Rapid Detection of Mycoplasma pneumoniae by Loop-Mediated Isothermal Amplification (LAMP) in Clinical Respiratory Specimens
Abstract
Background: Mycoplasma pneumoniae is a common cause of community-acquired pneumonia (CAP) worldwide, especially among children and debilitated populations. The present study aimed to investigate a loop-mediated isothermal amplification (LAMP) technique for rapid detection of M. pneumoniae in clini-cal specimens collected from patients with pneumonia.
Methods: Throat swabs were collected from 110 outpatients who suffered from pneumonia. Throat swab samples were obtained from patients referred to the hospital outpatient clinics of Tehran University hospitals, Iran in 2017. The presence of M. pneumoniae in the clinical specimens was evaluated by LAMP, PCR and culture methods. Sensitivity and specificity of the LAMP and PCR assays were also determined.
Results: Out of 110 specimens, LAMP assay detected M. pneumoniae in 35 specimens. Detection limit of the LAMP assay was determined to be 33fg /μL or ~ 40 genome copies/reaction. Moreover, no cross-reaction with genomic DNA from other bacteria was observed. Only 25 specimens were positive by the culture method. The congruence between LAMP assay and culture method was ‘substantial’ (κ=0.77). Specificity and sensitivity of LAMP assay were 88.2%, 100% in compare with culture. However, the con-gruence between LAMP assay and PCR assay was ‘almost perfect’ (κ=0.86). Specificity and sensitivity of LAMP assay were 92.5%, 100% in compare with PCR.
Conclusion: Overall, the LAMP assay is a rapid and cost-efficient laboratory test in comparison to other methods including PCR and culture. Therefore, the LAMP method can be applied in identification of M. pneumoniae isolates in respiratory specimens.
2. Aizawa Y, Oishi T, Tsukano S et al (2014). Clinical utility of loop-mediated isothermal amplification for rapid diagnosis of Mycoplasma pneumoniae in children. J Med Microbiol, 63(Pt2):248-51.
3. Centers for Disease Control and Prevention (2016). "Mycoplasma pneumoniae Infection." https://www.cdc.gov/pneumonia/atypical/mycoplasma/about/signs-symptoms.html (reviewed 13 April 2018).
4. Sauteur PMM, Jacobs BC, Spuesens EB et al (2014). Antibody responses to Mycoplasma pneumoniae: role in pathogenesis and diagnosis of encephalitis? PLoS pathog,10(6):e1003983.
5. Kashyap S, Sarkar M (2010). Mycoplasma pneumonia: Clinical features and management. Lung India, 27(2):75-85.
6. Petrone BL, Wolff BJ, DeLaney AA et al (2015). Isothermal detection of Mycoplasma pneumoniae directly from respiratory clinical specimens. J Clin Microbiol, 53(9):2970-6.
7. Saito R, Misawa Y, Moriya K et al (2005). Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma pneumoniae. J Med Microbiol, 54(Pt11):1037-41.
8. Ratliff AE, Duffy LB, Waites KB (2014). Comparison of the illumigene Mycoplasma DNA amplification assay and culture for detection of Mycoplasma pneumoniae. J clin microbiol, 52(4):1060-3.
9. Notomi T, Okayama H, Masubuchi H et al (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res, 28(12):E63.
10. Zhao N, Liu J, Sun D (2017). Detection of HCV genotypes 1b and 2a by a reverse transcription loop‐mediated isothermal amplification assay. J med virol, 89(6):1048-1054.
11. Gentilini F, Zanoni RG, Zambon E et al (2017). A comparison of the reliability of two gene targets in loop-mediated isothermal amplification assays for detecting leptospiral DNA in canine urine. J Vet Diagn Invest, 29(1):100-4.
12. Stratakos AC, Linton M, Millington S et al (2017). A loop‐mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenic Escherichia coli in beef and bovine faeces. J Appl Microbiol, 122(3):817-28.
13. Moustafa AM, Bennett MD (2017). Development of loop-mediated isothermal amplification–based diagnostic assays for detection of Pasteurella multocida and hemorrhagic septicemia–associated P multocida serotype B: 2. Am J Vet Res, 78(2):134-43.
14. Verma S, Singh R, Sharma V et al (2017). Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection. BMC Infect Dis, 17(1):223.
15. Gelaw B, Shiferaw Y, Alemayehu M et al (2017). Comparison of loop-mediated isothermal amplification assay and smear microscopy with culture for the diagnostic accuracy of tuberculosis. BMC Infect Dis,17(1):79.
16. Golmohammadi R, Ataee RA, Alishiri GH (2014). Molecular Diagnosis of Mycoplasma pneumoniae in Synovial Fluid of Rheumatoid Arthritis Patients. Iran J Med Microbiol, 8(1): 1-8.
17. Hadi N, Kashef S, Moazzen M et al (2011). Survey of Mycoplasma pneumoniae in Iranian children with acute lower respiratory tract infections. Braz J Infect Dis,15(2):97-101.
18. Oskooee MB, Karimi A, Shiva F et al (2014). Detection of Mycoplasma pneumoniae and Chlamydia trachomatis in Iranian children with acute lower respiratory infections by polymerase chain reaction. Asian Pac J Trop Dis, 4:S302-S306.
19. Arfaatabar M, Aminharati F, Azimi G et al (2018). High frequency of Mycoplasma pneumoniae among patients with atypical pneumonia in Tehran, Iran. Germs, 8(3):126-133.
20. Chanock R, James W, Fox H et al (1962). Growth of Eaton PPLO in broth and preparation of complement fixing antigen. Proc Soc Exp Biol Med, 110(4):884-9.
21. Parida M, Sannarangaiah S, Dash PK et al (2008). Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases. Rev med virol,18(6):407-21.
22. Chaudhry R, Sharma S, Javed S et al (2013). Molecular detection of Mycoplasma pneumoniae by quantitative real-time PCR in patients with community acquired pneumonia. Indian J Med Res,138:244-51.
23. Gotoh K, Nishimura N, Takeuchi S et al (2013). Assessment of the loop-mediated isothermal amplification assay for rapid diagnosis of Mycoplasma pneumoniae in pediatric community-acquired pneumonia. Jpn J Infect Dis, 66(6):539-42.
24. Kakuya F, Kinebuchi T, Fujiyasu H et al (2014). Genetic point‐of‐care diagnosis of Mycoplasma pneumoniae infection using LAMP assay. Pediatr Int, 56(4):547-52.
25. Matsuda C, Taketani T, Takeuchi S et al (2013). [Usefulness of the Loopamp Mycoplasma P detecting reagent kit developed based on the LAMP method]. Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi, 23(2):53-9.
26. Kakuya F, Kinebuchi T, Okubo H et al (2017). Comparison of oropharyngeal and nasopharyngeal swab specimens for the detection of Mycoplasma pneumoniae in children with lower respiratory tract infection. J Pediatr,189:218-21.
Files | ||
Issue | Vol 48 No 5 (2019) | |
Section | Original Article(s) | |
DOI | https://doi.org/10.18502/ijph.v48i5.1809 | |
Keywords | ||
Mycoplasma pneumoniae PCR Loop-mediated isothermal amplification (LAMP) Culture |
Rights and permissions | |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |