| Abstract|| |
Context: Chlamydophila pneumoniae is a common cause of community-acquired respiratory infections, including pneumonia, bronchitis, and upper respiratory tract infections. Since it is difficult to detect C. pneumoniae in clinical practice, specific etiological diagnosis is established only in a minority of cases. Aims: To investigate the role of C. pneumoniae in community-acquired lower respiratory tract infections (LRTIs) in children, with the use of serological tests and nested polymerase chain reaction (PCR) analysis. Settings and Design: One hundred children, age of 2 months to 12 years, hospitalized for community-acquired LRTIs were investigated for C. pneumoniae etiology. Materials and Methods: We investigated 100 children hospitalized for community-acquired LRTIs, using enzyme-linked immunosorbent assay for detecting anti-C. pneumoniae immunoglobulin M, and immunoglobulin G antibodies and nasopharyngeal aspirates for analysis of C. pneumoniae PCR. The demographic, clinical, and radiological findings for C. pneumoniae antibody positive and C. pneumoniae antibody negative cases were compared. Statistical Analysis Used: Data analysis was performed by Chi-square test and Fisher's exact tests using Epi Info (2002). Results: Clinical and radiological findings in both the groups were comparable. A relatively higher rate of C. pneumoniae infection in children was observed below 5 years of age. Serological evidence of C. pneumoniae infection was observed in 12 (12%) patients and nested PCR was positive in 5 (5%) children. Thirteen (13%) patients were diagnosed with C. pneumoniae infection by serology and/or nested PCR. Conclusions: Our study confirms that C. pneumoniae plays a significant role in community-acquired LRTIs in children of all ages, even in children aged <5 years.
Keywords: Chlamydophila pneumoniae, polymerase chain reaction, serology
|How to cite this article:|
Kumar S, Saigal SR, Sethi GR, Kumar S. Application of serology and nested polymerase chain reaction for identifying Chlamydophila pneumoniae in community-acquired lower respiratory tract infections in children. Indian J Pathol Microbiol 2016;59:499-503
|How to cite this URL:|
Kumar S, Saigal SR, Sethi GR, Kumar S. Application of serology and nested polymerase chain reaction for identifying Chlamydophila pneumoniae in community-acquired lower respiratory tract infections in children. Indian J Pathol Microbiol [serial online] 2016 [cited 2019 Jul 17];59:499-503. Available from: http://www.ijpmonline.org/text.asp?2016/59/4/499/191803
| Introduction|| |
Lower respiratory tract infections (LRTIs) are 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 and Haemophilus influenzae in young children and S. pneumoniae and Mycoplasma pneumoniae in older children. Respiratory infection with Chlamydophila pneumoniae occurs worldwide and in all age groups. Few data are available with regard to C. pneumoniae in pediatric age groups. Some studies have recently shown that M. pneumoniae and C. pneumoniae seem to play a more significant role than previously thought as causes of LRTIs in children of all ages. 
C. pneumoniae is a common cause of community-acquired respiratory infections, including pneumonia, bronchitis, and upper respiratory tract infections  and is held responsible in 6-20% of all community-acquired pneumonias. ,,,, Laboratory tests to detect Chlamydophila infections include the isolation in cell culture, direct detection of antigen by direct fluorescent antibody, enzyme-linked immunosorbent assay (ELISA), by DNA-RNA probes, and polymerase chain reaction (PCR). Isolation of C. pneumoniae by cell culture is difficult and its sensitivity is unknown. The microimmunofluorescence test could detect C. pneumoniae-specific antibodies, but the immune response against acute infection develops 2-4 weeks and 6-8 weeks after the first infection, as detected by immunoglobulin M (IgM) and immunoglobulin (IgG) respectively, or IgG titer rises 1-3 weeks after reinfection. 
Since it is difficult to detect C. pneumoniae in clinical practice, specific etiological diagnosis is established only in a minority of cases. The detection of antibodies in paired serum samples has been considered the standard laboratory diagnostic method, but PCR is considered to be a more reliable diagnostic method than other techniques for the laboratory diagnosis of C. pneumoniae. ,
The aim of this study was to investigate the role of C. pneumoniae in community-acquired LRTIs in Indian children, with the use of both serological tests and nested PCR analysis.
| Materials and Methods|| |
This was a prospective, single-center study designed to evaluate the incidence of C. pneumoniae infection in hospitalized children with community-acquired LRTIs and the sampling strategy and sample size estimation methodology adopted was based on prevailing estimated data on the prevalence of this infection and statistical considerations.
A total of 100 patients were enrolled comprising newly clinically diagnosed cases of acute LRTIs admitted to the pediatrics units of Lok Nayak Hospital and Maulana Azad Medical College, New Delhi, India, from 2009 to 2012. Protocols employed in this study were approved by the Ethical Committee of Maulana Azad Medical College and associated hospitals, New Delhi, India, and the work undertaken conformed to the provisions of the Declaration of Helsinki in 1995 (as revised in Edinburgh 2000).
