Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples

Strålin, Kristoffer; Bäckman, Anders; Holmberg, Hans; Fredlund, Hans; Olcén, Per

doi:10.1111/j.1600-0463.2005.apm1130203.x

Cited by 65 publications

(55 citation statements)

References 53 publications

(66 reference statements)

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“…PCR has previously been shown to identify a high rate of colonisation of H. influenzae in patients with chronic obstructive pulmonary disease [33], but H. influenzae was also identified in 22% by BAL culture and 37% by BAL mPCR in 27 patients who had never smoked. As mPCR has shown a high analytical specificity for H. influenzae [12], and as sputum mPCR was positive for H. influenzae in only two (7.7%) out of 26 CAP patients with definite aetiologies other than H. influenzae in the present authors' previous study [13], the high rate of H. influenzae in the present study probably represents colonisation. Hence, it would be interesting to test mPCR on BAL samples from another LRTI population and another control population with a lower smoking frequency.…”

Section: Discussionmentioning

confidence: 55%

“…By serial dilution of bacterial strains, the detection levels of the mPCR have been shown to be 10 2 cfu?mL -1 of sample for S. pneumoniae and H. influenzae, and 10 3 genome copies or inclusion-forming units per millilitre of sample for M. pneumoniae and C. pneumoniae [12].…”

Section: Multiplex Pcrmentioning

confidence: 99%

“…A detailed description of the development and procedures of the mPCR is presented in a previous paper [12]. mPCR was performed under blinded conditions for simultaneous identification of specific genes for S. pneumoniae (lytA, 229 bp), M. pneumoniae (P1, 483 bp), C. pneumoniae (ompA, 368 bp), and H. influenzae (16S rRNA, 538 bp).…”

Section: Multiplex Pcrmentioning

confidence: 99%

“…The method showed an analytical sensitivity of 100% (89 out of 89) and a specificity of 99% (167 out of 168) when 257 bacterial strains (37 different species) were tested [12]. mPCR has been evaluated on sputum samples and nasopharyngeal samples from adults with community-acquired pneumonia (CAP) and controls with promising results [13].…”

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confidence: 99%

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Evaluation of a multiplex PCR for bacterial pathogens applied to bronchoalveolar lavage

Strålin

Korsgaard²,

Olcén³

2006

European Respiratory Journal

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The present study assessed the diagnostic usefulness of a multiplex PCR (mPCR) for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae applied to bronchoalveolar lavage (BAL).Fibreoptic bronchoscopy was performed on 156 hospitalised adult patients with lower respiratory tract infection (LRTI) and 36 controls. BAL fluid was analysed with bacterial culture and mPCR.By conventional diagnostic methods, S. pneumoniae, H. influenzae, M. pneumoniae and C. pneumoniae were aetiological agents in 14, 21, 3.2 and 0% of the LRTI patients, respectively. These pathogens were identified by BAL mPCR in 28, 47, 3.2 and 0.6% of cases, respectively, yielding sensitivities of 86% for S. pneumoniae, 88% for H. influenzae, 100% for M. pneumoniae and 0% for C. pneumoniae, and specificities of 81, 64, 100 and 99% for S. pneumoniae, H. influenzae, M. pneumoniae and C. pneumoniae, respectively. Of the 103 patients who had taken antibiotics prior to bronchoscopy, S. pneumoniae was identified by culture in 2.9% and by mPCR in 31%. Among the controls, mPCR identified S. pneumoniae in 11% and H. influenzae in 39%.In lower respiratory tract infection patients, bronchoalveolar lavage multiplex PCR can be useful for identification of Streptococcus pneumoniae, Mycoplasma pneumoniae and Chlamydophila pneumoniae. The method appears to be particularly useful in patients treated with antibiotics.

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Section: Discussionmentioning

confidence: 55%

Section: Multiplex Pcrmentioning

confidence: 99%

Section: Multiplex Pcrmentioning

confidence: 99%

mentioning

confidence: 99%

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Evaluation of a multiplex PCR for bacterial pathogens applied to bronchoalveolar lavage

Strålin

Korsgaard²,

Olcén³

2006

European Respiratory Journal

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“…The aim of the present study was to estimate the diagnostic accuracy of respiratory culture and a single-run multiplex PCR (mPCR) for specific genes of Streptococcus pneumoniae (lytA), Haemophilus influenzae (16S rRNA, with verification by PCR for P6), Mycoplasma pneumoniae (P1), and Chlamydophila pneumoniae (ompA) (12) applied to respiratory samples in CAP patients.…”

mentioning

confidence: 99%

Etiologic Diagnosis of Adult Bacterial Pneumonia by Culture and PCR Applied to Respiratory Tract Samples

