Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis.

BACKGROUND: Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients. RESULTS: The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis) in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 105 genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively.In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both bacteria. CONCLUSIONS: The PCR provides increased sensitivity and the multiplex format facilitates diagnosis of S. pneumoniae, H. influenzae and N. meningitidis and the assay enable detection after antibiotic treatment has been installed. Quantification increases the specificity of the etiology for pneumonia.

Antibiotic treatment and the diagnosis of Streptococcus pneumoniae in lower respiratory tract infections in adults.

OBJECTIVE: To analyze the possible influence of antibiotic treatment on the results of different diagnostic tests for the diagnosis of lower respiratory tract infections with Streptococcus pneumoniae. MATERIAL AND METHODS: A prospective cohort of 159 unselected adult immunocompetent patients admitted to Silkeborg County Hospital in Denmark with community-acquired lower respiratory tract infections underwent microbiological investigations with fiber-optic bronchoscopy with bronchoalveolar lavage, blood and sputum culture and urine antigen test for type-specific polysaccharide capsular antigens of S. pneumoniae. RESULTS: When stratified for antibiotic treatment prior to microbiological sampling, three different groups of patients with documented or probable infection with S. pneumoniae could be identified. The first group comprised 14 patients who were culture positive in one or more culture tests, where most (11/14) did not receive any antibiotic treatment within 24 hours of sampling. The second group consisted of nine patients with a positive urine antigen test where 8/9 and 9/9 received antibiotic treatment 24 and 48 hours, respectively, prior to urine sampling. Only a single patient was positive in both systems, making a total of 22 patients with documented pneumococcal infection. As a positive culture test was dependent on the absence of antibiotic treatment, whereas a positive urine antigen test depended on antibiotic treatment within 48 hours, the two tests were complementary in the diagnosis of infection with S. pneumoniae. The third group of patients with probable pneumococcal infection were identified as 26% and 20% of the remaining 137 patients with unknown or known non-pneumococcal etiology, respectively, who received recent antibiotic treatment within 2-4 weeks of diagnostic sampling. By comparison, 0% (p < 0.01) with documented pneumococcal infection received antibiotic treatment in weeks 2-4 prior to microbiological sampling. As such a further eight patients should be expected to have infection with S. pneumoniae but would test negative in both culture tests and the urine antigen test because of antibiotic treatment within weeks 2-4 prior to sampling. CONCLUSION: The diagnosis of infection with S. pneumoniae is very dependent on whether or not recent (within 2-4 weeks) or immediate (within 48 hours) antibiotic treatment has been given prior to microbiological sampling of patients. The results suggest an optimized diagnostic strategy with, if possible, sampling for culture prior to antibiotic treatment, while sampling for pneumococcal antigens should wait 24-48 hours for antibiotic treatment.

Pulmonary rehabilitation in clinical routine: a follow-up study.

OBJECTIVE: The effect of rehabilitation for chronic obstructive pulmonary disease has been well-documented in randomized controlled trials. Evidence-based guidelines support rehabilitation programmes for chronic obstructive pulmonary disease, but knowledge of their outcome in clinical practice is limited. The aim of this study was to assess the outcome of a clinical routine rehabilitation programme for chronic obstructive pulmonary disease implemented by a Danish regional hospital. MATERIAL AND METHODS: Changes in walk-distance (6-min walk-distance test; 6MWD), dyspnoea (Medical Research Council dyspnoea scale; MRC) and health-related quality of life (Short-Form 36; SF-36) were compared between and within completers and non-completers from baseline to the end of clinical routine rehabilitation, and at 6 and 12 months. The 8-week clinical routine rehabilitation comprised bi-weekly 90-min sessions of patient education and physical training. RESULTS: The hospital treated 521 patients during the study period. Of these, 175 were invited to join the study, 148 participated at baseline, and 98 at the 12-month follow-up. Completers' 6MWD was sustained from baseline to the end of clinical routine rehabilitation, but had declined by 12 months. Dyspnoea and health-related quality of life did not change. Seventy-five percent of completers felt better or much better after rehabilitation. CONCLUSION: The failure of completers to achieve expected outcomes shows a need for a stronger implementation effort and continuous quality control. Successful implementation in clinical routine requires targeted recruitment and overall programme improvement in general, and a stronger focus on physical training and staff competences.

