Pneumocystis Carinii Detection And Genotyping Analysis

Pneumocystis carinii is an opportunistic Pathogenic pathogens. Chagas firstly found it in Guinea′s lung in 1909. Delanoe and his wife detected it in rat′s lung in1912, and named Pneumocystis carinii. It can infect with a variety of mammals, including animal and human. It Parasitic Normal immunity hos′s lung tissue in latent infection state does not cause symptoms, but when the host′s immune System damaged may cause a large number of Reproduction that cause deadly Pneumocystis carinii pneumonia (PCP). Because it is the most common opportunistic infections and the main cause of death for AIDS , So it is pay widespread attention. With the number of congenital or acquired immunodeficiency and patients with immunosuppressive agents, PCP has become a higher mortality with respiratory infectious diseases in these patients.Objective: In this paper, used Wright - Giemsa (Giemsa) stained, Gomori Methenamine silver (GMS) stained and polymerase chain (PCR) reaction for detection of Pneumocystis carinii (PC), to explore the chemical stained and the PCR method in detection of Pneumocystis carinii in the value of clinical. To establish a simple, stable, high sensitivity and specificity of laboratory tests for the clinical diagnosis of Pneumocystis carinii pneumonia. In addition, detection of Pneumocystis carinii′s mitochondrial large subunit (mtLSUrRNA), internal transcribed spacers (ITS1-5.8SrRNA-ITS2), dihydrofolate reductase (DHFR) gene to sequencing and genotype analysis, that provide molecular epidemiological data to better understand Pneumocystis carinii′s species and Biology.Methods: Experimental group is 30 cases of sputum from lung cancer with pulmonary infection ,bronchoalveolar lavage fluid (BALF) 20 cases and 10 cases lung tissues in patients with lung cancer surgery. Normal group is Normal human′s sputum 20 cases. Detect Pneumocystis carinii from sputum smear, BALF smear and lung Imprint by Wright - Giemsa (Giemsa) stained and Gomori Methenamine silver (GMS) stained. design mtLSUrRNA, ITS1-5.8SrDNA-ITS2, DHFR gene primer , extracted Pneumocystis carinii DNA from the sputum, BALF and lung tissue to polymerase chain reaction. PCR positive products were recovered , purification and gene sequencing. To search homology using Blast software with sequencing results, sequence Contrast and genotype analysis.Results:1 Wright - Giemsa (Giemsa) stained and Gomori Methenamine silver (GMS) stained method have low sensitivity. In the 80 specimens, PC detection rate is 0 (0 / 80).2 60 cases of the experimental group samples, ITS1-5.8SrRNA-ITS2 gene in sputum, BALF, lung tissue positive detection rates were 10% (1 / 10), 0 (0 / 30), 30% (6 / 20), mtLSUrRNA, DHFR gene BALF positive detection rates were 10% (2 / 20) and 5% (1 / 20), sputum, lung tissue were not amplified positive bands.3 With the normal control group and clinical common bacteria and fungi (such as E. coli DNA, Ac. baumannii DNA, Ps. aeruginosa DNA, S. aureus DNA, M. tuberculosis DNA, fungal DNA, etc) with mtLSUrRNA, ITS1-5.8SrRNA -ITS2, DHFR gene specific test, dose not appeared no specific band appeared.4 For ITS1-5.8SrRNA-ITS2 gene Nested PCR, from 1:1×101 to 1:1×105 different dilutions of DNA template, able to amplify fragments of the same length (about 550bp). Only amplificated the dilution 1:1×103 with a single PCR Under the same conditions.5 In this study 10 cases of positive products were amplified, mtLSUrRNA gene in 2 cases, ITS1-5.8SrRNA-ITS2 gene in 7 cases, DHFR gene in 1 case.6 After 10 cases of positive PCR products were recovered, purification, sequencing, to conduct the sequence Contrastion, sequences exist base′s absence, replacement and insertion.Conclusion:1 Wright - Giemsa stained, simple operation and low sensitivity. Gomori Methenamine silver (GMS) stained is complex and demand Operator have some operational experience, and low sensitivity.2 PCR is better sensitivly than chemical staining.3 Pneumocystis carinii′s mtLSUrRNA, ITS1-5.8SrRNA-ITS2, DHFR gene PCR detected high specificity.4 Nested PCR detection of Pneumocystis carinii better sensitivly than ordinary single-primer PCR.5 10 cases of PC genotype have higher homology with and gene banks Eg.6 Various host′s Pneumocystis carinii sequence exist differences sites, indicating the genetic diversity of Pneumocystis carinii.7 This study first time detect Pneumocystis carinii used PCR in Shijiazhuang, the primer of Pneumocystis carinii is highly sensitive for the detection of clinical patients.