Characteristics of asthmatic children with Mycoplasma pneumoniae infection in Vietnam

Introduction: Mycoplasma pneumoniae is regarded as an involved factor in refractory asthma exacerbations. The link between Mycoplasma pneumoniae infection and asthma is not fully understood, particularly regarding clinical and paraclinical characteristics. Objectives: To determine the frequency of Mycoplasma pneumoniae infection in asthmatic children, identify clinical and paraclinical characteristics along with clinical management in children aged 6 to 15 years at Can Tho Children's Hospital, Vietnam. Method: From March 2019 to April 2021, 124 patients participated in a cross-sectional study that was done. Mycoplasma pneumoniae was detected using an immunoglobulin M (IgM) serology test. Results: Mycoplasma pneumoniae -IgM serology was positive in 32.3% of asthmatic children. IgM-positive individuals had significantly greater percentages of accessory respiratory muscle contraction, coarse crackles, fever, and increased heart rate. Mycoplasma pneumoniae infection occurred in 42.3% of children who were hospitalized for the first time and had asthma diagnosis. Mycoplasma pneumoniae infection and the severity of acute asthma exacerbations had a positive association (p<0.05). A longer hospital stay (6.74±1.91 days) and a higher exacerbation recurrence rate (32.4%) were observed in the IgM-positive group (p<0.05). Conclusions: Mycoplasma pneumoniae infections were highly prevalent in asthmatic children. Severity of acute asthma exacerbations, first-time asthma diagnosis, and recurrence of asthma exacerbation were significantly


Introduction
While asthma can affect individuals of all age groups, it is more prevalent among children 1 .With careful monitoring, treatment, and control of asthma exacerbations, 80-90% of individuals affected by asthma can avoid a fatal outcome 2 .Mycoplasma pneumoniae (Mp) infection is the leading cause of community-acquired pneumonia 3 .In the past thirty years, Mp infection has been strongly linked to the development of acute asthma, especially in children, with rates as high as 64% 4,5 .Presence of Mp infection can pose difficulties in controlling asthma and have an adverse impact on the effectiveness of treatment, resulting in persistent symptoms during asthma exacerbations 3,6,7 .Clinical and paraclinical symptoms in asthmatic children with Mp infection are diverse and non-specific 8 .In clinical practice, even when physicians follow asthma treatment guidelines, neglecting Mp infection can hinder long-term patient recovery 9 .
In Vietnam, there is a lack of available evidence regarding the association between Mp infection and childhood asthma.To the best of our knowledge, no comprehensive study has been conducted to concurrently assess clinical manifestations, laboratory findings, and asthma control in Vietnam.This presents a challenge for doctors in terms of clinical decision-making and post-discharge monitoring.

Objectives
To determine the frequency of Mp infection in asthmatic children, identify clinical and paraclinical characteristics along with clinical management in children aged 6 to 15 years at Can Tho Children's Hospital, Vietnam.

Method
A cross-sectional study was carried out in Can Tho Children's Hospital, Vietnam from March 2019 to April 2021.

Inclusion and exclusion criteria:
We included all paediatric patients aged 6-15 years diagnosed with asthma exacerbation and who received diagnostic tests of Mpspecific immunoglobulin M (IgM) antibody.Children with other diseases in the lungs (tuberculosis, pulmonary fibrosis, broncho-pulmonary dysplasia, etc.), comorbid diseases (heart, liver, kidney diseases, cerebral palsy, etc.), recurrent wheezing of unknown cause, severe psychological disorder and those who had received macrolides within the 4 weeks before admission were excluded.

Sample size:
The required sample size for the study was estimated to be 115 participants, based on the Cochran formula with a Z1-α/2 value of 1.96, a confidence coefficient of 0.95, and a probability of type I error of 9%.The prevalence of Mp infection was 41%, based on data from a previous study (p=0.41) 10 .However, we ultimately collected a larger sample of 124 participants to increase the power of our analysis.
Diagnosis of asthma was based on: 1.Having been previously diagnosed with asthma or 2. Diagnostic criteria for asthma in children according to Global Initiative for Asthma (GINA) 2019 2 : History of variable respiratory symptoms with typical features: wheezing, shortness of breath, chest tightness, and cough and evidence of variable expiratory airflow limitation: peak flow test results with average daily diurnal PEF variability >13%.Asthma exacerbation and severity were also defined according to GINA 2019 2 .The x-ray was reported blindly by a doctor in the imaging department who was not involved in the data interpretation.

