Running Title: Mycoplasma pneumoniae IgA in school-age children

Transcription

1 CVI Accepted Manuscript Posted Online 19 October 2016 Clin. Vaccine Immunol. doi: /cvi Copyright 2016 American Society for Microbiology The role of serum Mycoplasma pneumoniae IgA, IgM, and IgG for the diagnosis of Mycoplasma pneumonia-related pneumonia in school-age children and adolescents Running Title: Mycoplasma pneumoniae IgA in school-age children Wei-Ju Lee 1, Eng-Yen Huang 2, Chih-Min Tsai 1, Kuang-Che Kuo 1*, Yi-Chuan Huang 1, Kai-Sheng Hsieh 1, Chen-Kuang Niu 1 and Hong-Ren Yu 1* Departments of Pediatrics 1 and Radiation Oncology 2, Chang Gung Memorial Hospital-Kaohsiung Medical Center; Graduate Institute of Clinical Medical Science, Chang Gung University College of Medicine, Taiwan Corresponding Author: Hong-Ren Yu, M.D., Ph.D. Department of Pediatrics, Kaohsiung Chang Gung Memorial Hospital, #123, Ta-Pei Road, Niao-Sung District, Kaohsiung, Taiwan. Tel: ext Fax: yuu2004taiwan@yahoo.com.tw *Hong-Ren Yu and Kuang-Che Kuo contributed equally to this article Abstract 1

2 24 Mycoplasma pneumoniae (M. pneumoniae) is an important causative pathogen of 25 community-acquired pneumonia in children. Rapid and reliable laboratory diagnosis of M. 26 pneumoniae infection is important so that appropriate antibiotic treatment can be initiated to 27 reduce the misuse of drugs and resistance rates. Anti-M. pneumoniae immunoglobulin M (IgM) is an indicator of recent primary infection, but can persist for several months after initial infection. It has been suggested that anti-m. pneumoniae immunoglobulin A (IgA) can be a reliable indicator for recent M. pneumoniae infection in adults. This study aimed to investigate the clinical diagnostic value of M. pneumoniae IgA in school-age children and adolescents with M. pneumoniae-related pneumonia. Eighty children with pneumonia and seropositive for M. pneumoniae IgM or with a four-fold increase of anti-m. pneumoniae immunoglobulin G (IgG) were enrolled from May 2015 to March The titers of M. pneumoniae IgA, IgM, and IgG, clinical features, and laboratory examinations of blood, C-reactive protein, and liver enzymes were analyzed. The initial positivity rates for M. pneumoniae IgM and IgA upon admission to the hospital were 63.6% and 33.8%, respectively. One week after admission, the cumulative positivity rates for M. pneumoniae IgM and IgA increased to 97.5% and 56.3%, respectively. Detection of M. pneumoniae IgM was more 40 sensitive than detection of M. pneumoniae IgA for the diagnosis of M. pneumoniae-related 41 pneumonia in school-age children and adolescents; however, paired sera are necessary for a 2

3 42 more accurate diagnosis. 43 KEYWORDS: Mycoplasma pneumonia IgA, Mycoplasma pneumonia IgM, paired sera, 44 pneumonia, school-age children 45 Downloaded from on July 4, 2018 by guest 3

4 46 Introduction 47 Mycoplasma pneumoniae (M. pneumoniae) is one of the most common atypical 48 pathogens causing pneumonia, especially in school-age children and adolescents. Cases occur in all seasons, but dominantly in spring and autumn. Although a significant cough without rhinorrhea is the typical manifestation, the symptoms can vary from an asymptomatic respiratory infection to severe pneumonia (1) M. pneumoniae is generally susceptible to macrolide or tetracycline antibiotics, but these treatments are not often immediately used for cases of community-acquired pneumonia if no optimal detecting tool for M. pneumoniae exists. Although M. pneumonia is also susceptive to fluoroquinolone, its utilization in children is limited due to its side effect of joint toxicity (2). In recent years, macrolide-resistant M. pneumoniae has become more and more prevalent across the world, partly due to the wide use of macrolides (3). The macrolide-resistance rate of M. pneumonia is about 10 20% in Taiwan (4, 5), and upwards of % in China, the neighboring country of Taiwan (3). Therefore, an optimal detection method is needed to allow for a more precise diagnosis, and thus the appropriate treatment of patients with M. pneumoniae-related 61 pneumonia, and reduce the misuse of antibiotics. 62 M. pneumoniae is difficult to culture and has a very slow growth rate, and thus, culture is 63 usually helpless clinically. The clinical use of polymerase chain reaction (PCR) assays for the 4

5 64 diagnosis of M. pneumoniae respiratory infections has been reported in both adults (6-9) and 65 children (10-14). However, inducing sputum samples is difficult in young children, and some 66 reports have shown no obvious differences between M. pneumoniae PCR positivity rates of 67 throat swabs from individuals with suspected cases of M. pneumoniae infection and those of healthy subjects. (15) This suggests that M. pneumoniae may colonize the upper airway in humans without inducing disease. Since the pathogen-induced immune response results in the production of antibodies, serological testing is a major diagnostic tool for the detection of M. pneumoniae infection. The complement fixation test has also been used; however, the complex nature of the technique and its high false-positivity rate limits its use (16, 17). The detection of M. pneumoniae IgM has been used to determine the presence of an acute infection. However, some patients do not have sufficient levels of M. pneumoniae IgM antibodies to allow for detection during the early stages of acute infection or re-infection (18), and some patients may have detectable levels of M. pneumoniae IgM for several months after M. pneumoniae infection (19). Both of these situations can lead to difficulties in making a clinical diagnosis. Only a few studies have reported on the use of M. pneumoniae IgA antibodies for the diagnosis of M. pneumoniae infection in the past two decades. One study 80 reported that detection of M. pneumoniae IgA is more sensitive than that of IgM in diagnosing 81 acute M. pneumoniae-related pneumonia in adults (20), whereas another reported lower 5

6 82 sensitivity in detecting M. pneumoniae IgA in young children (21). However, M. 83 pneumoniae-related pneumonia often occurs in school-age children and adolescents. To date, 84 no study has focused on the diagnostic value of M. pneumoniae IgA in school-age children. 85 Therefore, the aim of this study was to investigate the clinical significance and usefulness of detecting M. pneumoniae IgA in school-age children and adolescents with community-acquired pneumonia caused by M. pneumoniae. Downloaded from on July 4, 2018 by guest 6

7 89 Patients 90 We enrolled patients aged 3 years to 18 years who were admitted to Kaohsiung Chang 91 Gung Memorial Hospital between May 2015 and March All of the patients had a cough, fever, and chest X-ray-confirmed bronchopneumonia or lobar pneumonia, suggesting a lower respiratory tract infection. Fever was defined as a body temperature 38.0 C as measured by infrared tympanic membrane thermometers. All patients were diagnosed serologically, and exhibited seroconversion or an increase in antibody titers (two-fold increase of serum M. pneumoniae IgM or a four-fold increase in M. pneumoniae IgG) for at least two weeks following admission (22). The patients enrolled in this study were otherwise healthy without any underlying conditions that may alter their clinical course of disease. Methods All patients received macrolide antibiotic treatment, after which all improved clinically. The serum levels of M. pneumoniae IgA, IgM, and IgG were determined upon admission and at various time points during or after admission depending on their clinical condition. Serum 103 M. pneumoniae IgM and IgG were assayed using ELISA ImmunoWELL kits (Ben-Bio, San 104 Diego, CA, USA), and serum M. pneumoniae IgA was assayed using CHORUS kits (Diesse 105 Diagnostica Senese, Siena, Italy) according to the manufacturers instructions. The positive 7

8 106 cut-off values for M. pneumoniae IgA, IgM, and IgG were 18 AU/ml (with upper and lower 107 limits of 10 and 100 AU/ml), 950 AU/ml, and 320 AU/ml, respectively. The Institutional 108 Review Board of Kaohsiung Chang Gung Memorial Hospital approved the entire study 109 ( A3). The values and positivity rates for M. pneumoniae IgA, IgM, and IgG were correlated with the clinical features. Continuous variables are expressed as the mean ± standard error. The chi-square test was used to determine differences in initial positivity rates of M. pneumoniae IgA and IgM. Statistical correlations between M. pneumoniae IgM titers, clinical characteristics, and the results of laboratory analysis of blood, liver function, and C-reactive protein (CRP) were determined using Pearson s correlation test and multivariate logistic regression. All statistical tests were performed using SPSS version 19.0 for Windows (SPSS, Inc., Chicago, IL). A p value < 0.05 indicated statistical significance. 8

