Background
Acute respiratory tract infections (ARIs) include upper respiratory tract infections and lower respiratory tract infections (LRTIs), including pneumonia. ARIs, which are caused by a broad range of microbes, are a leading cause of hospitalization, morbidity, and mortality worldwide [
1,
2]. Recent studies examined the etiology of ARI using PCR-based molecular diagnosis techniques that can detect pathogens missed by conventional methods [
1‐
8]. Multiplex real-time PCR (rPCR) assays allow the rapid simultaneous and sensitive detection of multiple viruses, bacteria, and fungi [
9,
10]. Thus, multiple microbes are often detected simultaneously in the same clinical specimen. It is important to understand that the PCR-positive microbes are candidate disease-causing pathogens, but not all are necessarily associated with the particular disease [
11‐
14]. For bacteria and fungi in particular, it is difficult to gauge their relevance to the symptoms. Nevertheless, these assays provide epidemiological information that can be useful for treatment planning and prevention of infection.
The proportion of respiratory microbes detected by PCR varies between children and adults, inpatients and outpatients, regions, countries, and during epidemics [
15‐
20]. For hospitalized adult ARI patients, the main causative viruses are influenza A and B viruses, human rhinovirus, human parainfluenza virus, human adenovirus, respiratory syncytial virus, and human metapneumovirus [
18‐
20]. There are three genera of human influenza virus: type A (FluA), type B (FluB), and type C (FluC). Two subtypes of Flu A, A(H1N1)pdm09 virus (H1N1pdm) and A(H3N2) virus (H3), and two lineages of Flu B, B/Yamagata and B/Victoria, are currently co-circulating and have caused human epidemics every year [
21]. Several reports involving hospitalized patients suggest that H1N1pdm infection is associated with intensive care unit (ICU) admission or death [
22‐
25].
The primary objective of this study was to investigate 34 respiratory microbes that were simultaneously detected in airway specimens from hospitalized Vietnamese adult ARI patients. The secondary objective was to investigate associations between H1N1pdm detection and ICU admission or fatal outcome.
Methods
Study setting
This prospective observational study conducted between September 2015 and June 2017 at Bach Mai Hospital, a 3100 bed tertiary care hospital in Hanoi. It is the biggest government general hospital in Vietnam. Many patients are transferred from hospitals in various provinces of the country to receive more advanced care. The annual inpatient population is approximately 2000 each in the infectious diseases department (78 beds) and respiratory department (123 beds), and 450 in the ICU (73 beds). Patients presenting with severe lower respiratory tract infections, pneumonia, or acute respiratory distress syndrome (ARDS) require mechanical ventilation or extracorporeal membrane oxygenation (ECMO), and they are either admitted or transferred to the ICU.
Patients aged ≥15 years hospitalized in one of the aforementioned departments with one or more presenting symptoms, including shortness of breath, sore throat, runny nose, headache, and muscle pain/arthralgia in addition to cough and fever, were included. The cohort enrolled patients within 10 days of symptom onset, who were either newly hospitalized or those who developed these symptoms during their hospitalization for reasons other than ARI. Patients positive for human immunodeficiency virus (HIV)-1 and those with no available samples within 14 days of onset were excluded.
Specimens and data collection
Nasopharyngeal swabs (NPS) were collected from all patients. In addition, one sample comprising a throat swab (TS), sputum (SP) sample, or tracheal lavage aspirate (TLA) in case of intubated patients, was collected. The NPS, TS, and SP specimens were added to 1 ml of universal transport medium (Copan, Brescia, Italy), divided into aliquots, and stored along with TLA samples at − 80 °C until use. Demographics, clinical data, and outcomes were collected by retrospective review of patient charts (Table
1).
