Keywords
bacterial - viral - severe pneumonia - coinfection
Key Messages
This study underlined the pervasiveness of coinfections among young children with
severe viral pneumonia. Provision of effective antiviral treatment, especially for
respiratory syncytial virus (RSV), as well as the advancement of sensitive and rapid
diagnostic tools for screening pathogens of pneumonia, is critical to reducing the
burden of this disease.
Introduction
Globally, community-acquired pneumonia (CAP) has been well-recognized as a leading
cause of disease burden in children under 5 years. CAP is responsible for approximately
2 million infant deaths annually.[1]
[2] However, the management of this disease in the hospital still faces a significant
challenge due to critical knowledge gaps about the etiology and clinical manifestations
of CAP in young children.[3]
Viruses have been identified as the most common causes of CAP, with bacterial pneumonia
being less common.[1]
[3]
[4] Typical viruses include respiratory syncytial virus (RSV),[5] rhinovirus, influenza virus, and adenovirus. However, there are usually no specific
clinical symptoms to definitively diagnose the specific type of virus. Moreover, recent
advanced molecular techniques confirm the pervasiveness of virus-virus and virus–bacterium
coinfections.[6]
[7]
[8] Previous work figured out that approximately 14 to 35% of infants with CAP suffered
coinfections.[6]
[7]
[8]
[9]
[10] Younger age, admission to an intensive care unit, and comorbidity are significant
predictors of coinfection conditions.[4]
[11]
[12]
In Vietnam, the number of children with pneumonia accounts for 30 to 40% of cases
of medical examination and treatment in hospitals.[13] Additionally, 75% of deaths due to respiratory diseases and 30 to 35% of deaths
among children.[13] Studies of pneumonia in Vietnamese children have been performed previously[14]
[15]
[16]; however, evidence about coinfections and their risk factors has not been thoroughly
investigated. This study aims to identify the prevalence of coinfection and associated
factors Vietnamese children under 5 years with viral pneumonia.
Patients and Methods
Study Designs
A cross-sectional study was conducted of 202 children with severe viral pneumonia
who treated at the National Hospital of Pediatrics from January 2015 to March 2017,according
to World Health Organizations (WHO)-2013 standards.[17] Severe cases of viral pneumonia were defined as follows: (1) having cough or difficulty
breathing; (2) finding viruses in nasal fluid, phlegm, or sputum; and at least one
of the following main symptoms: (1) cyanosis or SpO2 <90%, (2) severe respiratory distress (moaning and intercostal muscle external retraction),
(3) inability to tolerate enteral fluids, (4) loss of consciousness or coma, and/or
(5) convulsions.
We excluded children who (1) age below 1 month or above 5 years; (2) had nonviral
pneumonia (for example pneumonia after drowning, chemical pneumonia, aspiration pneumonia);
(3) had chronic, associated congenital diseases, or not (e.g., airway malformation,
congenital lung disease, liver failure, kidney failure, cystic fibrosis, chronic granulomatous
disease, or immune deficiency); or (4) were eligible to participate in the study but
the parents or guardian did not agree to participate. This study was approved by the
Institutional Review Board of Vietnam Military Medical University (Code: 92/QĐ-HVQY,
January 21, 2015).
Data Collection
All participants after hospitalization were carefully examined clinical symptoms by
pediatrics. Demographic information and medical history were collected from parents
or guardians. The specimens were collected and sent to the laboratory within the first
1 hour, refrigerated, and performed at the Department of Molecular Biology, National
Hospital of Pediatrics. Also, participants underwent one laboratory draw for several
tests, including complete blood counts, interleukin (IL)-6, high-sensitivity C-reactive
protein (hsCRP), and procalcitonin (PCT).
