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Byun, Kim, Yoon, Park, and Kim: Molecular Diagnosis of Streptococcus pneumoniae in Middle Ear Fluids from Children with Otitis Media with Effusion
Original Article

Pediatr Infect Vaccine 2015;22(2):106-112.

Published online: 21 January 2015

DOI: https://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

Sung Wan Byun1, Han Wool Kim2, 3, Seo Hee Yoon2, 3, In Ho Park3, Kyung-Hyo Kim2, 3,

1Department of Otolaryngology—Head and Neck Surgery, Medical Research Institute, Ewha Womans University School of Medicine, Seoul, Korea

2Department of Pediatrics, Medical Research Institute, Ewha Womans University School of Medicine, Seoul, Korea

3Center for Vaccine Evaluation and Study, Medical Research Institute, Ewha Womans University School of Medicine, Seoul, Korea

Correspondence: Kyung-Hyo Kim Department of Pediatrics, Ewha Womans Univerisity School of Medicine, Seoul, Republic of Korea Tel: +82-02-2650-57O0 Fax: +82-2-2650-2817 EimaH: kaykim@ewha.ac.kr

Received 19 March 201530 June 2015       Accepted 1 July 2015

Copyright © 2015 The Korean Society of Pediatric Infectious Diseases

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

The longterm administration of antibiotics interferes with bacterial culture in the middle ear fluids (MEFs) ofyoung 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 0f 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 104 colony forming units (CFU), whereas that of LAMP was less than 10 CFU for the detection of s. pneumon/ae. Both PCR and LAMP did not amplify nucleic acid at over 106 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 pneumo—coccal 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

REFERENCES

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.
crossref
3. Bluestone CD, Stephenson JS, Martin LM. Ten-year review of otitis media pathogens. Pediatr Infect Dis J. 1992; 11:S7–11.
crossref
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.
crossref
5. Qvarnberg Y, Holopainen E, Palva T. Aspiration cytology in acute otitis media. Acta Otolaryngol. 1984; 97:443–9.
crossref
6. Rosenfeld RM. An evidence—based approach to treating otitis media. Pediatr Clin North Am. 1996; 43:1165–81.
crossref
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, Iackson GW, et al. Sensitive and specific method for rapid identification of Streptococcus pneumoniae using realtime fluorescence PCR. J Clin Microbiol. 2001; 39:3446–51.
10. Morrison KE, Lake D, Crook J, Carlene GM, Ades E, Facklam R, et al. Confirmation of pspA in all 90 serotypes of Streptococcus 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 DVV, 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.
crossref
13. Hoppe JE, Grieshaber , Hofler W. Colonization of Nigerian neonates with group B streptococci and its rapid detection. Infection. 1986; 14:74–8.
crossref
14. Prattes J, Koidl C, Eigl S, Krause R, Hoenigl M. Bronchoalveolar lavage fluid sample pretreatment with Sputasol (®) significantly reduces galactomannan levels. J Infect. 2015; 70:541–3.
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.
crossref
16. Black S, Shinefield HR, Fireman B, Lewis E, Ray P, Hansen JR, et al. Eflicacy, safety and immunogenicity of heptavalent pneumococcal conjugate vaccine in children. Pediatr Infect Dis J. 2000; 19:187–95.
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.
crossref
18. Berman S. Otitis media in children. N Eng J Med. 1995; 332:1560–5.
crossref
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.
20. Park JH, 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.
crossref
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.
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.
crossref

Fig. 1.
Sensitivities of polymerase chain reaction (PCR) and loop-mediated isothermal amplification assay (LAM P) against Streptococcus pneumoniae (SPECGB) spiked in sterile middle ear fluid. P and L indicate PCR and LAMP, respectively, and C (+) and C (—) indicate a positive control for pneumococcal DNA and a negative control, respectively. Bacteria was serially diluted in the middle ear fluid and used for two molecular diagnostic methods. The detection limits of PCR and LAMP are 104 CFU and less than 10 CFU, respectively.
piv-22-106f1.tif
Table 1.
Differential Detection of Streptococcus pneumoniae and Non-pneumococcal Reference and Clinical Isolates by Polymerase Chain Reaction (PCR) and Loop-Mediated Isothermal Amplification Assav (LAMP)
Strains and isolates PCR LAMP Referenre
Streptococcus pneumoniae serotype 4 TIGR + + 19)
Streptococcus pneumoniae nontvoable MNZ11/NCC1∗ + + 20)
Moraxella catarrhalis ATC C 25238 - - 21)
M cartarrhalis FMC 001-004 clinical isolates‡ - - This Studv
Staphylococcus aureus ATCC 12598 - - 22)
S. aureus EMC_OO1-OO4, clinical isolatest‡ - - this study
Haemophilus influenzae type b Eaqan† - - 23)
H. influenzae tVDe b EMC 001-004, clinical isolates‡ - - this study

∗ NCC1: null capsule clade1.

† Vaccine strain.

‡ Clinical isolates during 2000-2014.

Table 2.
List of Primers Used in this Study
Primer Description∗ Molecular assay DNA sequence (5→3') Reference
P5322 lytA840-860 PCR CAA CCG TAC AGA ATG AAG CGG 15)
P3322 lytA542-563 TTA TTC GTG CAA TAC TCG TGC G
sp5-FIP lytA826-847 LAMP asgav CCG CCA GTG ATA ATC CGC TTC A† CTC AAC TGG GAA TCC GC 12)
sp5-BIP lytA782-803 TCT CGC ACA TTG GGA AGG G†CC AGG CAC CAT TAT CAA CAG G
sp5-F3 lytA892-911 GCG TGC AAC CAT ATA GGC AA
Sp5-B3 lytA718-732 AGC ATT CCA ACC GCC
sp5-LB lytA 763-780 TCF ATC ATG CAG GTA GGA

∗ Numbers indicate nucleotide location in GenBank No. AEOO8540.

† The sequence of lytA gene is underlined.

Abbreviations: PCR, polymerase chain reaction; LAMP assay, Loop mediated isothermal amplification assay.

Table 3.
Detection ot Streptococcus pneumoniae by both Polymerase Chain Reaction (PCR) and Loop-Mediated Isothermal Amplification Assay (LAMP) in 22 Middle Ear Fluids from Otitis Media with Effusion
Result LAMP Total
Positive, n (%) Negative, n (%)
PCR positive, n (%) 3(13.6) 0(0) 3(13.6)
PCR negative, (°n) 9(40.9) 10(45.5) 19(86.4)
Total 12 (54.5) 10 (45.5) 22 (100)

Abbreviation: n number

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