Research Article
BibTex RIS Cite

Grup A Streptokokların Hızlı Moleküler Tanısında Loop-Mediated Isothermal Amplifi cation PCR (LAMP-PCR)

Year 2017, Volume: 1 Issue: 1, 11 - 16, 06.06.2017

Abstract

Amaç: Bakteriyel farenjit olgularının çoğunluğunu Grup A Streptokoklar (GAS) oluşturur. Bu nedenle akut tonsillofarenjit
olgularında tedavinin yönlendirilmesi açısından bakteriyel-viral etkenlerin ayrımı ve GAS’ın hızlı tanısı ön plana
çıkmaktadır. Bu çalışmada ise akut bakteriyel tonsillofarenjit düşünülen çocuk hastalarda, GAS hızlı tanısında LoopMediated
Isothermal Amplifi cation PCR (LAMP PCR) yönteminin etkinliğinin belirlenmesi ve konvansiyonel yöntem
olan kültür testleriyle uyumunun karşılaştırılması amaçlanmıştır.

Gereç ve Yöntemler Akut tonsillofarenjit yakınmaları ile Sakarya Üniversitesi Eğitim ve Araştırma Hastanesine başvuran hastaların posterior
farenks ve tonsillerinden alınan sürüntü örneklerinden izole edilen 36 GAS suşu, eş zamanlı boğaz kültürü GAS pozitif
olan 8 adet hastanın boğaz sürüntüsü ve bir adet standart Streptococcus pyogenes suşu (ATCC 19615) çalışma
kapsamına alınmıştır. İzole edilen GAS suşlarından LAMP PCR çalışılmıştır. Hedef bölge olarak Streptolizin B (speB)
gen bölgesi hedefl enmiştir. Ekstraksiyon, amplifi kasyon ve sonuçların analizi LAMP PCR cihazında (Genie II, Optigene,
UK) gerçekleştirilmiş olup, tüm bu işlemlerin 70 dakika içerisinde sonuçlandığı görülmüştür. LAMP PCR cihazında elde
edilen sonuç grafi kleri Genie Explorer v2.0.6.3 (Optigene, UK) programıyla değerlendirilmiştir.

Bulgular: LAMP PCR ile hasta örneklerinden izole edilmiş olan GAS suşlarının (n=36) % 97.2’si (n=35) pozitif olarak saptandı.
Eş zamanlı boğaz kültürü GAS yönünden pozitif olan 8 hastanın boğaz sürüntüsü örneğinden 7’si (% 87.5) LAMP PCR
ile GAS pozitif olarak saptandı. Duplike çalışılan standart suş örneği de pozitif bulundu. Enzimatik yöntem ile DNA
izolasyonu (~30 dakika) ve izotermal amplifi kasyon işlemi (~40 dakika) toplamda ~70 dakikada tamamlandı.

Sonuç: LAMP PCR; yüksek duyarlılık ve özgüllükte, ekstraksiyon prosedürü çok basit ve pratik olan, izolasyon ve amplifi kasyon
ile sonuçların analizinin taşınabilir özellikte küçük tek bir cihazda yapıldığı, 45-70 dk gibi çok kısa sürede sonuç
alınabilen, personel açısından kolay uygulanabilen, minimal laboratuvar donanımı ile gerektiren, sonuçların gözle de
değerlendirilmesi imkanı olan ve diğer moleküler testlere göre daha uygun fi yatı olan bir yöntemdir. LAMP PCR’ın bu
alanda kullanımı henüz çok yeni olduğundan yeni ve kapsamlı çalışmalara ihtiyaç vardır