9 119 Results 120 Clinical characteristics of enrolled patients 121 A total of 89 children with M. pneumoniae IgM positive serum were enrolled in this study, of whom nine (10.1%) were excluded due to persistently low positive M. pneumoniae IgM titers without a two- or four-fold increase in M. pneumoniae IgG titers after 2 weeks of follow-up. The remaining 80 children (seroconversion, n=26; IgM two-fold increase: n=72; IgG four-fold increase: n=31 (Figure 1)) (31 males, 49 females; mean age: 6.6 ± 3.3 years old, range 3 to 17 years old) were entered into the analysis and 215 serum samples were obtained. The duration of fever ( 38 C) before admission was 4.3 ± 2.7 days, as reported by the patient or a family member. The clinical characteristics including age, duration of fever, hospital stay, and laboratory analysis of blood, liver enzymes, and CRP are presented in Table 1. Detection of M. pneumoniae IgM was more sensitive than M. pneumoniae IgA in school-age children with M. pneumoniae-related pneumonia 133 Among the 80 cases, the positivity rates for M. pneumoniae IgA, IgM, and IgG upon 134 admission were 33.8% (27/80), 63.8% (51/80), and 32.5% (26/80), respectively (Figure 2). Of 9

10 135 note, 21 cases (26.2%) were initially serum negative for M. pneumoniae IgA, IgM, and IgG. 136 The chi-square test showed significant differences between the initial positivity rates of M. 137 pneumoniae IgA and IgM (p=0.019). Then, we analyzed the correlation between initial M. 138 pneumoniae IgA, IgM, and IgG values and the duration of fever before hospitalization, and the results showed that M. pneumoniae IgM but not IgA or IgG titers were significantly positively correlated with the duration of fever before hospitalization (r=0.377, p=0.002) (Figure 3). In order to determine the relevance between M. pneumonia antibody titer and fever duration before admission, we then divided the patients into three groups according to the duration of fever before hospitalization to compare the positivity rates of M. pneumoniae IgA and IgM as follows: (1) 0 to 3 days, (2) 4 to 6 days, and (3) 7 to 14 days. Most of the patients (40%; 32/80) had a fever duration of less than 3 days before hospitalization. Twenty-seven patients (33.8%) and 21 patients (26.3%) had a fever duration of 4 to 6 days and 7 to 14 days before hospitalization, respectively. As shown in Figure 4, both the M. pneumoniae IgA and IgM positivity rates were higher in the patients with a longer fever duration before hospitalization. In addition, the serum positivity rate of M. pneumoniae IgM was higher than M. pneumoniae IgA in all groups (Figure 4), suggesting that detection of M. pneumoniae IgM 151 is more sensitive than M. pneumoniae IgA detection at any stage of disease for school-age 152 children with M. pneumoniae-related pneumonia. 10

11 Paired serum follow-up is needed for patients who are initially M. pneumoniae IgM negative 155 but clinically symptomatic We then compared the cumulative positivity rates for M. pneumoniae IgA, IgM, and IgG within one month of hospitalization. Since the patients were at different time points in the disease course on admission to the hospital, blood samples were taken according to their clinical condition but not on the same schedule. In order to organize the serological data of the patients, blood sample data were divided into five groups according to the hospitalization stay after admission: (1) baseline (the day of admission); (2) 2 to 4 days; (3) 5 to 7 days; (4) 8 to 14 days; and (5) 15 to 28 days. The initial M. pneumoniae IgM positivity rate for all 80 patients was 63.8% (51/80) (Figure 5A). The cumulative positivity rates of M. pneumoniae IgM for groups 2 and 3 after admission were 85.0% and 97.5%, respectively. Twenty-six of the 80 cases (32.5%) had seroconversion of M. pneumoniae IgM 1 week after admission. These findings suggested that paired serum follow-up samples are needed for patients who initially M. pneumoniae IgM negative but are clinically symptomatic. The cumulative 168 positivity rate of M. pneumoniae IgM was always higher than that of M. pneumoniae IgA, 169 even though 25 of the 80 patients were still serum negative for M. pneumoniae IgA 15 to days after admission. 11

