A key unsolved question in the current coronavirus disease 2019 (COVID-19) pandemic is the duration of acquired immunity. Insights from infections with the four seasonal human coronaviruses might reveal common characteristics applicable to all human coronaviruses. We monitored healthy individuals for more than 35 years and determined that reinfection with the same seasonal coronavirus occurred frequently at 12 months after infection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus responsible for an ongoing pandemic. To date, there is limited evidence of reinfection by SARS-CoV-2, although it is generally assumed that reinfections by coronaviruses occur. To prepare for future waves of Coronavirus Disease 2019 (COVID-19), it is important to elucidate the duration of protection to reinfection for which the seasonal coronaviruses might serve as an informative model. There are four species of seasonal coronaviruses-HCoV-NL63, HCoV-229E, HCoV-OC43 and HCoV-HKU1-that all can cause respiratory tract infections but

A bacterial pathogen is identified in approximately one in five adult patients with LRTI in primary care, and a viral pathogen in just under half, with mixed infections in one in ten. Penicillin-resistant pneumococci and β-lactamase-producing H. influenzae are uncommon. These new findings support a restrictive approach to antibiotic prescribing for LRTI and the use of first-line, narrow-spectrum agents in primary care.

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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...

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 ...

Because of the absence of well-standardized both in-house and FDA-approved commercially available diagnostic tests, the reliable diagnosis of respiratory infection due to Mycoplasma pneumoniae remains difficult. In addition, no formal external quality assessment schemes which would allow to conclude about the performance of M. pneumoniae diagnostic tests exist. In this review, the current state of knowledge of M. pneumoniae-associated respiratory infections in the context of epidemiological studies published during the past 5 years is discussed, with particular emphasis on the diagnostic strategies used and their impact on results. The role of M. pneumoniae as a cause of respiratory tract infections (RTIs) differs from study to study due to geographical and epidemiological differences, as well as to the application of different diagnostic techniques and criteria used.

An external quality assessment (EQA) panel consisting of a total of 48 samples in bronchoalveolar lavage (BAL) fluid or transport medium was prepared in collaboration with Quality Control for Molecular Diagnostics (QCMD) (www.qcmd.org). The panel was used to assess the proficiency of the three laboratories that would be responsible for examining the 6,000 samples to be collected in the GRACE Network of Excellence (www.grace-lrti.org). The main objective was to decide on the best-performing testing approach for the detection of influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus (RSV), human metapneumovirus, coronavirus, rhinovirus, adenovirus, Chlamydophila pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila by nucleic acid amplification techniques (NAATs). Two approaches were chosen: (i) laboratories testing samples using their in-house procedures for extraction and amplification and (ii) laboratories using their in-house amplification procedures on centrally extracted samples. Furthermore, three commercially available multiplex NAAT tests-the ResPlex (Qiagen GmbH, Hilden, Germany), RespiFinder plus (PathoFinder, Maastricht, The Netherlands), and RespiFinder Smart 21 (PathoFinder) tests-were evaluated by examination of the same EQA panel by the manufacturer. No large differences among the 3 laboratories were noticed when the performances of the assays developed in-house in combination with the inhouse extraction procedures were compared. Also, the extraction procedure (central versus local) had little effect on performance. However, large differences in amplification efficacy were found between the commercially available tests; acceptable results were obtained by using the PathoFinder assays.

Mycoplasma pneumoniae (M. pneumoniae) belongs to the class Mollicutes and has been recognized as a common cause of respiratory tract infections (RTIs), including community-acquired pneumonia (CAP), that occur worldwide and in all age groups. In addition, M. pneumoniae can simultaneously or sequentially lead to damage in the nervous system and has been associated with a wide variety of other acute and chronic diseases. During the past 10 years, the proportion of LRTI in children and adults, associated with M. pneumoniae infection has ranged from 0 to more than 50%. This variation is due to the age and the geographic location of the population examined but also due to the diagnostic methods used. The true role of M. pneumoniae in RTIs remains a challenge given the many limitations and lack of standardization of the applied diagnostic tool in most cases, with resultant wide variations in data from different studies. Correct and rapid diagnosis and/or management of M. pneumoniae infections is, however, critical to initiate appropriate antibiotic treatment and is nowadays usually done by PCR and/or serology. Several recent reviews, have summarized current methods for the detection and identification of M. pneumoniae. This review will therefore provide a look at the general principles, advantages, diagnostic value, and limitations of the most currently used detection techniques for the etiological diagnosis of a M. pneumoniae infection as they evolve from research to daily practice.