Specimen Collection and Blood Tests
One milliliter of vascular blood was taken into the tube with ethylenediaminetetraacetic
(EDTA) upon hospitalization for hematological tests. The tests were run by ABX Micros
ES60. Meanwhile, biochemical tests were conducted by taking 2 mL of venous blood when
the patient was hospitalized, does not freeze, centrifuge, and is done. Specimens
were put into tubes without anticoagulants or with anticoagulants such as Li-Heparin
and K3-EDTA. After drawing blood, specimens were centrifuged to extract serum or plasma.
The level of hsCRP was determined via turbidity measurement using the Olympus AU 2700
machine. PCT concentration was measured via the luminescent immunization method using
the ADVIA Centaur machine of Siemens company. Meanwhile, IL-6 was measured by using
BioRad's Bio-Plex Protein Array System.
Viral Detection
Influenza virus A, influenza virus B, and RSV were detected via rapid tests that used
the immunochromatographic method. Rhinovirus and adenovirus were identified by utilizing
the real-time polymerase chain reaction (RT-PCR). Each patient received 1 mL of blood
and 2 mL of nasopharyngeal fluid upon admission. Samples collected were sent to the
laboratory within the first hour, refrigerated, and tested. The RT-PCR method was
conducted on RT-PCR ABI 7500 and ABI 7500 FAST machines, following the procedure of
the Department of Molecular Biology, National Hospital of Pediatrics.
Samples of respiratory fluid collected were used for total RNA/DNA extraction (MagNA
Pure LC Total Nucleic acid Isolation Kit, Roche) on MagNA Pure LC 2.0 automatic extraction
system (Roche). The forward primers were: GCC ACG GTG GGG TTT CTA AAC TT, the reverse
primers were GCC CCA GTG GTC TTA CATGCA CT C, and the probe sequences were carboxyfluorescein
(FAM), TGC ACC AGA CCC GGG CTC AGG TAC TCC GA, tetramethyl-6-carboxyrhodamine (TAMRA).
Reaction components include: 0.625 µL concentration of 10 pM of each primer; 0.5 µL
concentration of 10 pM probe; 12.5 µL 2X PCR master mix (Qiagen); 5 µL DNA and H2O, which were added in total reaction of 25 µL. Reaction program included: 500—2 minutes,
950—15 minutes, and 45 cycles of 950—15 seconds, and 580—1 minute. RT-PCR assays for
Adenovirus detection used oligonucleotide primers and dual-labeled hydrolysis probes
(Taqman). The estimated time for running samples was approximately 125 minutes. The
positive control was from the plasmid of Gothenburg University, Sweden, and the negative
control was the water component in the PCR reaction mixture. Results were read and
analyzed using a RT ABI 7500 Fast system.
Bacterial Detection
Bacterial testing was performed by the Vitek-2 machine. The bacterium was detected
by using the colorimetric method to identify the chemical, biological properties of
each bacteria via color change of environmental wells in the card. Moreover, an antibiotic
method was also utilized by using the minimum inhibitory concentration measurement
method, which measured turbidity to monitor the development of microorganisms in the
card wells. These two methods were performed according to the principle of light intensity
reduction. The system used wavelength 660, 568, and 428.
Statistical Analysis
Chi-squared and Kruskal–Wallis tests were utilized to compare demographic, clinical,
and paraclinical characteristics between children with pneumonia caused by one virus
only versus those in which more than one virus was identified (viral coinfection)
and versus those in which bacterial superinfection occurred. STATA software 15.0 was
used to analyze data. Multivariate logistic regression was performed to identify the
associated factors with mixed coinfections. A stepwise backward selection strategy
was used, using the p-value of a log-likelihood test of less than 0.2 as a threshold to select variables.
A two-tailed p-value of less than 0.05 was considered statistically significant.
