Clinical impact of respiratory virus in pulmonary exacerbations of children with Cystic Fibrosis.

BACKGROUNDS: Cystic Fibrosis (CF) is a genetic, multisystemic, progressive illness that causes chronic suppurative lung disease. A major cause of morbimortality in this condition are pulmonary exacerbations. Although classically attributed to bacterial infections, respiratory virus have been increasingly recognized in its ethiopathogeny.
METHODS: Nasopharyngeal swab samples were collected from children < 18 years old with CF in Rio de Janeiro, Brazil, with pulmonary exacerbation criteria. Samples were submitted to RT-PCR for Adenovirus, Influenza A and B, Parainfluenza Virus, Respiratory Syncytial Virus (RSV), Metapneumovirus and Rhinovirus. Virus positive and virus negative groups were compared in regards to clinical presentation, severity of exacerbation and bacterial colonization.
RESULTS: Out of 70 samples collected from 48 patients, 35.7% were positive for respiratory viruses. Rhinovirus were the most common (28% of all positive samples), followed by RSV. The virus positive group was associated with change in sinus discharge (p = 0.03). Considering only patients younger than five years old, positive virus detection was also associated with fever (p = 0.01). There was no significant difference in clinical severity or in bacterial colonization between virus positive and negative groups.
CONCLUSIONS: Prospective studies are still needed to assess the long term impact of viral infections in patients with CF, and their interaction with the bacterial microbiome in these patients.

Introduction

Cystic Fibrosis (CF) is a genetic multisystemic progressive condition, caused by mutations of the Cystic Fibrosis Transmembrane Regulation (CFTR) gene. Pulmonary disease is the main cause of mortality in CF patients [1]. The progressive lung damage is marked by episodes of acute worsening of symptoms, called pulmonary exacerbations (PE), which are associated with disease progression and have an important impact on patients’ quality of life [1, 2]. Bacterial infections have been historically acknowledged as the major cause of PE. However, with the popularization of molecular techniques for virus detection, several studies have demonstrated a large prevalence of virus in PE events [3-15], showing significant association between virus detection and PE symptoms [5, 8, 9, 12, 15]. Despite this growing identification, little is still known about the clinical impact of respiratory virus infection in CF patients, with studies often presenting conflicting results.

The aim of this study was to determine the prevalence of respiratory virus infections in children and adolescents with CF during PEs, and compare virus positive and negative groups in regards to clinical manifestations, severity of PE and bacterial colonization.

Material and methods

CF patients were recruited from the CF service, in Fernandes Figueira National Institute for Women, Children and Adolescent Health at Rio de Janeiro/ Brazil, from January to December 2018. The inclusion criteria were age < 18 years; regular follow up at the CF center and presence of PE, based on the Fuchs Criteria [16]. Shortly, this criteria defines an exacerbation by the presence of at least four of the following signs and symptoms: change in sputum pattern; new or worsening hemoptysis; increase in cough; dyspnea; malaise or fatigue; fever; anorexia or weight loss; sinus pain; change in sinus discharge; change in physical examination of the chest; decrease in pulmonary function by 10% of the forced expiratory volume, and radiographic changes indicative of pulmonary infection. Patients were excluded if they had other chronic pulmonary, cardiovascular, neurologic, digestive, or rheumatologic disease not related to CF.

Clinical data was collected from patients’ charts. The Cystic Fibrosis Clinical Score (CFCS) [17] was applied to evaluate PE severity and the Shwachman-Kulczycki Score [18] to evaluate CF severity. When a chest X-ray was not performed at the moment, the last X-ray performed was used to apply this score. Nutritional evaluation was performed according to the Cystic Fibrosis Nutritional Guidelines [19].

