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ORIGINAL ARTICLE  
Year : 2018  |  Volume : 61  |  Issue : 2  |  Page : 214-218
Detection of immunoglobulin M and immunoglobulin G antibodies to Mycoplasma pneumoniae in children with community-acquired lower respiratory tract infections


1 Department of Microbiology, Maulana Azad Medical College, New Delhi, India
2 Department of Pediatrics, Maulana Azad Medical College, New Delhi, India

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Date of Web Publication20-Apr-2018
 

   Abstract 


Context: Mycoplasma pneumoniae (M. pneumoniae) causes up to 40% of community-acquired pneumonia in children. It is impossible to identify M. pneumoniae infection on the basis of clinical signs, symptoms, and radiological features. Therefore, correct etiological diagnosis strongly depends on laboratory diagnosis. Aims: This study aims to investigate the role of M. pneumonia e in pediatric lower respiratory tract infections (LRTIs) employing enzyme-linked immunosorbent assays (ELISA) and particle agglutination (PA) test. Settings and Design: Two hundred and eighty children, age 6 months to 12 years with community-acquired LRTIs were investigated for M. pneumoniae etiology. Materials and Methods: We investigated 280 children hospitalized for community-acquired LRTIs, using ELISA and PA test for detecting M. pneumoniae immunoglobulin M (IgM) and immunoglobulin G antibodies. Statistical Analysis Used: The difference of proportion between the qualitative variables was tested using the Chi-square test and Fischer exact test. P ≤ 0.05 was considered as statistically significant. Kappa value was used to assess agreement between ELISA and PA test. Results: M. pneumoniae was positive in 51 (23.2%) <5 years and 33 (54.0%) children in ≥5 years of age group, and this difference was statistically significant (P < 0.001). Clinical and radiological findings in M. pneumoniae positive and negative groups were comparable. ELISA detected M. pneumoniae in 78 (27.8%) and PA test 39 (13.9%) patients; 33 (84.6%) ELISA positive and 6 (15.4%) ELISA negative. ELISA/PA test together detected M. pneumoniae infection in 84 (30%) children. Conclusions: Our data underline that M. pneumoniae plays an important role in children with community-acquired LRTIs and more particularly in children >5 years of age.

Keywords: Children, diagnosis, enzyme-linked immunosorbent assays, Mycoplasma pneumoniae, particle agglutination test

How to cite this article:
Kumar S, Garg IB, Sethi GR, Kumar S, Saigal SR. Detection of immunoglobulin M and immunoglobulin G antibodies to Mycoplasma pneumoniae in children with community-acquired lower respiratory tract infections. Indian J Pathol Microbiol 2018;61:214-8

How to cite this URL:
Kumar S, Garg IB, Sethi GR, Kumar S, Saigal SR. Detection of immunoglobulin M and immunoglobulin G antibodies to Mycoplasma pneumoniae in children with community-acquired lower respiratory tract infections. Indian J Pathol Microbiol [serial online] 2018 [cited 2018 Sep 22];61:214-8. Available from: http://www.ijpmonline.org/text.asp?2018/61/2/214/230540



   Introduction Top


Mycoplasma pneumoniae (M. pneumoniae) is implicated in a variety of respiratory tract infections such as bronchitis, bronchiolitis, pharyngitis, and croup,[1] causes 6%–20% of lower respiratory tract infections (LRTIs) and community-acquired pneumonia (CAP) both in older children and adults.[2]M. pneumoniae causes up to 40% of CAP in children and 18% of infections in patients requiring hospitalization.[3]M. pneumoniae infection is endemic in the larger communities of the world but epidemics lasting several months to years periodically occur every 3–7 years.[4] It is impossible to identify M. pneumoniae infection on the basis of clinical signs, symptoms, and radiological features not specific to M. pneumoniae infection.[2],[5] Empirical therapy is therefore adopted in most cases.[6]

A specific diagnosis of M. pneumoniae infection is important to initiate appropriate therapy. Lack of cell wall makes M. pneumoniae treatment with β-lactam antibiotics ineffective, whereas treatment with macrolides may markedly reduce the duration of illness.[6] However, macrolide resistance M. pneumoniae has been spreading worldwide, and macrolide-resistance rates have been rising up to 90%–100% in Asia, hindering the efficacy of common antibiotic regimens.[7]

Conventional assays for the detection of M. pneumoniae have their limitations. Culture methods have been the gold standard for diagnosis but are too insensitive producing a result after several weeks. Alternative diagnostic procedures such as detection of immunoglobulin M (IgM) and/or immunoglobulin G (IgG) by enzyme-linked immunosorbent assays (ELISA), antigen detection by immunochromatography and nucleic acid amplification techniques, mainly polymerase chain reaction (PCR) were developed. Serological methods, in particular, ELISA are most widely used to diagnose M. pneumoniae infection. However, the most convincing evidence of an ongoing infection is a significant increase in IgG or an IgG seroconversion in paired sera, collected 3–4 weeks apart.[8] Particle agglutination (PA) tests use a mixture of M. pneumoniae antigens to detect both IgG and IgM simultaneously.[3]

The present study was conducted for the detection of M. pneumonia e in pediatric LRTIs employing ELISA and PA test.