Results
Of 202 children with severe viral pneumonia, overall, the mean age of patients was
8.6 months (standard deviation [SD] = 9.6, range: 1.0–48.7 months). Among them, 59.9%
of study participants were male. Also, there was 19.8% of children were born with
low birth weight, 19.8% were born premature, 52% suffered from malnutrition, and 25.7%
were not fully. [Table 1] shows that the majority of patients had RSV (36.1%), followed by influenza virus
A (24.3%) and adenovirus (19.8%). There were 53 children (26.2%) coinfected with bacteria
or/and other viruses.
Table 1
Etiology of viral pneumonia
|
Characteristics
|
Single virus (n = 149)
|
Coinfection with bacteria (n = 34)
|
Coinfection with virus (n = 11)
|
Coinfection with bacteria and virus (n = 8)
|
Total (n = 202)
|
|
n (%)
|
n (%)
|
n (%)
|
n (%)
|
n (%)
|
|
Influenza virus A
|
36 (73.5)
|
5 (10.2)
|
6 (12.2)
|
2 (4.1)
|
49 (24.3)
|
|
Influenza virus B
|
10 (71.4)
|
4 (28.6)
|
0 (0.0)
|
0 (0.0)
|
14 (6.9)
|
|
RSV
|
59 (80.8)
|
11 (15.1)
|
2 (2.7)
|
1 (1.4)
|
73 (36.1)
|
|
Adenovirus
|
28 (70.0)
|
7 (17.5)
|
3 (7.5)
|
2 (5.0)
|
40 (19.8)
|
|
Rhinovirus
|
16 (61.5)
|
7 (26.9)
|
0 (0.0)
|
3 (11.5)
|
26 (12.9)
|
Abbreviation: RSV, respiratory syncytial virus.
[Table 2] illustrated that among children with bacteria superinfection, the incidence of Haemophilus influenza was the highest (45.2%), followed by Klebsiella pneumoniae and Pseudomonas aeruginosa (with 19.1%).
Table 2
Type of bacterium among viral pneumonia children with coinfections
|
Type of bacterium
|
n
|
Children with bacterial coinfections (%)
|
Percentage in entire sample
|
|
Haemophilus influenza
|
19
|
45.2
|
9.4
|
|
Klebsiella pneumoniae
|
8
|
19.1
|
4.0
|
|
Pseudomonas aeruginosa
|
8
|
19.1
|
4.0
|
|
Streptococcus pneumoniae
|
7
|
16.7
|
3.5
|
|
Acinetobacter baumannii
|
4
|
9.5
|
2.0
|
|
Burkholderia cepacia
|
1
|
2.4
|
0.5
|
|
Staphylococcus aureus
|
1
|
2.4
|
0.5
|
The distribution of coinfection status, according to sociodemographic characteristics,
is depicted in [Table 3]. No difference was found among these coinfection conditions regarding age, gender,
new-born weight, nutritional status, and immunization status (p > 0.05).
Table 3
Patients' characteristics according to coinfection status
|
Characteristics
|
Single virus (n = 149)
|
Coinfection with bacteria (n = 34)
|
Coinfection with virus (n = 11)
|
Coinfection with bacteria and virus (n = 8)
|
p-Value