Participation in pulmonary rehabilitation in routine clinical practice.

BACKGROUND AND AIMS: Denmark offers COPD rehabilitation to enable patients to tackle the consequences of COPD, but only a minority of the patients complete these programs. To increase the completion rate, an follow-up study was performed, to characterize COPD patients and to identify potential differences between those who complete and those who do not complete rehabilitation or do not even get a rehabilitation offer in daily clinical routine. METHODS: In- and out COPD-patients who participated in baseline tests were compared in terms of completion of rehabilitation, drop-out, and no rehabilitation offer. We obtained data on basic characteristics, co-morbidity, lung-function (FEV1), dyspnea (MRC), six-minute walkg-distance (6MWD), and quality of life (SF36). RESULTS: The source population counted 521 COPD patients of whom 256 were excluded (diagnosis withdrawn, death, moved away, long-term oxygen, severe illness). Patients who completed rehabilitation had a 15% longer 6MWD than patients not offered rehabilitation and a 10% longer 6MWD than drop-outs despite a significant lower subjective perception of physical function among completers than in the two other groups. Patients not offered rehabilitation had a slightly better lung function than the other two groups. This suggests that lower physical performance with the same (drop-outs) or even higher (not offered) lung function indicates a lower chance of completion. CONCLUSION: COPD patients who could potentially benefit most from completing rehabilitation seem to be deselected. A mere 9% completed rehabilitation within the study period and 23% ever completed. This demonstrates that the political target that 60% of COPD patients should be offered rehabilitation is still far away.

Does pulmonary rehabilitation work in clinical practice? A review on selection and dropout in randomized controlled trials on pulmonary rehabilitation.

AIM: To analyze randomized controlled trials (RCTs) on pulmonary rehabilitation (PR) to determine whether the patients who complete PR form a representative subset of the chronic obstructive pulmonary disease (COPD) target population and to discuss what impact this may have for the generalizability and implementation of PR in practice. MATERIAL AND METHODS: A review of 26 RCTs included in a Cochrane Review 2007. We analyzed the selection at three different levels: 1) sampling; 2) inclusion and exclusion; 3) and dropout. RESULTS: Of 26 studies only 3 (12%) described the sampling as the number of patients contacted. In these studies 28% completed PR. In all we found, that 75% of the patients suitable for PR programs were omitted due to sampling exclusion and dropout. Most of the study populations are not representative of the target population. CONCLUSION: The RCTs selected for the Cochrane review gave sparse information about the sampling procedure. The demand for high internal validity in studies on PR reduced their external validity. The patients completing PR programs in RCTs were not drawn from a representative subset of the target population. The ability to draw conclusions relevant to clinical practice from the results of the RCTs on PR is impaired.

Comparison of two different levels of physical training in patients with moderate to severe COPD.

Our objective was to compare the effect of a 4-week homebased low and middle intensity and frequency training program in patients with moderate to severe chronic obstructive pulmonary disease. From 124 patients hospitalized with chronic obstructive lung disease (COPD) in an 18-month period 65 fulfilled the inclusion criteria and were invited to participate. Only 31 (48%) accepted and among these only 20 patients (31% of invited) completed the 4-week study period. The walking time in seconds in a standardized treadmill walking test was unchanged after 4 weeks of low intensity training 60 minutes per week for two weekly training sessions. In contrast, the walking time in seconds increased 55% (p < 0.001) from 321 seconds to 499 seconds in 9 patients who completed 4 weeks of middle intensity training which comprised 2 1/2 hours of training per week for 5 weekly training sessions. There was no change in lung function over the 4 weeks but the combined score for physical quality of life (physical component summary) measured by SF-36 increased (p < 0.05) with both low intensity and middle intensity physical training. In conclusion, homebased physical training, which aims at improvements in patient performance and quality of life as part of pulmonary rehabilitation programs, is only accepted by about one-third of unselected patients with moderate to severe COPD. The minimum training time necessary to improve physical performance is 2-3 hours per week of middle intensity training.