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Atypical Bacterial Pathogen Infection in Children with Acute Bronchiolitis in Northeast Thailand

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Atypical Bacterial Pathogen Infection in Children with Acute Bronchiolitis in Northeast Thailand Journal of Microbiology, Immunology and Infection (2011) 44,95e100 available at www.sciencedirect.com journal homepage: www.e-jmii.com ORIGINAL ARTICLE Atypical bacterial pathogen infection in children with acute bronchiolitis in northeast Thailand Chamsai Pientong a,*, Tipaya Ekalaksananan a, Jamree Teeratakulpisarn b, Sureeporn Tanuwattanachai c, Bunkerd Kongyingyoes d, Chulaporn Limwattananon e a Department of Microbiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand b Department of Pediatrics, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand c Pediatrics Unit, Khon Kaen Hospital, Khon Kaen, Thailand d Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand e Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand Received 1 October 2009; received in revised form 10 November 2009; accepted 11 February 2010 KEYWORDS Background: Atypical bacterial pathogensdincluding Mycoplasma pneumoniae, Chlamydophi- Bronchiolitis; la pneumoniae, and Chlamydia trachomatisdare important infectious agents of the respira- Chlamydia tory system. Most current information pertains to adults and little is known about the role trachomatis; of these organisms in lower respiratory tract infections among young children with acute Chlamydophila bronchiolitis. pneumoniae; Methods: This study detected these pathogens in the nasopharyngeal secretions of children Mycoplasma between 1 month and 2 years of age admitted with acute bronchiolitis to hospitals in Khon pneumoniae Kaen, northeast Thailand. The M pneumoniae and C pneumoniae in the nasopharyngeal secre- tions were detected using multiplex and nested-polymerase chain reaction (PCR), whereas PCR and restriction fragment length polymorphism were used to investigate C trachomatis. These samples were also tested by multiplex reverse transcriptase PCR for respiratory viruses, including respiratory syncytial virus (RSV), influenza A, influenza B, and human metapneumo- virus. Results: Of the 170 samples taken from hospitalized children with acute bronchiolitis, 12.9% were infected with atypical bacteria and 85.3% with respiratory viruses. RSV was the most common causative viral agents found in 64.7% of the samples. Mpneumoniaewas the most common atypical bacterial pathogen (14/170, 8.2%) and most of the patients infected with it were between 6 and less than 12 months of age (71 cases). Of the infected cases in this age group, 7 of 14 were infected with Mpneumoniaeand 4 of 4 with Cpneumoniae.Both * Corresponding author. Department of Microbiology, Faculty of Medicine, Khon Kaen University, Ambhur Muang, Khon Kaen 40002, Thailand. E-mail address: [email protected] (C. Pientong). 1684-1182/$36 Copyright ª 2011, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. All rights reserved. doi:10.1016/j.jmii.2010.02.001 96 C. Pientong et al. M pneumoniae (13/14) and Cpneumoniae(4/4) had etiologies indicating viral coinfections. Four (2.4%) of all of the cases had C trachomatis infections and all of these were infected with RSV, including three patients less than 6 months of age. Conclusion: These results suggest that in children with virus-induced acute bronchiolitis coin- fection with M pneumoniae, C pneumoniae,orC trachomatis can be expressed differently in each age group. These atypical bacteria may be the important infectious agents that induce severe illness of acute bronchiolitis. Copyright ª 2011, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. All rights reserved. Introduction culture techniques or a long period of incubation before their presence can be confirmed or excluded. The inter- Acute bronchiolitis is the most common lower respiratory pretation of serological tests used for C pneumoniae, tract infection and the most frequent cause of hospitali- C trachomatis, and M pneumoniae diagnoses is also prob- lematic because a large proportion of the population has zation in young children. The most common pathogen is 9 respiratory syncytial virus (RSV), which occurs as a yearly preexisting IgG antibodies from prior exposure(s) ; there- winter epidemic with various symptoms, ranging from mild fore, diagnosis of infections caused by these organisms is upper respiratory tract infection to severe bronchiolitis usually confirmed with the polymerase chain reaction (PCR) with hyperinflated lungs and hypoxemia.1 technique. The atypical bacterial pathogensdMycoplasma pneu- To our knowledge, there are few reports about atypical moniae, Chlamydophila pneumoniae, and Chlamydia tra- bacterial pathogen infection in children with acute bron- chomatisdare recognized as respiratory pathogens. They chiolitis, especially in northeast