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Revista chilena de pediatría

versión impresa ISSN 0370-4106

Rev. chil. pediatr. vol.91 no.3 Santiago jun. 2020

http://dx.doi.org/10.32641/rchped.v91i3.1476 

ORIGINAL ARTICLE

Seroprevalence of anti-Mycoplasma pneumoniae antibodies in otherwise healthy children

Gabriela Sanluis Fenelli1 

María José Chiolo2 

Fernando Adrián Torres1 

Jeanette Balbaryski3 

Paula Dominguez1 

María Fabiana Ossorio1 

María José Rial4 

Fernando Ferrero5 

1 Department of Education and Research, Hospital General de Niños Pedro de Elizalde, Buenos Aires, Argentina.

2 Department of Surgery, Hospital General de Niños Pedro de Elizalde, Buenos Aires, Argentina.

3 Division of Immunology, Hospital General de Niños Pedro de Elizalde, Buenos Aires, Argentina.

4 Division of Laboratory Medicine, Hospital General de Niños Pedro de Elizalde, Buenos Aires, Argentina.

5 Department of Medicine, Hospital General de Niños Pedro de Elizalde, Buenos Aires, Argentina.

Abstract:

Introduction:

Mycoplasma pneumoniae (Mypn) infection could be occurring at an earlier age due to social pheno mena such as attending daycare centers more frequently and earlier than decades ago.

Objective:

to estimate the prevalence of anti-Mypn antibodies in children aged 0-12 years, and to explore whether age, attendance to daycare center/school, overcrowding or the presence of children aged below 12 years in the households increase the risk of seropositivity.

Patients and Method:

Cross-sectional stu dy including healthy children aged 0-12 years which required blood draws for routine laboratory tests. In all cases, the aforementioned variables were recorded and anti-Mypn IgG was determined by enzyme immunoassay. The association between predictors and seropositivity was assessed in a logistic regression model.

Results:

We included 232 patients (average age 56.4 ± 40.0 months). 56.9% attended a daycare center/school, 63.8% co-habited with children under 12 years old, and 15.9% lived in overcrowded households. The prevalence of anti-Mypn antibodies was 14.6%. There were no significant differences between seropositive and seronegative children regarding age (63.1 ± 40.7 vs. 55.4 ± 41.3 months), school/day-care attendance (64.7% vs. 55.5%), overcrowding (14.7% vs. 14.9%), or co-habiting with children (64.7% vs. 63.6%). Age was not an independent predictor of seropositivity in the multivariate model.

Conclusion:

The prevalence of anti-Mypn antibodies in children was 14.6% and age was not a predictor of seropositivity.

Keywords: Acute respiratory infection; Mycoplasma pneumoniae; pneumonia; prevalence; serology; child

What do we know about the subject matter of this study?

Mycoplasma pneumoniae infection can occur in children with both pulmonary and extra-pulmonary manifestations. Such manifesta tions have been reported in numerous studies, using specific labo ratory tests.

What does this study contribute to what is already known?

This study provides information on the presence of antibodies aga inst Mycoplasma pneumoniae in healthy children from an early age, showing that this infection is not exclusive to adolescents or young adults.

Introduction

Acute respiratory infections are a prevalent cause of morbidity and mortality in childhood1, where Myco plasma pneumoniae (Mypn) is one of the pathogens frequently involved in these conditions2. Mypn can cause pneumonia3, even highly severe4.

Prevalence data on Mypn infection are variable and can be influenced not only by local epidemiology but also by the method used to identify the infection. Therefore, the presence of IgG antibodies has been re ported in 50% of children under 12 years, and at least 80% of adults5. In Barakaldo, Spain, there is 37.5% of prevalence in children aged between 1 and 5 years6; in Diyarbakir, Turkey, it is 10%7; and in Buenos Aires, Argentina, it reaches 6.9% in children under 58.

While Mypn infection was considered typical of school-age children, adolescents and/or young adults for many years, the new evidence points out that it may occur at younger ages. This could be related to social changes, such as attending day-care centers more fre quently and at younger ages9.