12 171 Among the 80 patients with pneumonia due to M. pneumoniae, 26 (32.5%) who were 172 initially negative for M. pneumoniae IgM upon admission had seroconversion after 2 weeks 173 of follow-up (Figure 5B). These findings showed the natural course of M. pneumoniae 174 antibodies in these patients. Among the 26 patients who were initially negative for M pneumoniae IgM, four (15.4%) were initially positive for M. pneumoniae IgA; however, the cumulative positivity rate for M. pneumoniae IgM was also higher than that for M. pneumoniae IgA after 2 to 4 days of follow-up (61.5% vs. 46.2%) in all enrolled cases (Figure 5B). These findings also suggest the clinical importance of paired serum follow-up. We also analyzed the levels of M. pneumoniae IgA, IgM, and IgG after one month of follow-up, and all of the patients were still positive for all antibodies after 15 to 28 days of follow-up (Figure 6). Fever duration before admission and initial platelet count were positively correlated with initial M. pneumoniae IgM value 185 In order to establish what factors can influence the M. pneumoniae IgM titer, we 186 analyzed the correlation between the patients initial M. pneumoniae IgM value, their clinical 187 features, and the results of the laboratory analysis of blood, liver function, and CRP using 12

13 188 Pearson s correlation analysis. We found that fever duration before admission and initial 189 platelet count were positively correlated with initial M. pneumoniae IgM titers (Table 1 and 190 Figure 7A, 7B). Then, the associations between the clinical characteristics, laboratory analysis 191 of blood cells, liver enzymes, and CRP, and M. pneumoniae IgM titer were tested again with multivariate logistic regression. This result also indicated that fever duration before admission and platelet count were positive correlated with M. pneumoniae IgM (p=0.005 and p=0.001, respectively). However, the correlation between the fever duration before admission and platelet count was not significant (r=0.075, p=0.638) (Figure 7C). This suggests that both fever duration before admission and initial platelet count are independent factors correlated with M. pneumoniae IgM titer. 13

14 199 Discussion 200 In this study, we investigated longitudinal changes in M. pneumoniae IgA, IgM, and IgG 201 titers in school-age children with pneumonia caused by M. pneumoniae infection. We found that the initial positivity rates for M. pneumoniae IgM and IgA upon admission to hospital were 63.8% and 33.8%, respectively. The duration of fever before admission and platelet count were positively correlated with initial M. pneumoniae IgM titers, and the cumulative positivity rates for M. pneumoniae IgM and IgA increased to 97.5% and 56.3%, respectively, one week after admission. Detection of M. pneumoniae IgM was more sensitive than detection of M. pneumoniae IgA in the diagnosis of school-age children and adolescents with pneumonia caused by M. pneumoniae. The progression of Mycoplasma infection is often gradual and slow, and in adults, the illness is usually present for more than 1 week before the patient seeks medical assistance (20). However, our results showed that children with pneumonia caused by M. pneumoniae were mostly admitted to hospital within the first week of infection (mean: 4.3 ± 2.7 days). This may be because fever is the major symptom in children rather than the prolonged cough 214 typically seen in adults infected with M. pneumoniae. Children are therefore brought to the 215 hospital sooner than adults due to the concerns of the caregiver. For this reason, we found that 216 many cases (21/80, 26.3%) were negative for M. pneumoniae-specific antibodies upon 14

15 217 admission. In order to avoid missing false negative cases, we collected paired sera for M. 218 pneumoniae IgA, IgM, and IgG after admission. However, since fever duration before 219 admission was reported by the patients or family members, there is a limitation for the 220 accuracy of this value. Beersma reported high background rates of seropositivity for M pneumoniae IgM in some adult populations, ranging from 0 to 51% depending on the assay used (23). In the current study, nine children were excluded due to persistently low positive M. pneumoniae IgM titers with no obvious increase in the paired sera. The background seropositivity rate for M. pneumoniae IgM was 10.1% in the children in this study. In previous studies, the detection of M. pneumoniae IgA has been reported to be a good indicator of recent M. pneumoniae infection in predominantly adult populations (24-26). Detection of M. pneumoniae IgA has been reported to be more sensitive than detection of M. pneumoniae IgM for the diagnosis of M. pneumoniae infection in adults (20). However, conflicting results have been reported in toddlers, for whom M. pneumoniae IgA was reported to be a poor indicator of M. pneumoniae infection (21). In this study, we investigated the diagnostic value of M. pneumoniae IgA in diagnosing pneumonia caused by M. pneumoniae 232 in school-age children, the most common age for infection with this pathogen. Our results 233 showed positivity rates for M. pneumoniae IgM and IgA on the first day of hospital admission 234 of 63.8% and 33.8%, respectively, and these rates were positively correlated with the duration 15