|
|
n (%)
|
n (%)
|
n (%)
|
n (%)
|
|
Age group (mo)
|
|
|
|
|
|
|
0–6
|
88 (76.5)
|
16 (13.9)
|
6 (5.2)
|
5 (4.4)
|
0.19
|
|
> 6–12
|
31 (77.5)
|
8 (20.0)
|
0 (0.0)
|
1 (2.5)
|
|
> 12–24
|
14 (51.9)
|
8 (29.6)
|
3 (11.1)
|
2 (7.4)
|
|
> 24
|
16 (80.0)
|
2 (10.0)
|
2 (10.0)
|
0 (0.0)
|
|
Gender
|
|
|
|
|
|
|
Female
|
63 (77.8)
|
13 (16.1)
|
2 (2.5)
|
3 (3.7)
|
0.46
|
|
Male
|
86 (71.1)
|
21 (17.4)
|
9 (7.4)
|
5 (4.1)
|
|
Birth weight (g)
|
|
|
|
|
|
|
< 2,500
|
26 (65.0)
|
9 (22.5)
|
2 (5.0)
|
3 (7.5)
|
0.38
|
|
≥ 2,500
|
123 (75.9)
|
25 (15.4)
|
9 (5.6)
|
5 (3.1)
|
|
Nutritional status
|
|
|
|
|
|
|
Normal
|
51 (75.0)
|
11 (16.2)
|
5 (7.4)
|
1 (1.5)
|
0.65
|
|
Malnutrition
|
75 (71.4)
|
18 (17.1)
|
6 (5.7)
|
6 (5.7)
|
|
Overweight/obesity
|
23 (79.3)
|
5 (17.2)
|
0 (0.0)
|
1 (3.5)
|
|
Immunization
|
|
|
|
|
|
|
Full
|
114 (76.0)
|
26 (17.3)
|
7 (4.7)
|
3 (2.0)
|
0.08
|
|
Missing
|
35 (67.3)
|
8 (15.4)
|
4 (7.7)
|
5 (9.6)
|
Clinical characteristics of different coinfection conditions are shown in [Table 4]. Fever, cyanosis, and not hepatosplenomegaly were significantly different across
groups (p < 0.05). The total white blood cell count, neutrophils cell count, concentration
of hsCRP, and PCT were significantly higher in patients with bacterial coinfections
or bacterial and viral coinfections (p < 0.05).Invasive mechanical ventilation was significantly pervasive in infants with
both bacterial and viral coinfections (p < 0.05). They also had a significantly higher length of stay compared with other
groups (p < 0.05).
Table 4
Clinical characteristics regarding coinfection status
|
Characteristics
|
Single virus (n = 149)
|
Coinfection with bacteria (n = 34)
|
Coinfection with virus (n = 11)
|
Coinfection with bacteria and virus (n = 8)
|
p-Value
|
|
n (%)
|
n (%)
|
n (%)
|
n (%)
|
|
|
Fever
|
100 (67.1)
|
30 (88.2)
|
10 (90.9)
|
6 (75.0)
|
0.04
|
|
Rapid heart pulse
|
80 (53.7)
|
20 (58.8)
|
10 (90.9)
|
4 (50.0)
|
0.11
|
|
Runny nose
|
61 (40.9)
|
14 (41.2)
|
4 (36.4)
|
1 (12.5)
|
0.45
|
|
Wheezing
|
125 (83.9)
|
29 (85.3)
|
9 (81.8)
|
6 (75.0)
|
0.91
|
|
Grunting
|
9 (6.0)
|
1 (2.9)
|
2 (18.2)
|
2 (25.0)
|
0.07
|
|
Poor feeding
|
117 (78.5)
|
23 (67.7)
|
10 (90.9)
|
8 (100.0)
|
0.14
|
|
Excessive crying
|
36 (24.2)
|
11 (32.4)
|
1 (9.1)
|
4 (50.0)
|
0.17
|
|
Convulsions
|
9 (6.0)
|
0 (0.0)
|
1 (9.1)
|
1 (12.5)
|
0.37
|
|
Cyanosis
|
35 (23.5)
|
7 (20.6)
|
5 (45.5)
|
8 (100.0)
|
<0.01