References

  • 1. Walker MJ, Barnett TC, McArthur JD, Cole JN, Gillen CM, Henningham A, Sriprakash KS, Sanderson-Smith ML, Nizet V. Disease manifestations and pathogenic mechanisms of group A Streptococcus. Clin Microbiol Rev 2014;27(2):264-301.
  • 2. Ralph AP, Carapetis JR. Group a streptococcal diseases and their global burden. Curr Top Microbiol Immunol 2013;368:1-27.
  • 3. Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000;13(3):470-511.
  • 4. Bisno AL. Acute pharyngitis: etiology and diagnosis. Pediatrics 1996;97:949-54.
  • 5. Ebell MH, Smith MA, Barry HC, Ives K, Carey M. The rational clinical examination. Does this patient have strepthroat? JAMA 2000;284:2912-8.
  • 6. Kim DW, Kilgore PE, Kim EJ, et al. The enhanced pneumococcal LAMP assay: a clinical tool for the diagnosis of meningitis due to Streptococcus pneumoniae. PLoS One 2012;7:e42954.
  • 7. Nagamine K, Watanabe K, Ohtsuka K, Hase T, Notomi T. Loopmediated Isothermal Amplifi cation Reaction Using a Non denatured Template. Clinical Chemistry 2001; 9: 47.
  • 8. Dhama K, Karthik K, Chakraborty S, Tiwari R, Kapoor S, Kumar A, Thomas P. Loop-mediated isothermal amplifi cation of DNA (LAMP): a new diagnostic tool lights the world of diagnosis of animal and human pathogens: a review. Pak J BiolSci. 2014;17(2):151-66.
  • 9. Niessen L. Current state and future perspectives of loop-mediated iso thermal amplifi cation (LAMP) – based diagnosis of fi lamentous fungi and yeasts. Appl Microbiol Biotechnol 2015;99:553–574
  • 10. Han ET, Watanabe R, Sattabongkot J, Khuntirat B, Sirichaisinthop J, Iriko H, Jin L, Takeo S, Tsuboi T. Detection of Four Plasmodium Species by Genus- and Species-Specifi c Loop-Mediated Isothermal Amplifi cation for Clinical Diagnosis. J Clin Microbiol. 2007;45(8):2521-8.
  • 11. Saito R, Misawa Y, Moriya K, Koike K,Ubukata K, Okamura N. Development and evaluation of a loop-mediated isothermal amplifi cation assay for rapid detection of Mycoplasma pneumoniae. J Med Microbiol. 2005; 54:1037-1041.
  • 12. Uemura N, Makimura K, Onozaki M, Otsuka Y, Shibuya Y, Yazaki H, Kikuchi Y, Abe S, Kudoh S. Development of a loop-mediated iso thermal amplifi cation method for diagnosing Pneumocystis pneumonia. J Med Microbiol. 2008;57(Pt 1):50-7.
  • 13. Kamachi K, Toyoizumi-Ajisaka H, Toda K, Soeung SC, Sarath S, Nareth Y, Horiuchi Y, Kojima K, Takahashi M, Arakawa Y. Development andevaluation of a loop-mediated iso thermal amplifi cation method for rapid diagnosis of Bordetella pertussis infection. J Clin Microbiol. 2006;44(5):1899-902.
  • 14. Jayaratne P, andRutherford C. Detection of Group A streptococci (GAS) by Loop-Mediated Isothermal Amplifi cation (LAMP) directly from specimens: a rapid, simple and cost-effective alternative to culture. P0684.25th European Congress of Clinical Microbriology and Infectious Diseases (ECCMID 2015) Kopenhag, Danimarka
  • 15. Kimura K Yanagisawa H Wachino Jet al Rapid and reliable loop-mediated isothermal amplifi cation method for detecting Streptococcus agalactiae Jpn J Infect Dis 2013; 66: 5468.
  • 16. Kılıç S, Ötgün SN, Özgümüş GG, Turan M, Ketre C, Kolukırık M. A Grubu Beta Hemolitik Streptokoklar (AGBHS) Hızlı Tanısı İçin LAMP Tabanlı Bir Yöntem Geliştirilmesi. S.26. 37. TMC Kongresi, 16-20 Kasım 2016, Antalya

Loop-Mediated Isothermal Amplifi cation PCR (LAMP-PCR) For Rapid Molecular Diagnosis of Group A Streptococci

