Browse > Article
http://dx.doi.org/10.14776/piv.2015.22.2.106

Molecular Diagnosis of Streptococcus pneumoniae in Middle Ear Fluids from Children with Otitis Media with Effusion  

Byun, Sung Wan (Department of Otolaryngology-Head and Neck Surgery, Medical Research Institute, Ewha Womans University School of Medicine)
Kim, Han Wool (Department of Pediatrics, Medical Research Institute, Ewha Womans University School of Medicine)
Yoon, Seo Hee (Department of Pediatrics, Medical Research Institute, Ewha Womans University School of Medicine)
Park, In Ho (Center for Vaccine Evaluation and Study, Medical Research Institute, Ewha Womans University School of Medicine)
Kim, Kyung-Hyo (Department of Pediatrics, Medical Research Institute, Ewha Womans University School of Medicine)
Publication Information
Pediatric Infection and Vaccine / v.22, no.2, 2015 , pp. 106-112 More about this Journal
Abstract
Purpose: The long-term administration of antibiotics interferes with bacterial culture in the middle ear fluids (MEFs) of young children with otitis media with effusion (OME). The purpose of this study is to determine whether molecular diagnostics can be used for rapid and direct detection of the bacterial pathogen in culture-negative MEFs. Methods: The specificity and sensitivity of both polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to the lytA gene of Streptococcus pneumoniae were comparatively tested and then applied for pneumococcal detection in the clinical MEFs. Results: The detection limit of the PCR assay was approximately $10^4$ colony forming units (CFU), whereas that of LAMP was less than 10 CFU for the detection of S. pneumoniae. Both PCR and LAMP did not amplify nucleic acid at over $10^6$ CFU of H. influenzae or M. catarrhalis, both of which were irrelevant bacterial species. Of 22 culture-negative MEFs from children with OME, LAMP positivity was found in twelve MEFs (54.5%, 12/22), only three of which were PCR-positive (25%, 3/12). Our results showed that the ability of LAMP to detect pneumococcal DNA is over four times higher than that of PCR (P<0.01). Conclusions: As a high-resolution tool able to detect nucleic acid levels equivalent to <10 CFU of S. pneumoniae in MEFs without any cross-reaction with other pathogens, lytA -specific LAMP may be applied for diagnosing pneumococcus infection in OME as well as evaluating the impact of a pneumococcal conjugate vaccine against OME.
Keywords
Streptococcus pneumoniae; Otitis media with effusion (OME); Loop-mediated isothermal amplification (LAMP); Polymerase chain reaction (PCR); Molecular diagnosis;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Howie VM, Ploussard JH, Lester RL, Jr. Otitis media: a clinical and bacteriological correlation. Pediatrics 1970;45:29-35.
2 Luotonen J, Herva E, Karma P, Timonen M, Leinonen M, Makela PH. The bacteriology of acute otitis media in children with special reference to Streptococcus pneumoniae as studied by bacteriological and antigen detection methods. Scand J Infect Dis 1981;13:177-83.   DOI
3 Bluestone CD, Stephenson JS, Martin LM. Ten-year review of otitis media pathogens. Pediatr Infect Dis J 1992;11:S7-11.   DOI
4 Kilpi T, Herva E, Kaijalainen T, Syrjanen R, Takala AK. Bacteriology of acute otitis media in a cohort of Finnish children followed for the first two years of life. Pediatr Infect Dis J 2001;20:654-62.   DOI
5 Qvarnberg Y, Holopainen E, Palva T. Aspiration cytology in acute otitis media. Acta Otolaryngol 1984;97:443-9.   DOI
6 Rosenfeld RM. An evidence-based approach to treating otitis media. Pediatr Clin North Am 1996;43:1165-81.   DOI
7 Rosenfeld RM, Schwartz SR, Pynnonen MA, Tunkel DE, Hussey HM, Fichera JS, et al. Clinical practice guideline: Tympanostomy tubes in children. Otolaryngol Head Neck Surg 2013;149:S1-35.
8 Saleem M, Naz M, Waris A, Muneer B, Khurshid R. Screening of pneumococcal pneumonia by amplification of pneumolysin gene in children visiting hospitals in lahore, pakistan. Iranian J Pediatr 2012;22:524-30.
9 McAvin JC, Reilly PA, Roudabush RM, Barnes WJ, Salmen A, Jackson GW, et al. Sensitive and specific method for rapid identification of Streptococcus pneumoniae using real-time fluorescence PCR. J Clin Microbiol 2001;39:3446-51.   DOI
10 Morrison KE, Lake D, Crook J, Carlone GM, Ades E, Facklam R, et al. Confirmation of pspA in all 90 serotypes of Streptococ cus pneumoniae by PCR and potential of this assay for identification and diagnosis. J Clin Microbiol 2000;38:434-7.
11 Rintamaki S, Saukkoriipi A, Salo P, Takala A, Leinonen M. Detection of Streptococcus pneumoniae DNA by using polymerase chain reaction and microwell hybridization with Europium-labelled probes. J Clin Microbiol 2002;50:313-8.
12 Kim DW, Kilgore PE, Kim EJ, Kim SA, Anh DD, Dong BQ, et al. The enhanced pneumococcal LAMP assay: a clinical tool for the diagnosis of meningitis due to Streptococcus pneumoniae . PLoS One. 2012;7:e42954.   DOI
13 Hoppe JE, Grieshaber, Höfler W. Colonization of Nigerian neonates with group B streptococci and its rapid detection. Infection 1986;14:74-8.   DOI
14 Prattes J, Koidl C, Eigl S, Krause R, Hoenigl M. Bronchoalveolar lavage fluid sample pretreatment with Sputasol (R) significantly reduces galactomannan levels. J Infect 2015;70:541-3.   DOI
15 Nagai K, Shibasaki Y, Hasegawa K, Davies TA, Jacobs MR, Ubukata K, et al. Evaluation of PCR primers to screen for Streptococcus pneumoniae isolates and beta-lactam resistance, and to detect common macrolide resistance determinants. J Antimicrob Chemother 2001;48:915-8.   DOI
16 Black S, Shinefield HR, Fireman B, Lewis E, Ray P, Hansen JR, et al. Efficacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Pediatr Infect Dis J 2000;19:187-95.   DOI
17 Eskola J, Kilpi T, Palmu A, Jokinen J, Haapakoski J, Herva E, et al. Efficacy of a pneumococcal conjugate vaccine against acute otitis media. N Eng J Med 2001;344:403-9.   DOI
18 Berman S. Otitis media in children. N Eng J Med 1995;332:1560-5.   DOI
19 Tettelin H, Nelson KE, Paulsen IT, Eisen JA, Read TD, Peterson S, et al. Complete genome sequence of a virulent isolate of Streptococcus pneumoniae . Science 2001;293:498-506.   DOI
20 Park IH, Kim KH, Andrade AL, Briles DE, McDaniel LS, Nahm MH. Nontypeable pneumococci can be divided into multiple cps types, including one type expressing the novel gene pspK. mBio 2012;3:e00035-12.
21 Approved lists of bacterial names. Med J Aust 1980;2:3-4.
22 Ankerst J, Christensen P, Kjellen L, Kronvall G. A rountine diagnostic test for IgA and IgM antibodies to rubella virus: absorption of IgG with Staphylococcus aureus. J Infect Dis 1974;130:268-73.   DOI
23 Crisel RM, Baker RS, Dorman DE. Capsular polymer of Haemophilus influenzae , type b. I. Structural characterization of the capsular polymer of strain Eagan. J Biol Chem 1975;250:4926-30.