| Objective:On the basis of cryptococcus neoformans infection in the bloodstream of the rat model,using the real-time PCR detection system(real-time fluorescent quantitative PCR, FQ-PCR) which was established by previous studies, the sample of blood, bronchoalveolar lavage fluid, cerebrospinal fluid were detected respectively by FQ-PCR, blood culture, serum capsular antigen detection, Comparison of the sensitivity and specificity of various detection methods, and provide laboratory evidence for the clinical application of cryptococcus neoformans FQ-PCR detection.Method:1.The 60 male Sprague-Dawley (SD) rats, SPF grade,6-8 weeks, weighing 200±20g, were randomly divided into four groups,Group A was immunosuppressive infection group (n=20), group B was normal infection group (n=20), group C was immunosuppressive control group (n=10), D group was normal control group (n=10);Odd-numbered rats in 0,12,48 hour, even-numbered rats in 6,24,72 hour were collected blood specimens about 2.5ml each.On 4th day the group A and group B rats were tail vein injected by lml Cryptococcus neoformans suspension of 1×106 concentration,group C and B rats were injected by normal saline.2.After establishing a successful infection model in rats, blood samples were collected from ocular venous plexusat at three different time period, centrifugal separated and stored at -20℃.Sampling time were 0,6,12,24,48,72h. The sixth day rats were all sacrificed, aseptic operation, collected alveolar lavage fluid, cerebrospinal fluid from 4 groups of rats, and stored at -20℃ to be measured,Meanwhile the lung, liver, spleen, brain tissue and 10% formaldehyde solution fixed overnight, HE staining and PAS staining under paraffin,observed under an optical microscope.3.Extraction DNA from plasma, alveolar lavage fluid, using the real-timePCRdetection system to detect all these samples, And simultaneously blood culture,histopathology, ink stain, capsular agglutination test, compared different methods of detection.Results:1.Cyclophosphamide immunosuppressed rats infected with Cryp-tococcus neoformans, prone to systemic infections, including lung, brain, spleen, liver tissue infections, not only found by the naked eye anatomy, lung appeared multiple nodular changes and various pathological PAS staining were found a large number of round or oval cryptococcal body, confirmed bloodstream infection in rats model were successed established.2. Culture of blood group A was most 30%, the positive rate was compared between the four groups, no significant difference (P>0.05). Group A capsular antigen positive rate was 80%, the positive detection rate higher than that of B, C, D groups at 48 hours, the difference was statistically significant (P<0.05).3. The positive rate of Cryptococcus neoformans FQ-PCR detection system in A group after 12h compared with B, C, D group, the positive detection rate is high, the difference was statistically significant (P<0.05), in the blood samples, the positive rate of FQ-PCR detection method is obviously better than that of blood culture and serum capsular antigen method, which can be used to quantitate, the difference was statistically significant(P<0.05).4.Cryptococcus neoformans FQ-PCR detection system can detect pathogens of lavage specimens, higher positive rate than blood culture, ink staining, the difference was statistically significant (P<0.05).5. Cryptococcus neoformans FQ-PCR detection system can detect pathogens in cerebrospinal fluid, higher than the ink stain positive rate, the difference was statistically significant (P<0.05).Conclusion:1. The immunosuppressive tail vein injection of Cryptococcus bacteria suspension can construct animal model of rat blood infection;2. Cryptococcus neoformans FQ-PCR reaction system can detect pathogens in the blood of rats, with the extension of the infection time, the positive rate gradually increased, its sensitivity is higher than that of blood culture, serumcapsular antigen detection;3. Method for the detection of cryptococcus neoformans FQ-PCR can detect pathogens earlier compared with blood culture and serum capsular antigen examination;4. The new FQ- PCR system can detect pathogenic bacteria in rat bronchoalveolar lavage fluid and cerebrospinal fluid. |
