Bacteriology
Quantitative detection of Streptococcus pneumoniae from sputum samples with real-time quantitative polymerase chain reaction for etiologic diagnosis of community-acquired pneumonia

Parts of the content of the manuscript was presented at the 17th European Congress of Clinical Microbiology and Infectious Diseases, Munich, Germany 2007.
https://doi.org/10.1016/j.diagmicrobio.2007.10.011Get rights and content

Abstract

We assessed the clinical usefulness of a real-time quantitative polymerase chain reaction (RQ-PCR) method applied on sputum samples to identify Streptococcus pneumoniae in 184 consecutive patients admitted to hospital with community-acquired pneumonia. Induced sputum samples were analyzed by culture and RQ-PCR. In total, 70/184 patients (38%) were diagnosed with S. pneumoniae. Cultures from blood and nasopharyngeal secretions were positive in 27/179 (15%) and 42/158 (27%) cases, respectively. Pneumococcal antigen was detected in 33/169 (20%) urine specimens. In sputum samples, culture was significantly positive in 19/128 (15%), whereas a significant concentration of DNA was found by RQ-PCR in 34/127 (27%) cases (P < 0.001). In 28/34 (82%) patients with RQ-PCR–positive sputum samples, S. pneumoniae was also detected with other methods. In the 34 RQ-PCR–positive sputum samples, 17 were negative by sputum culture, out of which 14 were obtained from patients treated with antibiotics prior to sampling. S. pneumoniae may be rapidly diagnosed by analyzing induced sputum samples by RQ-PCR and may be particularly valuable in patients in whom antibiotic therapy has been initiated.

Introduction

Community-acquired pneumonia (CAP) is a common disease (Jokinen et al., 1993, Macfarlane et al., 1993, Woodhead et al., 1987) representing a major cause of hospital admission and mortality of infectious origin among adults, also in high-income countries (Almirall et al., 1993, Jokinen et al., 1993, Lave et al., 1996, Marrie, 1990, Marston et al., 1997). Streptococcus pneumoniae is the leading cause of CAP, especially among young children, elderly people, and persons with underlying chronic diseases (British Thoracic Society, 2001). S. pneumoniae is also the most common pathogen causing fatal CAP (Fine et al., 1996).

Many well-designed studies of patients with CAP however have succeeded in identifying the etiologic agent in only 40% to 60% of the cases (British Thoracic Society, 2001, Fang et al., 1990). More recent studies, using polymerase chain reaction (PCR) assays on specimens from lung aspirates (Ruiz-Gonzalez et al., 1999) and blood (Menendez et al., 1999), have suggested that pneumococci probably also cause a majority of CAP cases, with undefined etiology.

In the management of patients with CAP, rapid identification of the microbiologic cause is important to permit antibiotic selection directed at the causative agent. However, rapid methods to determine a pneumococcal etiology have been limited to Gram stain and polysaccharide antigen detection, and a clear need for improved diagnostic testing in CAP, most likely using molecular methodology rather than culture, has recently been recognized by the US National Institutes of Health (Mandell et al., 2007). In the last few years, PCR has been adopted to detect airway pathogens. The main challenge with regular PCR methods from airway samples has been the difficulties in distinguishing colonization from infection because these assays usually do not generate quantitative data (Murdoch, 2004). This obstacle could potentially be overcome by the use of a quantitative PCR assay. In the last few years, several studies have shown promising results in detecting S. pneumoniae from lower respiratory tract secretions by real-time quantitative PCR (RQ-PCR) (Apfalter et al., 2005, Greiner et al., 2001, Kais et al., 2006, Saukkoriipi et al., 2004, Yang et al., 2005).

The aim of this prospective study was to estimate the accuracy of a recently developed RQ-PCR method for identification of pneumococci in sputum, among patients with CAP treated in hospital.

Section snippets

Patients

All patients with CAP admitted to the Department of Infectious Diseases at Karolinska University Hospital in Solna, Stockholm, Sweden, from September 13, 2004, to September 12, 2005, were reviewed for inclusion in a prospective study. A total of 184 patients were included in the study.

CAP was defined as clinical signs of acute lower respiratory tract infection with onset before admission and presence of new infiltrates on a chest radiograph. All patients were assessed by 1 of the study

Pneumococcal etiology diagnosed by different methods

In 70 patients (38%), S. pneumoniae was identified by 1 or more methods (Table 1). Blood cultures provided the diagnosis in 27 patients (15%), whereas pneumococcal antigen was detected in urine from 33 patients (20%). A total of 19 patients (15%) produced at least 1 sputum specimen with significant growth (≥105 CFU/mL) of S. pneumoniae. S. pneumoniae DNA (corresponding to ≥105 CFU/mL) with sputum RQ-PCR was detected in 34 patients (27%) (P < 0.0001 compared with sputum culture).

In 15 of the

Discussion

Accurate diagnosis of pneumococcal pneumonia is limited by lack of sensitive methods and difficulties in obtaining specimens appropriate for examination. Blood culture definitely provides an etiologic diagnosis, but bacteremia occurs in less than 30% of all CAP cases (Chalasani et al., 1995, Nohynek et al., 1991, Skerrett, 1999) and almost never in less severe cases. Urinary antigen detection in adults is specific, but sensitivity is less satisfactory, 50% to 80% (Dominguez et al., 2001,

Acknowledgments

The authors thank Dr. Rima Dandachi for her help with the data compilation. We also thank the respiratory physiotherapists Susanna Wennman and Annette Idengren for their help with the collection of sputum samples. The study was supported by grants from Karolinska Institutet.

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      They potentially could provide results within a few hours, detect low levels of pneumonia pathogens in clinical specimens, and finally, they don't depend on the viability of the targeted pathogen and so, they are probably less affected by previous antimicrobial therapy than culture-based diagnostic methods. Indeed, in a study by Johansson et al., within 184 admitted patients with CAP diagnosis, 80% of cases with a positive PCR test and negative sputum culture had been treated with antibiotics prior to sputum sampling [20]. PCR has positive impact in acute-care settings where timely and accurate diagnostic tools are critical for patient treatment decisions and outcomes.

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    Address of the institution at which the work was performed: Infectious Diseases Unit, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, SE-171 76, Solna, Stockholm, Sweden.

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