Total nucleic acid was extracted from combined nasopharyngeal swabs using QIAamp® Viral RNA mini kit (QIAGEN, Hamburg, Germany), generating 80uL of purified nucleic acid in the final step. The Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) for Rhinovirus (RV), Adenovirus (AdV), Respiratory Syncytial Virus (RSV), Influenza A and B, human metapneumovirus (hMpV) and Parainfluenza (PIV) 1, 2 and 3 detection was performed using Go Taq® Probe 1-Step RT-qPCR System kit (Promega, USA) in a ABI 7500 real time thermocycler. The cycling protocol was: 45°C/30min, 95°C/5min followed of 45 cycles of 95°C/15s, 55°C/30, with data collection in the last stage. All molecular biology procedures and analysis were performed in the Respiratory Virus and Measles Laboratory at IOC/FIOCRUZ. Real time results were made available to the CF Center’s physicians and to the patients right after their analysis.

Oropharyngeal swabs or sputum samples for bacterial culture were also collected at each visit following the CF Center’s protocol, and its results were made available to the researches through patients’ charts. This study was approved by both institutions’ Research Ethics Committee (protocol n. 79277117.0.0000.5269) and informed consent was obtained from all participants and their parents/guardians.

Statistical analysis was performed using the program R, version 3.5.2 ® (2018). Virus positive and negative groups were compared using chi-squared and Fisher’s Exact Test for categorical variables, Students T test for normally-distributed numerical variables and Mann- Whitney`s U Test for skewed data. Results were considered statistically significant when p<0.05.

Results

During the year of 2018, 183 patients were followed by the CF Center, totaling 706 appointments. Forty-eight of these patients presented PE during appointments, in 71 different occasions. One case was excluded because the patient had chronic hypoxic ischemic encephalopathy, leaving a total of 47 patients and 70 episodes of PE included in the study. All 70 swab samples were tested by real time RT-PCR, and 25 (35.7%) were positive, as shown in Table 1. Two samples tested positive for more than one virus: one case of PIV 1 + RSV and one of PIV 2 + HMpV. Epidemiological characteristics of the patients included in the study are described in Table 2. Clinical characteristics of virus positive and negative groups are shown in Table 3.

Table 1

Results of real time RT-PCR respiratory viruses detection.

Virus	Positive Samples (n = 25)
Rhinovirus	7 (28%)
Respiratory Syncytial Virus	6 (24%)
Adenovirus	5 (20%)
human Metapneumovirus	4 (16%)
Parainfluenza virus	3 (12%)1 PIV1, 1 PIV 2, 1 PIV3
Influenza A	2 (8%)1 H1N1 and 1 H3N2

Table 2

Baseline characteristics of the 47 patients enrolled in the study.

Characteristics	Patients (n = 47)
Gender (male:female)	24:23
Homozygus F508del	12 (25.5%)
Heterozygus F508del	13 (27.6%)
Pancreatic Insufficiency	41 (87.7%)
Diabetes Mellitus	1 (2.1%)
Influenza Immunization	25 (53.2%)

Table 3

Clinical and epidemiological characteristics of virus positive and negative groups.

	Virus Positive (n = 25)	Virus Negative (n = 45)	p value
Median age in years (range +- SD)	3.9 (0.3–17,9; +- 4.9)	5,5 (0.3–17; +- 4.6)	0.3a
Median Days of symptoms (range+- SD)	6 (1–21; +-5.3)	6 (0–30; +-5.5)	0.7 a
Clinical Features
Change in Sinus Discharge	21 (84%)	26 (57.8%)	0.03
Worsening cough	25 (100%)	44 (97.8%)	1
Change in sputum pattern	22 (88%)	38 (84.4%)	1
Worsening dyspnea	14 (56%)	27 (60%)	0.8
Hemoptysis	1 (4%)	1 (2.2%)	1
Fatigue	9 (36%)	10 (22.2%)	0.21
Fever	16 (64%)	19 (42.2%)	0.08
Anorexia/weight loss	14 (56%)	26 (57.8%)	0.89
Sinus pain	1 (4%)	2 (4.4%)	1
Low oxygen saturation	7 (28%)	15 (33.3%)	0.65
Abnormal Chest Auscultation	20 (80%)	42 (93.3%)	0.1
Treatment
Antibiotics	19 (76%)	40 (88.9%)	0.18
Oseltamivir	1 (4%)	0	0.35
Oxygen therapy	6 (24%)	15 (33.3%)	0.41
Noninvasive Ventilation	3 (12%)	4 (8.9%)	0.69
Hospital Admission	10 (40%)	21 (46.7%)	0.11
Mean length of hospital stay in days (range +- SD)	14.6 (7–21; +- 3.8)	16,5 (8–34; +- 5.6)	0.45 a
Severity Scores
Mean CFCSc (range +- SD)	30.3 (19–39; +- 5.4)	29.4 (18–41; +- 5.9)	0.25b
Mean Shwachman-Kulczycki Score (range +- SD)	72.2 (45–95; +-11.9)	71.6 (35–90; +-12.4)	0.42 b

