A Study On Real-time Quantitative PCR Method For Diagnosis Of Mycoplasma Pneumonia Septicemia In Children

Subject:A Study on Real-time Quantitative PCR Method for Diagnosis of Mycoplasma Pneumonia Septicemia in ChildrenMajor:PediatricsAuthor:Wang HongboDirector:Cheng HuanjiMycoplasma pneumoniae (MP) is the major pathogen of the lower-respiratory tract infections and community-acquired pneumonia (CAP). It is among the smallest microorganisms, intermediate in size between bacteria and viruses, and due to its small genome and size, has caused diagnostic difficulties. MP causes pneumonia that can be fatal in all age groups, and in some, this same organism has the ability to produce invasive infection resulting in variable clinical manifestations. In recent years, the number of MP pneumonia cases has increased in China. The death toll caused by primary atypical pneumonia attracted more and more attention to determine its source. The causal agent remained unidentified in as many as 50% of cases. Mycoplasma pneumoniae is also the name for a syndrome associated with high fever and erythema with an infection by MP. While, until now, no publications accredit or verify this association, some physicians just presume that the hypothesis is true, choose an antibiotic with high plasma concentrations, like erythromycin, to treat the "mycoplasma septicemia," and believe that erythromycin is more effective than azithromycin.The traditional clinical diagnosis for mycoplasma infection is serology, whose sensitivity and specificity is from 55% to 100%, as statistics show. During the early stage of the MP infection, the antibody of specificity does not exist, and the result of serological test is usually negative or weakly positive. It is only during the recovery period and acute stages that the results of serology testing are more meaningful. So, with its hysteretic nature, serology testing is not instructive in confirming the pathogenesis. However, polymerase chain reaction (PCR) testing is a valuable technique for diagnosing the pathogeny of MP pneumonia at the early stage of the infection.The PCR method involves throat swabs, sputum, and bronchi-alveolar lavage fluid (BALf). For different reasons, these samples when tested by PCR also have their limitations, since MP pneumonia does not have a propensity for staying in the blood. So, if one can find a way to test blood by PCR, it will be very important for the diagnosis of mycoplasma pneumonia in its early stage. Presently, there are fewer testing results for mycoplasma pneumonia by whole blood, plasma and serum. Among the results, the positive rate is even lower, so the clinical treatment of mycoplasma infection can only be based on experience. PCR has the potential of increasing the sensitivity and rapidity, and many testing methods based on RNA VL are developed for a quick diagnosis of mycoplasma pneumonia.Compared with routine PCR, real-time fluorescent PCR has been widely applied with high specificity, pollution free and a high-degree of automation, and it can be measured with exceptional quality. The result can be obtained within 1 hour, which is very fast. The identity of the pathogeny could be measured with immediate feedback to the clinic. My research aims at investigating the possibility of measuring mycoplasma septicemia by PCR, studying the sensitivity and practicability of real-time fluorescent PCR for mycoplasma infection in children, and hoping that the result can be considered for clinical treatment in the differential diagnosis of pneumonia in all age groups.Method:â‘ Select 24 in-patients in our hospital,16 male,8 female, with ages ranging from 2 to 15. The average age was 6.2. They are diagnosed as mycoplasma pneumoniae pneumonia patients by immu-serological tests or/and throat swab tests, which are accorded with the standardization of in-patient procedure for mycoplasma pneumoniae in children, as regulated by Ministry of Health.â‘¡Test-group and control-group:24 patients in test group, and 20 in control group. The children in control-group do not catch respiratory tract infection during the test and half of a year before the test. Control group:13 male,7 female, with ages ranging from 2 to 13. There is no difference in gender and age between test-group and control-group. The samples are collected from the children before they eat in the morning, and the children are not treated with antibiotics, which are sensitive to mycoplasma pneumoniae, like azithromycin and erythromycin.â‘¢Instrument PE GeneAmp 5700, centrifugal machine, and kits are purchased from Da'an Gene Co., Ltd. Zhongshan University. The sensitivity is 1* 104copy/mL.â‘£ Before the samples are collected, the patients have had an empty stomach for more than 4 hours. The purple sample tubes from 3M are used. Once the samples are collected, they are centrifuged to separate the plasma and blood cells. Then, DNA is abstracted from plasma and blood cells respectively, and a PE GeneAmp 5700 is used for the PCR test. The negative control material, the gradient of positive control reference, and the conditions for the PCR reaction are established, followed by saving the file and beginning the reaction. After an hour, the reaction ends, and the results are saved and analyzed by the computer automatically.Results:8 of the patients showed positive in blood specimen by PCR test; 3 serum and lymphocyte specimens were positive by PCR; 2 serum specimens were positive by PCR test; 3 blood lymph-ocytes specimens were positive by PCR test.The relevance ratio of mycoplasma pneumoniae for serum was 5/24 (20.8%). In lymphocyte extract, it was 6/24 (25.0%). No obvious or significant statistical difference between the positive rate of the serum and lymphocyte specimens. Among the 8 patients with positive results,8 had high fever; sternums of 4 patients showed expanding pulmonary inflammation and a change to croupous pneumonia; 2 of the 4 patients with atelectasis, tested by fiberoptic bronchoscopy, and the mycoplasma in bronchoalveolar lavage fluid was positive as tested by PCR; among the other 4 patients,1 sternum showed pleural effusion,1 without obvious pleural effusion, but with syndrome of chest pain with breathing, which showed that pleurisy existed; 1 patient with cerebritis. While among the 16 patients with negative test results,12 with high fever,3 sternums of 4 patients showed croupous pneumonia,1 with atelectasis and the mycoplasma in bronchoalveolar lavage fluid was positive as tested by real-time PCR. No patients caught pleurisy or cerebritis. The 20 patients of the control group tested by PCR had no positive serum result. However,1 positive result was found in the lymphocyte extract.Conclusion:1. Using Real-time PCR to test the MP sections of specialty in the blood of the Children with MP pneumonia indicates the existence of mycoplasma septicemia. 2. The patient with mycoplasma septicemia tested by Real-time PCR for blood has the more prominent symptoms and severe clinical features.3. Real-time PCR blood testing for mycoplasma pneumoniae is helpful for the clinical diagnosis.