Year 2017, Volume: 1 Issue: 1, 11 - 16, 06.06.2017

Abstract

Objective: Most bacterial pharyngitis is caused by Group A Streptococci (GAS). Therefore, discrimination between bacterial-viral
causes, and rapid diagnosis of GAS are important in terms of guiding treatment in acute tonsillopharyngitis cases. The
present study aims to assess the effi cacy ofLoop-Mediated Isothermal Amplifi cation PCR (LAMP PCR) test for rapid
diagnosis of GAS, and compare its correlation with culture tests as conventional methods in children with suspected
acute bacterial tonsillopharyngitis.

Materials and Methods: We studied 36 GAS strains isolated from swab samples obtained from posterior pharynges and tonsils of patients
presenting to Sakarya University Training and Research Hospital with acute tonsillopharyngitis symptoms, pharyngeal
swab samples from 8 patients who had concurrent positive pharyngeal culture results for GAS, and one standard
Streptococcus pyogenesstrain (ATCC 19615). Isolated GAS strains were analyzed with LAMP PCR. As target region,
Streptoplysin B (speB) gene region was selected. Extraction, amplifi cation and analysis of the results were performed
with LAMP PCR analyzer (Genie II, Optigene, UK), and all these procedures were completed within 70 minutes. Result
graphics obtained from LAMP PCR analyzer were evaluated with Genie Explorer v2.0.6.3 (Optigene, UK) program.

Results: LAMP PCR method yielded positive results for 97.2% (35/36) of GAS strains isolated from patient samples. Pharyngeal
swab samples of 8 patients who had concurrent positive culture results for GAS were analyzed, and 7 of them
(87.5%) had positive GAS results with LAMP PCR. Standard strain sample which was analyzed with duplicate run was
also detected as positive. With enzymatic method, DNA isolation (~30 minutes) and isothermal amplifi cation process
(~40 minutes) were completed in ~70 minutes.

Conclusion: LAMP PCR has high sensitivity and specifi city with pretty simple and practical extraction procedure. A single small
portable device is suffi cient for isolation, amplifi cation and analysis of the results, with the whole process taking only
as short as 45-70 minutes. It is a convenient method requiring minimal laboratory equipment, and the results can be
evaluated visually. It is more affordable compared to other molecular tests. Since LAMP PCR has been introduced
recently to clinical practice, more comprehensive studies are necessary.