aStudent’s T test.

bMann Whitney’s U test.

cCystic Fibrosis Clinical Score.

The number of X-ray exams ordered and the finding of new radiologic images suggesting infection did not differ between virus positive and negative groups. Bacterial colonization status previous to the PE events are described in Table 4. The bacterial culture from sputum or oropharyngeal swabs of the moment of the PE were tested for all samples enrolled in the study, but two (Table 5).

Table 4

Previous bacterial colonization.

Previous Bacterial Colonization	Virus Positive (n = 25)	Virus Negative (n = 45)	P value
Negative or Staphyloccocus aureus	6 (24%)	11 (24.4%)	0.97
Intermittent MRSAa	3 (12%)	9 (20%)	0.52
Intermittent Pseudomonas aeruginosa	12 (48%)	20 (44.4%)	0.77
Chronic Pseudomonas aeruginosa	4 (16%)	4 (8.9%)	0.44
Intermittent Burkholderia cepacia	3 (12%)	5 (11.1%)	1
Chronic Burkholderia cepacia	1 (4%)	5 (11.1%)	0.41

aMethicillin-resistant Staphylococcus aureus.

Table 5

Bacterial culture results from sputum or oropharyngeal swab of virus positive and negative patients.

Bacterial Culture Results	Virus Positive (n = 24)	Virus Negative (n = 44)	p Value
Negative	3 (12.5%)	7 (15.9%)	1
S. aureus	13 (54.2%)	25 (56.8%)	0.83
MRSAa	3 (12.5%)	6 (13.6%)	1
K. pneumoniae	0	1 (2.3%)	1
P. aeruginosa	11 (45.8%)	12 (27.3%)	0.12
B. cepacia	1 (4.2%)	8 (18.2%)	0.14
A. xylosoxidans	1 (4.2%)	0	0.35
S. maltophilia	2 (8.3%)	0	0.12
K. pneumoniae	0	1 (2.3%)	1
NFGNBb	1 (4.2%)	2 (4.5%)	1

aMethicillin-resistant Staphylococcus aureus.

bNon-fermentative Gram-Negative Bacilli, Pseudomonas and Burkholderia excluded.

A subgroup analysis was conducted with 37 samples from children <5 years. Fourteen samples (37.8%) were positive for virus detection, namely: six RSV, four RV, two hMpV, one Influenza A(H1N1)pdm09, one PIV 1, one AdV. Clinical and epidemiological characteristics of these patients are shown in Table 6.

Table 6

Clinical and epidemiological characteristics of virus positive and negative patients < 5 years.

Variable	Virus Positive (n = 14)	Virus Negative (n = 22)	p value
Median Age in Years (range +- SD)	2.3 (0.3–4.9; +-1.3)	2.6 (0.3–4.3; +-1.2)	0.29a
Clinical and Radiological Features
Change in sinus discharge	12 (85.7%)	13 (59.1%)	0.14
Worsening cough	14 (100%)	22 (100%)	1
Change in sputum pattern	11 (78.6%)	15 (68.2%)	0.71
Worsening dyspnea	9 (64.3%)	16 (72.7%)	0.72
Hemoptysis	0	0	1
Fatigue	6 (42.9%)	3 (13.6%)	0,11
Fever	9 (64.3%)	5 (22.7%)	0,01
Anorexia / weight loss	8 (57.1%)	11 (50%)	0,68
Oxygen therapy	4 (8.6%)	7 (31.8%)	1
Abnormal Chest Auscultation	13 (92.9%)	21 (95.4%)
Hospital Admission	6 (42.9%)	10 (45.4%)
Severity Scores
CFCSb (mean)	30.3 (19–38; +-5.7)	28.1 (19–37; +-4.4)	0.12a
Mean Shwachman-Kulczycki Score (range +- SD)	78.2 (65–95; +-8.5)	76.4 (60–90: +-7.4)	0.25 a

aStudent’s T test.

