m. pneumoniae easily attaches to ciliated epithelial cells of the respiratory tract and causes damage ( 4 , 5 ) . for diagnosis of m. pneumoniae , culture and serological diagnosis are common methods in clinical practice , but insensitivity , time - consuming or cross - reactions limit their application ( 6 - 10 ) . recently , pcr method was used to directly detect m. pneumoniae dna , which has high specificity and sensitivity in clinical detection of m. pneumoniae ( 11 , 12 ) . but in clinical practice , co - infections are common in children with pneumonia and direct detection of m. pneumoniae is not efficient in distinguishing it from pneumonia ( 8) . our previous study suggested that th1/th2 balance plays a significant part in anti - infectious immunity and th1/th2 cytokines are useful biomarkers in diagnosis and treatment of bacterial and viral infection ( 13 - 15 ) . in this study , we wanted to confirm the value of th1/th2 cytokines in diagnosis and treatment of m. pneumoniae pneumonia . subjects : from may 2012 to june 2014 , a total of 13161 throat swab specimens were collected . the age of the 13161 patients ranged from 3 months to 10 years . all these children had been primarily diagnosed with pneumonia ( 16 ) and had received no clinical treatment . detection of m. pneumoniae : m. pneumoniae pcr kit ( daan gen co. , ltd . guangzhou , china ) was used in dna extraction and m. pneumoniae dna detection ( 4 ) . amplication and data analysis were carried out with an applied biosystems 7500 real - time pcr system ( applied biosystems , inc . , ca , usa ) under this condition : 93c for 2 minutes and 40 cycles of 93c for 45 seconds and 55c for 60 seconds . specific tests were used to detect other pathogens , such as immunofluorescence to detect respiratory viruses including adenovirus , human metapneumovirus , respiratory syncytial virus , parainfluenza virus and influenza virus ; blood and sputum culture for bacteria ; and real - time pcr to detect human cytomegalovirus , epstein - barr virus , chlamydia pneumoniae , ureaplasma urealyticum and chlamydia trachomatis . if any of these assays was positive , the patient was excluded from the study . cytokine determination : the protocol of cytokine measurement was reported in our previous study ( 13 ) as follows : the clotted blood samples were centrifuged at 1,000 g , 20c for 20 minutes . after that , the supernatant was collected and the levels of th1/th2 cytokines by 320 flow cytometry detected . il-2 , il-4 , il-6 , il-10 , tnf- and ifn- were detected quantitatively by cba kit - bdtm cba human th1/th2 cytokine kit ii ( bd biosciences , san jose , ca ) . after collecting the sample data on a facscaliburtm flow cytometer ( becton dickinson , san jose , ca , usa ) , we used the bd cba software ( bd biosciences , san jose , ca , usa ) to display the results in tabular and graphical format . 1.0 pg / ml was the lowest detection limit for these six cytokines , while the highest was 5,000 pg / ml . statistical analysis : we used  or fisher s exact test and mann - whitney u test in spss statistics 19.0 software . it was considered that p < 0.05 is statistically significant . we used receiver operating characteristic ( roc ) curve to estimate the value of cytokines in diagnosing m. pneumoniae pneumonia by spss statistics19.0 software . patients characteristics : between may 2012 and june 2014 , 13161 throat swab samples were collected and tested for m. pneumoniae , including 1353 from outpatients and 11808 from hospitalized children . 8082 samples were from boys and 5079 from girls , yielding a male - to - female ratio of 1.59:1 . 2188 were tested positive for m. pneumoniae , with a positive rate of 16.62% . among 2188 m. pneumoniae positive samples , 1277 were from boys and 911 from girls , giving positive rates of 15.80% in boys and 17.93% in girls . as shown in figure 1 , positive rate for m. pneumoniae infection was at the peak in children aged 5 - 9 years ( 42.46% - 46.92% , compared with other group , p < 0.01 ) . it steadily declined with increasing or decreasing age . among age groups , children younger than 1 year had the lowest ( 7.39% ) positive rate , compared with the other group ( p < 0.01 ) . m. pneumoniae infection occurred all year round , the monthly positive rates for m. pneumoniae infection ranged from 7.65% to 27.35% , with a peak in june and august ( compared with other group p < 0.01 ) , and steadily declined in the previous and the following months ( figure 2 ) . co - infections were found in 1662 ( 75.96% ) m. pneumoniae positive children , which were higher than in mono - infection children . inflammatory cytokine levels : to evaluate the levels of these six cytokines in healthy children and children with m. pneumoniae pneumonia . 