For Mp infection diagnosis LIAISON® Mp-IgM
technique based on serology method was used.This method has a diagnostic sensitivity of 99.1% and a specificity of 97.8% 11 .A venous blood sample (2 ml) was obtained from each patient on days 5-10 after onset of asthma.Blood sample was collected immediately upon admission.Collected blood samples were centrifuged at 2,000 rpm for 10 minutes at 4℃. Serum was separated and stored at 2-8℃ for 8 days or frozen at -20℃ until sent for antibody testing.Samples were analysed by chemiluminescence immunoassay for Mp-specific IgM antibodies using the LIAISON® Mp-IgM analyser.As per the manufacturer's procedure, the IgM antibody value ≥10AU/mL obtained in a single determination was considered positive.
We recorded the prevalence of Mp infection through the Mp-IgM positivity in asthma exacerbation in children.The clinical characteristics of asthmatic children with and without Mp infection were assessed with the appearance of the clinical symptoms of asthma exacerbation, including cough, wheezing, fever, tachypnoea, increased pulse (the child's pulse is over the normal range of age), and accessory respiratory muscle contraction, coarse crackles, moist rales.When a patient comes in with acute asthma, we diagnose the exacerbation severity according to GINA 2019 and treat it as per the guidelines of GINA.In addition, with a non-severe asthma exacerbation, if the child does not respond to three doses of salbutamol inhalation, the attack would also be defined as severe.We also recorded the patient's history with asthma as first-time diagnosed asthma and pre-existing asthma with exacerbations.

Laboratory characteristics of IgM-positive asthmatic patients:
White blood cell count (aged normal range; increased); neutrophil count (<8000/mm 3 ; ≥8000mm 3 ); eosinophil count (<400/mm 3 ; ≥400/mm 3 ) 12 ; posteroanterior chest x-rays (normal; abnormal with the image of air trapping, air-space lesions, or interstitial lesions) were recorded in comparison with IgM-negative asthmatic patients.Asthma management in a patient was assessed with 2 clinical indicators: recurrence of asthma exacerbation during hospitalization and length of hospitalstay.The recurrence of asthma exacerbation was defined as when the patient again revealed symptoms of asthma exacerbation and needed reliever medication during the hospital stay.
Ethical issues: This study was approved by the Ethics Committee in Biological Research of Can Tho University of Medicine and Pharmacy (Approval number: 026/PCT-HĐĐĐ on February 19, 2019).Written informed consent was obtained from the parents of the participants and assent from the older children.

Statistical analysis:
Data analyses were performed using Statistical Package for the Social Sciences version 20.0 (SPSS 20.0).The difference between the two ratios and the difference between 2 means were analysed by the Chisquare test and the t-test.p-values <0.05 were considered statistically significant.

Results
During the study period 124 patients were recruited, of whom 40 (32.3%) had positive Mp-IgM.The proportion of children aged 6-10 years in the IgM positive group was significantly higher than in the negative group.In contrast, the proportion of children aged 11-15 years in the IgM positive group was lower than in the negative group.The difference in prevalence of IgM positive between the two age groups was statistically significant (p <0.05) (Table 1).The age group in this study was divided based on references from prior studies 10,13 .Fever, increased pulse, accessory respiratory muscle contraction, and sibilant wheeze were significantly higher in the IgM positive group compared to the negative group (Table 2).The prevalence of severe asthma exacerbation was significantly higher in the IgM positive group compared to the negative group (Table 2).IgM positive prevalence in the children with first-time diagnosed asthma was significantly higher than that of patients with pre-existing asthma (p <0.05).In asthmatic patients with abnormal chest x-rays, children having positive Mp-IgM had significantly more interstitial lesions compared to children in the IgM negative group (Table 2).  1 Asthma was diagnosed for the first time in this admission, where the children were admitted with asthma and an adequate history to diagnose asthma (according to GINA diagnostic criteria). 2Children have been diagnosed with asthma and had exacerbations.
Recurrent exacerbation was recorded in 27.5% of asthmatic patients having positive Mp-IgM, which was significantly higher compared to the IgM negative group (10.7%) ( 2 =5.64, p <0.05).Mp-IgM positive patients had a longer hospital stay (6.58±1.91 days) when compared with the negative group (5.7±1.9 days); the difference was statistically significant with p<0.05 (Table 3).