Although our current treatment schemes are ap propriate to address community-acquired pneumonia10,11,12, there is a need for monitoring epidemiological changes that may require re-evaluation.

Moreover, in Buenos Aires, previous data from our institution8,13 allow us to objectively compare the seroprevalence of anti-Mypn antibodies throughout 25 years, providing useful information to monitor the epidemiology of this microorganism.

Therefore, the objectives of this study are to estima te the prevalence of anti-Mypn antibodies in children aged between 0 and 12 years and to explore whether age, attendance at a day-care center/school, overcrow ding, or living with children under 12 years of age in crease the risk of seropositivity for Mypn.

Patients and Method

Cross-sectional study, including healthy children aged between 0 and 12 years who required blood sam pling for pre-surgical studies of planned surgery at the Hospital General de Niños Pedro de Elizalde during the study period, and whose mother, father or legal guar dian signed informed consent to participate in the stu dy (and child’s assent, where applicable). We excluded children with acute infectious condition, any known chronic or acute pathology, or previously known im mune disorders.

All patients in the waiting room of the hospital’s blood collection service were called consecutively, identifying those with pre-surgical routines for plan ned surgeries such as inguinal hernias, tonsillar hyper trophy, phimosis, cryptorchidism, among others. The parent or guardian of each child was invited to partici pate, signing the respective informed consent.

One of the researchers interviewed the caregiver in order to record information related to the variables un der study. Patients were included between 23/08/2018 and 27/02/2019 until reaching the calculated sample size.

Variables

The following variables were established:

Prediction variable: Age in months (calculated based on birth date and blod sampling date). In the logistic regression model, this variable was consi dered as dichotomous (< 5 years and ≥ 5 years).

Primary outcome variable: Serology (IgG) for Mypn (considered positive or negative according to the cut-off value described in the section related to the diagnostic test).

Variables to be monitored:

- Attendance to day-care center/school (atten dance of 4 hours a day for at least 3 times a week, in the last 2 months).

- Overcrowding (more than 3 people per room other than the bathroom and kitchen).

- Living with children under 12 years of age (the presence of cohabitants from this age group was recorded).

Laboratory procedure

From the blood sample collected by venipuncture for the requested pre-surgical studies, at least 1.5 mL of blood was taken. The resulting serum was divided into two aliquots, one was at least 0.3 mL. The samples were stored at - 20°C until processing.

The determination of anti-Mypn antibodies (igG) was carried out through enzyme immunoassay (EiA), using Vircell SL equipment. (Granada, Spain).

The optical density (OD) was measured with an EiA reader at 450 nm, along with the bichromatic measurement at a 620 nm reference wavelength. Each assay uses a positive control, a negative one, and a cut off. The OD of the controls must be in the following ranges: positive control > 0.9; negative control < 0.5; cut-off > 0.55 and < 1.5 (otherwise the test is ruled out). Antibody index = (OD of sample/serum OD cut off) x 10: < 9 negative; 9 - 11 uncertain; > 11 positive. Kit reference: G1002. The manufacturer reports 98% of sensitivity and 97% of specificity (http://peramed.com/peramed/docs/G1002_EN.pdf).

According to the protocol, the results of the sero logy were reported to the patients on request, when available.

Sample size determination

It was determined based on the known prevalence of anti-Mypn antibodies (igG), for 2 different age ran ges. Data were collected from a previous study, where it was observed that the prevalence of seropositivity was different for those over and under 5 years of age (7% and 20%, respectively) (10). Given the large variability of the topic in the literature, a confidence interval bet ween 12% and 27%, respectively, was considered. To calculate the sample size, we considered approximately 1,000 potentially enrollable patients who visit the sur gery service annually (data from the last 3 years), with an age distribution of 65% younger and 35% older than 5 years. Therefore, with a 95% confidence inter val, 121 subjects aged 0-4 years and 92 aged 5-12 years are required. Assuming that up to 5% of serums may not be evaluable, we decided to incorporate 130 and 100 subjects, respectively.