   Materials and Methods Top


A total of 280 patients with acute LRTIs which were admitted to Pediatrics ward of Lok Nayak Hospital, Maulana Azad Medical College, New Delhi, India, were selected for this study. The study was approved by the institutional ethics committee, Maulana Azad Medical College, New Delhi, India.

Inclusion criteria

Previously healthy male and female children aged 6 months to 12 years, who had been hospitalized for signs and symptoms of community-acquired LRTIs were considered eligible for inclusion. The presence of cough and fever with breathlessness of <30 days duration, increased respiratory rate (with or without features of respiratory distress) on examination, presence of signs of consolidation, or bronchopneumonia with or without wheeze on auscultation.

Criteria for exclusion

Hospital-acquired pneumonia, i.e., pneumonia that developed 72 h after hospitalization or within 7 days of discharge.

Enrollment and evaluation of patients

Written informed consent from the parents or legal guardian was taken of young children (aged 6 months to 1 year) and older children (aged 13 months to 12 years) before being enrolled for this study and collecting clinical specimens. Detailed history and clinical examination were performed on admission, and chest X-ray was conducted in all children, the details of which were noted in a predesigned pro forma. Blood specimens (1–2 ml) were collected for the detection of IgM and IgG antibodies against M. pneumoniae. A convalescent-phase sera was obtained after 4–6 weeks of enrollment.

Serology

Mycoplasma pneumoniae enzyme-linked immunosorbent assays for immunoglobulin M and immunoglobulin G antibodies

ELISA was performed for serum IgM and IgG antibodies against M. pneumoniae using commercially available ELISA based kits following the manufacturer's instructions (Calbiotech Inc. Austin Dr Spring Valley, CA, Germany). The interpretative criteria were consistent with the recommendations of the manufacturer as outlined on the package insert.

Particle agglutination test

PA test (Serodia Myco II, Fujirebio, Japan) was performed according to the manufacturer's instructions. Serodia-Myco II is an in vitro diagnostic test for the detection of antibodies to M. pneumoniae which is manufactured using artificial gelatin particles sensitized with cell-membrane components of M. pneumoniae (Mac strain). Briefly, 25 μL of serum was diluted twofold to give dilutions of 1 in 10–1 in 10,240. Unsensitized particles were added to 1 in 10 serum dilution to give a final dilution of 1 in 20. Twenty-five μL of the sensitized particle suspension was then added to the remaining wells, giving final dilutions of 1 in 40–1 in 20,480. The plates were shaken for 30 s and then covered and left undisturbed on a level surface at room temperature for 3 h. The test was initially calibrated using the control serum dilution series. Definite compact button in the center of the well with a smooth round outer margin was read as negative result and a definite large ring with firmly agglutinated particles spread within the ring as positive result. Titers of ≥40 were regarded as positive for M. pneumoniae antibody.

Statistical analysis

Data analysis was performed using the statistical software Epi Info version 3.5.3, CDC, Atlanta, GA, USA. The difference of proportion between the qualitative variables was tested using the Chi-square test and Fischer exact test. P ≤ 0.05 was considered as statistically significant. Kappa value was used to assess agreement between ELISA and PA test.


   Results Top


Demographic profile

The maximum number 219 (78.2%) children were <5 years and 61 (21.7%) children were ≥5 years of age group. In the age group of <5 years, 51 (23.2%) children were positive for M. pneumoniae, whereas in ≥5 years of age group 33 (54.0%) children were positive for M. pneumoniae, and this difference was statistically significant [P < 0.001; [Table 1]. There were 175 (62.5%) males and 105 (37.5%) females and male-to-female ratio was 1.66 with a male preponderance. The percentage of M. pneumoniae positive patients were higher in 34 (32.3%) females than in 50 (28.5%) males, and this difference was statistically insignificant [P = 0.59; [Table 1].
Table 1: Association of Mycoplasma pneumoniae infection with age and sex in 280 children with community-acquired lower respiratory tract infections

