Early detection of Pseudomonas aeruginosa – comparison of conventional versus molecular (PCR) detection directly from adult patients with cystic fibrosis (CF)

Jiru Xu,John E Moore,J Stuart Elborn,Philip G Murphy,B Cherie Millar

doi:10.1186/1476-0711-3-21

Abstract

BackgroundPseudomonas aeruginosa (PA) is the most important bacterial pathogen in patients with cystic fibrosis (CF) patients. Currently, routine bacteriological culture on selective/non- selective culture media is the cornerstone of microbiological detection. The aim of this study was to compare isolation rates of PA by conventional culture and molecular (PCR) detection directly from sputum.MethodsAdult patients (n = 57) attending the regional adult CF centre in Northern Ireland, provided fresh sputum following airways clearance exercise. Following processing of the specimen with sputasol (1:1 vol), the specimen was examined for the presence of PA by plating onto a combination of culture media (Pseudomonas isolation agar, Blood agar & McConkey agar). In addition, from the same specimen, genomic bacterial DNA was extracted (1 ml) and was amplified employing two sequence-specific targets, namely (i) the outer membrane protein (oprL) gene locus and (ii) the exotoxin A (ETA) gene locus.ResultsBy sputum culture, there were 30 patients positive for PA, whereas by molecular techniques, there were 35 positive patients. In 39 patients (22 PA +ve & 17 PA -ve), there was complete agreement between molecular and conventional detection and with both PCR gene loci. The oprL locus was more sensitive than the ETA locus, as the former was positive in 10 more patients and there were no patients where the ETA was positive and the oprL target negative. Where a PCR +ve/culture -ve result was recorded (10 patients), we followed these patients and recorded that 5 of these patients converted to being culture-positive at times ranging from 4–17 months later, with a mean lag time of 4.5 months.ConclusionsThis study indicates that molecular detection of PA in sputum employing the oprL gene target, is a useful technique in the early detection of PA, gaining on average 4.5 months over conventional culture. It now remains to be established whether aggressive antibiotic intervention at this earlier stage, based on PCR detection, has any significant benefits on clinical outcome.

Highlights

Cystic fibrosis [cystic fibrosis (CF)] is the most commonly inherited fatal disease in persons originating from a white and European background, currently affecting approximately 30,000 adults and children in the US [1]
It is an autosomal recessive condition whereby two alleles carrying a polymorphism in the cystic fibrosis transmembrane conductance regulator [CFTR] gene phenotypically manifest the disease state through a variety of multiorgan problems, associated with a pharmacological disfunction to regulate sodium and chloride secretion across cell membranes
These infections are usually dominated by Gram-negative organisms, especially by the pseudomonads, including Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia

Summary

Introduction

Cystic fibrosis [CF] is the most commonly inherited fatal disease in persons originating from a white and European background, currently affecting approximately 30,000 adults and children in the US [1]. The defective gene carrying the mutation is carried in one in every 31 Americans [one in 28 Caucasians], equating to more than 10 million people being a symptomless carrier of the defective gene [1] It is an autosomal recessive condition whereby two alleles carrying a polymorphism in the cystic fibrosis transmembrane conductance regulator [CFTR] gene phenotypically manifest the disease state through a variety of multiorgan problems, associated with a pharmacological disfunction to regulate sodium and chloride secretion across cell membranes. CF patients continue to suffer from recurrent and chronic respiratory tract infections and most of their morbidity and mortality is due to such infections throughout their life [3] These infections are usually dominated by Gram-negative organisms, especially by the pseudomonads, including Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. The aim of this study was to compare isolation rates of PA by conventional culture and molecular (PCR) detection directly from sputum

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