16 235 of fever before admission (Figure 2). We also found that the positivity rate for M. pneumoniae 236 IgM was higher than that of M. pneumoniae IgA in each duration of fever before admission 237 group (Figure 3). This suggests that detection of M. pneumoniae IgA is less sensitive than 238 detection of M. pneumoniae IgM for the diagnosis of M. pneumoniae-related pneumonia in school-age children and adolescents. The reason why M. pneumoniae IgA is a good marker in adults but not children with M. pneumoniae-related pneumonia is unclear. The developing immune system may partly explain this phenomenon. Human serum IgA and IgM concentrations change with age, with lower concentrations at birth. Serum IgM reaches an adult concentration at 4 to 5 years of age, while serum levels of IgA mature slowly until puberty (27, 28). Additional studies are needed to investigate this issue further. In the current study, the positivity rate for M. pneumoniae IgM upon hospital admission was 63.8% with a mean duration of illness of 4.3 ± 2.7 days prior to admission. This is consistent with another study in which the M. pneumoniae IgM positivity rate in children was 62.2% in the first week (29) and 70.9%~81.8% in the second week (30, 31) of the illness. The sensitivity of serological tests can be affected by the timing of specimen collection, the 250 standard of diagnosis, and the assay used (32). This could explain the higher sensitivity of M. 251 pneumoniae IgM in patients with a longer duration of illness prior to admission (Figure 2B 252 and Figure 3). We found a positive correlation between the M. pneumoniae IgA positivity rate 16

17 253 and the duration of illness prior to admission, although this correlation was less significant 254 than that for M. pneumoniae IgM (Figure 2A). 255 A four-fold increase in M. pneumoniae IgG titers in acute and convalescent sera is considered to be the gold standard for the diagnosis of acute M. pneumoniae respiratory infection (33). The rate of four-fold increase in M. pneumoniae IgG in paired sera varies in different studies. Biljana et al. reported that 90% of cases had a four-fold increase in M. pneumoniae IgG in patients with positive throat swabs via PCR (30). In comparison, Ma et al. reported that only 2.4% of cases had a four-fold increase in M. pneumoniae IgG in patients with positive IgM or positive nasopharyngeal swabs via PCR (31). In the current study, 31 cases (38.8%) had a four-fold increase in M. pneumoniae IgG in paired sera. Since M. pneumoniae IgG in paired sera cannot provide an instant diagnosis and as obtaining second blood samples from children can be difficult in the convalescence phase, M. pneumoniae IgG does not seem to be an optimal tool for the early diagnosis of M. pneumonia infection. In addition to the duration of fever before admission, we found a positive correlation between M. pneumoniae IgM titers and platelet count upon hospital admission. This may be 268 explained by the production of immunoglobulin after an illness. However, this correlation has 269 not previously been reported in the literatures. It has been reported that chronic inflammation 270 is often associated with reactive thrombocytosis (34). Youn et al. reported a correlation 17

18 271 between platelet count and serum status of M. pneumoniae IgM, where the platelet count was 272 higher in a subgroup with increased M. pneumonia IgM than in those with seroconversion 273 (35). In the present study, the platelet count was also higher in the subgroup with increased M. 274 pneumonia IgM than in that of the seroconverted cases (292,090 ± 73,530/μL vs. 227,850 ± ,730/μL, respectively; p < 0.001). Furthermore, we found that the platelet count was significantly positively correlated with the M. pneumonia IgM titer, which could explain the lower platelet count in the seroconverted cases. In conclusion, detection of M. pneumoniae IgM was more sensitive than detection of M. pneumoniae IgA for the diagnosis of M. pneumoniae-related pneumonia in school-age children and adolescents. If only one serum sample for M. pneumoniae IgM was tested at initial admission, there was a 10.1% false positivity rate and a 32.5% false negativity rate. For a more accurate diagnosis of M. pneumoniae-related pneumonia in school-age children, testing of paired sera of M. pneumoniae IgM is still necessary. 18

19 285 Acknowledgments This study was supported in part by grants CMRPG8E0011 (W. J. Lee) and MOST B MY2 (H. R. Yu) from the Ministry of Science and Technology, Taiwan. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication Downloaded from on July 4, 2018 by guest 19

