Identification of some microorganisms directly from clinical sample by multiplex PCR

Abstract

Background and Aims: Infectious diseases are affecting people more often than before and that is the main cause of morbidity and mortality in developed and developing countries. Taking the necessary measures by diagnosing earlier and using appropriate antibiotics made significant contributions to the control of the infections. In our study we aimed to develop a method for the diagnosis of some of pathogen gram-negative bacteria by using multiplex PCR (Polymerase Chain Reaction) from a positive blood culture bottles. Methods: Extraction method was applied from the blood cultures in which growth was detected. After mono¬plex PCR experiments were performed and images were obtained, multiplex PCR experiments were performed. Considering the product sizes and primers for multiplex PCR, a quadruple bacterial set was prepared by matching Acinetobacter baumannii, Escherichia coli, Serratia marcescens and Pseudomonas aeruginosa. Experiment conditions and tube concentration were adjusted by making serial trial experiments. Results: Multiplex-PCR-based methods have the advantage of defining multiple pathogens in a single PCR reaction. These are used for identifying respiratory infections, bacteraemia, and meningitis which are common bacterial and viral diseases. Type-specific genes and molecular techniques including identification of infectious diseases with nucleic acid amplification are promising tools for the verification of accurate diagnosis. The feasibility of this assay in routine diagnostic testing was evaluated using 146 positive blood cultures and 40 negative blood culture samples. Conclusions: Early diagnosis and intervention with effective antimicrobial therapy is important in reducing mortality that is associated with bloodstream infections.

References

1. 1.Jarvis WR. Selected aspects of the socioeconomic impact of nosocomial infections: Morbidity, mortality, cost and prevention. Infect Control Hosp Epidemiol. 1996 Aug; 17: 552-57.
2. 2.Öztürk R. Tanı yöntemlerindeki gelişmelerin infeksiyon hastalıkları kliniğine yansıması. ANKEM Dergisi 2005 May; 19 (2): 8-13.
3. 3.Eraç B, 2006. Hastane kökenli gram-negatif bakterilerde per-1 enziminin moleküler epidemiyolojisi. PhD, Dokuz Eylül Üniversitesi, İzmir, Türkiye, 2006.
4. 4.Kuyucu N. Çocuklarda dirençli gram negatif bakteri enfeksiyonları ve tedavisi. Çocuk Enf Dergisi 2009 May; 3 (Special Issue 1): 70-4.
5. 5.Rodríguez-Baño J, Navarro MD, Romero L, Muniain MA, de Cueto M, Gálvez J et al. Risk-factors for emerging bloodstream infections caused by extended-spectrum β-lactamase-producing Escherichia coli. Clin Micro-biol Infect. 2008 Nov 15; 14:180-3.
6. 6.Küçükbasmacı Ö, Çalışkan AR, HamançaÖ,ÇelikŞ,KöksalF,GönüllüN, Saması M. Kan örneklerinden üretilen Gram negatif çomakların antibiyotiklere duyarlılıkları. Türk Mikrobiyol Majmaah Journal of Health Sciences ,Vol. 11, Issue 3, July - 2023, DhuAl-Hijjah - 1444 69 Identification of some microorganisms directly from clinical sample by multiplex PCR Cem Dergisi 2007 Dec; 37: 201–3.
7. 7.Er H,Aşık G,Yoldaş Ö, Demir C, Keş- li R. Kan kültürlerinde izole edilerek tanımlanan mikroorganizmaların ve antibiyotik direnç oranlarının belirlenmesi. Türk Mikrobiyol Cem Derg. 2015 Nov; 45(1):48-54.
8. 8.Yüksekkaya Ş, Opuş A, Güvenç Hİ ve ark. 2009- 2013 yılları arasında Konya Eğitim veAraştırma Hastanesi’nde kan kültüründen izole edilen Staphylococcus aureus suşlarının antimikrobiyal ajanlara duyarlılıklarının değerlendirilmesi. ANKEMDerg.2017;31(1):1-6).