Ethical considerations

The study was carried out according to the stan dards of good clinical practice, the Declaration of Hel sinki, and the regulations in force of the Government of Buenos Aires. The study was approved by the Re search Ethics Committee and the institutional Review Board of the Hospital and the project was registered in the Health Research Council of the Government of Buenos Aires. in all cases, the patients’ guardian signed the informed consent to participate in the study and we ask those patients older than 7 years to give their assent. The confidentiality of the subjects’ identity was guaranteed according to the procedures established in the protocol.

Statistical processing

The prevalence of seropositivity for Mypn was determined for the whole population and each age group (Ci 95%). To examine possible factors asso ciated with seropositivity for Mypn, we used the Chi- Square test for categorical variables and the Student’s t-test in independent samples for continuous variables (after verifying goodness of fit for normality using the Kolmogorov-Smirnov test). Age (over and under 5 years) was assessed as a predictor of seropositivity through logistic regression in a model that in cluded day-care-center/school attendance, overcrow ding, and living with children as control variables. A significance level < 0.05 was considered. In addition, we evaluated, using the ROC curve (including the area under the curve -AUC- and its Ci 95%), if there was a better age cut-off point to predict seropositi vity. Data processing was performed with iBM SSPS Statistics 20.0 software.

Results

We included 232 patients (129 < 5 years and 103 ≥ 5 years), with mean age 56.4 ± 40.0 months. One hundred and thirty-two patients attended day-care center/school (56.9%), 148 lived with children under 12 years of age (63.8%), and 37 patients lived in over crowded households (15.9%). Anti-Mypn antibodies (igG) were present in 14.6% (Ci 95% 10.6-19.7) of our patients (Table 1).

Table 1 Characteristics of the population in which IgG antibodies to Mycoplasma pneumoniae were screened. 

When stratified by age group, the proportion of seropositivity in the group under 5 years was 13.9%, and 15.5% in the group aged > 5 years (OR: 1.1 CI 95% 0.5-2.3; p = 0.8). This age cut-off point for identifying seropositivity presented 47.1% of sensitivity (Ci 95% 30.1-64.6), 56.1% of specificity (Ci 95% 48.8-63.1), 15,3% of positive predictive value (Ci 95% 9.4-24.3), 86.1% of negative predictive value (Ci 95% 78.5-91.3), positive likelihood ratio 1.1 (Ci 95% 0.7-1.5), and negative likelihood ratio 0.9 (Ci 95% 0.6-1.3).

The seropositive children did not show significant differences with the seronegative ones regarding the mean age (63.1 ± 40.7 vs. 55.4 ± 41.3 months; p = 0.5), day-care center/school attendance (64.7% vs. 55.5%; p = 0.3), overcrowding (14.7% vs. 14.9%; p = 0.5), or living with children under 12 years of age (64.7% vs. 63.6%; p = 0.5).

In the multivariate analysis, after controlling by day-care center/school attendance, cohabitants under 12 years of age, and overcrowding, older age also does not appear as an independent predictor of seropositivity for Mypn (Table 2).

Table 2 Multivariate analysis including potential predictors and seropositivity to Mycoplasma pneumoniae. 

In order to assess if there was a better age cut-off point to predict seropositivity for Mypn, we used the ROC curve which also showed very low predictive abi lity (aucROC = 0.53; Ci 95% 0.4-0.6).

Discussion

Mycoplasma pneumoniae is a common cause of res piratory infections in childhood, so one would expect a significant number of healthy children to have spe cific antibodies (IgG). In our study, there was 14.6% of prevalence of anti-Mypn antibodies in children aged between 0 and 12 years.

There is relatively little information on the seroprevalence of anti-Mypn antibodies in healthy children. The figures of our study are similar to that reported by Lezcano et al. in Buenos Aires (12.4%) in 20088 and to that referred by Tuuminen et al. in Finland, who re ported a prevalence between 13 and 19% in children under 4 years of age14.

