Application Of PCR Detection Method To The Diagnosis And Treatment In Children Legionaires' Disease

In 1976 when an outbreak of infectious disease with pneumonia as its major symptoms occurred among veterans attending a convention of American Legion in Philadelphia in the USA. The following year, American CDC isolated the pathogen and named it Legionella. As its main manifestaion is pneumonia, in a narrow sense, legionnaires's disease is also called legionella pneumonia. Legionnaires'disease (LD) accounts for an estimated 8000 to 18,000 cases of hospitalized community acquired pneumonia in the United States annually. Riskfactors for LD include advanced age and chronic illness such as diabetes, cancer, and renal disease. Legionella is a cytozoic gram-negative bacillus. The latest report has found that it has 48 strains and 70 serogroups. Among them legionella pneumophila has the closest relationship with human diseases. Legionella pneumophila infection with atypical pneumonia frequently manifests itself more systemic injury, clinical manifestations not typical, easy Misdiagnosis, Outbreak, epidemic as well as infectivity are significant characteristics of Legionnaires's disease. Clinic features of legionnaires's disease include fever, cough, dyspnea and a sheet of shade changes on chest X-ray. Besides, outbreak, epidemic as well as infectivity are also significant characteristics of Legionnaires's disease. Pediatrician responsible for their vigilance and attention.The infection of legionella pneumophila usually occur in adults and related reports are also common, while there are little studies on children's infection of legionella. It become urgent and necessary for us to know about the clinic features of children's legionella infection and establish quick and sensitive detection techniques.Purpose: Create a PCR method for detecting Legionella pneumophila by analyzing legionella peumophila mip gene, associated with serological TAT, to find out the clinic features of children's legionella infection. and treatment so as to provide a reliable basis.Methods: Legionella pneumophila microphage infectivity potentiator (mip) gene sequence Was amplified by PCR method ,using traditional serology TAT antibody detection methods, analysis of its specificity and sensitivity.1.Establishment of PCR method:Primers were designed according to legionella peumophila mip gene sequence. Streptococcus pneumoniae and non-legionella pneumophila LM were used as evaluation system for PCR detection.The tested bacteria were used as positive control, and the blank tubes were used as negative control.2.PCR techniques were adopted to detect the sputum and pharynx secretion of 95 children patients with respiratory inflammationThe DNA of the clinic specimens and experimental bacteria were extracted by classic saturated phenol-chloroform method. The primers were designed according to the legionella pneumophila mip gene sequence and then 628bp mip gene segments were amplified. The gene segments of LP1—14 and other strains as well as clinic specimens were also amplified by PCR method. The amplification products were electrophoresed on 2% agarose gel with EB in it and the results were observed under the ultraviolet light.3.The blood samples from the patients were collected and the serums were isolated to be tested by TAT Results:628bp specific segments were found in the PCR products of legionella pneumophila type 1-14, while no such amplification segments were found in the PCR products of Streptococcus pneumoniae and non-legionella pneumonia LM; and the sensitivity of legionella peumophila DNA was2.1pg/ul. Among the 95 specimens, there were 9 in whose PCR production 628bp gene segments were found, and the positive rate was 9.47%. Among the 9 cases, 6 of them were TAT tested positively, and the other 3 were negatively.From the above results, we believe that:1. Legionella pneumophila exist in infectious diseases of the respiratory system in paediatrics. In this study the infectious rate is 9.47%.2. The PCR method established by legionella pneumophila mip gene for the detection of legionella pneumophila has high specificity and strong sensitivity, and it can be widely used. It is valuable to the early diagnosis of legionlella pneumonia.For all the LP1-14, 628bp specific segments could be amplified using the primers designed according to the legionella pneumophia mip gene sequence, while for the non-legionella peumophila LM and Streptococcus pneumoniae, no such segments could be found. Among the 85 children cases that PCR detection were negative, there were 2 cases whose specimens were cultured were found aeruginosus Bacillus and Staphylococcus aureus, however no specific gene segments were found in their PCR products. It suggested that this pair of primers only amplified legionella pneumophia gene. In addition, the sensitivity of this experiment has reached the level of 2.1pg/ul DNA, which sensitivity is higher than that of traditional serological antibody detection. Since the detection of PCR method is not limited to antibody producing, it can be used to early diagnosis with quickness, sensitivity and specificity.We detected 95 children patients with respiratory inflammation. There were 9whose sputum and pharynx secretion could be amplified 628bp gene segments, we consider that legionella can not be neglected as a common pathogen among the respiratory track infectious pathogens in paediatrics. When we come into the clinic manifestations such as persistent fever, sever pneumonia and refractory pneumonia, we should think of the possible infection of legionella. Only in this way can we reduce misdiagnosise and missed diagnosise and decrease the mortality of legionella pneumonia. Legionella pneumophila pneumonia early treatment, timely treatment and prognosis, quickly identify pathogens, and thus choosing the right drugs to prevent bacteria's tolerance for the drug and reduce cost of treatment, the most critical thing is to reduce children suffering.3 There are no effective treatment programmes to Legionella bacteria, recognized at home and abroad, erythromycin treatment of Legionella bacteria is the first choice of antibiotics, is early diagnosis and treatment, strengthen support for symptomatic treatment.