Quantitative detection of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in lower respiratory tract samples by real-time PCR

doi:10.1016/j.diagmicrobio.2006.01.007

Diagnostic Microbiology and Infectious Disease

Volume 55, Issue 3, July 2006, Pages 169-178

https://doi.org/10.1016/j.diagmicrobio.2006.01.007 Get rights and content

Abstract

The limitation of polymerase chain reaction (PCR) in diagnosis of lower respiratory tract infections (LRTIs) caused by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis has been a distinguishing colonization from infection. We assess here the usefulness of real-time quantitative PCR (RQ-PCR) performed on lower respiratory tract samples to overcome this problem.

Consecutive respiratory tract samples from patients with and without signs of infection (n = 203) were subjected to RQ-PCR, targeting the genes pneumolysin (S. pneumoniae), fumarate reductase (H. influenzae), and outer membrane protein B (M . catarrhalis). DNA from positive controls with predefined colony forming units (CFUs) per milliliter were included to allow estimation of CFU per milliliter for the test samples. In parallel, assessment of quantitative cultures from all samples was performed.

In the group of patients with LRTI, significant pathogens (≥10⁵ CFU/mL) were found in 32/135 samples (23.7%) with culture, in 51/135 (37.7%) with RQ-PCR, and in 59/135 (43.7%) when combining the methods.

Introduction

Respiratory tract infection, mainly in the form of pneumonia, is an important cause of morbidity and mortality, especially in children and the elderly (Hedlund and Örtqvist, 2002). Establishing a microbiologic diagnosis is considered an important measure for a proper treatment, but even in the most rigorous studies, it is difficult to establish an etiologic diagnosis in 50% of cases of community-acquired pneumonia (CAP) (Reimer and Carroll, 1998). The most frequently detected pathogens are Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae (File, 2003). Conflicting evidence exists regarding the importance of pathogens such as Moraxella catarrhalis, and the high rates of colonization has lead to a general skepticism toward the role of this pathogen (Reimer and Carroll, 1998, Karalus and Campagnari, 2000), though some reports suggest an important role for this bacterium in selected patient groups, such as patients with chronic obstructive pulmonary disease (COPD) (Karalus and Campagnari, 2000, Hedlund et al., 1997).

Lower respiratory tract cultures have so far been the most important method for diagnosing respiratory tract infections. Recently, immunochromatographic urinary antigen detection assays for pathogens such as S. pneumoniae and Legionella pneumophila have shown promising results as supplementary tests with relatively high sensitivities and specificities (Dominguez et al., 1999, Murdoch et al., 2001). However, the need for still more sensitive methods has lead to studies evaluating the role of nucleic acid amplification methods such as polymerase chain reaction (PCR) (Murdoch, 2003). For pathogens such as M. pneumoniae, Chlamydia pneumoniae, and L. pneumophila, PCR is already available (Welti et al., 2003). However, for S. pneumoniae, H. influenzae, and M. catarrhalis, which are frequent colonizers, the main challenge with regular PCR assays has been the difficulties in distinguishing colonization from infection because these assays do not generate quantitative data (Murdoch, 2004).

Potentially, this obstacle could be overcome by the use of a quantitative PCR assay. The diagnostic properties of real-time quantitative PCR (RQ-PCR) have been evaluated in a few studies of both upper and lower respiratory tract samples. Two studies on the detection of S. pneumoniae in nasopharyngeal secretion by targeting the pneumolysin gene showed high sensitivity, specificity, and reproducibility, as well as good correlation with quantitative cultures (Greiner et al., 2001, Saukkoriipi et al., 2004). Similar conclusions could be drawn from a study of the detection of M. catarrhalis in nasopharyngeal secretions (Greiner et al., 2003). Two recent studies have also evaluated RQ-PCR for the detection of S. pneumoniae in sputum and bronchoalveolar lavage fluid (BAL) (Apfalter et al., 2005, Yang et al., 2005). In both studies, the novel assays were found to yield rapid and accurate diagnosis of pneumococcal pneumonia.

