|Year : 2020 | Volume
| Issue : 2 | Page : 152-156
Diagnostic utility of serology and polymerase chain reaction for detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae in paediatric community-acquired lower respiratory tract infections
Sanchit Kumar1, Bineeta Kashyap1, Surinder Kumar1, Seema Kapoor2
1 Department of Microbiology, Maulana Azad Medical College, New Delhi, India
2 Department of Pediatrics, Maulana Azad Medical College, New Delhi, India
|Date of Submission||04-Apr-2020|
|Date of Decision||03-Jun-2020|
|Date of Acceptance||11-Aug-2020|
|Date of Web Publication||29-Aug-2020|
Dr. Surinder Kumar
Director Professor (Ex), Department of Microbiology, Maulana Azad Medical College, New Delhi - 110 002
Source of Support: None, Conflict of Interest: None
Purpose: Mycoplasma pneumoniae (M. pneumoniae) and Chlamydophila pneumoniae (C. pneumoniae) play a significant role in children of all ages with lower respiratory tract infections (LRTIs). This study was conducted to detect M. pneumoniae and C. pneumoniae in children with community-acquired LRTIs employing serology, polymerase chain reaction (PCR) and nested PCR analysis. Material and Methods: This study included 75 children with acute LRTIs for detection of M. pneumoniae and C. pneumoniae. Blood was obtained for M. pneumoniae and C. pneumoniae antibodies and nasopharyngeal aspirates for M. pneumoniae PCR and C. pneumoniae nested PCR. Results: M. pneumoniae infection was positive in 9 (64.21%) children aged 2–6 months and in 5 (35.79%) aged 7 months–12 years, and this difference was statistically significant (P = 0.002). C. pneumoniae infection was comparable within the age group and statistically insignificant (P = 0.43). Clinical and radiological profiles of M. pneumoniae- and C. pneumoniae-positive and negative patients were numerically comparable. Serology and PCR together detected M. pneumoniae infection in 14 (18.6%) children. The sensitivity, specificity and positive and negative predictive values of serology were 77.78%, 92.42%, 58.33% and 96.83%, respectively. C. pneumoniae infection was positive in 11 (14.6%) children by serology and nested PCR with 50% sensitivity, 87.67% specificity, 10% positive predictive value and 98.46% negative predictive value. Conclusions: Our study confirms that M. pneumoniae and C. pneumoniae play a significant role in community-acquired LRTIs and a combination of serology and nested PCR is useful for its diagnosis.
Keywords: Chlamydophila pneumoniae, lower respiratory tract infections, Mycoplasma pneumoniae, polymerase chain reaction, serology
|How to cite this article:|
Kumar S, Kashyap B, Kumar S, Kapoor S. Diagnostic utility of serology and polymerase chain reaction for detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae in paediatric community-acquired lower respiratory tract infections. Indian J Med Microbiol 2020;38:152-6
|How to cite this URL:|
Kumar S, Kashyap B, Kumar S, Kapoor S. Diagnostic utility of serology and polymerase chain reaction for detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae in paediatric community-acquired lower respiratory tract infections. Indian J Med Microbiol [serial online] 2020 [cited 2020 Oct 24];38:152-6. Available from: https://www.ijmm.org/text.asp?2020/38/2/152/293898
| ~ Introduction|| |
Lower respiratory tract infections (LRTIs) are considered a common cause of morbidity and mortality among young children, an overwhelming majority occurring in developing countries. The causative organisms of LRTIs are believed to be mainly respiratory viruses, Streptococcus pneumoniae (S. pneumoniae) and Haemophilus influenzae in young children and S. pneumoniae and Mycoplasma pneumoniae (M. pneumoniae) in older children. M. pneumoniae and Chlamydophila pneumoniae (C. pneumoniae) seem to play a more significant role than previously thought as causes of LRTIs in children of all ages. M. pneumoniae and C. pneumoniae can cause mild, moderate or severe acute respiratory infections. M. pneumoniae clinical manifestations may range from mild cases of tracheobronchitis to severe atypical pneumonia which can be followed by a broad spectrum of extrapulmonary complications. M. pneumoniae can cause as much as 40% of cases of community-acquired pneumonia (CAP) in children over 5 years of age. A specific diagnosis is important because β-lactam antibiotic treatment of infections due to these atypical pathogens is ineffective. Empirical therapy is, therefore, is given in most cases. C. pneumoniae is another agent associated with CAP that can also be involved in pharyngitis, bronchitis and sinusitis.
