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Evaluation of real-time PCR with serology for diagnosis of community acquired pneumonia caused by Mycoplasma pneumoniae

Open AccessPublished:June 02, 2022DOI:https://doi.org/10.1016/j.cegh.2022.101084

      Abstract

      Objective

      To evaluate the utility of real-time polymerase chain reaction (PCR) against serology (IgM) for the diagnosis of community acquired pneumonia (CAP) caused by Mycoplasma pneumoniae (M. pneumoniae).

      Methods

      Hospitalized children from 1 month to 18 years of age with symptoms and findings consistent with CAP as per World Health Organization (WHO) criteria were enrolled. Mycoplasma IgM serology (Acute phase) was done in all children using enzyme-linked immunosorbent assay (ELISA). Real-time PCR was done using a commercial FDA approved Multiplex Real-time PCR kit for M. pneumoniae.

      Results

      M. pneumoniae was confirmed in 15% (10 out of 66 patients) of the hospitalized children with CAP. Of those with confirmed Mycoplasma infection, 90% (9 out of 10) patients were serology reactive while PCR was positive in only 40% (4 out of 10). Both IgM and PCR were positive in 30% (3 out of 10 patients) and only one real-time PCR positive patient was negative for IgM.

      Conclusion

      In our study, real-time PCR had a lower sensitivity compared to serology. However, this test has the advantage of detecting early infection prior to the development of an immune response (presence of IgM). Clinicians could consider real-time PCR testing if the patient presents early after onset of symptoms and prior to the use of empirical antibiotics and serology should be the test of choice at late presentations.

      Keywords

      1. Introduction

      Community acquired pneumonia (CAP) is a common cause of morbidity and mortality among children.
      • Kumar S.
      • Saigal S.R.
      • Sethi G.R.
      Detection of IgM and IgG antibodies to Chlamydophila pneumoniae in pediatric community-acquired lower respiratory tract infections.
      Mycoplasma pneumoniae is an important cause of human respiratory disease, accounting for 15–20% of total CAP.
      • Maheshwari M.
      • Kumar S.
      • Sethi G.R.
      • Bhalla P.
      Detection of Mycoplasma pneumoniae in children with lower respiratory tract infections.
      Clinical diagnosis of M. pneumoniae is difficult because it simulates the clinical picture of viral and other pneumonias.
      • Shenoy V.D.
      • Upadhyaya S.A.
      • Rao S.P.
      • Shobha K.L.
      Mycoplasma pneumoniae infection in children with acute respiratory infection.
      In a typical clinical setting, due to relative unavailability of rapid, sensitive and specific laboratory tests, an etiologic diagnosis is not established, thereby necessitating the use of empirical therapy. An etiologic diagnosis will help avoid empiric therapy, reduce antibiotic misuse and allow early initiation of specific antibiotics effective against M. pneumoniae.
      The clinical course of mycoplasma infections in humans is thought to be mediated by host immune and inflammatory responses, rather than direct cytopathological effects initiated by mycoplasmal cell components.
      • Kannan T.R.
      • Baseman J.B.
      ADP-ribosylating and vacuolating cytotoxin of Mycoplasma pneumoniae represents unique virulence determinant among bacterial pathogens.
      Classically, the symptoms are worse than the signs would suggest.
      British Thoracic Society Standards of Care Committee
      British thoracic society guidelines for the management of community acquired pneumonia in childhood.
      Although the main burden of infection is found in school-age children (5 years and above), M. pneumoniae has also been noted as a significant cause of respiratory tract infection in children under the age of five.
      • Gadsby N.J.
      • Reynolds A.J.
      • McMenamin J.
      • et al.
      Increased reports of Mycoplasma pneumoniae from laboratories in Scotland in 2010 and 2011-impact of the epidemic in infants.
      ,
      • Waites K.B.
      • Talkington D.F.
      Mycoplasma pneumoniae and its role as a human pathogen.
      Due to the lack of a cell wall, all mycoplasma species are innately resistant to beta-lactams and glycopeptides. Fluoroquinolones have been shown to be bactericidal for M. pneumoniae, whereas macrolides and tetracyclines are primarily bacteriostatic.
      • Waites K.B.
      • Talkington D.F.
      Mycoplasma pneumoniae and its role as a human pathogen.
      ,
      • Waites K.B.
      • Balish M.F.
      • Atkinson T.P.
      New insights into the pathogenesis and detection of Mycoplasma pneumoniae infections.
      Serology is the mainstay of laboratory diagnosis of M. pneumonia.
      • Waites K.B.
      • Balish M.F.
      • Atkinson T.P.
      New insights into the pathogenesis and detection of Mycoplasma pneumoniae infections.
      Commercial enzyme immunoassays (EIA) for IgM detection are employed in tertiary care centers. In specialized centers, real-time PCR can also be performed for diagnosis; it is useful especially in early presentation where typical symptoms may not manifest
      • Waites K.B.
      • Balish M.F.
      • Atkinson T.P.
      New insights into the pathogenesis and detection of Mycoplasma pneumoniae infections.
      ,
      • Kumar S.
      • Maria A.
      • Saigal S.R.
      • Maheshwari M.
      Mycoplasma pneumoniae as a cause of non-resolving pneumonia in a neonate.
      and has been considered as a gold standard test for diagnosis of Mycoplasma infections.
      • Chang Hsin-Yu
      • Chang Luan-Yin
      • Shao Pei-Lan
      • et al.
      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.
      ,
      • Li1 Qing-Ling
      • Dong He-Ting
      • Sun Hui-Ming
      • et al.
      The diagnostic value of serological tests and real-time polymerase chain reaction in children with acute Mycoplasma pneumoniae infection.
      The present study evaluated the utility of a real-time PCR against serology for the diagnosis of M. pneumoniae and describes the frequency of M. pneumoniae in children with CAP comparing clinical presentation with the results of Real Time PCR and serology.