|
|
Diarrhea
|
44 (29.5)
|
11 (32.4)
|
1 (9.1)
|
1 (12.5)
|
0.34
|
|
No hepatosplenomegaly
|
138 (92.6)
|
30 (88.2)
|
8 (72.7)
|
5 (62.5)
|
0.01
|
|
Invasive mechanical ventilation
|
11 (7.4)
|
7 (21.2)
|
2 (18.2)
|
5 (62.5)
|
<0.01
|
|
Mean (SD)
|
Mean (SD)
|
Mean (SD)
|
Mean (SD)
|
|
|
Time from illness onset to admission (d)
|
7.2 (6.6)
|
5.9 (4.8)
|
5.5 (3.9)
|
7.6 (5.2)
|
0.93
|
|
Length of stay (d)
|
13.2 (13.6)
|
23.4 (33.1)
|
17.4 (14.5)
|
33.5 (22.3)
|
<0.01
|
|
Hemoglobin (g/dL)
|
105.2 (14.3)
|
105.2 (13.4)
|
105.5 (8.5)
|
94.6 (9.6)
|
0.13
|
|
White blood cell (G/L)
|
11.1 (5.1)
|
14.8 (7.7)
|
9.1 (2.8)
|
14.4 (6.2)
|
<0.01
|
|
Neutrophils (G/L)
|
4.7 (3.6)
|
8.3 (6.4)
|
4.1 (1.8)
|
7.9 (5.2)
|
<0.01
|
|
Lymph (G/L)
|
4.7 (2.6)
|
4.6 (2.4)
|
3.3 (1.2)
|
5.1 (2.5)
|
0.30
|
|
Mono (G/L)
|
1.3 (1.1)
|
1.5 (1.0)
|
1.2 (0.7)
|
1.3 (0.6)
|
0.72
|
|
Platelet (G/L)
|
382.6 (155.6)
|
417.7 (230.1)
|
324.7 (163.8)
|
437.1 (190.9)
|
0.42
|
|
hsCRP (mg/dL)
|
12.7 (25.6)
|
32.5 (51.8)
|
8.1 (10.8)
|
9.3 (10.6)
|
0.02
|
|
PCT (ng/mL)
|
1.1 (1.7)
|
3.4 (5.9)
|
0.8 (1.1)
|
6.8 (15.1)
|
<0.01
|
|
IL-6 (pg/mL)
|
26.0 (82.1)
|
22.1 (36.9)
|
13.8 (12.4)
|
98.9 (171.1)
|
0.13
|
Abbreviations: hsCRP, high sensitivity C-reactive protein; IL-6, interleukin-6; PCT,
procalcitonin; SD, standard deviation.
The multivariate regression model showed that in toddlers ages12 to 24 months with
severe viral pneumonia (odds ratio [OR] = 3.37, 95% confidence interval [CI]: 1.22–9.33),
the higher concentration level of PCT (OR = 1.16; 95% CI: 1.00–1.34), and neutrophils
(OR = 1.13; 95% CI: 1.04–1.22) were associated with a higher risk of suffering from
any type of coinfection ([Table 5]).
Table 5
Associated factors with mixed coinfections
|
Factor
|
Mixed coinfection (yes/no)
|
|
OR
|
p
|
95%CI
|
|
Age group (mo)
|
|
|
|
|
|
0–6
|
REF
|
|
|
|
|
> 6–12
|
0.90
|
0.84
|
0.32
|
2.49
|
|
> 12–24
|
3.37
|
0.02
|
1.22
|
9.33
|
|
> 24
|
1.02
|
0.97
|
0.27
|
3.83
|
|
Immunization
|
|
|
|
|
|
Full injection
|
REF
|
|
|
|
|
Missing injection
|
2.02
|
0.11
|
0.86
|
4.73
|
|
Cyanosis
|
|
|
|
|
|
No
|
REF
|
|
|
|
|
Yes
|
2.08
|
0.07
|
0.93
|
4.63
|
|
No hepatosplenomegaly
|
|
|
|
|
|
No
|
REF
|
|
|
|
|
Yes
|
0.38
|
0.06
|
0.14
|
1.06
|
|
PCT (ng/mL)
|
1.16
|
0.045
|
1.00
|
1.34
|
|
Neutrophil (G/L)
|
1.13
|
< 0.01
|
1.04
|
1.22
|
Abbreviation: CI, confidence interval; OR, odds ratio; PCT, procalcitonin; REF, reference.