Selection of cases
Previously healthy male and female children who had been hospitalized for signs and symptoms of community-acquired LRTIs and fulfilling the following criteria were included in this study: children aged 2 months to 12 years; 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; radiological findings suggestive of consolidation; bronchopneumonia or interstitial infiltrates with or without hyperinflation.
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 was taken from the parents or legal guardian of infants (aged 2 months to 1 year) and older children (aged 13 months to 12 years) before enrolling them in the study and collecting clinical specimens, in conformation with standard bioethical norms. Detailed history and clinical examination was performed on admission, and chest X-rays were conducted in all children, the details of which were noted in a redesigned proforma. The laboratory samples taken at enrollment included venous blood specimens (1-2 ml) for serum to assay for antibodies to C. pneumoniae and nasopharyngeal aspirates for the detection of C. pneumoniae DNA. A second serum sample was obtained after 4-6 weeks to assay for convalescent C. pneumoniae antibody titers.
The serum samples were stored at −20 0 C and later each sample was tested for IgM and IgG antibodies to C. pneumoniae by commercially available ELISA-based kits (Calbiotech Inc., CA, 91978, USA). Tests were performed and results interpreted according to manufacturer's instructions.
Nested polymerase chain reaction for Chlamydophila pneumoniae
The nasopharyngeal aspirate specimens were collected and transported to the laboratory within an hour of collection at 4°C and if viscous, was diluted with normal saline, vortexed by agitation for homogenization and centrifuged at 8000 revolutions per minute and stored at −20°C later for nested PCR analysis.
Polymerase chain reaction amplification and gel electrophoresis
Nasopharyngeal aspirates were taken up for DNA extraction by Qiamp 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 major outer membrane protein gene (CPl (sense) 5' TTA CAA GCC TTG CCT GTA GG 3'; CP2 (anti-sense) 5' GCG ATC CCA AAT GTT TAA GGC 3').
Extracted DNA solution (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 of Taq DNA polymerase. 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 μL of the diluted PCR product amplified by the outer primers was transferred to a new 50-μL PCR reaction mix for a second amplification using the inner primers CPC (sense) 5' TTA TTA ATT GAT GGT ACA ATA 3'; CPD (anti-sense) 5' ATC TAC GGC AGT AGT ATA GTT 3') to amplify internal 207 base pair region employing 30 PCR cycles at 94°C, 50°C, 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 analyzed by agarose gel electrophoresis, gel stained with ethidium bromide and visualized under a UV transillumination to observe specific amplified bands of PCR products.
Data analysis was performed using Epi Info 2002. Chi-square test and Fisher's exact test were applied to determine any difference between the proportions. P < 0.05 was considered as statistically significant.
| Results|| |
This study included 72 (72%) males and 28 (28%) females in the 100 cases investigated. The presence of C. pneumoniae positive children was higher in 10 (76.9%) males than in 3 (23.1%) females though this difference was statistically insignificant (P = 0.6) [Table 1]. The differential presence of C. pneumoniae in LRTIs across age groups 2-6 months (Group 1), aged 7-12 months (Group 2), aged 13 months to 2 years (Group 3), aged 25-60 months (Group 4), and age 6-12 years (Group 5) was statistically insignificant (P = 0.7, P = 0.05, P = 0.7, P = 0.8, and P = 0.13, respectively) [Table 2].
|Table 1: Sex distribution in 100 children with lower respiratory tract infections|
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All the clinical characteristics at baseline were comparable between C. pneumoniae positive and C. pneumoniae negative cases. Fever, cough, coryza, and tachypnea were present in all C. pneumoniae positive and C. pneumoniae negative cases [Table 3]. No correlation was found between C. pneumoniae positive and C. pneumoniae negative cases with past history, wheezing, crepitations, supracostal retraction/intracostal retraction, central vascular system/central nervous system abnormalities, and differences were statistically insignificant in both groups [Table 3].
|Table 3: Clinical findings in 13 Chlamydophila pneumoniae positive children with lower respiratory tract infections|
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[Table 4] summarizes the radiological profile in C. pneumoniae positive and in C. pneumoniae negative associated LRTIs. Documentation of hyperinflation, infiltration, consolidation, collapse bronchopneumonia, and normal chest X-ray was numerically comparable and differences were statistically insignificant in C. pneumoniae positive and C. pneumoniae negative categories [Table 4].
|Table 4: Radiological findings in children with lower respiratory tract infections|
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Chlamydophila pneumoniae serology
Serological evidence of C. pneumoniae infection was observed in 12 (12%) children; specific IgM antibodies in acute phase were detected in 10 (83.33%) patients; specific IgG antibodies in both acute and paired sera in 2 (16.67%) childern. A 4-fold rise in IgG titer in a convalescent serum specimen in comparison to that in acute phase serum was considered as seroconversion for C. pneumoniae in LRTIs.
