Growth in serum-free medium improves isolation of Chlamydia pneumoniae

Maass, Matthias; Essig, Andreas; Marre, R.; Henkel, W

doi:10.1128/jcm.31.11.3050-3052.1993

Cited by 19 publications

(8 citation statements)

References 23 publications

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“…The cell growth medium was Eagle's minimal essential medium with nonessential amino acids and 2 mM glutamine (EMEM) (GIBCO/BRL GmbH, Eggenstein, Germany), supplemented with 10% fetal calf serum (Biochrom KG, Berlin, Germany). Chlamydiae were continuously cultured as previously described (12,14,16,19) by centrifugation (2,000 x g at 35°C for 45 min) of infectious inocula onto host cell monolayers in multiwell tissue culture plates. Supernatants were replaced by the chlamydial isolation medium consisting of EMEM with 1 ,ug of cycloheximide (Sigma Chemical Co., St. Louis, Mo.)…”

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confidence: 99%

Comparison of sample preparation methods for detection of Chlamydia pneumoniae in bronchoalveolar lavage fluid by PCR

Maass

Dalhoff

1994

J Clin Microbiol

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Amplification inhibitors can lead to false-negative results for PCR. In order to evaluate the reliability of PCR for the detection of Chlamydia pneumoniae, the presence of PCR inhibitors in 75 bronchoalveolar lavage specimens was assessed after treatment by various sample preparation methods. Specimens were collected from patients with acute respiratory infections, including four cases of proven C. pneumoniae infection. Substances inhibitory to the amplification of chlamydial DNA continued to be present in 12% of the samples treated according to the commonly used single-step proteinase K digestion and in 31% of the samples processed by heat treatment. However, the complexing of DNA-contaminating proteins and polysaccharides from digested specimens to cetyltrimethylammonium bromide (CTAB) followed by DNA extraction efficiently removed inhibitors from all experimental samples and provided subsequent identification of all positive clinical samples by PCR. The CTAB method and proteinase K treatment had comparable detection limits of

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confidence: 99%

Comparison of sample preparation methods for detection of Chlamydia pneumoniae in bronchoalveolar lavage fluid by PCR

Maass

Dalhoff

1994

J Clin Microbiol

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“…Those values indicate markedly lower survival rates than were achieved in our study by the use of FCS-supplemented transport media. FCS apparently must not be omitted from transport media, although its use in the chlamydial isolation medium for cell culture is not obligatory (13). As in our study, a stabilizing effect of FCS during prolonged storage and a decreasing viability with elevated temperatures and extended holding periods have been noted by Theunissen et al (24), but some results in their investigation, such as the 100% retrieval reported to have occurred in SPG with 10% FCS after 48 h at 5 to 20ЊC, differ from those of our study.…”

Section: Discussionmentioning

confidence: 99%

“…Host cells were grown in Eagle's minimal essential medium with nonessential amino acids and 2 mM glutamine (EMEM) (GIBCO/ BRL GmbH, Eggenstein, Germany), and 10% fetal calf serum (FCS) (Biochrom KG, Berlin, Germany). Chlamydiae were continuously cultured in an adaptation of recent protocols (13,14,18,26): briefly, infectious inocula of the stock strains were centrifuged at 2,000 ϫ g at 35ЊC for 45 min onto HEp-2 monolayers, and then supernatants were replaced by chlamydial isolation medium consisting of EMEM with 1 g of cycloheximide per ml (Sigma Chemical Co., St. Louis, Mo.). After 3 days of incubation at 35ЊC in 5% CO 2 , chlamydial growth was demonstrated by staining inclusions with a C. pneumoniae-specific mouse monoclonal antibody and fluorescein isothiocyanate-coupled secondary antibody according to the manufacturer's instructions (Cellabs Diagnostics Pty Ltd., Sydney, Australia).…”

Section: Methodsmentioning

confidence: 99%

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Transport and storage conditions for cultural recovery of Chlamydia pneumoniae