      2. Methodology

      2.1 Study design

      It is a prospective cross sectional observational study performed in children between 1 month and 18 years of age. The overall study population included children who were hospitalized for symptoms and findings consistent with CAP, as per WHO criteria - pneumonia with fast breathing and/or chest indrawing warranting admission and oral antibiotics or severe pneumonia with any danger signs (e.g., lethargy, inability to drink, persistent vomiting, stridor or convulsions) requiring injectable therapy. The population excluded children hospitalized in the past 14 days for any other illness. Baseline investigations were done for all. Chest radiography was done where required; results were interpreted according to WHO categorization - Class I = consolidation/pleural effusion, Class II = interstitial pattern/infiltrate, Class III = no consolidation/infiltrate/effusion, Class IV = radiograph quality not sufficient for reading.

      2.2 Serology

      Mycoplasma IgM serology (Acute phase) was done in all children using Nova Lisa- M. pneumoniae IgM ELISA, (NOVATEC Immunodiagnostica, GmbH, Germany). This is an indirect IgM ELISA intended for the qualitative determination of IgM antibodies against M. pneumoniae.

      2.3 Real-time PCR

      Nasopharyngeal (NP) swab was collected from all enrolled subjects in viral transport medium (VTM). Total nucleic acids were extracted from NP samples using the Versant 1.0 Reagent kit (Siemens, Belgium). Automated extraction (VERSANT kPCR, Siemens) was done if load was more than 10 patient samples. Real-time PCR was done using Fast Track Diagnostics (FTD) kit, Respiratory pathogen 21 plus kit (FTD Jung linster, Luxembourg) which is a multiplex real-time PCR and can detect 20 viral and 5 bacterial pathogens. Real-Time PCR was used as the gold standard test for this study as paired sera was not available and culture was not feasible.
      Informed consent was obtained from the parents of the children or the children themselves (as per age), who were included for the study. The study commenced in January 2020 and continued till March 2020. Subsequently, enrollment had to be halted due to the COVID-19 pandemic. Enrollment was re-initiated a year later in March 2021 and continued till May 2021. This study was cleared by the Institutional Ethics Committee (IEC) for its scientific content and ethics.

