Detection of respiratory syncytial virus & Mycoplasma pneumoniae in paediatric lower respiratory tract infections.

Background & objectives: Respiratory syncytial virus (RSV) and Mycoplasma pneumoniae are considered common cause of lower respiratory tract infections (LRTIs) in children. The present study was conducted to detect M. pneumoniae and RSV in paediatric LRTIs employing serology, polymerase chain reaction (PCR) and reverse transcriptase PCR (RT-PCR) analysis.
Methods: Seventy five children aged one month to five years with acute LRTIs were investigated for M. pneumoniae antibodies and RSV antigen using immunochromatographic test, RT-PCR for RSV and M. pneumoniae by PCR on nasopharyngeal aspirates.
Results: RSV infection was observed in 33 (44%) and M. pneumoniae was positive in 26 (35%) children. No significant difference in infection was noted between male and female children. Clinical and radiological features among RSV and M. pneumoniae positive and negative cases were similar. Considering RT-PCR for RSV as gold standard, RSV antigen immunochromatography was 90.90 per cent sensitive and 100 per cent specific. Interpretation & conclusions: Our study showed the presence of RSV and M. pneumoniae infection in 44 and 35 per cent children, respectively with community-acquired LRTIs and aged less than five years.

Lower respiratory tract infections (LRTIs) are known for morbidity and mortality among children12. Viruses are the most common aetiological agents for childhood acute respiratory tract illnesses3. Respiratory syncytial virus (RSV) infection is a major cause of serious lower respiratory disease in infancy and early childhood4. Diagnosis is not possible on clinical grounds alone5. Mycoplasma pneumoniae is a common cause of respiratory tract infections in all age groups. Clinical manifestations range from mild cases of tracheobronchitis to severe atypical pneumonia and can be followed by extrapulmonary complications6. Serological tests are more sensitive than culture for detection of acute M. pneumoniae infection, but polymerase chain reaction (PCR) can detect M. pneumoniae earlier than serology with the potential to produce rapid, sensitive and specific results7.

The aim of this study was to detect RSV by employing chromatographic assay and reverse transcriptase PCR (RT-PCR) and M. pneumoniae by serological tests and PCR analysis in children with community-acquired LRTIs.

Material & Methods

A prospective study was designed to detect RSV and M. pneumoniae in 75 consecutive children aged one month to five years (57 male, 18 female) with community-acquired LRTIs presenting to the department of Pediatrics, Maulana Azad Medical College, New Delhi, India, for a period of two months (July to August 2014). Inclusion criteria were the presence of cough and fever with chest indrawing of <30 days duration, respiratory rate increase (with or without features of respiratory distress) on examination and the presence of signs of consolidation or bronchopneumonia with or without wheeze on auscultation. Exclusion criteria were hospital-acquired pneumonia i, e., pneumonia that developed 72 h after hospitalization or within seven days of discharge.

Blood specimens (1 ml) were collected for enzyme-linked immunosorbent assay (ELISA) for IgM and IgG antibodies to M. pneumoniae (ELISA kit, Calbiotech Inc., CA, USA) and nasopharyngeal aspirates (NPAs) for RSV antigen (Binax NOW RSV Card, Alere, USA), RT-PCR for RSV and PCR for M. pneumoniae on admission before starting antibiotics. Convalescent serum samples were obtained for antibodies to M. pneumoniae 4-6 wk after enrolment. The study protocol was approved by the Institutional Ethics Committee and written informed consent was obtained from parents.

RNA was extracted from NPA specimens using RNeasy Mini Kit 120 (Qiagen GmbH, Hilden, Germany) followed by reverse transcription and PCR amplification to amplify a 287 bp fragment using the primers: 5'-GCAGCAACAATCCAACCTGCTGG-3', 5'-ATCGGAGGAGGTTGAGTGGAGGG-3'8. A negative and positive control was run with each batch of PCR reaction.

DNA for M. pneumoniae PCR was extracted from NPA using proteinase K method9. A 345 bp fragment on P1 gene of M. pneumoniae was amplified employing the following primers1011: P4A 5'-AGGCTCAGGTCAATCTGGCGTGGA-3', P4B 5'-GGATCAAACAGATCGGTGACTGGGT-3'. Negative control included all PCR components] excepting DNA extract which was replaced by addition of sterile distilled water. Positive control included all PCR components and DNA extract from M. pneumonia (ATCC 29342).