It is difficult to clinically distinguish M. pneumoniae from C. pneumoniae infections, so specific etiologic diagnoses are established in only a minority of cases. The diagnosis of M. pneumoniae and C. pneumoniae infections relies on culture, serology and polymerase chain reaction (PCR), all of which are clinically impractical. Culture is a very insensitive diagnostic technique, given the fastidiousness of these pathogens. Therefore, serological tests for detection of antibodies in paired serum samples have been considered the standard laboratory diagnostic method. PCR may facilitate the detection of both C. pneumoniae and M. pneumoniae which is a highly sensitive and specific diagnostic technique., The aim of this study was to detect M. pneumoniae and C. pneumoniae in children with community-acquired LRTIs by serological tests, PCR and nested PCR analysis.
| ~ Material and Methods|| |
This prospective study was designed to evaluate the prevalence of M. pneumoniae and C. pneumoniae in community-acquired LRTIs in 75 children aged 2 months to 12 years. The patients enrolled comprised fresh clinically diagnosed cases of acute LRTIs admitted to the paediatric wards of Lok Nayak Hospital and Maulana Azad Medical College, New Delhi, India. LRTI is defined as a child with fever or cough with fast breathing/chest indrawing (fast breathing is present when respiratory rate is ≥60 breaths/min in a child of <2 months, ≥50 breaths/min in a child aged 2–12 months and ≥40 breaths/min in a child aged 12–60 months).
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 and radiological findings suggestive of consolidation, bronchopneumonia or interstitial infiltrates with or without hyperinflation.
Neoplasia, kidney or liver disease, immunodepression, cardiovascular disease and hospital-acquired pneumonia, i.e., pneumonia developing 72 h after hospitalisation or within 7 days of discharge.
Enrolment and evaluation of patients
Written informed consent was taken from the parents/legal guardian in younger children, whereas for the older children, their assent was taken before enrolling them in the study. At the time of admission, a detailed history of the patients was noted, clinical examination performed on all the children and details noted on a predesigned pro forma. After a complete physical examination, chest radiographs were obtained and the findings of the chest radiographs were correlated clinically. Blood specimens (1–2 mL) were collected from children on enrolment for IgM and IgG antibodies to M. pneumoniae and C. pneumoniae and nasopharyngeal aspirates (NPAs) within 24 h of admission before receipt of antibiotics for PCR of M. pneumoniae and nested PCR for C. pneumoniae. Convalescent serum samples were obtained to assay for antibodies to M. pneumoniae and C. pneumoniae after 4–6 weeks of enrolment.
Sera were collected in labelled Eppendorf tubes and stored at −20°C for serological studies. Each serum sample was tested for IgM and IgG antibodies to M. pneumoniae and C. pneumoniae using commercially available ELISA-based kits (Calbiotech Inc., CA, 91978, USA) following manufacturer's instructions.
Polymerase chain reaction for Mycoplasma pneumoniae
DNA extraction from the NPAs was carried out according to centrifugation–proteinase K method. The partially purified DNA was used immediately or stored at −10°C or lower.
A 543 base pair (bp) region of the P1 gene of M. pneumoniae was amplified employing following primers: Mpn 1 CAA GCC AAA CAC GAG CTC CGG CC and Mpn 2 CCA GTG TCA GCT GTT TGT CCT TCC CC. A 50 μL PCR reaction mixture containing DNA extract, 50 mM MgCl2, 0.02% w/v gelatin, 200 mM each dNTP, 0.6 μM of both primers and 2U Taq DNA polymerase was set up.
Positive control with the use of a DNA extract from M. pneumoniae ATCC 29085 and negative PCR control with the use of sterile distilled water instead of DNA were included in each assay. Thirty PCR cycles (PTC-0150 MiniCycler TM MJ Research, USA) were performed with each PCR cycle consisting of 1 min at 94°C, 1 min at 55°C and 2 min at 72°C. PCR products were electrophoresed on 2% ethidium bromide-stained agarose gel, along with 100 bp DNA molecular weight marker. Electrophoresis was carried out at 80 volts for 2 h, and a band at 543 bp was taken to be a positive result.
Nested polymerase chain reaction for Chlamydophila pneumoniae
NPAs were taken up for DNA extraction by QIAamp DNA mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. A touchdown nested PCR for the detection of C. pneumoniae DNA was performed with the use of primers  designed to detect the 333 bp region on major outer membrane protein gene CPl (sense) 5' TTA CAA GCC TTG CCT GTA GG 3' and CP2 (antisense) 5' GCG ATC CCA AAT GTT TAA GGC 3'. Extracted DNA (10 μL in a total volume of 50 μL) was used in the first PCR round consisting of 40 PCR cycles. PCR mixtures consisted of PCR buffer with 0.4 μM each of CP1 and CP2 primers, 200 μM each of dATP, dTTP, dCTP and dGTP and 2 units Taq DNA polymerase. Positive control in PCR included PCR components and purified DNA from Chlamydophila species. Negative control included all PCR components and sterile distilled water instead of DNA extract. PCR amplification was carried out using the technique of touchdown, wherein the annealing temperature was lowered by 1°C for every two cycles starting from 65°C till the temperature touched down to 55°C, and then, at 55°C, 20 more cycles were carried out to complete the PCR. The denaturation and extension temperatures were kept constant at 94°C and 72°C, respectively, for 1 min duration. Ten microlitres of the diluted PCR product amplified by the outer primers was transferred to a new 50-μL PCR reaction mix for a second (nested) amplification using the inner primers CPC (sense) 5' TTA TTA ATT GAT GGT ACA ATA 3' and CPD (antisense) 5' ATC TAC GGC AGT AGT ATA GTT 3' to amplify internal 207 bp region employing 30 PCR cycles at 94°C, 50°C and 72°C, each for 1 min each. Positive control in PCR included PCR components and purified DNA from Chlamydophila species. Negative control included all PCR components and sterile distilled water instead of DNA extract. PCR products were analysed by agarose gel electrophoresis, gel stained with ethidium bromide and visualised under a UV transillumination to observe specific amplified bands of PCR products.
Data analysis was performed using SPSS Inc. Released 2008. SPSS statistics for Windows, Version 17.0, Chicago: SPSS Inc. The difference of proportion between qualitative variables was tested using the Chi-square and Fisher's exact tests. P < 0.05 was considered as statistically significant.
| ~ Results|| |
M. pneumoniae was positive in 9 (64.21%) children aged 2–6 months and in 5 (35.79%) children aged 7 months–12 years, and this difference was statistically significant (P = 0.002). C. pneumoniae was positive in 10 (90.90%) children aged 2–6 months and in 1 (9.10%) child aged 6 months–12 years, and this difference was statistically insignificant (P = 043). M. pneumoniae and C. pneumoniae infections in male and female groups were comparable and were statistically insignificant [Table 1].
|Table 1: Age and sex distribution in children with lower respiratory tract infections|
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No correlation was found between M. pneumoniae- and C. pneumoniae-positive and negative patients with regard to wheezing, crepitations and supracostal retractions/intracostal retractions, and the differences were numerically comparable and statistically insignificant. In radiology, documentation of hyperinflation, interstitial infiltrates, consolidation, pleural effusion, bronchopneumonia and normal X-rays were numerically comparable, and differences were statistically insignificant across M. pneumoniae- and C. pneumoniae-positive and negative patients [Table 2].
|Table 2: Clinical and radiological features in children with lower respiratory tract infections|
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Serological evidence of M. pneumoniae was observed in 12 (16%) patients: specific IgM antibodies alone in 5 (41.66%), both IgM and IgG antibodies in 5 (41.66%) and IgG antibodies in acute and convalescent sera in 2 (16.68%) children. PCR for M. pneumoniae was positive in 7 (9.3%) patients: 5 (71.4%) serologically proven and 2 (28.6%) serologically unproven. Together, serology and PCR detected M. pneumoniae infection in 14 (18.6%) patients with 77.78% sensitivity, 92.42% specificity, 58.33% positive predictive value and 96.83% negative predictive value considering PCR as the diagnostic standard [Table 3].
|Table 3: Sensitivity, specificity and positive and negative predictive values of Mycoplasma pneumoniae serology using polymerase chain reaction as diagnostic standard|
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Serological evidence of C. pneumoniae LRTIs was observed in 10 (13.3%) children: specific IgM antibodies alone in 2 (20%) and specific IgG antibodies in 8 (80%) children in acute and convalescent serum specimens. Touchdown nested PCR for C. pneumoniae was positive in 2 (2.6%) children: 1 (50%) serologically proven and 1 (50%) serologically unproven. Together, serology and touchdown nested PCR detected C. pneumoniae infection in 11 (17.18%) patients. The sensitivity of C. pneumoniae serology was 50%, specificity 87.67%, positive predictive value 10% and negative predictive value 98.46% considering nested PCR as the diagnostic standard [Table 4].