Table 1
Demographic and clinical characteristics of the study participants
Sex (Male, %) | 155 (57.6) | 35 (50.7) | 120 (60.0) | 16 (64.0) | 139 (57.0) |
Age; Median (IQR); years | 51 (33–65) | 54 (40–67) | 50 (31–64) | 56 (47–67) | 50 (31–65) |
Duration from onset to sampling; Median (IQR); days | 5 (3–7) | 5 (4–8) | 5 (3–7) | 6 (4–9) | 5 (3–7) |
Dead patients | 25 (9.3) | 23 (33.3) | 2 (1.0) | | |
Comorbidities or conditions | 162 (60.2) | 45 (65.2) | 117 (58.5) | 19 (76.0) | 143 (58.6) |
Lung disease | 34 (12.6) | 6 (8.7) | 28 (14.0) | 1 (4.0) | 33 (13.5) |
Hypertension | 27 (10.0) | 10 (14.5) | 17 (8.5) | 3 (12.0) | 24 (9.8) |
Diabetes mellitus | 24 (8.9) | 12 (17.4) | 12 (6.0) | 4 (16.0) | 20 (8.2) |
Liver disease | 24 (8.9) | 5 (7.2) | 19 (9.5) | 4 (16.0) | 20 (8.2) |
Cardiovascular disease | 22 (8.2) | 10 (14.5) | 12 (6.0) | 3 (12.0) | 19 (7.8) |
Pregnancy | 19 (7.1) | 3 (4.3) | 16 (8.0) | 0 (0) | 19 (7.8) |
Kidney disease | 19 (7.1) | 6 (8.7) | 13 (6.5) | 1 (4.0) | 18 (7.4) |
Collagen disease | 11 (4.1) | 5 (7.2) | 6 (3.0) | 3 (12.0) | 8 (3.3) |
Cancer | 9 (3.3) | 3 (4.3) | 6 (3.0) | 0 (0) | 9 (3.7) |
Tuberculosis | 6 (2.2) | 2 (2.9) | 4 (2.0) | 1 (4.0) | 5 (2.0) |
HBV/HCV | 6 (2.2) | 1 (1.4) | 5 (2.5) | 1 (4.0) | 5 (2.0) |
Hematological disease*1 | 5 (1.9) | 5 (7.2) | 0 (0) | 4 (16.0) | 1 (0.4) |
Bronchial asthma | 4 (1.5) | 3 (4.3) | 1 (0.5) | 0 (0) | 4 (1.6) |
Immunosuppressive agent usage*2 | 4 (1.5) | 4 (5.8) | 0 (0) | 4 (16.0) | 0 (0) |
Others | 16 (5.9) | 4 (5.8) | 12 (6.0) | 2 (8.0) | 14 (5.7) |
Multiplex real-time PCR
To collect from as wide an area as possible, two different airway specimens were acquired: NPS (100 μl) and another specimen (TS or SP or TLA; 100 μl). The specimens were mixed and nucleic acids were extracted from the 200 μl mixture using the QIAamp® MinElute Virus Spin kit (Qiagen, Hilden, Germany). Nucleic acids were eluted in 100 μl of RNase-free water and subjected to multiplex rPCR using the Fast Track Diagnostics Respiratory pathogens 33 (FTD33, Fast Track Diagnostics, Esch-sur-Alzette, Luxembourg) plus the CFX96 Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). In addition, for Flu A-positive patients, subtypes (H1N1pdm or H3) were confirmed using FTD-Flu differentiation (Fast Track Diagnostics). FTD33 combined with FTD-Flu differentiation allowed the detection of 34 respiratory microbes (
Supplemental Table). Flu A, H1N1pdm, H3, Flu B, influenza C virus (Flu C), respiratory syncytial virus A/B (RSV A/B), human coronavirus NL63 (NL63), human coronavirus 229E (229E), human coronavirus OC43 (OC43), human coronavirus HKU1 (HKU1), human rhinovirus (HRV), human parainfluenza virus, 1, 2, 3, and 4 (HPIV-1, 2, 3 and 4), human metapneumovirus A/B (HMPV A/B), human bocavirus (HBoV), human adenovirus (HAdV), enterovirus (EV), human parechovirus (HPeV),
Mycoplasma pneumoniae (
M. pneumoniae),
Chlamydophila pneumoniae (
C. pneumoniae),
Streptococcus pneumoniae (
S. pneumoniae),
Klebsiella pneumoniae/Klebsiella variicola (
K. pneumoniae/K. variicola),
Haemophilus influenzae (
H. influenzae),
Haemophilus influenzae type B (
H. influenzae B),
Staphylococcus aureus (
S. aureus),
Salmonella species (
Salmonella. spp.),
Moraxella catarrhalis (
M. catarrhalis),
Legionella pneumophila/longbeachae (
L. pneumophila/L. longbeachae),
Pneumocystis jirovecii (
P. jirovecii), and
Bordetella species (
Bordetella. spp.)This assay used Equine arteritis virus as an internal control. The internal control was added directly to the lysis buffer during each extraction step.
Statistical analysis
To identify the association between outcomes (ICU admission or fatal outcome) and microbes and other host factors, the univariate associations between the two independent groups were analyzed using the Chi-square test or Fisher’s exact test (categorical variables) and the Mann-Whitney U test (continuous variables). Multiple comparisons were made after Bonferroni correction to obtain adjusted p-values.