bCystic Fibrosis Clinical Score.

Discussion

The prevalence of respiratory virus in this study was 35.7%. In agreement with previous studies, picornaviridae viruses were the most frequently detected viruses, regardless of age [4–9, 12, 20, 21]. RSV was the second most frequent virus, and had the lowest hospital admission rate, even though it has been suggested that CF patients could be at risk for more severe infection by this agent [22]. One possible explanation is that severe infections are more common in patients under two years of age, whereas the median age of the patients in our study was 4.6 years old. Noteworthy, the mean age of RSV positive patients was two years old.

Regarding clinical findings, change in sinus discharge was associated with viral detection (p 0.03), as would be expected and has been described by other authors [8, 9]. In patients under five years old, fever had a significant association with virus detection (p = 0.01), as also previously described [3]. Influenza has been found to be particularly associated with presence of fever [9]. In our study, the two influenza positive cases were older than five years and had this symptom indeed.

Regarding treatment decisions, antibiotic prescription was not significantly different in patients with and without virus detection, corroborating that these situations may be difficult to clinically distinguish. On the other hand, oseltamivir prescription was extremely low, even when clinical symptoms were indicative for its empiric use. Only one patient received the anti-viral treatment, which was started empirically and was later confirmed to have Influenza A H3N2. The second patient with influenza A was not treated because the drug was not started empirically and viral detection results were only available after 48 hours of symptoms, when the patient was already clinically improving. It is important to note that the vaccination rate for influenza was also low (58.1%), as previously reported [14, 15]. CF is an important risk factor for severe infection by influenza [23], and adequate treatment and prevention of this condition must be encouraged.

In contrast to other studies which showed PE to be more severe in cases with viral detection [3, 8, 14, 24], our findings didn’t show a significant difference between virus positive and negative groups, measured by the CFCS and by dyspnea, low oxygen and hospital admission rates. However, median age in these studies was older, including adolescents and adults, and it must also be stressed that the limited size of the sample may have impacted the power of statistical analysis.

Viral infections have been shown to increase susceptibility to bacterial colonization of the respiratory tract [25, 26]. Since this colonization plays a major role in the progression of lung disease in CF, the relationship between these agents in CF is an important object of study. RSV has been shown to promote Pseudomonas colonization in CF patients [27, 28], and the same has been suggested for picornavirus [11]. In patients already chronically colonized by this bacteria, RV may increase liberation of planktonic bacteria from the biofilm [29] which is associated with new bacterial infection. On the other hand, a study by Chin et al didn’t find an increase in Pseudomonas density in sputum during viral infections [30]. As in most previous studies [9, 10, 22, 31] we found no difference of pseudomonas prevalence in patients with viral detection.

It has also been suggested that the presence of bacteria in the respiratory tract of CF patients may favor viral infection [25], but more studies regarding the specific role of Pseudomonas in this interaction are still needed. In the present study, virus detection did not significantly differ between different types of previous bacterial colonization. It is important to consider that in developing countries such as Brazil, the age of Pseudomonas colonization tends to be younger than in wealthier countries where most of these cited studies were performed. In our population, 57,1% of samples belonged to children with previous intermittent or chronic P. aeruginosa colonization.

The prevalence of respiratory virus, although significant, was smaller than previously reported in some prior studies that also applied molecular technics [3–6, 11]. An explanation for this difference is the broader PE definition criteria [3, 5, 6] and the inclusion of samples from patients with upper respiratory tract infections without PE [4, 11]. In our perspective, a more open inclusion criteria eventually included milder cases, less important for the course of the disease or the clinical conduct.