Although RQ-PCR has been applied on lower respiratory tract samples for the detection of S. pneumoniae, no available assay exists for multiple traditional pathogens. We present here a comparison of the properties of quantitative culture and a novel RQ-PCR assay for quantitative detection of S. pneumoniae, H. influenzae, and M. catarrhalis in lower respiratory tract samples.

Section snippets

Bacterial strains

S. pneumoniae CCUG 28588, M. catarrhalis CCUG 18284, and H. influenzae CCUG 23969 (Culture Collection, University of Gothenburg, Sweden) were used as positive controls.

Specificity of the real-time PCR assay for S. pneumoniae was tested with reference strains Streptococcus anginosus CCUG 27298, Streptococcus milleri NCTC 10708 (National Collection of Type Cultures, London, UK), Streptococcus mitis ATCC 15912 (American Type Culture Collection, Manassas, VA), as well as the strains Streptococcus

Sensitivity and amplification efficiency of the RQ-PCR assay

Tenfold serial dilutions from a concentration of 10⁸ CFU/mL of S. pneumoniae, H. influenzae, and M. catarrhalis were used to construct standard curves for determination of the sensitivity of the real-time PCR assay. The concentration of DNA corresponding to 10⁸ CFU/mL was approximately 40 ng/μL for S. pneumoniae, 100 ng/μL for H. influenzae, and 200 ng/μL for M. catarrhalis. Genomic equivalents (GEQ) per milliliter were calculated for the 3 species and were 1.8 × 10⁹, 4.7 × 10⁹, and 9.4 × 10⁹

Discussion

In this material consisting of samples from patients with various clinical conditions, RQ-PCR substantially improved significant pathogen detection. Conflicting evidence exist regarding the impact of bacteriologic investigations on therapeutic outcome in pneumonia. In the case of nonsevere pneumonia, several authors have come to the conclusion that no beneficial effect is detectable (Levy et al., 1988, Sanyal et al., 1999, Theerthakarai et al., 2001). A common trait for these studies has been

Acknowledgments

The authors thank Martin Vondracek, Kristina Fahlander, Hector Roldan, Siri Wahlquist, Inga Karlsson, and Carina Bengtsson for excellent technical assistance.

We would also like to thank Professor Richard Moxon, Oxford, for technical advice.

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BacteriologyQuantitative detection of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in lower respiratory tract samples by real-time PCR

Abstract

Introduction

Section snippets

Bacterial strains

Sensitivity and amplification efficiency of the RQ-PCR assay

Discussion

Acknowledgments

Lancet

Lancet

Microbial. Infect.

Chest

Microb. Pathog.

Diagn. Microbiol. Infect. Dis.

Chest

Mol. Cell. Probes

Chest

Diagn. Microbiol. Infect. Dis.

Mapping of a protective epitope of the CopB outer membrane protein of Moraxella catarrhalis

Infect. Immun.

Community-acquired bacteria frequently detected by means of quantitative polymerase chain reaction in nosocomial early-onset ventilator-associated pneumonia

Crit. Care Med.

Practice guidelines for the management of community-acquired pneumonia in adults. Infectious Diseases Society of America

Clin. Infect. Dis.

Bronchoscopic diagnosis of pneumonia

Clin. Microbiol. Rev.

Evaluation of a rapid immunochromatographic assay for the detection of Legionella antigen in urine samples

Eur. J. Clin. Microbiol. Infect. Dis.

Assessment of the usefulness of sputum culture for diagnosis of community-acquired pneumonia using the PORT predictive scoring system

Arch. Intern. Med.

An overview of real-time quantitative PCR: applications to quantify cytokine gene expression

Methods

Quantitative detection of Moraxella catarrhalis in nasopharyngeal secretions by real-time PCR

J. Clin. Microbiol.

Bacteriology
Quantitative detection of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in lower respiratory tract samples by real-time PCR