20 292 References Ali NJ, Sillis M, Andrews BE, Jenkins PF, Harrison BD The clinical spectrum and diagnosis of Mycoplasma pneumoniae infection. Q J Med 58: Bradley JS, Jackson MA, Committee on Infectious D, American Academy of P The use of systemic and topical fluoroquinolones. Pediatrics 128:e Pereyre S, Goret J, Bebear C Mycoplasma pneumoniae: Current Knowledge on Macrolide Resistance and Treatment. Front Microbiol 7: Wu PS, Chang LY, Lin HC, Chi H, Hsieh YC, Huang YC, Liu CC, Huang YC, Huang LM Epidemiology and clinical manifestations of children with macrolide-resistant Mycoplasma pneumoniae pneumonia in Taiwan. Pediatr Pulmonol 48: Wu HM, Wong KS, Huang YC, Lai SH, Tsao KC, Lin YJ, Lin TY Macrolide-resistant Mycoplasma pneumoniae in children in Taiwan. J Infect Chemother 19: Gnarpe J, Lundback A, Gnarpe H, Sundelof B Comparison of nasopharyngeal and throat swabs for the detection of Chlamydia pneumoniae and Mycoplasma pneumoniae by polymerase chain reaction. Scand J Infect Dis Suppl 104: Raty R, Ronkko E, Kleemola M Sample type is crucial to the diagnosis of Mycoplasma pneumoniae pneumonia by PCR. J Med Microbiol 54: Reznikov M, Blackmore TK, Finlay-Jones JJ, Gordon DL Comparison of nasopharyngeal aspirates and throat swab specimens in a polymerase chain reaction-based test for Mycoplasma pneumoniae. Eur J Clin Microbiol Infect Dis 14: Waris ME, Toikka P, Saarinen T, Nikkari S, Meurman O, Vainionpaa R, Mertsola J, Ruuskanen O Diagnosis of Mycoplasma pneumoniae pneumonia in children. J Clin Microbiol 36: Dorigo-Zetsma JW, Zaat SA, Wertheim-van Dillen PM, Spanjaard L, Rijntjes J, van Waveren G, Jensen JS, Angulo AF, Dankert J Comparison of PCR, culture, and serological tests for diagnosis of Mycoplasma pneumoniae respiratory tract infection in children. J Clin Microbiol 37:

21 Maltezou HC, La-Scola B, Astra H, Constantopoulou I, Vlahou V, Kafetzis DA, Constantopoulos AG, Raoult D Mycoplasma pneumoniae and Legionella pneumophila in community-acquired lower respiratory tract infections among hospitalized children: diagnosis by real time PCR. Scand J Infect Dis 36: Michelow IC, Olsen K, Lozano J, Duffy LB, McCracken GH, Hardy RD Diagnostic utility and clinical significance of naso- and oropharyngeal samples used in a PCR assay to diagnose Mycoplasma pneumoniae infection in children with community-acquired pneumonia. J Clin Microbiol 42: Skakni L, Sardet A, Just J, Landman-Parker J, Costil J, Moniot-Ville N, Bricout F, Garbarg-Chenon A Detection of Mycoplasma pneumoniae in clinical samples from pediatric patients by polymerase chain reaction. J Clin Microbiol 30: Templeton KE, Scheltinga SA, Graffelman AW, Van Schie JM, Crielaard JW, Sillekens P, Van Den Broek PJ, Goossens H, Beersma MF, Claas EC Comparison and evaluation of real-time PCR, real-time nucleic acid sequence-based amplification, conventional PCR, and serology for diagnosis of Mycoplasma pneumoniae. J Clin Microbiol 41: Spuesens EB, Fraaij PL, Visser EG, Hoogenboezem T, Hop WC, van Adrichem LN, Weber F, Moll HA, Broekman B, Berger MY, van Rijsoort-Vos T, van Belkum A, Schutten M, Pas SD, Osterhaus AD, Hartwig NG, Vink C, van Rossum AM Carriage of Mycoplasma pneumoniae in the upper respiratory tract of symptomatic and asymptomatic children: an observational study. PLoS Med 10:e Ponka A, Ponka T, Sarna S, Penttinen K Questionable specificity of lipid antigen in the Mycoplasma pneumoniae complement fixation test in patients with extrapulmonary manifestations. J Infect 3: Kleemola M, Kayhty H Increase in titers of antibodies to Mycoplasma pneumoniae in patients with purulent meningitis. J Infect Dis 146: Sillis M The limitations of IgM assays in the serological diagnosis of Mycoplasma pneumoniae infections. J Med Microbiol 33: Thacker WL, Talkington DF Analysis of complement fixation and commercial enzyme immunoassays for detection of antibodies to Mycoplasma pneumoniae in human serum. Clin Diagn Lab Immunol 7:

