Sequential changes of Legionella antigens and bacterial load in the lungs and urines of a mouse model of pneumonia.

Legionella pneumophila is an important cause of community- and hospital-acquired pneumonia. In spite of the introduction of the urinary antigen detection method, Legionella pneumonia may be still underdiagnosed. We performed kinetic and quantitative analysis of diagnostic markers, such as bacterial loads, DNA assays, and antigen titers, in a 28-day time course murine model of L. pneumophila pneumonia. L. pneumophila replicated approximately 100-fold in the lungs of A/J mice in the first 48 h, and then became undetectable on day 14. Unexpectedly, pathogens other than L. pneumophila were consistently recovered from the lungs and livers at the acute phases, although those numbers were far below Legionella loads in the lungs. The peaks of specific antigen titer were observed on 48 h in the lungs, bronchoalveolar lavage (BAL) fluids, and urines and sustained positive even at 28 days after the infection. Especially, the lung homogenates and BAL fluids demonstrated 16 to 64 times higher levels of antigen titer than the urines by the end of observation. Legionella-specific DNA in the lungs was detected by polymerase chain reaction and loop-mediated isothermal amplification methods until 7 and 14 days after the infection, respectively. The inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, interleukin 6, and MIP-2, exhibited a peak on the acute phase, whereas the maximal production of high mobility group box 1 in the serum was observed on day 7. These results characterized the kinetic nature of diagnostic markers in L. pneumophila pneumonia. The present data suggested prolonged and compartmentalized deposition of antigen in the lungs, which may have an impact on the diagnosis of L. pneumophila pneumonia, especially in missed cases even after recovery from disease.

[Sensitive and rapid detection of Mycoplasma pneumoniae by loop-mediated isothermal amplification].

For establishment of a method for rapid diagnosis of Mycoplasma pneumoniae in clinical specimens, we designed and evaluated a loop-mediated isothermal amplification (LAMP) assay, using a set of primers targeting the SDC1 repetitive element of the M. pneumoniae genome. The method showed rapid and specific amplification for all the strains of M. pneumoniae tested (Type I and II), within 1 hour at 65 degrees C. No cross-reactivity with the most common causative organisms bacterial pneumonia was observed. The detection limit was shown as 6 copies, which was equal to or higher than that of the two nested PCRs used as references. Two hundred four clinical samples, comprising sputum samples, throat swabs, etc., were tested by both LAMP and PCR, and determination of the correlations revealed complete agreement individually (24 positives). The LAMP assay, a simple procedure in a closed system, allowed rapid amplification and accurate detection consistently; therefore we considered that it could become an caeasily available diagnostic method suitable for clinical situations requiring quick and appropriate decisions for treatments and care.

[Rapid and simple detection of Legionella species by LAMP, a mew DNA amplification method].

Legionella is a common cause of community-acquired respiratory tract infections and occasionally causes nosocomial pneumonia. Rapid and accurate detection of legionellae is important for diagnosis and treatment of patients. In order to detect legionellae, a new DNA amplification method was designed and evaluated. Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity, and rapidity under isothermal conditions at 65 degrees C. This method employs a DNA polymerase with strand displacement activity and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. The primers targeting 16S rRNA gene were designed in order to detect a wide range of Legionella species. We could specifically detect Legionella species including Legionella pneumophila, Legionella anisa, Legionella bozemanii, Legionella dumoffii, Legionella erythra, Legionella feeleii, Legionella gormanii, Legionella longbeachae, Legionella micdadei, Legionella oakridgensis, and Legionella sainthelensi. The detection limit of the assay was 6 cfu per test of L. pneumophila strain. Furthermore, all of the positive LAMP results could be obtained within 50 minutes. The LAMP method was able to detect a wide range of Legionella species with high specificity, sensitivity, rapidity, and a simple procedure.