Chi-square and the Fischer's exact tests were used for testing the difference of proportion between the qualitative variables.

Results & Discussion

RSV is the most common viral pathogen causing LRTIs in young children1112. In the present study, RSV infection was positive in 20 (60.60%) children aged up to one year and 13 (39.40%) children aged 2-5 yr. No significant difference in RSV positivity was observed between male and female children (Table I). Fattouh et al13 also reported similar findings with 64 per cent RSV-positive patients less than six months of age. The clinical and radiological profiles were similar in RSV-positive and RSV-negative children (Table II) in agreement with a previous study which reported no significant difference among RSV-positive and RSV-negative cases in LRTIs14. Considering RT-PCR as a gold standard, the sensitivity of RSV antigen by immunochromatography was 90.90 per cent, specificity 100 per cent, positive predictive value 100 per cent and negative predictive value 93.3 per cent.

Table I

Age and sex distribution in respiratory syncytial virus (RSV) and Mycoplasma pneumonia infections in children with lower respiratory tract infections

Characteristics	Total (n=75), n (%)	RSV		M. pneumoniae
		Positive (n=33), n (%)	Negative (n=42), n (%)	Positive (n=26), n (%)	Negative (n=49), n (%)
Age
1 month - 1 year	45 (60)	20 (60.60)	25 (59.52)	15 (57.69)	30 (61.22)
2-5 year	30 (40)	13 (39.39)	17 (40.47)	11 (42.30)	19 (38.77)
Sex
Male	57 (76)	23 (69.69)	34 (80.95)	18 (69.23)	39 (79.59)
Female	18 (24)	10 (30.30)	8 (19.04)	8 (30.76)	10 (20.40)

Table II

Clinical and radiological findings in respiratory syncytial virus (RSV) and Mycoplasma pneumoniae infection in children with lower respiratory tract infections

Clinical findings	RSV		M. pneumoniae		Total (n=75), n (%)
	Positive (n=33), n (%)	Negative (n=42), n (%)	Positive (n=26), n (%)	Negative (n=49), n (%)
Wheeze	20 (60.60)	30 (71.42)	18 (69.23)	32 (65.30)	50 (66.66)
Rhonchi	22 (66.67)	33 (78.57)	20 (76.92)	35 (71.42)	55 (73.33)
Crepitations	19 (52.57)	32 (76.19)	17 (65.38)	34 (69.38)	51 (68)
Radiological profile
Bronchopneumonia	3 (9.09)	3 (7.14)	2 (7.69)	4 (8.16)	6 (8)
Interstitial infiltrates	4 (12.12)	6 (14.28)	8 (30.76)	14 (28.57)	10 (13.33)
Consolidation	5 (15.15)	6 (14.28)	4 (15.38)	7 (14.28)	11 (14.67)
Hyperinflation	8 (24.24)	12 (28.57)	7 (26.92)	13 (26.53)	20 (26.67)
Pleural effusion with collapse	5 (15.15)	5 (11.90)	4 (15.38)	6 (12.24)	10 (13.33)
Within normal limits	8 (24.24)	10 (23.80)	6 (23.07)	12 (24.48)	18 (24)

M. pneumoniae infection was documented in 15 (57.69%) children aged up to one year and in 11 (42.4%) children aged 2-5 yr; the difference was insignificant. The difference in the presence of M. pneumoniae among male and female children with LRTIs was insignificant (Table I). Kashyap et al15 reported no association between sex of the patient and the incidence of M. pneumoniae infection. Clinical and radiological profiles across M. pneumoniae positive and negative children were comparable (Table II). An earlier report has cast doubts on the specificity of clinical and radiological features in predicting the microbial cause of LRTIs16. Serological evidence of M. pneumoniae infection was observed in 24 (32%) children. M. pneumoniae PCR was positive in eight (10.66%) patients - six with serologically proven and two serological unproven for M. pneumoniae infections. Kumar et al16 reported 10 per cent PCR positivity in children with acute LRTIs. Together, serology and PCR detected M. pneumoniae in 26 (34.66%) children in concordance with earlier studies which reported 34 and 30 per cent M. pneumoniae infection in children1617. Considering PCR as a diagnostic standard, serology sensitivity was 75 per cent, specificity 73.3 per cent, positive predictive value 25 per cent and a negative predictive value 96 per cent.

In conclusion, our study showed the presence of RSV and M. pneumoniae infection in community-acquired LRTIs in children aged less than five years.