Strålin¹,

Tornqvist

Kaltoft

et al. 2006

J Clin Microbiol

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Respiratory culture and multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, and Chlamydophila pneumoniae were applied to sputum, nasopharyngeal swabs, and nasopharyngeal aspirates from 235 adult patients with community-acquired pneumonia and 113 controls. Both culture and multiplex PCR performed well with the different samples and appear to be useful as diagnostic tools.As an etiologic agent can rarely be identified in more than 50% of patients with community-acquired pneumonia (CAP) (9), further development of diagnostic methods has been encouraged (4, 10).The aim of the present study was to estimate the diagnostic accuracy of respiratory culture and a single-run multiplex PCR (mPCR) for specific genes of Streptococcus pneumoniae (lytA), Haemophilus influenzae (16S rRNA, with verification by PCR for P6), Mycoplasma pneumoniae (P1), and Chlamydophila pneumoniae (ompA) (12) applied to respiratory samples in CAP patients.(The study was approved by the ethics committee of the Ö rebro County Council [868-1999; 556-2000].)In a prospective study described previously (14), 235 hospitalized CAP patients with X-ray infiltrates were enrolled. Their median age was 71 years (age range, 18 to 96 years), 40% belonged to severity risk classes IV and V, 14% had chronic obstructive pulmonary disease, and 22% were smokers. In 82% of the patients, the interval from the onset of illness to admission was 0 to 7 days.During the study period, 113 adult controls (median age, 69 years) without respiratory symptoms were enrolled. They were hospitalized for skin infection (n ϭ 14), urinary tract infection (n ϭ 14), arthritis or spondylitis (n ϭ 6), or planned orthopedic or urological surgery (n ϭ 79).The results of the respiratory cultures and mPCR analyses performed with specimens from the patients and the controls are shown in Table 1. Sputum samples were analyzed if there were more than five leukocytes per squamous epithelial cell (6). To obtain a nasopharyngeal aspirate (NpA), secretions from the nasopharynx were aspirated by a catheter connected to an electronic suction device. About 1 ml NaCl (0.85%) was then aspirated to collect the secretions situated within the catheter. Culture and mPCR of sputum, nasopharyngeal swabs (NpSs), and NpAs were performed as described previously (12). Multiplex PCR was performed blindly with samples previously frozen at Ϫ70°C.To establish the definite etiologies of CAP, we performed the following: blood culture for 235 patients, the NOW S. pneumoniae urinary antigen test (Binax) for 215 patients, the complement fixation test for M. pneumoniae (8) with paired serum specimens for 216 patients, the microimmunofluorescence test for C. pneumoniae (3) with paired serum specimens for 216 patients, and the indirect immunofluorescence test for H. influenzae (13) with paired serum specimens for 48 patients. Our criteria for definite CAP etiologies were as follows: for S. pneumoniae, a positive blood culture or a positive urinary antigen test (only visible result lines at l...

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Widespread of non‐typeable Haemophilus influenzae with high genetic diversity after two decades use of Hib vaccine in China

Dong

Shi

Cheng

et al. 2019

Clinical Laboratory Analysis

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This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. AbstractBackground: The aim of this study was to analyze the microbiological characteristics of nasopharyngeal carriage Haemophilus influenzae isolates collected from children with respiratory infections in Beijing hospital and Youyang Hospital of China. Methods:The serotypes of all isolates were determined using latex agglutinated antisera (a-f). The minimum inhibitory concentrations (MICs) of 11 antibiotics were determined using E-test strips. For the beta-lactamase-negative ampicillin-resistant (BLNAR) isolates, ftsI gene was sequenced based on fragments amplified by PCR. STs of H influenzae isolates were determined by multi-locus sequence typing. Results:The overall carriage rate of H influenzae in the study population was 9.1% (362/3984). One hundred and ninety H influenzae isolates which were selected in our study were non-typeable (NTHi) and 44 (23.2%) of them were positive for β-lactamase.All isolates were susceptible to ceftriaxone and levofloxacin. Susceptibility rates to erythromycin and sulfamethoxazole-trimethoprim in Beijing were significantly higher than Youyang (P < .05). Thirty-six BLNAR isolates were identified. The MLST analysis showed 108 STs in 190 isolates, the most common of which were ST408 (11, 5.8%), ST914 (10, 5.3%), ST57 (9, 4.7%), and ST834 (6, 3.2%). Twelve STs were detected in both of the study sites, which covered 63 isolates. Conclusions:All isolates in the present study were NTHi, which suggested widespread of this type in China. The BLNAR isolates were detected more frequently than before. Because high genetic diversity of NTHi isolates of H influenzae exists worldwide, it is important to continuously monitor these bacteria in the future.

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Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples

Cited by 65 publications

References 53 publications

Evaluation of a multiplex PCR for bacterial pathogens applied to bronchoalveolar lavage

Evaluation of a multiplex PCR for bacterial pathogens applied to bronchoalveolar lavage

Etiologic Diagnosis of Adult Bacterial Pneumonia by Culture and PCR Applied to Respiratory Tract Samples

Widespread of non‐typeable Haemophilus influenzae with high genetic diversity after two decades use of Hib vaccine in China

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