      2.4 Statistical analysis

      Statistical analysis was performed by the statistical software STATA 11.0. Continuous variables were represented as Mean (SD), and categorical variables were represented as Frequency (percentage). Chi-square test or Fisher's exact tests were used to assess differences in categorical data. Mann Whitney U test were used for differences in means of independent data. Sensitivity and specificity were measures of a test's ability to correctly classify a person as having a disease or not having a disease.

      3. Results

      This study enrolled 66 children diagnosed with CAP. Ten patients were positive for M. pneumoniae (Table 1). Nine of 10 were positive for IgM and four were positive by real-time PCR. Both IgM and real-time PCR were positive in three out of 10 patients and only one patient was serology negative but real time PCR positive.
      Table 1Demography of subjects enrolled, M. pneumonia positive patients and M. pneumoniae negative patients.
      Subjects enrolled N = 66 (%)M. pneumoniae positive patients N = 10 (%)M. Pneumoniae negative patients N = 56 (%)
      Age:
       Mean (±SD)3.8 (±3.6) years6.2 (±3.5) years3.43 ± 3.45
       Median (Range)2.6 years (1 month–13 years)6.6 years (14 months–11 years)1.95 years (1 month–13 years)
      Age distribution:
       ≤1 year18 (27.27)0 (0%)18(32.14%)
       >1 to ≤ -5 years28 (42.42)3 (30)25(44.64%)
       >5 to ≤ 10 years14 (21.21)5 (50)9(16.07%)
      >10 to <18 years6 (9.09)2 (20)4(7.14%)
      Gender:
       Male41 (62.12)5 (50)36 (64.29%)
       Female25 (37.88)5 (50)20 (35.71%)
      Symptoms:
       Short duration fever35 (53.03)2 (20)33 (58.92)
       High grade fever36 (54.54)7 (70)29 (51.78)
       Preceding URI
      Upper Respiratory Tract Infection.
      34 (51.51)2 (20)32 (57.14)
       Sore throat3 (4.54)1 (10)2 (3.57)
       Cough <7 days48 (72.72)6 (60)42 (75)
       Non-Productive cough55 (83.33)10 (100)45 (80.35)
       Cough severity15 (22.72)3 (30)12 (21.42)
       Systemic symptoms32 (48.48)3 (30)29 (51.78)
      Signs:
       Tachypnea41 (62.12)4 (40)37 (66.07)
       Dyspnea19 (28.78)2 (20)17 (30.35)
       Hypoxia17 (25.75)0 (0)17 (30.35)
       Increased work of breathing30 (45.45)4 (40)26 (46.42)
       Systemic signs of consolidation18 (27.27)5 (50)13 (23.21)
       Other respiratory signs42 (63.63)4 (40)38 (67.85)
      Laboratory and radiological investigations:
       Elevated WBC counts31 (46.96)3 (30)28 (50)
       Neutrophilic predominance49 (74.24)8 (80)41 (73.21)
       WHO categorization of chest radiography
       Class I37 (56.06)7 (70)30 (53.57)
       Class II12 (18.18)1 (10)11 (19.64)
       Class III11 (16.66)2 (20)9 (16.07)
      Duration of hospitalization5.93 ± 3.597.1 ± 3.955.73 ± 3.51
      a Upper Respiratory Tract Infection.
      The age and gender distribution among the overall enrolled population and in the subset of M. pneumoniae positive patients is provided in Table 1. The mean and median age of the M. pneumoniae positive patients (6.2 and 6.6 years) was observed to be higher than the overall population (3.8 and 2.6 years respectively). In the youngest (≤1 year) age group, no child was positive for M. pneumoniae, but as age advances, the percentage of children positive for M. pneumoniae becomes higher than children negative for M. pneumoniae.
      Most of the children were not critical at presentation and did not require intensive care. The typical presentation of Mycoplasma was prolonged high-grade fever (70%) associated with non-productive, short duration cough (60%), not associated with tachypnea, dyspnea or persistent vomiting. Other systemic findings were not prominent. Of the 10 confirmed cases of M. pneumoniae, more than half the patients (70%) presented with Class I WHO categorization of chest radiography suggestive of consolidation/pleural effusion. Other investigations were suggestive of normal white blood cell count with neutrophilic predominance. The duration of hospitalization was approximately 7 days in majority of the patients.
      Table 2 presents the comparison of real-time PCR versus serology (Acute IgM) for Mycoplasma among the 66 cases. Using real-time PCR as the “gold standard” the sensitivity and specificity of IgM were estimated to be 75% (3 out of 4) and 90.32% (56 out of 62), respectively. Positive predictive value of IgM was 33.3% (3 true positive out of 9 overall positive) and negative predictive value was 98.25% (56 true negative out of 57 overall negative).
      Table 2Comparison of NP swab real-time PCR vs serology (acute IgM).
      Acute IgMReal Time PCR
      PositiveNegativeTotal
      Positive369
      75%9.68%13.64%
      Negative15657
      25%90.32%86.36%
      Total46266
      100%100%100%