In order to account for viral seasonality patterns, samples were collected through 12 consecutive months. On the other hand, some studies were conducted only throughout autumn and winter seasons, when there’s a higher circulation of respiratory virus, which may contributed to higher viral detection rates [3, 4, 7].

The study’s main limitations were the size of the sample and timing of sample collection. In a previous study in which patients were trained to self-collect samples as soon as the first signs of respiratory disease appeared and mail them to the laboratory, virus detection rates reached 81% of 43 samples investigated [6].

In addition, we did not test for coronavirus or bocavirus. Even though these agents have been usually shown to have a small prevalence in PE [3, 5–7, 10, 12, 20], this may have had some impact on the total viral detection rates, and especially on the seven samples in which neither virus nor bacteria were identified.

Conclusions

Our findings corroborate that respiratory virus have a significant prevalence in pulmonary exacerbations in CF. Their detection was associated with change in sinus discharge and in children <5 years with fever. Routine testing for these agents may help to better guide PE treatment and antibiotic use. Furthermore, as PEs have a great impact on morbidity and mortality in CF patients, the recognition of respiratory virus as an important agent in these conditions proves how fundamental it is to increase preventive strategies such as isolation protocols for patients and immunization. Unfortunately, there still are no effective vaccines, prophylaxis and treatments for most respiratory virus, however the viral diagnostic supported the rational use of antibiotics, avoiding its misuse. Longitudinal studies are still needed to better understand the relationship between virus and bacterial colonization in CF.

De-identified data set.

(XLSX)

Click here for additional data file.

17 Aug 2020

PONE-D-20-16036

Clinical impact of respiratory virus in pulmonary exacerbations of children with Cystic Fibrosis

PLOS ONE

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This is a very simple retrospective analysis of data that I think is flawed in combining static, patient-specific features with variable event-specific features. I would encourage evaluation by a statistician.

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Reviewer #1: Although the manuscript is an original work, it is more like an observational study, in which the findings reported are very similar to previously published articles, at least in some form.

The conclusion of this study is very similar to other published reports and thus it does not fulfill the criteria of Replication studies described in the Plos One Guidelines for Reviewers.

I suggest to submit it as an 'Observational study' instead of 'original article'

Reviewer #2: The authors have completed a prospective collection of samples and data on a cohort of subjects with cystic fibrosis. They have determined the viral infection at the time of pulmonary exacerbation and identified factors that differentiate those that are virus positive compared with virus negative.

The authors have provided a comprehensive introduction and discussion with reference to the established literature. They authors have used well established assessment tools in CF research. As such, the study provides some useful information.

I have a few major and minor comments to make that could be addressed by the authors.

Major

1. Since the number of positive subjects is quite limited, I would like to know the power to detect statistical differences between virus positive and negative groups for each outcome. This could be added as an online supplement. I am concerned this study does not have the statistical power to detect differences based on the subject numbers and that is why so few outcomes are significant.

2. If a result is not statistically significant (or even close to p-0.05) then there is no relationship. For example, the authors claim that they virus positive group were younger, but statistically they were not (p=0.3), therefore they are NOT younger. This is described in the results and the discussion. This also includes fever, fatigue antibiotic prescription, P. aeruginosa prevalence, acquisition of P. aeuginosa, intermittent or chronic P. aeruginosa in the whole group or the children <5 years of age (outlined in the discussion).

3. It would be helpful if the authors identified which previous studies mentioned in the discussion (for example: when the authors are discussing the lack of difference in CF severity between virus positive and negative groups) were of a similar age and which were from older children.

Minor

1. Although the article is quite well written and perfectly understandable – the article requires further editing for English.

2. It would be helpful if standard acronyms were used such as URTI (upper respiratory tract illness) instead of URTS.

Reviewer #3: The manuscript by Correa Meyer et al describes an analysis of respiratory virus infection in persons with CF during episodes of pulmonary exacerbation (PE). The aim of the study was to compare virus-positive and virus-negative groups in regards to clinical manifestations, severity of PE and bacterial infection. The authors conclude that prospective studies are needed to assess the impact of viral infection in CF.