Multivariate logistic regression analysis was used to identify associations between H1N1pdm detection and ICU admission or fatal outcomes after adjusting for age, comorbidities, and bacterial/
P. jirovecii co-infection. These independent variables were selected based on the reports that comorbidities, age, and secondary bacterial or fungal superinfection are risk factors associated with the severity of influenza [
26‐
28]. Odds ratios (ORs) and 95% confidence intervals (CIs) for each factor were derived from logistic regression analysis. The goodness of fit of the multivariate logistic regression model was determined using the Hosmer-Lemeshow test. All statistical analyses were performed using IBM SPSS statistics for Windows (Version 26.0 J; IBM Corp., Chicago, IL, USA). The level of significance was set at
p < 0.05, except when Bonferroni correction was applied.
Discussion
This study enrolled 269 hospitalized adult ARI patients. Twenty-two viruses, 11 bacteria, and one fungus (P. jirovecii) in respiratory specimens were examined using a commercial multiplex rPCR assay. Specimens were collected from the nasal cavity and pharynx or trachea. The use of multiplex rPCR allows the rapid and sensitive detection of potential pathogens that are the major causes of symptoms. The assay also screens for other pathogens and can prevent misdiagnosis, even during an epidemic caused by one specific pathogen.
Several reports documented the viral etiology of respiratory infections in hospitalized children and/or adults using multiplex rPCR [
15‐
17]. The virus detection rate (50.9%) in this study is similar to that in a previous report of adults (58.5%) [
20], but is lower than that reported in pediatric studies (70–82%) [
16,
17]. One possible reason for the difference is that adults already have antibodies against various viruses. In the pediatric studies, the most commonly detected virus varied according to country and ongoing epidemic during the study period. Several adult studies (including this study) most frequently detected influenza virus. The detection could be affected by whether the flu season in the study period occurred during an epidemic [
12,
18‐
20]. Influenza viruses mutate very quickly, so adults who have been infected in the past can be infected again and are at risk of hospitalization [
21].
The rate of bacteria/
P. jirovecii detection (56.1%) was higher than that of viruses, even though the number of screened bacteria/
P. jirovecii was almost half that of the screened viruses. It is worth remembering that multiplex rPCR also detects the genomes of asymptomatic microbes, persistently infectious viruses, and normal bacterial flora. Therefore, the test does not necessarily signify clinical etiology [
11‐
14]. As an example, a report that analyzed microbes in 312 non-acute specimens from military trainees using the same FTD33 kit showed that viruses and bacteria were present in 13.8 and 93.3% of throat and nasal swabs, respectively, from asymptomatic examinees [
11]. It is difficult to verify the clinical significance of the detected microbes using only multiplex rPCR. Etiology should be considered in combination with additional information, such as changes in microbe load and symptoms over time, results of virus isolation or bacterial culture, and changes in serum antibody levels against the microbe. Thus, it is reasonable that detection of multiple microbes in a single patient was not always associated with disease severity (Table
2).
On the other hand, Influenza viruses are not persistent or latent infections, and are rarely detected in asymptomatic controls [
11‐
13]. Several reports have compared disease severity in hospitalized patients with laboratory-confirmed H1N1pdm and H3 infections [
22‐
25,
29,
30]. Focusing on the differences in ICU admission, a recent analysis of a large cohort (696 H1N1pdm and 388 H3 patients from 2010 to 2016) showed that H1N1pdm patients required ICU admission more frequently than those with H3 (
p < 0.01) [
30]. Presently, H1N1pdm detection was significantly associated with both ICU admission and fatal outcome after adjusting for the confounding factors of the presence of comorbidities, bacteria/
P. jirovecii co-detection, and age. Influenza vaccination coverage is not high in Vietnam. The influenza vaccine prevents severe illness and is reported to be more effective against H1N1pdm infection [
31]. If H1N1pdm is associated with severe disease, vaccination should be recommended. Currently, subtyping is not important in a clinical setting, even in ICU-related cases. However, if the attending physician is aware that H1N1pdm can lead to more severe complications (such as ARDS) than H3 and influenza B, the timing of respiratory management and the introduction of anti-inflammatory therapy could be significantly improved. This suggests that subtype diagnosis of influenza may be not only epidemiologically important but also clinically beneficial, although not always mandatory.
This study has several limitations. First, it was a single center study. Second, the number of enrolled patients was small. Third, some control data from asymptomatic adults in Vietnam was unavailable. These adults might harbor bacterial colonization and latent infection. Fourth, the mixing of nasal swabs and lower respiratory tract specimens made it impossible to assess the clinical pathogenicity of LRTI. It might be necessary to take specimens from the alveolar region [
32,
33] to more precisely and accurately detect microbes that cause pneumonia. However, sampling of the LRT is very invasive and was therefore was limited to TLA from intubated patients or sputum. Fifth, a patient with a cough and fever, but with a non-infectious respiratory illness, might have been enrolled. Sixth, this was an observational study with limited epidemiological, socioeconomic, and clinical information, and the residual confounders were inevitable limitations. Further studies are needed to establish these associations.
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