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Clinical profile

Wheezing (audible/auscultable) was present in 43 (35.2%) M. pneumoniae-positive and in 79 (64.7%) M. pneumoniae-negative patients (P = 0.12); coryza was present in 48 (26.8%) M. pneumoniae-positive and 131 (73.1%) M. pneumoniae-negative patients (P = 0.15); chest pain in 9 (52.9%) M. pneumoniae-positive and 8 (47.0%) M. pneumoniae-negative patients (P = 0.06); crepitations in 40 (31.4%) M. pneumoniae-positive and 84 (66.1%) M. pneumoniae-negative patients (P = 0.54); and rhonchi in 18 (23.3%) M. pneumoniae-positive and 59 (76.6%) M. pneumoniae-negative patients (P = 0.17) of the study group. There was not any statistically significant association between M. pneumoniae infection and clinical signs and symptoms [Table 2].
Table 2: Clinical profile in the study group

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Radiological findings

Documentation of consolidation, consolidation and pleural effusion, bronchopneumonia, hyperinflation, interstitial infiltrates, hyperinflation/bronchopneumonia/infiltrates, and normal chest X-ray were numerically comparable and differences were statistically insignificant in M. pneumoniae positive and negative categories [Table 3].
Table 3: Radiographic findings in children with community-acquired lower respiratory tract infections

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Microbiological profile

In the present study, ELISA detected M. pneumoniae infection in 78 (27.8%) children; specific IgM antibodies alone in 23 (29.4%) in acute sera; specific IgM and IgG antibodies (acute phase) in 7 (8.9%); IgG antibodies (acute phase) in 18 (23%); IgG antibodies in convalescent phase sera with four-fold rise in titer in comparison to that in acute phase sera in 30 (38.4%); 17 (56.6%) were IgM positive; and 13 (43.3%) IgM negative. Convalescent sera were available only in 113 (40.3%) patients. The association between IgG convalescent serum positivity and IgM positivity in acute phase serum was moderate (kappa value = 0.358). PA test was positive in 39 (13.9%) patients; 33 (84.6%) ELISA positive, and 6 (15.4%) ELISA negative. ELISA and PA test together detected M. pneumoniae infection in 84 (30%) children [Table 4].
Table 4: Serological diagnosis of Mycoplasma pneumoniae infection by enzyme-linked immunosorbent assays and particle agglutination test

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The sensitivity, specificity, positive, and negative predictive values of ELISA for M. pneumoniae infection were 84.62%, 81.33%, 42.31%, and 97.03%, respectively when PA test was assumed to be the diagnostic standard [Table 5]. When ELISA was assumed to be the diagnostic standard for M. pneumoniae infection sensitivity, specificity, positive, and negative predictive values of PA test were 42.31%, 97.03%, 84.62%, and 81.33%, respectively [Table 6]. The agreement between PA test and ELISA was moderate (kappa value = 0.465).
Table 5: Sensitivity, specificity, positive, and negative predictive values of Mycoplasma pneumoniae enzyme-linked immunosorbent assays using particle agglutination test as a diagnostic standard

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Table 6: Sensitivity, specificity, positive, and negative predictive values of Mycoplasma pneumoniae particle agglutination test using enzyme –linked immunosorbent assays as a diagnostic standard

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   Discussion Top


M. pneumoniae is an important cause of acute respiratory tract infection, especially as a potential etiology of the clinical entity termed atypical pneumonia. To improve the identification of patients in need of treatment and to avoid unnecessary antibiotic therapy, early diagnosis of M. pneumoniae infection is essential. Serology is probably the most frequently used method to diagnose M. pneumoniae infections. Moreover, serologic tests are more sensitive indicators of mycoplasma infection than culture of the organism because in many instances antibody responses can be detected when the organism cannot be recovered.[9] Therefore, the current routine methods for laboratory diagnosis of M. pneumoniae infection are based primarily on serologic analysis.[9],[10] Among the various serological tests available for detecting M. pneumoniae infections, ELISA has been described to be the most sensitive and specific.[11],[12],[13]

Our study shows higher prevalence of M. pneumoniae in children ≥5 years of age, and this was found to be statistically significant (P < 0.001). Our findings are in agreement with the previous studies which reported a higher rate of M. pneumoniae infections in children >5 years of age,[3],[14] in contrast Kumar et al. reported M. pneumoniae infection relatively higher in patients <1 year old with LRTIs.[2] The percentage of M. pneumoniae-positive patients were higher in 34 (32.3%) females than in 50 (28.5%) males and the incidence of M. pneumoniae infection with sex was found to be statistically insignificant (P = 0.59). Our study is in agreement with Vervloet et al.[15] and is in contrast to previous findings by Kumar et al.[2]