Discussion
According to this study, Mp was present in about one-third of hospitalised children with asthma.Thus, patients with acute asthma are advised to undergo the Mp diagnostic test at the start of their hospitalisation, especially if they are aged between 6 and 10 years or have just been diagnosed as asthma.Prevalence of Mp infection with positive Mp-IgM (over 40%) was lower than in previous studies 12,13 , but higher than 14.3% reported by Nguyen TVA, et al 14 using polymerase chain reaction for Mp detection.Local climatic factors like temperature and humidity were associated with Mp epidemic [15][16][17] .Positive Mp-IgM was mainly in the 6-10-year-old age group, while prevalence in males and females did not differ.This is similar to studies by Biscardi S, et al 6 and Le TMH, et al 12 .
Infection with Mp can worsen severity of asthma exacerbation 7,13 .In our study, prevalence of Mp-IgM positivity in patients with severe asthma exacerbation was 53.8%.Also, Community-Acquired Respiratory Distress syndrome (CARDs) toxin produced during Mp infection is related to pathogenesis of asthma [18][19][20] .We also found an association between Mp infection and severity of the acute respiratory failure, which is consistent with studies by Ou CY, et al 7  The results of chest x-rays showed that asthmatic children with positive Mp-IgM had a significantly higher rate of interstitial lesions compared to the negative group.However, as the radiographic feature is influenced by radiologists' interpretation, it is necessary to rely on other markers to approach the Mp infection diagnosis 8,22 .In contrast to Huynh's findings in asthmatic children with raised IgE levels of only 9.1% 23 , this study found a greater rate of exacerbation recurrence in asthmatic children with positive Mp-IgM.A retrospective study considered Mp infection an independent risk factor for re-admission in asthmatic patients, and Mp infection doubled the chances of early re-admission due to asthma exacerbation.The authors, therefore, emphasized the importance of longterm post-discharge follow-up goals for these children 24 .
Mp may also play a role in triggering recurrent wheezing in children 25,26 .In children who have had their first asthma exacerbation and are infected with Mp, absence of macrolide use increases risk of asthma recurrence and more severe exacerbation requiring admission to intensive care unit 27 .Therefore, asthma patients admitted to the hospital with Mp infection need to be closely monitored.
In our study, length of hospital stay of children with asthma exacerbation and positive Mp-IgM was similar to a previous study in Vietnam with an average of 6.2±2 days 28 .In a study by Wood PR, et al 29 asthmatic children infected with Mp had a longer hospital stay than children with asthma alone but this was not statistically significant.
The study had some limitations.This is a single hospitalbased study.We utilised a single determination in a semiquantitative IgM antibody analysis to diagnose Mp infection.As IgM antibodies can remain in the body for months after an initial infection, Mp-IgM positivity at admission may not be a reliable indicator of whether the child had Mp at the time of admission or had already recovered.A more precise diagnosis of acute Mp infection needs paired sera and an increase in antibody level.Convalescent-phase serum samples, however, are not usually accessible 30,31 .Additionally, a limited sensitivity (13%) for identification of Mp infection by PCR on nasopharyngeal aspirates was found 30 .We only examined patients while they were hospitalised; therefore, a very small recurrence in hospitalised patients was noted when assessing the recurrence of asthma exacerbations.In order to better generalise results due to the aforementioned restrictions, additional research should be conducted across various centres with bigger samples of children.

Conclusions
Mycoplasma pneumoniae infections were highly prevalent in asthmatic children.Severity of acute asthma exacerbations, first-time asthma diagnosis, and recurrence of asthma exacerbation were significantly associated with Mycoplasma pneumoniae infection.

Table 3 : Asthma management in a hospitalized patient with Mycoplasma pneumoniae (Mp) -IgM positivity Characteristic Total (n=14) Mp-IgM positive (n=40) Mp-IgM negative (n=84) p
62d Cosentini R, et al21.Similarly, Kassisse E, et al13revealed a significant increase in Mp-IgM level in severe exacerbation compared to moderate or mild exacerbation.Therefore, children with acute asthma need to be closely monitored and more intensively treated if having Mp infection.The higher rate of fever in asthmatic children having positive Mp-IgM, compared with the negative group in our study is consistent with studies by Hong SJ 3 and Le TMH, et al12.Fever should be considered an additional marker of Mp infection in patients with acute asthma exacerbation.Inaddition, results of our study showed that Mp played an important role in asthma exacerbations, especially in first-time diagnosed asthma.This was similar to the conclusions of Ou CY, et al 7 and Biscardi S, et al 6 .Biscardi S, et al6found that Mp might have a role in the onset of asthma in children and in recurrent wheezing.