      4. Discussion

      M. pneumoniae is an important pathogen causing CAP. Although literature shows the prevalence of Mycoplasma to be up to 40%,
      • Kannan T.R.
      • Baseman J.B.
      ADP-ribosylating and vacuolating cytotoxin of Mycoplasma pneumoniae represents unique virulence determinant among bacterial pathogens.
      the usual range is between 15 and 20%.
      • Maheshwari M.
      • Kumar S.
      • Sethi G.R.
      • Bhalla P.
      Detection of Mycoplasma pneumoniae in children with lower respiratory tract infections.
      Likewise, this study enrolled 66 children, of which 10 were confirmed positive for M. pneumoniae (an incidence of 15%).
      Literature suggests that M. pneumoniae has a peak incidence at 5 years and above
      • Kumar S.
      • Saigal S.R.
      • Sethi G.R.
      Detection of IgM and IgG antibodies to Chlamydophila pneumoniae in pediatric community-acquired lower respiratory tract infections.
      ,
      • Maheshwari M.
      • Kumar S.
      • Sethi G.R.
      • Bhalla P.
      Detection of Mycoplasma pneumoniae in children with lower respiratory tract infections.
      Consistent with that, we observed that the mean and median age of the M. pneumoniae positive patients (6.2 and 6.6 years) was higher than the overall population (3.8 and 2.6 years) afflicted with CAP.
      The clinical features of M. pneumoniae range from inapparent infection to upper respiratory-tract disease to bronchopneumonia.
      • Chanok R.M.
      • Dienes L.
      • Eaton M.D.
      • et al.
      Mycoplasma pneumoniae: proposed nomenclature for atypical pneumonia organism (Eaton agent).
      The most common clinical symptoms are cough (non-productive at the start), fever, chills, sore throat, headache, hoarseness, myalgia and general malaise.
      • Kumar S.
      Mycoplasma pneumoniae: a significant but underrated pathogen in paediatric community-acquired lower respiratory tract infections.
      Consistent with literature, in our study too, we had symptoms with prolonged high-grade fever and non-productive cough, not associated with tachypnea, dyspnea or persistent vomiting. Other systemic findings, though present in few, were not prominent. More than half of the patients (N = 7/10; 70%) infected with Mycoplasma had radiological evidence of lobar consolidation and all except one, were in the age group from 5 to 10 years which corresponds with the data from other studies.
      • Kumar S.
      Mycoplasma pneumoniae: a significant but underrated pathogen in paediatric community-acquired lower respiratory tract infections.
      Confirmation of M. pneumoniae infection is clinically challenging. Culture of this pathogen is difficult to perform and time consuming. Detection of antibody conversion or four-fold rise in antibody titers (IgG) in two consecutive sera samples collected 2 weeks apart indicates an acute infection. However, this is generally infeasible, since many patients do not return for follow-up, and even in positive cases, it provides only retrospective confirmation of diagnosis.
      • Chang Hsin-Yu
      • Chang Luan-Yin
      • Shao Pei-Lan
      • et al.
      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.
      To allow prospective diagnosis in our study we performed serology as well as real-time PCR analysis for all cases of CAP. Real-time PCR was considered the ‘gold standard’ against which serology was compared, an approach used in earlier studies as well
      • Chang Hsin-Yu
      • Chang Luan-Yin
      • Shao Pei-Lan
      • et al.
      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.
      ,
      • Li1 Qing-Ling
      • Dong He-Ting
      • Sun Hui-Ming
      • et al.
      The diagnostic value of serological tests and real-time polymerase chain reaction in children with acute Mycoplasma pneumoniae infection.
