Comparison of three different techniques for the isolation of viral RNA in sputum.

BACKGROUND: Respiratory infections are a major cause of morbidity and mortality worldwide. A high percentage of all respiratory tract infections are caused by RNA viruses. Real-time PCR is a highly sensitive method for the detection of respiratory viruses in clinical samples. A good RNA isolation protocol is of high importance, since RNA is more unstable than DNA and many clinical samples contain RNAses. OBJECTIVES: To evaluate the performance of three different RNA extraction protocols for the extraction of respiratory viral RNA from sputum samples obtained from patients with the suspicion of a viral respiratory tract infection. STUDY DESIGN: A total of 50 sputum samples, PCR positive for a respiratory RNA virus, were used for viral RNA isolation with the phenol/chloroform method, RTP(®) DNA/RNA virus mini kit and the automated MagNa Pure LC (MPLC) extraction system. After isolation, real-time PCR was performed for the detection of viral RNA in the sputum samples. RESULTS: The MPLC extraction increased the detection probability from 82% (phenol/chloroform) and 86% (RTP(®) DNA/RNA virus mini kit) to 94%. In 16% the RTP(®) DNA/RNA virus mini kit resulted in lower Ct values compared to the phenol/chloroform method, while in 32% the phenol/chloroform resulted in lower Ct values. CONCLUSIONS: The extraction of viral RNA performed with the MPLC extraction method was superior to the extraction with the RTP(®) DNA/RNA virus mini kit and to the extraction with phenol/chloroform. In general, there was no difference in the detection of viral RNA between the phenol/chloroform extraction method and the RTP(®) DNA/RNA virus mini kit.

Mimivirus is not a frequent cause of ventilator-associated pneumonia in critically ill patients.

Acanthamoeba polyphaga mimivirus (APMV) belongs to the amoebae-associated microorganisms. Antibodies to APMV have been found in patients with pneumonia suggesting a potential role as a respiratory pathogen. In addition, positive serology for APMV was associated with an increased duration of mechanical ventilation and intensive care unit stay in patients with ventilator-associated pneumonia. The aim of the present study was to assess the presence of APMV in bronchoalveolar lavage fluid samples of critically ill patients suspected of ventilator-associated pneumonia. The study was conducted in the intensive care unit of the Maastricht University Medical Centre. All consecutive bronchoalveolar lavage fluid samples obtained between January 2005 and October 2009 from patients suspected of ventilator-associated pneumonia were eligible for inclusion. All samples were analyzed by real-time PCR targeting the APMV. A total of 260 bronchoalveolar lavage fluid samples from 214 patients (139 male, 75 female) were included. Bacterial ventilator-associated pneumonia was confirmed microbiologically in 105 out of 260 (40%) suspected episodes of ventilator-associated pneumonia (86 patients). The presence of APMV DNA could not be demonstrated in the bacterial ventilator-associated pneumonia positive or in the bacterial ventilator-associated pneumonia negative bronchoalveolar lavage fluid samples. Although suspected, APMV appeared not to be present in critically ill patients suspected of ventilator-associated pneumonia, and APMV does not seem to be a frequent cause of ventilator-associated pneumonia.

Infections with mimivirus in patients with chronic obstructive pulmonary disease.

INTRODUCTION: Antibodies against mimivirus, a recently discovered giant virus, have been found in patients presenting with pneumonia suggesting a potential role for this virus as a respiratory pathogen. Several bacterial and viral pathogens have been associated with the occurrence of acute exacerbations in COPD. However, a large part of these exacerbations have an unknown cause. In the present study we evaluated the presence of mimivirus in sputum samples of COPD patients. METHODS: From March 2009 until January 2010 all sputum samples collected during stable conditions and during exacerbations of COPD patients, referred for pulmonary rehabilitation, were included. All sputum samples were analysed by real-time PCR targeting mimivirus. Furthermore, serum samples were analysed for the presence of antibodies against mimivirus. RESULTS: A total of 220 sputum samples from 109 patients were eligible for inclusion. None of the sputum samples showed the presence of mimivirus DNA. Antibodies against mimivirus were detected in 3 serum samples from 3 patients, of which one showed an increase in antibody-titre. CONCLUSIONS: Although mimivirus was suggested as a potential respiratory pathogen, its presence could not be confirmed in the present study-population of patients with COPD.