Detection of
            <i>Mycoplasma pneumoniae</i>
            in Spiked Clinical Samples by Nucleic Acid Sequence-Based Amplification

Loens, Katherine; Ursi, D.; Ieven, M.; Aarle, Pierre van; Sillekens, Peter; Oudshoorn, P.; Goossens, Herman

doi:10.1128/jcm.40.4.1339-1345.2002

Cited by 46 publications

(37 citation statements)

References 42 publications

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“…The NASBA assay, targeted at 16S rRNA, followed by an enzyme-linked gel assay (ELGA) was used to type M. pneumoniae (55). Later on, NASBA in combination with ELGA and electrochemiluminescence detection was used to detect M. pneumoniae RNA in nucleic acid extracts from respiratory specimens (47). The Q␤ replicase assay was applied to detect synthetic M. pneumoniae 16S rRNA transcripts and seems to be less sensitive than PCR (63).…”

Section: Technical Aspectsmentioning

confidence: 99%

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Molecular Diagnosis of Mycoplasma pneumoniae Respiratory Tract Infections

et al. 2003

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2Mycoplasma pneumoniae is responsible for 10 to 20% of the cases of community-acquired pneumonia and has been associated with acute exacerbations of asthma (22). M. pneumoniae is also implicated in mild acute respiratory infections, such as sore throat, pharyngitis, rhinitis, and tracheobronchitis (2).Correct diagnosis of M. pneumoniae infections is important to allow the appropriate antibiotic treatment of patients, since it is impossible to identify a M. pneumoniae infection solely on the basis of clinical signs and symptoms. It should decrease inappropriate use of antibiotics, influence the patient outcome by reduction of morbidity and mortality, and improve our knowledge of the prevalence of the causes of so-called atypical pneumonia.Conventional assays for the detection of M. pneumoniae have their limitations, resulting in the need for more accurate diagnostic methods. Culture is time-consuming and relatively insensitive, because M. pneumoniae grows slowly in vitro, requiring 2 to 5 weeks for colonies to become visible. Serological methods, particularly the complement fixation (CF) test, are most widely used. The sensitivity of these assays depends on whether the first serum sample is collected early or late after the onset of disease and on the availability of paired serum samples collected with an interval of 2 to 3 weeks. Immunoglobulin M (IgM) assays which are more sensitive than the CF test have been developed, but the IgM response may be nonspecific (61) or absent, particularly in adults (70). Hybridization with DNA probes has also been proposed as a rapid and specific procedure to replace culture, but it lacks sensitivity (35).Nucleic acid amplification techniques (NAATs) have the potential to produce rapid, sensitive, and specific results, allowing early appropriate antibiotic therapy.In the absence of a reference method, the so-called "gold standard" for the diagnosis of an M. pneumoniae infection, either an expanded gold standard or the technique of latent class analysis (LCA) should be applied to calculate the sensitivity and specificity of the available diagnostic tests. The technique of LCA can be used if at least three independent techniques can be compared. Thus far, only a few PCR tests and a limited number of studies applying culture, serology, and several NAATs targeting different genes to detect M. pneumoniae have been adequately evaluated.Since NAATs targeting DNA can detect both viable and nonviable organisms, detecting RNA by reverse transcriptase PCR (RT-PCR) or nucleic acid sequence-based amplification (NASBA) may be a useful method to identify productive M. pneumoniae infections.The possible long-term carrier state of M. pneumoniae in the respiratory tract may hinder the evaluation of different diagnostic tests for the diagnosis of acute infections.An overview of the peer-reviewed literature on the use of NAATs to detect M. pneumoniae since 1989 is given. Search combinations were M. pneumoniae and PCR, M. pneumoniae and diagnosis, and M. pneumoniae and amplification. This minireview...

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Section: Technical Aspectsmentioning

confidence: 99%

“…Specimens should be transported to the laboratory as soon as possible and stored at 4°C or frozen at Ϫ70°C. RNA specimens should be processed or frozen at Ϫ70°C as soon as possible to prevent RNA degradation (6,47).…”

Section: Technical Aspectsmentioning

confidence: 99%

Molecular Diagnosis of Mycoplasma pneumoniae Respiratory Tract Infections

et al. 2003

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“…The sensitivity of the NASBA assay for C. pneumoniae 16S rRNA was studied by using 10-fold dilutions of suspensions of C. pneumoniae in physiologic water or dilutions of wild-type 16S rRNA generated in vitro in water. The sensitivity was also studied by using 10-fold dilutions of C. pneumoniae added in quadruplicate to protease-treated samples (20) of the respiratory specimen pools.…”