A concern with the design and analysis of data in this study is that patient-specific (static) and exacerbation event-specific (variable) characteristics are combined and compared (Table 2). That is, some patient-specific features (eg CFTR genotype, gender pancreatic insufficiency) are counted as independent features for each episode of exacerbation. This seems to introduce confounding. Eg, if an individual heterozygous for F508del had three exacerbation episodes, but by virtue of lack of siblings or age, none of these were associated with viral infection, the three episodes would inflate the number of virus-negative events in the F508del heterozygous group… supporting that viral infection is less common in persons heterozygous for F508del. A more robust multivariable analysis with attention to interactions between variables seems to be required.

On a more minor note: There are implications of causality, when associations are all that can be assessed. Eg: Abstract: do exacerbations cause morbidity in CF … or are these events a sign of morbidity? Eg: Intro: do exacerbations impact disease progression…or are these events a manifestation of lung disease progression? Eg, have the studies cited (refs 3-15) show that viral infection is ‘responsible for’ exacerbation events?

Reviewer #4: This is a study from Brazil in pediatric patients with CF that explores whether viral DNA/RNA can be found during a CF exacerbation. The authors found that 36% of 70 respiratory CF exacerbations were associated with retrieval of viral genetic material from NP swabs.

Comments:

1) Unfortunately this is a relatively small study, and statistical power is quite limited. Therefore the investigators were not able to show significant differences between viral-associated exacerbations and non-viral exacerbations.

2) I think that the study abstract and conclusions need to stress lack of statistical power as a major limitation of this study, this may explain why significant differences between viral-associated exacerbations and non-viral exacerbations was not found.

3) The fact that viral exacerbations were associated with upper resp tract symptoms is of course not surprising, and this may need to be made clearer in the abstract and conclusions.

4) Tables 2 and 5 are far too long and hard to read and all statistical comparisons are non-significant. Please limit these tables to important variables only- please shorten them to 5-6 variables.

5) The discussion should include mention of a study that is highly relevant : Chin M, De Zoysa M, Slinger R, Aaron SD. Acute effects of viral respiratory tract infections on sputum bacterial density during CF pulmonary exacerbations. Journal of Cystic Fibrosis, 14: 482-489, 2015.

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21 Sep 2020

We thank Reviewer #1 for their considerations, but we checked with the journal office and PLOS ONE does not have a 'Observational study' article type. Therefore we resubmitted the manuscript as a "Research Article", which include observational studies.

We appreciate Reviewer #2 for their considerations. The study was reviewed by a statistician and we have included a minimal anonymized data set from the Study as a Supporting Information File. The discussion was also rewritten. All sections describing relationships that were not statistically significant were reviewed and corrected to show that in fact no relationship could be found. The limited size of the sample was also stressed as a potential limitation for statistical analysis. The manuscript was also thoroughly reviewed and edited for English.

We appreciate reviewer #3's comments. In fact, we reviewed the study with a statistician and excluded patient-specific features from the comparison of virus positive and virus negative exacerbations. These features were separately described in Table 2. Due to the limited size of the sample a more robust multivariable analyses was not possible.

We thank Reviewer #4 for their comments. We included the suggested article in the discussion, shortened the tables and stressed potential limitations of the study.

Submitted filename: Response do Reviewers.docx

Click here for additional data file.

28 Sep 2020

Clinical impact of respiratory virus in pulmonary exacerbations of children with Cystic Fibrosis

PONE-D-20-16036R1

Dear Dr. Viviane Mauro Correa Meyer,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Abdelwahab Omri, Pharm B, Ph.D

Academic Editor

PLOS ONE

7 Oct 2020

PONE-D-20-16036R1

Clinical impact of respiratory virus in pulmonary exacerbations of children with Cystic Fibrosis

Dear Dr. Meyer:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Abdelwahab Omri

Academic Editor

PLOS ONE