      Among the 10 confirmed M. pneumoniae cases, 9 were positive by serology and 4 were positive by real-time PCR. In the 4 real-time PCR positive cases, 3 were positive by serology too (75%). Six of the serologically diagnosed patients were negative by real-time PCR. The reason for this could be that M. pneumoniae had decreased to undetectable levels in the throat at the time of sampling, possibly either due to initiation of antibiotics or the sampling was done more than a week or 10 days after the onset of symptoms due to late presentation. Molecular tests perform best when samples are collected within a week after onset of illness. Other possibilities include presence of unrecognized PCR inhibitors. An error in sample collection (i.e., insufficient scraping of the pharyngeal wall) is also possible, however, this is unlikely since collection was performed by well-trained nurses. All patients who were positive by real-time PCR for M. pneumoniae were investigated around 7–8 days of illness and were not administered prior antibiotics. In contrast, patients negative by real-time PCR for M. pneumoniae but positive by serology had a more delayed presentation, about 9 days and beyond after the onset of symptoms. These findings correlate with other studies.
      • Kumar S.
      Mycoplasma pneumoniae: a significant but underrated pathogen in paediatric community-acquired lower respiratory tract infections.
      This emphasizes that the methodology of identifying Mycoplasma by real-time PCR will be effective when done during early infection period and when the patient has not had any prior antibiotics.
      Literature suggests that in order to diagnose an acute M. pneumoniae infection, a combination of PCR and IgM serology is the most sensitive and convenient method. However, in our study, 6 (60%) of the 10 patients with M.pneumoniae infection were identified on the basis of serology alone. Without serology the diagnostic yield would have been meagre. Most of the studies done for comparing real time PCR with serology also had a sensitivity range of serology from 62% to 78%.
      • Kumar S.
      • Saigal S.R.
      • Sethi G.R.
      Detection of IgM and IgG antibodies to Chlamydophila pneumoniae in pediatric community-acquired lower respiratory tract infections.
      ,
      • Maheshwari M.
      • Kumar S.
      • Sethi G.R.
      • Bhalla P.
      Detection of Mycoplasma pneumoniae in children with lower respiratory tract infections.
      ,
      • Chang Hsin-Yu
      • Chang Luan-Yin
      • Shao Pei-Lan
      • et al.
      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.
      ,
      • Li1 Qing-Ling
      • Dong He-Ting
      • Sun Hui-Ming
      • et al.
      The diagnostic value of serological tests and real-time polymerase chain reaction in children with acute Mycoplasma pneumoniae infection.
      Studies have also compared PCR and serological tests for the diagnosis of M. pneumoniae.
      • Chang Hsin-Yu
      • Chang Luan-Yin
      • Shao Pei-Lan
      • et al.
      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.
      ,
      • Li1 Qing-Ling
      • Dong He-Ting
      • Sun Hui-Ming
      • et al.
      The diagnostic value of serological tests and real-time polymerase chain reaction in children with acute Mycoplasma pneumoniae infection.
      Hsin-Yu Chang et al.
      • Chang Hsin-Yu
      • Chang Luan-Yin
      • Shao Pei-Lan
      • et al.
      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.
      evaluated the sensitivity and specificity of IgM keeping PCR as the gold standard which was found to be 62.2% and 85.5%. Our results were in line with these earlier reports.
      Both serology and real-time PCR have their pros and cons. The key advantages of serology are: specialized equipment is not necessary, small volumes of serum is adequate,
      • Waites K.B.
      • Balish M.F.
      • Atkinson T.P.
      New insights into the pathogenesis and detection of Mycoplasma pneumoniae infections.
      and M. pneumoniae-specific IgM elevation is adequate evidence of acute infection.
      • Kumar S.
      • Maria A.
      • Saigal S.R.
      • Maheshwari M.