In the present study, clinical profile was compared between M. pneumoniae positive and negative groups, and none of the signs and symptoms were statistically associated with M. pneumoniae infection which is in accordance with previous studies which cast doubt on the specificity of clinical and laboratory features for predicting the microbial cause of LRTIs and emphasizing the need for laboratory confirmation.[16],[17]

Radiographic findings can be very much variable and mimic different lung diseases either a viral or bacterial pneumonia. In M. pneumoniae, LRTIs radiologic changes are reported as nonspecific where M. pneumoniae positive and negative cases were not possible to differentiate on the basis of radiological picture of chest.[2]

Serological tests are more sensitive for the detection of acute M. pneumoniae infection than culture. The development of antibody to M. pneumoniae infection is performed by a range of serological methods which include immunofluorescence assays, enzyme immunoassays (EIA), and PA assays. These tests are easy to use, their sensitivities and specificities are also improved and have largely replaced the older complement fixation test which was popular in the past as the primary method for detection of M. pneumoniae antibodies. M. pneumoniae infection by ELISA detected 78 (27.8%) children. PA test was positive in 39 (13.9%) children; 33 (84.6%) were ELISA positive; and 6 (15.4%) ELISA negative. ELISA/PA test together were positive in 84 (30%) children [Table 4]. The association between IgG convalescent serum positivity and IgM positivity in acute phase serum was moderate. Our results are in agreement with previous study, in which Kumar et al.[2] reported 34% and Kashyap et al.[17] 21.3%. M. pneumoniae positivity by serology in children with community-acquired LRTIs. The antigenic compositions used in serologic diagnostic kits differ among manufacturers, therefore, the positivity rates of M. pneumoniae-specific IgM and IgG antibodies determined by each method can differ significantly during the acute stage of M. pneumoniae infection.[8],[18] Rapid EIAs for IgM detection of acute infection are available where a single serum specimen is employed.[19] IgM or IgA and PCR in a combination may be an optimum diagnostic approach for M. pneumoniae infection [19] but adding considerable cost to laboratory testing.

Sensitivity, specificity, positive, and negative predictive values of ELISA for M. pneumoniae were 84.62%, 81.33%, 42.31%, and 97.03%, respectively, while sensitivity, specificity, positive, and negative predictive values of PA test for M. pneumoniae were 42.31%, 97.03%, 84.62%, and 81.33%, respectively. The agreement between PA test and ELISA was moderate. PCR similar to culture characterize high specificity but low or variable sensitivity thus PCR cannot replace serology although may be performed in conjunction with serology.[20]


   Conclusions Top


Our data underline that M. pneumoniae plays an important role in children with community-acquired LRTIs and more particularly in children >5 years of age.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Jain S, Williams DJ, Arnold SR, Ampofo K, Bramley AM, Reed C, et al. Community-acquired pneumonia requiring hospitalization among U.S. Children. N Engl J Med 2015;372:835-45.  Back to cited text no. 14
    
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Vervloet LA, Camargos PA, Soares DR, Oliveira GA, Oliveira JN. Clinical, radiographic and hematological characteristics of Mycoplasma pneumoniae pneumonia. J Pediatr (Rio J) 2010;86:480-7.  Back to cited text no. 15
    
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Maheshwari M, Kumar S, Sethi GR, Bhalla P. Detection of Mycoplasma pneumoniae in children with lower respiratory tract infections. Trop Doct 2011;41:40-2.  Back to cited text no. 16
    
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Kashyap B, Kumar S, Sethi GR, Das BC, Saigal SR. Comparison of PCR, culture and serological tests for the diagnosis of Mycoplasma pneumoniae in community-acquired lower respiratory tract infections in children. Indian J Med Res 2008;128:134-9.  Back to cited text no. 17
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18.
Beersma MF, Dirven K, van Dam AP, Templeton KE, Claas EC, Goossens H, et al. 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 2005;43:2277-85.  Back to cited text no. 18
    
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Atkinson TP, Waites KB. Mycoplasma pneumoniae infections in childhood. Pediatr Infect Dis J 2014;33:92-4.  Back to cited text no. 19
    
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Sobieszczańska BM, Kasprzykowska U, Duda-Madej A, Secewicz A, Marciniak J, Gościniak G, et al. Relevance of serology for Mycoplasma pneumoniae infection among children with persistent cough. Adv Clin Exp Med 2014;23:185-90.  Back to cited text no. 20
    

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Correspondence Address:
Surinder Kumar
Department of Microbiology, Maulana Azad Medical College, New Delhi - 110 002
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_21_17

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