References

  • 1. Walker MJ, Barnett TC, McArthur JD, Cole JN, Gillen CM, Henningham A, Sriprakash KS, Sanderson-Smith ML, Nizet V. Disease manifestations and pathogenic mechanisms of group A Streptococcus. Clin Microbiol Rev 2014;27(2):264-301.
  • 2. Ralph AP, Carapetis JR. Group a streptococcal diseases and their global burden. Curr Top Microbiol Immunol 2013;368:1-27.
  • 3. Cunningham MW. Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 2000;13(3):470-511.
  • 4. Bisno AL. Acute pharyngitis: etiology and diagnosis. Pediatrics 1996;97:949-54.
  • 5. Ebell MH, Smith MA, Barry HC, Ives K, Carey M. The rational clinical examination. Does this patient have strepthroat? JAMA 2000;284:2912-8.
  • 6. Kim DW, Kilgore PE, Kim EJ, et al. The enhanced pneumococcal LAMP assay: a clinical tool for the diagnosis of meningitis due to Streptococcus pneumoniae. PLoS One 2012;7:e42954.
  • 7. Nagamine K, Watanabe K, Ohtsuka K, Hase T, Notomi T. Loopmediated Isothermal Amplifi cation Reaction Using a Non denatured Template. Clinical Chemistry 2001; 9: 47.
  • 8. Dhama K, Karthik K, Chakraborty S, Tiwari R, Kapoor S, Kumar A, Thomas P. Loop-mediated isothermal amplifi cation of DNA (LAMP): a new diagnostic tool lights the world of diagnosis of animal and human pathogens: a review. Pak J BiolSci. 2014;17(2):151-66.
  • 9. Niessen L. Current state and future perspectives of loop-mediated iso thermal amplifi cation (LAMP) – based diagnosis of fi lamentous fungi and yeasts. Appl Microbiol Biotechnol 2015;99:553–574
  • 10. Han ET, Watanabe R, Sattabongkot J, Khuntirat B, Sirichaisinthop J, Iriko H, Jin L, Takeo S, Tsuboi T. Detection of Four Plasmodium Species by Genus- and Species-Specifi c Loop-Mediated Isothermal Amplifi cation for Clinical Diagnosis. J Clin Microbiol. 2007;45(8):2521-8.
  • 11. Saito R, Misawa Y, Moriya K, Koike K,Ubukata K, Okamura N. Development and evaluation of a loop-mediated isothermal amplifi cation assay for rapid detection of Mycoplasma pneumoniae. J Med Microbiol. 2005; 54:1037-1041.
  • 12. Uemura N, Makimura K, Onozaki M, Otsuka Y, Shibuya Y, Yazaki H, Kikuchi Y, Abe S, Kudoh S. Development of a loop-mediated iso thermal amplifi cation method for diagnosing Pneumocystis pneumonia. J Med Microbiol. 2008;57(Pt 1):50-7.
  • 13. Kamachi K, Toyoizumi-Ajisaka H, Toda K, Soeung SC, Sarath S, Nareth Y, Horiuchi Y, Kojima K, Takahashi M, Arakawa Y. Development andevaluation of a loop-mediated iso thermal amplifi cation method for rapid diagnosis of Bordetella pertussis infection. J Clin Microbiol. 2006;44(5):1899-902.
  • 14. Jayaratne P, andRutherford C. Detection of Group A streptococci (GAS) by Loop-Mediated Isothermal Amplifi cation (LAMP) directly from specimens: a rapid, simple and cost-effective alternative to culture. P0684.25th European Congress of Clinical Microbriology and Infectious Diseases (ECCMID 2015) Kopenhag, Danimarka
  • 15. Kimura K Yanagisawa H Wachino Jet al Rapid and reliable loop-mediated isothermal amplifi cation method for detecting Streptococcus agalactiae Jpn J Infect Dis 2013; 66: 5468.
  • 16. Kılıç S, Ötgün SN, Özgümüş GG, Turan M, Ketre C, Kolukırık M. A Grubu Beta Hemolitik Streptokoklar (AGBHS) Hızlı Tanısı İçin LAMP Tabanlı Bir Yöntem Geliştirilmesi. S.26. 37. TMC Kongresi, 16-20 Kasım 2016, Antalya
There are 16 citations in total.

Details

Subjects Health Care Administration
Journal Section Research Article
Authors

Mustafa Altındiş

Bahri Elmas

Ümit Kılıç This is me

Ferhat Gürkan Aslan This is me

Gökhan Küçükkara

Mehmet Köroğlu

Publication Date June 6, 2017
Acceptance Date June 22, 2017
Published in Issue Year 2017 Volume: 1 Issue: 1

Cite

AMA Altındiş M, Elmas B, Kılıç Ü, Aslan FG, Küçükkara G, Köroğlu M. Loop-Mediated Isothermal Amplifi cation PCR (LAMP-PCR) For Rapid Molecular Diagnosis of Group A Streptococci. J Biotechnol and Strategic Health Res. June 2017;1(1):11-16.
  • Dergimiz Uluslararası hakemli bir dergi olup TÜRKİYE ATIF DİZİNİ, TürkMedline, CrossREF, ASOS index, Google Scholar, JournalTOCs, Eurasian Scientific Journal Index(ESJI), SOBIAD ve ISIindexing dizinlerinde taranmaktadır. TR Dizin(ULAKBİM), SCOPUS, DOAJ için başvurularımızın sonuçlanması beklenmektedir.