The Pathogenesis Of Mycoplasma Pneuoniae Infection And Molecular Diagnosis Study

Part1The pathogenesis of Mycoplasma pneumoniae infectionBackground:Mycoplasma pneumoniae (M. pneumoniae) was first isolated from the sputum of a patient suffered from primary atypical pneumonia by Eaton et al. in1944. It is one of the most common pathogens causing respiratory infection worldwide in all aged people. M. pneumoniae induces upper respiratory tract infection, tracheitis, bronchitis, bronchiolitis, community-acquired pneumonia, and may induce or aggravates asthma. M. pneumoniae infections are basically considered as self-limited with good prognosis. However, severe and refractory M. pneumoniae pneumonia increased these years. Some patients even develop life-threatening complications. The mechanisms of M. pneumoniae pathogenesis include cytadherence, intracellular localization, cytotoxicity, and immune response. However, the mechanism of pathogenesis still needs to be elucidated.Our previous studies have shown that M. pneumoniae induced reactive oxygen species (ROS) in A549cells, which in turn led to DNA damage. Can M. pneumoniae induce similar effect of DNA damage in different cell lines? Cytadherence is considered as an important mechanism in the pathogenesis of M. pneumoniae. Thus, is cytadherence required for ROS induced by M. pneumoniae? Moreover, M. pneumoniae induced vimentin upregulation after infection in A549cells, then what is the function of vimentin in the pathogenesis of M. pneumoniae?In light of the above information, this study focused on the following issues:the difference in DNA damage induced by M. pneumoniae in different cell lines; the relationship between cytadhesion and M. pneumoniae induced ROS in A549cells; the signal transduction pathways in the upregulation of vimentin in M. pneumoniae-infected A549cells; the function of vimentin in A549cells during M. pneumoniae infection.Methods:1. DNA damage was detected by immunofluorescent microscopy and comet assay.2. ROS levels were detected by flow cytometry.3. The change of vimentin was observed by immunofluorescent microscopy.4.The molecules in MAPK and Ca2+/CaM-CaMK Ⅱ pathways were detected by western blot or fluorescence probe method.5. The expression of vimentin in A549cells was modulated by siRNA technique.Results:1. In immunofluorescence assay, M. pneumoniae induced γH2AX foci formation, an indicator for DNA damage, after12h and24h, but not4h in A549cells. It also induced γH2AX foci formation after4h, but not12h or24h in HEK293cells. The comet assay results indicated that M. pneumoniae induced DNA damage in A549cells after6h and12h.2. M. pneumoniae with normal or decreased cytadherence ability induced similar levels of ROS in A549cells.3. M. pneumoniae induced vimentin fluorescence intensity.4. The levels of ERK1/2, p-ERK1/2, p38, p-p38and Ca2+concentration were not changed after M. pneumoniae infection in A549cells.5. Knockdown of vimentin in A549cells did not affect cell morphology and viability, however, lower γH2AX foci formation was found in such cells after M. pneumoniae infection compared to normal A549cells. Conclusions:1. M. pneumoniae induced DNA damage in both A549cells and HEK293cells with cell-type specific responses.2. M. pneumoniae induced ROS generation was cytadherence independent.3. M. pneumoniae induced upregulation of vimentin. MAPK (ERK1/2and p38) and Ca+/CaM-CaMK Ⅱ pathways did not participate in this event.4. Vimentin may affect the extent of DNA damage in M. pneumoniae-infected A549cells. Part2The molecular diagnosis of Mycoplasma pneumoniae in different respiratory specimensBackground:For the reasons that the manifestations of M. pneumoniae infection are non-specific, and treatment of M. pneumoniae infection with P-lactam antibiotics is ineffective, correct laboratory diagnosis is extremely important. However, laboratory diagnosis is difficult by lacking standardized, sensitive and specific methods for detection of M. pneumoniae. Several diagnostic methods are used to detect M. pneumoniae infection, including culture, serology and molecular-based detection assays. Now serology and PCR assay are the two main laboratory tests for M. pneumoniae infection. Nasopharyngeal aspirate (NPA) and bronchoalveolar lavage (BAL) could be both used for PCR assay. Little information was compared about the sensitivity and specificity of PCR using different specimens including bronchoalveolar lavage (BAL) and nasopharyngeal aspirate (NPA). The aim of the present study was to evaluate diagnostic values of different specimens by fluorescence quantitative real-time PCR and to find clinical features helpful to diagnose M. pneumoniae pneumonia (MPP).Methods:1. Four hundred and six hospitalized pneumonia children were studied. M. pneumoniae DNA in NPA and BAL samples were detected by fluorescence quantitative real-time PCR. M. pneumoniae-specific IgM was tested by ELISA.2. Records of all patients were reviewed. Important information, including complete blood count, immunoglobulins and T lymphocyte were collected.Results:1. MPP were diagnosed based on positive M. pneumoniae-specific IgM in101(24.9%) children. The median ages of MPP and non-MPP children were4.1and2.4years, respectively, with significant difference between them (p<0.001).2. Laboratory results including leukocyte count, neutrophil percentage, immunoglobulins, except serum IgM, subgroups of T lymphocyte, and BAL cell count had no significant differences in MPP and non-MPP.3. BAL macrophage cell percentage was lower in BAL-PCR positive children (p=0.003), while BAL neutrophil percentage was higher in BAL-PCR positive children (p=0.007). 4. PCR from NPA and BAL were similar in diagnostic parameters, including sensitivity, specificity. PPV, and NPV (78.6%,63.4%,39.8%, and90.6%for NPA-PCR, respectively;70.3%,58.7%,36.0%, and85.6%for BAL-PCR, respectively).Conclusions:1. Age is a prefigurative factor in MPP.2. BAL is useful in defining local inflammatory condition.3. NPA is better than BAL as PCR sample in MPP diagnosis for similar performance in PCR assay, cheap, and less invasive.