Mycoplasma pneumoniae and viruses in acute respiratory tract infections in children were studied during the winter of 1992-1993 in Antwerp, Belgium. M. pneumoniae was diagnosed in nasopharyngeal aspirates by culture and polymerase chain reaction (PCR). For this, amplification of a fragment of the PI adhesin gene in samples prepared by two methods was compared in two laboratories, and in one laboratory, a fragment of the 16S rRNA gene was amplified. The sensitivity of culture versus PCR was 61.5%. Provided a specific internal control is used, sample preparation by freeze-boiling combined with PCR for the PI gene and amplicon detection by visual inspection of the electrophoresis gel can be recommended, although maximal results are obtained after hybridization. M. pneumoniae was present in 0.5% of patients <2 years old and 6.9% of patients >2. M. pneumoniae was second to respiratory syncytial virus or detected equally in lower respiratory infections.

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...

The objectives of this study were to evaluate the performance of the NucliSens easyMAG platform for nucleic acid extraction from different clinical specimens compared to NucliSens miniMAG platform and manual QIAGEN extraction. The NucliSens easyMAG and the NucliSens miniMAG showed equal performance on 215 throat swabs since real-time nucleic acid sequence-based amplification scored the same samples positive for Mycoplasma pneumoniae (n ‫؍‬ 9) and Chlamydia pneumoniae (n ‫؍‬ 5) RNAs, although internal control RNA was slightly better detected with the NucliSens easyMAG (99.3% versus 96.8%). NucliSens easyMAG extracted nucleic acids more efficiently (higher recovery and/or fewer inhibitors) compared to QIAGEN extraction by showing, on average, lower Ct values in real-time LightCycler PCR, although 4 individual specimen out of 45 were found positive only with QIAGEN. For nine M. pneumoniae-positive throat swabs, the mean difference in Ct values between NucliSens easyMAG extraction and QIAGEN extraction was ؊2.26 (range, ؊5.77 to ؉0.60); for the detection of five C. pneumoniae-positive throat swabs, the average difference in Ct values between the two methods was ؊3.38 (range, ؊6.62 to ؊2.02); and for the detection of cytomegalovirus in 24 blood samples, the mean difference in Ct values between the two methods was ؊0.95 (range, ؊5.51 to ؉1.68). The NucliSens easyMAG is considerably easier to perform, efficiently extracts nucleic acids from throat swabs and whole blood, is automated, and has high throughput.Numerous nucleic acid amplification tests are performed daily in an increasing number of clinical laboratories because of their high sensitivities and specificities. Further developments in the field have decreased the turnaround time and hands-on time. Nucleic acid extraction systems with high flexibilities in the type and number of samples to be handled and with a wide range of sample and elution volumes and short turnaround times provide a further advantage to adapt amplification techniques to clinical diagnostic requirements.A high-quality nucleic acid extract is expected to be free of amplification inhibitors and other substances that might affect enzyme substrates, and the target should be optimally recovered.The NucliSens easyMAG platform (bioMérieux, Boxtel, The Netherlands) is a second-generation system for automated isolation of nucleic acids from clinical samples based upon silica extraction technology (3). It is a benchtop instrument with the same reagents as the manual version, i.e., NucliSens miniMAG platform (bioMérieux) (4,14,17,19). Manual steps are limited to the loading of samples, reagents, and disposables. One to 24 samples can be analyzed in one run. The extraction method is universal and can be applied to a broad range of different specimens such as blood, sputum, serum, and throat swabs. The instrument can be used in combination with different amplification methods such as nucleic acid sequence-based amplification (NASBA) or PCR. Limited data are available on the NucliSens easyMAG extraction ...

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Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Mycoplasma pneumoniae. M. pneumoniae RNA prepared from a plasmid construct was used to assess the sensitivity of the assay, and an internal control for the detection of inhibitors was constructed. The sensitivity of the NASBA assay was 10 molecules of wild-type M. pneumoniae RNA generated in vitro and 5 color-changing units (