Discussion
This study contributes to global literature regarding the etiology and clinical characteristics
of severe viral pneumonia among infants in Vietnam. Results indicated that RSV and
influenza virus A was the most common viral pathogens, and one-fifth of patients identified
coinfections. Age and level of PCT and Neutrophils were important markers to predict
coinfection conditions.
The pattern of viral pathogens in the current study differs from other previous studies.
In Pakistan, a study of 817 infants and children under 2 years of age showed that
the prevalence of enterovirus/rhinovirus infection was 51.7%, followed by influenza
viruses (8.3%) and RSV (5.7%).[18] A study in China showed that the most common causative viruses were enterovirus/rhinovirus
(54.1) %), RSV (51.1%), Human bocavirus (33.8%), parainfluenza viruses type 3 (PIV3;
15.4%), and adenovirus (ADV; 13.0%).[19] Another study in China indicated a similar finding with our study that RSV was the
most common pathogen,[4] which was consistent with other studies in the United States of America[7] and Spain.[20]
Coinfection was common among children with severe viral pneumonia in our study, approximately
25.3% of our patients experienced coinfection in any type. This rate was a slightly
lower than that reported in previous studies in China and the United States, which
found that 34.6% of infants with CAP suffering from coinfections,[4]
[21] but was similar to another nationally representative study in the United States,
which indicated that 26% had coinfections.[7] Pavia et al found that among 58 pediatric patients with pneumonia, 35% were coinfected.[22] Nascimento-Carvalho et al in a group of 25 pneumonia children with pleural effusion,
showed that 22% of cases had viral–bacterial coinfections.[23] Juvén et al indicated that approximately 30% of cases suffered from viral–bacterial
coinfections.[24] Moreover, the study results showed that the incidence of H. influenza was the highest (9.4% of the entire sample), followed by K. pneumoniae and P. aeruginosa (with 4.0% of the entire sample). Streptococcus pneumoniae had been found in 3.5% of the sample, which was consistent with prior studies in
China (3.0%) and the United States (4%).[4]
[7] However, 9.4% of our study population were infected with another virus, which was
much lower compared with the previous study in the United States (26.0%).[7] The disparities might be attributable to the seasonal or geographical factors.
The results of this study showed that the majority of clinical characteristics could
not distinguish between coinfection groups except fever, cyanosis, hepatosplenomegaly,
and invasive mechanical ventilation. Similarly, analysis of paraclinical indicators
indicated that the total white blood cell count, neutrophils cell count, concentration
of hsCRP, and PCT were significantly higher among infants with bacterial superinfection
versus single virus. This result was in line with prior study in the United States,
which revealed that the prevalence of fever, as well as the level of neutrophils,
were the lowest among patients having viral infections only or viral coinfections.[21] Similarly, the study in China found that infants with mixed infections had a higher
rate of fever than that of patients who were infected with a single virus.[4] We conjecture that pediatric patients having viral coinfections presented a higher
level of inflammation than those having an only single viral infection.[4] Additionally, the regression model confirmed the associations between PCT and neutrophils
with mixed coinfections after adjusting to other covariates, suggesting potential
biomarkers to predict coinfections among young children with severe viral pneumonia.
Limitations
This study had several limitations. The study was conducted at the hospital; therefore,
the results of the study were only conclusive for the pneumonia children treated at
the National Hospital of Pediatrics, and we could not extrapolate to the community.
Our sample size was small and conveniently recruited, which thus might reduce our
generalizability. Moreover, data on several variables in the study, such as nutritional
status and premature birth, were significantly high or not allowed to categorize into
more details. The cross-sectional design is a limitation because of its limitations
due to its nature.
Conclusion
To conclude, this study underlined the pervasiveness of coinfections among infants
and young children with severe viral pneumonia. Provision of effective antiviral treatment,
especially for RSV, as well as the advancement of sensitive and rapid diagnostic tools
for screening pathogens of pneumonia, is critical to reducing the burden of this disease.