526 patients with single m. pneumoniae infection were used as m. pneumoniae pneumonia group and 30 healthy children acted as the control group . as shown in table 1 , comparisons between m. pneumoniae infection group and normal control group revealed no difference of inflammatory cytokine ( il-6 and tnf- ) levels between the two groups ( median levels , pg / ml : il-6:14.27 vs. 4.54 , p = 0.057 ; tnf- : 3.56 vs. 2.21 , p = 0.182 ) . the level of il-2 was significantly lower in serum from m. pneumoniae pneumonia patients than in serum from normal controls ( median levels , pg / ml : il-2 : 3.19 vs. 5.72 , p = 0.00 ) , while the levels of il-4 , il-10 and ifn- in m. pneumoniae pneumonia patients were significantly higher than in the normal controls ( median levels , pg / ml : il-4 : 3.23 vs. 1.46 , p = 0.00 ; il-10 : 5.56 vs. 2.53 , p = 0.001 ; ifn- : 20.35 vs. 4.83 , p = 0.001 ) . roc - analysis : to confirm value of inflammatory cytokines in diagnosis of m. pneumoniae pneumonia , we used roc - analysis to evaluate the abilities of the six cytokines in identifying the possibility of m. pneumoniae pneumonia . the aucs were 0.922 ( 95% ci , 0.878 to 0.966 ) , 0.954 ( 95% ci , 0.928 to 0.979 ) , 0.819 ( 95% ci , 0.758 to 0.880 ) and 0.928 ( 95% ci , 0.892 - 0.963 ) for il-2 ( lower than 4.5pg / ml ) , il-4 ( greater than 2.5 pg / ml ) , il-10 ( greater than 3.0 pg / ml ) and ifn- ( greater than 5.5 pg / ml ) respectively , with sensitivity and specificity above 80% ( table 2 ) . these results indicated that il-2 , il-4 , il-10 and ifn- could be effective biomarkers to identify m. pneumoniae pneumonia . m. pneumoniae pneumonia is common in children , in clinical practice it is important to accurately diagnose m. pneumoniae infection ( 1 , 2 ) . in this article , we used real time pcr to detect m. pneumoniae in throat swab samples . our results showed that positive rate for m. pneumoniae was highest among children aged 5 - 9 years and summer was the m. pneumoniae season in china . there are common methods to detect m. pneumoniae in clinical practice , such as pcr , culture and serological test . interestingly , co - infections were found in 1662 ( 75.96% ) of the m. pneumoniae positive cases . it was difficult to estimate the true association between the clinical manifestations and m. pneumoniae infection ( 8) . so , in this study only children with m. pneumoniae infection were evaluated with th1/th2 cytokines level . our previous study has shown that the useful biomarkers to diagnose bacterial infection in children were il-2 , il-6 and il-10 ( 14 ) . in contrast to the normal controls , the levels of il-2 , il-4 , il-10 and ifn- in children with m. pneumoniae pneumonia had significantly changed and had high sensitivity and specificity . among them , il-2 can fight against infection and plays a unique role in generating and maintaining regulatory t - cells ( 15 ) . after m. pneumoniae infection , changing il-2 level was reported in previous studies with controversial results ( 16 , 17 ) . our study supports findings indicating that il-2 level decreases after m. pneumoniae infection in children . il-10 has pleiotropy , which can limit antigen - presenting cell function and primarily inhibits antigen - presenting cells from releasing chemokines and proinflammatory cytokines ( 13 , 14 ) . furthermore , it also can directly suppress t - cell proliferation , function and cytokine production to limit inflammation ( 14 ) . il-10 increases and plays an important role as anti - inflammation agent ( 18 , 19 ) . this is the underlying mechanism of regulating il-2 and il-10 during m. pneumoniae infection in children . cytokines which are produced by th1 cells can be blocked by il-4 produced by th2 cells ( 18 ) . il-4 also plays a vital role of switching to ige which induces allergic diseases ( 20 , 21 ) . additionally , increase of ifn- during m. pneumoniae infection was also shown in a previous report ( 22 ) . m. pneumoniae is a most common pathogen which causes pneumonia in children in hangzhou , china . our study suggests that the effective biomarkers which can be used to diagnose whether children with pneumonia are afflicted by m. pneumoniae are il-2 , il-4 , il-10 and ifn-.
background : the levels of th1/th2 cytokine can alter in pathogenic infection in children with pneumonia.objectives:to evaluate th1/th2 cytokine profile and its diagnostic value in m. pneumoniae pneumonia in children.patients and methods : children with m. pneumoniae mono - infection and 30 healthy children were tested with cytokines assay . 
 we used real time pcr to detect m. pneumoniae in children with pneumonia.results:m . 
 pneumoniae test was positive in 2188 ( 16.62% ) out of 13161 pneumonia children . 
 children aged 5 - 9 years had the highest rate and summer was a season with high rate of m. pneumoniae incidence in zhejiang province . during the course of study , in 526 pneumonia children with m. pneumoniae mono - infection and 30 healthy children 
 cytokines assay was performed . 
 il-2 level of m. pneumoniae pneumonia children was lower than that of healthy children ( median levels , pg / ml : il-2 : 3.2 vs. 5.7 , p = 0.00 ) , while il-4 , il-10 and ifn- were higher than in healthy children ( median levels , pg / ml : il-4 : 3.2 vs. 1.5 , p = 0.00 ; il-10 : 5.6 vs. 2.5 , p = 0.001 ; ifn- : 20.4 vs. 4.8 , p = 0.001).conclusions : il-2 decreases and il-4 , il-10 and ifn- increase in children with m. pneumoniae pneumonia , which has a promising prospect in diagnosis of this disease in clinical practice .