22 Granstrom M, Holme T, Sjogren AM, Ortqvist A, Kalin M The role of IgA determination by ELISA in the early serodiagnosis of Mycoplasma pneumoniae infection, in relation to IgG and mu-capture IgM methods. J Med Microbiol 40: Yamazaki T, Narita M, Sasaki N, Kenri T, Arakawa Y, Sasaki T Comparison of PCR for sputum samples obtained by induced cough and serological tests for diagnosis of Mycoplasma pneumoniae infection in children. Clin Vaccine Immunol 13: Lee SC, Youn YS, Rhim JW, Kang JH, Lee KY Early Serologic Diagnosis of Mycoplasma pneumoniae Pneumonia: An Observational Study on Changes in Titers of Specific-IgM Antibodies and Cold Agglutinins. Medicine (Baltimore) 95:e Beersma MF, Dirven K, van Dam AP, Templeton KE, Claas EC, Goossens H Evaluation of 12 commercial tests and the complement fixation test for Mycoplasma pneumoniae-specific immunoglobulin G (IgG) and IgM antibodies, with PCR used as the "gold standard". J Clin Microbiol 43: Lieberman D, Lieberman D, Ben-Yaakov M, Shmarkov O, Gelfer Y, Varshavsky R, Ohana B, Lazarovich Z, Boldur I Serological evidence of Mycoplasma pneumoniae infection in acute exacerbation of COPD. Diagn Microbiol Infect Dis 44: Lieberman D, Lieberman D, Korsonsky I, Ben-Yaakov M, Lazarovich Z, Friedman MG, Dvoskin B, Leinonen M, Ohana B, Boldur I A comparative study of the etiology of adult upper and lower respiratory tract infections in the community. Diagn Microbiol Infect Dis 42: Watkins-Riedel T, Stanek G, Daxboeck F Comparison of SeroMP IgA with four other commercial assays for serodiagnosis of Mycoplasma pneumoniae pneumonia. Diagn Microbiol Infect Dis 40: Jolliff CR, Cost KM, Stivrins PC, Grossman PP, Nolte CR, Franco SM, Fijan KJ, Fletcher LL, Shriner HC Reference intervals for serum IgG, IgA, IgM, C3, and C4 as determined by rate nephelometry. Clin Chem 28: Kilian M, Mestecky J, Russell MW Defense mechanisms involving Fc-dependent functions of immunoglobulin A and their subversion by bacterial 22

23 382 immunoglobulin A proteases. Microbiol Rev 52: Chang HY, Chang LY, Shao PL, Lee PI, Chen JM, Lee CY, Lu CY, Huang LM Comparison of real-time polymerase chain reaction and serological tests for the confirmation of Mycoplasma pneumoniae infection in children with clinical diagnosis of atypical pneumonia. J Microbiol Immunol Infect 47: Medjo B, Atanaskovic-Markovic M, Radic S, Nikolic D, Lukac M, Djukic S Mycoplasma pneumoniae as a causative agent of community-acquired pneumonia in children: clinical features and laboratory diagnosis. Ital J Pediatr 40: Ma YJ, Wang SM, Cho YH, Shen CF, Liu CC, Chi H, Huang YC, Huang LM, Huang YC, Lin HC, Ho YH, Mu JJ, Taiwan Pediatric Infectious Disease A Clinical and epidemiological characteristics in children with community-acquired mycoplasma pneumonia in Taiwan: A nationwide surveillance. J Microbiol Immunol Infect 48: Busson L, Van den Wijngaert S, Dahma H, Decolvenaer M, Di Cesare L, Martin A, Vasseur L, Vandenberg O Evaluation of 10 serological assays for diagnosing Mycoplasma pneumoniae infection. Diagn Microbiol Infect Dis 76: Gavranich JB, Chang AB Antibiotics for community acquired lower respiratory tract infections (LRTI) secondary to Mycoplasma pneumoniae in children. Cochrane Database Syst Rev doi: / cd pub2:cd Klinger MH, Jelkmann W Role of blood platelets in infection and inflammation. J Interferon Cytokine Res 22: Youn YS, Lee KY, Hwang JY, Rhim JW, Kang JH, Lee JS, Kim JC Difference of clinical features in childhood Mycoplasma pneumoniae pneumonia. BMC Pediatr 10:

24 407 Figure Legends 408 Figure 1. Venn diagram showing serologically diagnostic criteria for the 80 children with 409 Mycoplasma pneumoniae-related pneumonia. The intersection areas among these three circles show cases that simultaneously fit the different diagnostic criterion. Figure 2. Venn diagram showing the case numbers initially positive for Mycoplasma pneumoniae IgA (27, 33.8%), IgM (51, 63.8%), and IgG (26, 32.5%) upon hospital admission. Twenty-one cases were negative for M. pneumoniae IgA, IgM, and IgG upon admission. Figure 3. Correlation of initial Mycoplasma pneumoniae IgA, IgM, and IgG values with duration of fever upon admission. M. pneumoniae IgM, but not IgA or IgG values were significantly positively correlated with the duration of fever (Pearson s correlation coefficient, IgA: r=0.240, p=0.052; IgM: r=0.377, p=0.002; IgG: r=-0.016, p=0.902). Figure 4. Positivity rates for Mycoplasma pneumoniae IgA, IgM, and IgG with different durations of fever upon admission. After dividing the patients into three groups based on their duration of fever upon admission (0~3, 4~6, and 7~14 days), all of them had a higher 421 positivity rate for M. pneumoniae IgM than IgA. 422 Figure 5. The cumulative positivity rate of Mycoplasma pneumoniae IgA, IgM, and IgG. (A) 24

25 423 The cumulative positivity rate for M. pneumoniae IgA, IgM, and IgG among all 80 patients 424 was calculated at 5 time intervals after admission; 0, 2 4, 5 7, 8 14, and days. (B) The 425 cumulative positivity rates for M. pneumoniae IgA, IgM, and IgG for 26 cases with 426 seroconversion. Twenty-six cases were initially negative for M. pneumoniae IgM and then experienced seroconversion after 2 weeks. Only four cases (15.4%) were initially positive for M. pneumoniae IgA. The cumulative positivity rate for M. pneumoniae IgM was higher than for IgA after 2 days (61.5% vs. 46.2%, respectively). Figure 6. The titers of Mycoplasma pneumoniae IgA, IgM, and IgG after follow-up for 1 month. The means and standard errors of M. pneumoniae IgA, IgM, and IgG titers were classified into five time intervals: 0, 2 4, 5 7, 8 14 and days after admission. None of the titers showed any significant decline after 1 month of follow-up. Figure 7. Correlation between the duration of fever before admission, platelet count, and Mycoplasma pneumoniae IgM. (A) There was no significant correlation between fever duration before admission and platelet count. (p=0.638). (B) There was significant correlation between M. pneumoniae IgM and platelet count. (p=0.004). (C) There was significant 438 correlation between M. pneumoniae IgM and fever duration before admission. (p=0.014)

26 441 Table 1. The clinical characteristics and laboratory results of blood, liver function, and 442 C-reactive protein (CRP) of the 80 patients with Mycoplasma pneumoniae 443 Clinical characteristics Mean ± SD (range) p-value* 444 Age (years) 6.6 ± 3.3 (3~17) Gender (M:F) 31:49 Fever duration before admission (days) 4.3 ± 2.7 (0~10) Total fever duration (days) 5.7 ± 3.4 (0~14) Hospitalization duration (days) 7.3 ± 5.0 (3~19) Red blood cell (million/µl) 4.6 ± 0.4 (3.4~5.7) Hemoglobin (g/dl) 12.6 ± 1.0 (10.1~14.4) Platelet (1000/µl) ± 76.4 (114.0~454.0) White blood cell (1000/µl) 9.2 ± 3.9 (3.8~20.7) Segment (%) 63.7 ± 13.2 (28.0~91.0) Lymphocyte (%) 26.3 ± 11.3 (5.0~65.0) Eosinophil (%) 1.9 ± 2.8 (0.0~8.6) Basophil (%) 0.3 ± 0.4 (0.0~3.0) Monocyte (%) 7.0 ± 3.0 (1.8~14.0) AST (u/l) 34.6 ± 11.3 (22.0~65.0) ALT (u/l) 20.2 ± 13.2 (9.0~66.0) CRP(mg/L) 43.5 ± 42.6 (1.4~215.4) *The p-value of Pearson s correlations with initial Mycoplasma pneumoniae IgM values. The 462 fever duration before admission and platelet count were significantly correlated with 463 Mycoplasma pneumoniae IgM titers. 26

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