|Table 4: Sensitivity, specificity and positive and negative predictive values of Chlamydophila pneumoniae serology using nested polymerase chain reaction as diagnostic standard|
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| ~ Discussion|| |
M. pneumoniae and C. pneumoniae seem to play a more significant role than previously thought as causes of LRTIs in children of all ages and are increasingly being recognised as prominent causative agents in community-acquired pneumonia worldwide. The present study showed a higher prevalence of M. pneumoniae in children 2–6 months of age group, and this was found to be statistically significant (P = 0.002). This is in agreement with the previous studies which reported M. pneumoniae infection in infants and preschool children with LRTIs  and in contrast to the previous studies which reported a higher rate of M. pneumoniae infections in children >5 years of age and that school-age children 5–15 years of age are the most affected.,
The percentage of M. pneumoniae-positive patients was higher in males than females but showed no statistically significant association between the sex of the patient and the incidence of M. pneumoniae infection (P = 0.53) in agreement to previous findings which have reported the male preponderance.
A relatively higher rate of C. pneumoniae infection was observed in children 2-6 months of age but was statistically insignificant. This is in contrast to previous reports where the prevalence was highest in the school going age group. Seroprevalence studies from. Seroprevalence studies from different parts of the world suggest a low antibody prevalence during childhood, which increases during the teenage years, and relatively high values in middle age and old age. No relationship was observed between sex of the children and C. pneumoniae infection.
Previous studies cast doubt on the specificity of clinical and laboratory features for predicting the microbial cause of LRTIs and emphasising the need for laboratory confirmation.,, In M. pneumoniae LRTIs, radiographic findings can be variable and mimic different lung diseases (viral or bacterial pneumonia). Normal X-rays were more frequent in M. pneumoniae-negative group as compared to M. pneumoniae-positive group which could be due to the fact that, in the M. pneumoniae-negative group, viral LRTIs are more common.,
Radiological features in C. pneumoniae-positive and C. pneumoniae-negative cases were comparable. It has been reported that classic C. pneumoniae respiratory tract infection shows unilateral infiltration. Another study compared radiological features in patients with pneumonia due to M. pneumoniae and C. pneumoniae and cases where aetiology was unknown, and a higher and statistically significant proportion of C. pneumoniae-positive cases showed bilateral patchy features and also infiltration in comparison to the category of unknown aetiology.
A combination of PCR and acute-phase serum immunoglobulin IgM assay is the most sensitive and prompt method for diagnosing M. pneumoniae pneumonia in children., In this study, serological evidence of M. pneumoniae acute infection was observed in 12 (16%) children similar to a study by Kumar et al. IgM antibodies to M. pneumoniae were detected in 40 (20%) patients which is in agreement with an earlier study.
The sensitivity of nucleic acid amplification techniques (NAATs) is almost always superior to that of traditional procedures, and they are more and more thought of 'new gold standard'. PCR for M. pneumoniae was positive in 7 (9.3%): 5 (71.4%) serologically proven and 2 (28.6%) serologically unproven M. pneumoniae infection. M. pneumoniae PCR-positive and serology-negative findings could be due to a false-negative serology despite an active infection, detection of carrier state not manifesting systemic antibody responses or a false-positive PCR result. M. pneumoniae infection was positive in 14 (18.6%) patients with serology and PCR. The sensitivity of M. pneumoniae was 77.78%, specificity 92.42%, positive predictive value 58.33% and negative predictive value 96.83% considering PCR as the diagnostic standard.
Together, serology and nested PCR detected C. pneumoniae infection in 11 (17.18%) patients in agreement with a previous study which reported 13% C. pneumoniae infection using serology and nested PCR. PCR-negative and serology-positive results could be due to some technical factors such as the presence of some inhibitors in the NPA, poor specimen quality or low organism load. The sensitivity of C. pneumoniae serology was 50%, specificity 87.67%, positive predictive value 10% and negative predictive value 98.46% considering nested PCR as the diagnostic standard.
Limitation of this study was time point of the first serum specimen because specific serum IgM antibodies emerge within 1 week after initial infection and about 2 weeks before IgG antibodies. Although IgG paired serum seroconversion and/or rise in antibody titre is a definitive diagnosis of M. pneumoniae/C. pneumoniae infection, a convalescent-phase serum is not always available.
| ~ Conclusions|| |
Serology and PCR in combination is useful for detection of M. pneumoniae and C. pneumoniae in children with community-acquired LRTIs and M. pneumoniae even in children aged <6 months.
We are thankful to the Indian Council of Medical Research (ICMR) for funding this project vide short-term studentship (STS), Reference ID: 2015-04650.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]