      Mycoplasma pneumoniae as a cause of non-resolving pneumonia in a neonate.
      IgM antibody typically appears within 1 week of infection
      • Kumar S.
      • Maria A.
      • Saigal S.R.
      • Maheshwari M.
      Mycoplasma pneumoniae as a cause of non-resolving pneumonia in a neonate.
      and is a reliable indicator of infection in pediatric patients older than 5 years and those with a disease duration of 1–2 weeks.
      • Li1 Qing-Ling
      • Dong He-Ting
      • Sun Hui-Ming
      • et al.
      The diagnostic value of serological tests and real-time polymerase chain reaction in children with acute Mycoplasma pneumoniae infection.
      On the other hand, the key advantages of real-time PCR are: potential for rapid testing (it can be completed in 1 day),
      • Kumar S.
      • Saigal S.R.
      • Sethi G.R.
      Detection of IgM and IgG antibodies to Chlamydophila pneumoniae in pediatric community-acquired lower respiratory tract infections.
      it may be positive earlier during infection than serology, and is perhaps more sensitive (detection of even a single organism may be possible).
      • Waites K.B.
      • Talkington D.F.
      Mycoplasma pneumoniae and its role as a human pathogen.
      ,
      • Waites K.B.
      • Balish M.F.
      • Atkinson T.P.
      New insights into the pathogenesis and detection of Mycoplasma pneumoniae infections.
      Limitations with PCR include: the need for rigorous specificity testing to ensure that nonpathogenic commensal mycoplasma species in the respiratory tract do not affect the test.
      • Waites K.B.
      • Balish M.F.
      • Atkinson T.P.
      New insights into the pathogenesis and detection of Mycoplasma pneumoniae infections.
      Another key limitation is that PCR may be negative when empiric antibiotics are started prior to etiologic confirmation, a consequence of indiscriminate use of antibiotics.
      Study related limitations include inability to use nasopharyngeal aspirate in lieu of NP swab. One may therefore argue that NP swab real-time PCR assay may not be the ideal gold standard due to the limitations cited above. Of concern was, majority of our study patients presented to us during late infection and/or were administered empirical antibiotics elsewhere and referred here. Lastly, our sample size was relatively small, and further research with larger sample size is needed to validate our findings.
      Taken together, it is important to establish etiologic diagnosis of CAP, to ensure judicious use of antibiotics. However, early and indiscriminate antibiotic use vitiates the assessment of etiologic diagnosis. Notwithstanding these issues, clinicians should endeavor to confirm the etiology of CAP soon after presentation and in cases with suspected M. pneumoniae infection, serology or real-time PCR should be chosen as per the time of assessment for confirmation of diagnosis.
      Clinicians must prefer real-time PCR as the first line investigation when there is suspicion of M. pneumoniae in patients with CAP who presents early without prior use of empirical antibiotics and serology should be opted when they present at a later stage. Confirmation of diagnosis helps ensure appropriate management and avoid emergence of resistant organisms.

      Funding sources

      Authors acknowledged intramural funding support from The CHILDS Trust Medical Research Foundation (CTMRF), Chennai, Tamil Nadu, India.

      Ethical approval

      The study was approved by the Institutional Review Board (IRB) Ethics committee of Kanchi Kamakoti CHILDS Trust Hospital & The CHILDS Trust Medical Research Foundation (KKCTH-CTMRF: IEC- 36/July 2019 (IRB Min. dt. July 18, 2019).

      Declaration of competing interest

      None.

      Acknowledgments

      Dr.K.Dhanalakshmi, Dr.Sumanth Amperayani and Dr.P.Sulochana Putli Bai for providing clinical and laboratory support data. Mr.Robinson Peter for technical assistance in performing the laboratory tests.

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