Maass

Dalhoff

1995

J Clin Microbiol

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Chlamydia pneumoniae is characterized by rapidly decreasing viability outside the host cell, and efficient preservation of its infectivity is a prerequisite for subsequent cell culture recovery. Extracellular survival of three C. pneumoniae stock strains and three wild-type strains subjected to simulated conditions of transport was therefore examined in order to establish recommendations for transport and storage of clinical specimens. The presence of fetal calf serum in transport media as well as refrigeration distinctly improved chlamydial retrieval during prolonged transport. Loss of infectivity was kept to a minimum in Eagle's minimal essential medium or sucrose-phosphate-glutamine medium. Storage at 22؇C permitted a stock strain recovery of >81% after 12 h. When refrigeration to 4؇C was provided, recovery rates of >74% could be achieved after 48 h. Though the strains were from different geographic regions, requirements for good survival were comparable and should therefore apply worldwide. The results indicate that the laboratory strains are not extremely labile. However, comparative examination of the wild-type strains showed less stability: primary isolates were not satisfactorily retrievable beyond 4 h at 22؇C or beyond 24 h at 4؇C. Further extension of storage times resulted in rapidly decreasing recovery, indicating a requirement to freeze samples at ؊75؇C to preserve viability. Adherence to the shorter storage periods suggested by the data obtained with primary isolates is recommended to ensure successful transport until more extensive testing with clinical materials is available.

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“…In a pilot study with asthma patients, an elevated IgG antibody level with a concomitant elevated IgA antibody level proved to be a useful marker of chronic C. pneumoniae infection (13). Unlike for Chlamydia trachomatis infections, culture and direct antigen detection with clinical specimens have proven to be unsuccessful in both acute and chronic C. pneumoniae infections (6,9,17,27). The PCR test is promising, but it is expensive and not yet available for routine analysis.…”

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Measurement of sputum antibodies in the diagnosis of acute and chronic respiratory infections associated with Chlamydia pneumoniae

Hertzen

Leinonen

Surcel

et al. 1995

Clin Diagn Lab Immunol

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The aim of this study was to develop methods for the measurement of sputum antibodies in the laboratory diagnosis of acute and chronic lower respiratory tract infections caused by Chlamydia pneumoniae. Paired serum specimens, sputum specimens, and pharyngeal or nasopharyngeal swabs were obtained from 97 patients; 51 of them had community-acquired pneumonia, and 46 had chronic obstructive pulmonary disease (COPD). C. pneumoniae-specific serum immunoglobulin G (IgG), IgA, and IgM antibodies were measured by the microimmunofluorescence (micro-IF) test. For sputa, specific IgA and IgG antibodies were measured by the micro-IF test and secretory IgA (sIgA) was measured by enzyme immune assay (EIA) with C. pneumoniae elementary bodies as the antigen. Sputum IgA and sIgA antibodies to C. pneumoniae were found, respectively, in 52 and 51% of the COPD patients. Elevated levels of stable serum IgG and IgA antibodies (IgG titer of >128 and IgA titer of >40), suggesting chronic infection, were found in 54% of the COPD patients. The sensitivity for the sputum IgA micro-IF test compared with elevated serum antibody levels was 87.5%, and that for the sputum sIgA EIA was 88%; the respective specificities were 90 and 95%. Acute C. pneumoniae infection was diagnosed in seven pneumonia patients, and two (29%) of these patients were positive by sputum EIA antibody measurements. Two pneumonia patients without acute infection had stable elevated IgG and IgA levels in their sera, and both of them were sputum antibody positive. We conclude that the measurement of IgA antibodies to C. pneumoniae in sputum is a useful additional diagnostic tool for chronic C. pneumoniae infections.

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Growth in serum-free medium improves isolation of Chlamydia pneumoniae

Cited by 19 publications

References 23 publications

Comparison of sample preparation methods for detection of Chlamydia pneumoniae in bronchoalveolar lavage fluid by PCR

Comparison of sample preparation methods for detection of Chlamydia pneumoniae in bronchoalveolar lavage fluid by PCR

Transport and storage conditions for cultural recovery of Chlamydia pneumoniae

Measurement of sputum antibodies in the diagnosis of acute and chronic respiratory infections associated with Chlamydia pneumoniae

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