Nested polymerase chain reaction for Chlamydophila pneumoniae
C. pneumoniae DNA was detected in 5 (5%) patients; 4 (80%) in serologically proven C. pneumoniae; and 1 (20%) in serologically unproven C. pneumoniae LRTIs. Together, nested PCR and/or ELISA detected C. pneumoniae in 13 (13%) children.
| Discussion|| |
C. pneumoniae has recently been recognized as a common and important intracellular bacterium, implicated in upper and LRTIs in humans worldwide. The isolation of the organism is difficult because of poor growth in culture.  The detection of C. pneumoniae by molecular biology-based amplification methods has recently been introduced as a diagnostic aid but most reports are still based on serology. , Respiratory tract infection related to C. pneumoniae is typically an endemic disease, but epidemics have also been described among military recruits. Antibody prevalence studies have shown that almost everybody is infected with C. pneumoniae at some time during their lifetime and that reinfection is common.
A majority of 89 (89%) cases investigated were aged <5 years, whereas 73 (73%) were aged <1 year. Relationship between age and sex group, and the presence of C. pneumoniae positive across the five age groups in children were statistically insignificant. A relatively higher rate of infection in children below 5 years of age though was in minor contrast to western reports where the prevalence of C. pneumoniae antibodies was highest in the school going age group and adolescents with acute respiratory disease. , 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. 
In the present study, wheezing was observed in significant 6 (46.15%) C. pneumoniae positive cases. C. pneumoniae infection has been known to trigger acute episodes of wheezing in asthma.  It is likely that M. pneumoniae and C. pneumoniae trigger the wheezing process or act as cofactors in genetically predisposed subjects.  None of the signs, symptoms, or laboratory parameters for the diagnostic tests for C. pneumoniae considered in this study seem to be unique to C. pneumoniae infections which suggests that they are not useful in therapeutic decision-making,  which is in line with other data casting doubt on the sensitivity and specificity of clinical and laboratory features in predicting the etiology of community-acquired pneumonia in children, emphasizing the need for effective laboratory diagnosis.
Literature reports reveal that classic C. pneumoniae respiratory tract infection shows unilateral infiltration.  In other study  which compared radiological features in patients with pneumonia due to M. pneumoniae, C. pneumoniae and cases where etiology was unknown, 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 etiology. In contrast to findings of these studies, radiological features in C. pneumoniae positive and C. pneumoniae negative cases were comparable, and no feature was significantly associated with positive C. pneumoniae infection in our study.
Overall, 12 (12%) children documented serological evidence of C. pneumoniae infection, spreading across age groups in children, which is in line with earlier reports, though the methods of diagnosis of this infection have been disproportionately variable. , Indian studies are scattered, conducted on small population and have employed different methods of diagnosis of this infection.  In a recent Indian study in 200 children, age 2 months to 12 years, serological evidence of C. pneumoniae infection was observed in 12 (6%) patients; specific IgM antibodies were detected in 11 (91.67%; specific IgG antibodies in acute and paired sera in 1 (8.33%) child.  Serological testing of C. pneumoniae remains problematical because of difficulties in obtaining paired serum samples, discrepancies in the antibody values of a healthy population from different geographical locations, and the lack of standardized tests and reagents. It is, therefore, of crucial importance to establish a background for the particular population, which a laboratory intends to diagnose.
PCR and serological tests shed light on the present LRTIs caused by C. pneumoniae. Nested PCR is, in general, more sensitive than is single-step PCR because of the two-step amplification and the use of two sets of primers. Though in house PCR methods are available for the detection of C. pneumoniae, the sensitivity and specificity of the majority of these tests remain unknown. In this study, C. pneumoniae nested PCR on nasopharyngeal aspirates was positive in 5 (5%) patients; 4 (80%) in serologically proven C. pneumoniae; and 1 (20%) in serologically unproven C. pneumoniae LRTIs. PCR negative and serology positive results could be due to some technical factors such as the presence of some inhibitors in the nasopharyngeal aspirate, poor specimen quality, or low organism load.  Together PCR and/or ELISA detected C. pneumoniae in 13 (13%) cases; nested PCR could increase detection in patients who have begun antibiotic therapy before diagnosis in serologically negative cases.
| Conclusion|| |
Our study confirms that C. pneumoniae plays a significant role in community-acquired LRTIs in Indian children of all ages, even in children aged <5 years and more particularly those aged <1 year.
We are thankful to the Indian Council of Medical Research, New Delhi - 110 029, India, for financial help for this research scheme vide Letter No. 5/3/3/4/2007-ECD-1, dated 27-08-2009.
Financial support and sponsorship
Indian Council of Medical Research, New Delhi, India.
Conflicts of interest
There are no conflicts of interest.
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Director Professor, Department of Microbiology, Maulana Azad Medical College, New Delhi - 110 002
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]