplex varying from 73.1% to 11.% for different viruses.16 Combination of multiplex test and monoplex PCR tests can give 100% recovery rate.17 Virus copy numbers have been seldom reported. High copy number would support causative role of the virus detected. It must be remembered that MRVD have been found only by NAATs. NAATs do not necessarily reflect active virus replication. In addition to acute infection, positive NAATs may reflect prolonged shedding after acute infection (eg, human bocavirus), latent chronic infection (eg, adenovirus) or asymptomatic acute infection (eg, human rhinovirus). The increase of specific immunoglobulin G antibodies between acute and convalescent serum samples would confirm acute infection. The difficulty with human rhinovirus is the paucity of serologic tests. Innate immunity gene expression studies may give new and valuable information in MRVD. It has just been shown that symptomatic human rhinovirus infection induced a robust and reproducible transcriptional signature, whereas identification of human rhinovirus in asymptomatic children was not associated with significant systemic transcriptional immune responses. Therefore, transcriptional profiling represents a useful tool to discriminate between active infection and incidental virus detection.18
The real clinical role of MRVD remains to be clarified. It is possible that different pathogenic mechanisms may be triggered by different viruses, which may potentiate or inhibit each other’s effect. It appears that severity between SRVD and MRVD may differ by virus pathogen. At present, there is no convincing evidence that patients admitted with viral coinfections are at higher risk of increased disease severity than patients with SRVD. Prospective longitudinal studies, including more sensitive NAATs, serial respiratory sampling for viruses and bacteria, host transcriptome analysis and serologic tests, will hopefully be more informative.
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respiratory infection; respiratory virus; respiratory virus coinfection; viral infection