Thailand. We, therefore, are all small bacteria and cannot be detected using routine conducted our study using a PCR technique to estimate the culturing methods. prevalence of C pneumoniae, C trachomatis, and M pneu- M pneumoniae is a well-known childhood pathogen and moniae infections in pediatric patients with acute bron- is highly transmissible. Most infections caused by this chiolitis admitted to either of two hospitals in Khon Kaen organism are relatively minor (including pharyngitis, tra- province, Thailand. cheobronchitis, bronchiolitis, and croup) with one-fifth being asymptomatic. Acute infections with this organism Methods may promote the exacerbation of asthmatic symptoms and may be accompanied by wheezing in children not having Subjects and sample collection asthma.2 C pneumoniae was also reported in acute lower respi- This study was approved by the Khon Kaen University Ethics ratory infection with mild dyspnea and wheezing in the Committee for Human Research, as per the Helsinki pediatric population.3 Acute C pneumoniae infections have Declaration. This study was part of a randomized, clinical been associated with the presence and/or exacerbation of trial evaluating the efficacy of dexamethasone for the asthma in children.4 treatment of acute bronchiolitis (registered in C trachomatis is transmitted by infected women to their ClinicalTrials.gov identification number: NCT00122785). infants at birth via contact with infected cervicovaginal Between April 2002 and August 2004, children with acute secretion. If the infection is not detected and treated, such bronchiolitis hospitalized at either of two hospitals in Khon infected infants may develop conjunctivitis, bronchiolitis, Kaen, northeast Thailand, were recruited with informed, and pneumonia.5 It should therefore not be ruled out in written, parental consent. Acute bronchiolitis was defined as infants less than 6 months of age with clinical symptoms of the first episode of wheezing associated with tachypnea, lower respiratory tract disease for which no other pathogen increased respiratory effort, and an upper respiratory tract can be found. infection. Patients were eligible for the study if they met the One of the main difficulties in dealing with lower respi- following criteria: being between 4 weeks and 24 months of ratory tract infections in pediatric patients is correctly age and being admitted with their first episode of wheezing identifying the infecting agent. Culture, antigen screening, within 7 days. Patients were excluded from the study if they and serological methods are only helpful in about one of had (1) a known history of asthma; (2) a previous history of three cases.6 Chlamydia and mycoplasma are common intubation; (3) a history of bronchopulmonary dysplasia or bacteria that may cause rhinitis, pharyngitis, bronchitis, chronic lung disease; (4) an underlying congenital heart and pneumonia,7 and bronchiolitis may predispose some disease or any immunodeficiency; or (5) been on any steroid infants to developing childhood asthma or asthma exacer- treatment within the previous 2 weeks. bation. Despite their ubiquity, chlamydia and mycoplasma On admission, a sample of nasopharyngeal secretion was are among the least frequently diagnosed respiratory taken from each child’s nostril by a previously described pathogens in the clinical setting mainly because of the lack technique.10 The secretion obtained was immediately put of standardized, rapid, and specific diagnostic tests.8 into a tube containing viral transport media and then was Chlamydia and mycoplasma are fastidious and difficult sent to a laboratory for processing. Aliquots of samples to grow in culture, so they require either specialized cell were stored at À70C until used. Atypical bacteria in acute bronchiolitis 97 Atypical bacteria study Madison, WI, USA). Digestion was performed by incubating 10 mL of PCR product with 1 U of enzyme, 2 mLof10Â buffer, DNA extraction and 7 mL of water for 1 hour at 37 C. The PCR products were DNA was extracted from a 200-mL aliquot of sample using analyzed using electrophoresis on a 4% agarose gel, stained a PUREGENE DNA purification kit (Gentra Systems, Minne- with ethidium bromide, and compared with the predicted apolis, MN, USA) as per the manufacturer’s instructions. fragments. For Ctrachomatis, the fragment lengths of 158, The integrity of the DNA was confirmed by amplification of 119 and 114, and 84 and 77 bp were accurately predicted. a housekeeping gene (b-globin) from the DNA samples, using the PC04/GH20 primers (Invitrogen Life Technologies, Virus study Carlsbad, CA, USA). RNA extraction Multiplex and nested PCRs for detection of M pneumoniae An aliquot of the same samples was also detected for and C pneumoniae respiratory viruses, including RSV, influenza A, influenza B, Multiplex PCR was used for simultaneous detection
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