Section: Methodsmentioning

confidence: 99%

“…Nucleic acids were extracted as described by Boom et al (3) by using the NucliSens basic kit extraction module (bioMérieux). Briefly, 100 l of all protease-treated clinical specimens, protease-treated respiratory specimen pools (20), or aliquots of a bacterial culture was added to a guanidinium thiocyanate (GuSCN) lysis solution, pH 6.2, and mixed vigorously for rapid lysis. Fifty microliters of activated silica was added.…”

Section: Methodsmentioning

confidence: 99%

“…The authors concluded that the specimens could be kept at 4°C, provided that the transportation time was as short as possible, since most RNA degradation is likely to occur during transportation of the specimens. Furthermore, specimens should be processed on the day of sampling and should be stored at Ϫ70°C, thus stabilizing the RNA for at least 6 months, as found previously for NASBA assays for M. pneumoniae (20) and human rhinoviruses (19).…”

Section: Vol 44 2006mentioning

confidence: 99%

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Development of Conventional and Real-Time Nucleic Acid Sequence-Based Amplification Assays for Detection of Chlamydophila pneumoniae in Respiratory Specimens

et al. 2006

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Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Chlamydophila pneumoniae 16S rRNA by using the NucliSens basic kit (bioMérieux, Boxtel, The Netherlands). The assay was originally developed as a conventional NASBA assay with electrochemiluminescence detection and was subsequently adapted to a real-time NASBA format by using a molecular beacon. C. pneumoniae RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild-type C. pneumoniae RNA and 0.1 inclusion-forming unit (IFU) of C. pneumoniae. In spiked respiratory specimens, the sensitivity of the C. pneumoniae NASBA assay varied between 0.1 and 1 IFU/100 l sample, depending on the type of specimen. Finally, conventional and real-time NASBA were applied to respiratory specimens previously tested by PCR. A 100% concordance between the test results was obtained.Chlamydophila pneumoniae is a common etiologic agent of respiratory tract infections in humans and is responsible for 3% of all cases of community-acquired pneumonia and 43% of all cases during an isolated miniepidemic (8,13,16,23,24). It has been associated with asthma (12), chronic obstructive pulmonary disease (21), chronic pharyngitis (9), a wide range of mild to serious extrapulmonary complications (11), and atherosclerosis (5).In the past, diagnosis of infection by this organism was based on the detection of fourfold rises in antibody titers in the microimmunofluorescence test or an elevated immunoglobulin G (IgG) or IgM titer when only a single serum sample was available. Culture confirmation of the diagnosis of C. pneumoniae infection, followed by the propagation of clinical isolates, has proven difficult to date. Therefore, nucleic acid amplification techniques have been introduced. PCR was shown to be considerably more sensitive than culture for the detection of C. pneumoniae (1, 2).Real-time nucleic acid sequence-based amplification (NASBA; bioMérieux, Boxtel, The Netherlands) is targeted at RNA. NASBA makes use of the simultaneous enzymatic activities of avian myeloblastosis virus reverse transcriptase, RNase H, and T7 RNA polymerase and molecular beacons (15) under isothermal conditions.The whole process of amplification and detection runs in a fluorescent reader. The same amplicons can also be used for conventional electrochemiluminescence (ECL) detection (14). The real-time NASBA technique has already been successfully applied for the quantification of human immunodeficiency virus type 1 isolates (7) and for the detection of Mycoplasma pneumoniae (18).The aim of this study was to develop both a conventional assay and a real-time NASBA assay for the detection of C. pneumoniae in respiratory specimens based on NASBA amplification of a 16S rRNA target sequence by using the NucliSens basic kit (10, 17) and to compare the results of the two assays. MATERIALS AND METHODSBacterial strains. C. pneumoniae (ATCC VR-1355) was grown on HEp-2 cells. Shell v...

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Bacterial Infections of the Lower Respiratory Tract

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Detection of Mycoplasma pneumoniae in Spiked Clinical Samples by Nucleic Acid Sequence-Based Amplification

Cited by 46 publications

References 42 publications

Molecular Diagnosis of Mycoplasma pneumoniae Respiratory Tract Infections

Molecular Diagnosis of Mycoplasma pneumoniae Respiratory Tract Infections

Development of Conventional and Real-Time Nucleic Acid Sequence-Based Amplification Assays for Detection of Chlamydophila pneumoniae in Respiratory Specimens

Bacterial Infections of the Lower Respiratory Tract

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