In a previous study, we reported that seroprevalence increased significantly with age, however, we could not corroborate it in this one. In such a study, we found that 6.9% of the children under 5 years of age and 24.7% of those over that age had anti-Mypn antibodies, a difference statistically significant8. In this study, the mean age showed no difference between se ropositives and seronegatives, nor did the proportion of seropositives between the two age groups studied (0-4 and 5-12 years). This could be explained because seroconversion currently occurs at younger ages, as a result of social changes that are difficult to demostrate9,15.

We evaluated three variables related to this pheno menon, attendance at day-care centers/schools, coha bitants under 12 years of age, and overcrowding, but we could not verify the change. Moreover, the propor tion of subjects attending day-care centers/schools in the group under 5 years of age was similar between this study and that referred to by Lezcano et al. (56.9% and 57.6%, respectively).

A similar result was observed concerning cohabi tants under 12 years of age (63.8% and 70.1%) and overcrowding (15.9% and 27.3%)8. This is consistent with a study carried out in Chile, which found that over 10 years (2003-2014) seropositivity for Mypn (IgM, probably secondary to acute infection by the mi croorganism) in children aged under 5 years increased from 8.6% to 30% and that this change was especially evident in the 2 to 5-year-old group (33%)16. It also could be that, an increase in Mypn resistance to macrolides could facilitate its circulation, affecting a younger population17, but there are no local reports that sup port this option.

There are reports that Mycoplasma pneumoniae infection has increased, making it a relevant issue. Although the most frequent age of presentation of pneumonia due to Mypn ranges from 6 to 10 years, almost 15% of cases of pneumonia in children under 3 years of age may be due to this microorganism18. A report from Vietnam found that more than 50% of Mypn cases of pneumonia can occur in children under 5 years of age19.

A limitation of our study is that it was not develo ped as a population-based study. However, the diver sity of patients that are usually seen at the institution20 probably reflects, at least, the population of the Buenos Aires Metropolitan Area, where approximately one- third of the country’s population resides21.

It could be argued that the sample size calcula tion caused some kind of bias by assuming the age of 5 years as the cut-off point for seropositivity. Howe ver, in a previous experience using the same diagnostic methodology, we found that this fact was a strong pre dictor of seropositivity8. Furthermore, the difference in the prevalence between both groups in our study is so small that, if we maintain the observed proportions, it will not be enough to quadruple the sample to find a statistically significant difference, proving that the lack of significance is not due to a power problem.

Despite potential limitations, this study provides updated data on one aspect of Mycoplasma pneumo niae behavior in the region, confirming childhood seroprevalence values. Although it may be appropriate to carefully evaluate the results, it is essential to continue surveillance to allow a proper understanding of the changes observed with higher precision, and to moni tor epidemiological changes that could lead us to re evaluate the community-acquired pneumonia current antibiotic treatment22.

In conclusion, in our study, the prevalence of anti-Mypn antibodies in children aged 0-12 years was 14.6%. We could not demonstrate that age would act as a predictor of it. The age of seroconversion may be lower than that observed in previous decades.

Ethical Responsibilities

Human Beings and animals protection: Disclosure the authors state that the procedures were followed ac cording to the Declaration of Helsinki and the World Medical Association regarding human experimenta tion developed for the medical community.

Data confidentiality: The authors state that they have followed the protocols of their Center and Local regu lations on the publication of patient data.

Rights to privacy and informed consent: The authors have obtained the informed consent of the patients and/or subjects referred to in the article. This docu ment is in the possession of the correspondence author.

Conflicts of Interest: Authors declare no conflict of interest regarding the present study.

Financial Disclosure: Partially supported by a research grant from the Health Research Council (MS-GCABA).

Aknowledgments: We thank lab technicians for their help on blood sam pling.

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Received: October 24, 2019; Accepted: March 09, 2020

Correspondence: Gabriela Sanluis Fenelli. E-mail: gabisanluis@gmail.com.

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