| Objective To investigate the clinical manifestations,opportunistic infection status and therapeutical effect of AIDS patients combined with PCP(pneumocystis pneumonia).And to analyze the relationship between lymphocyte subsets counts and PCP or opportunistic infections in AIDS patients combined with PCP.To detect the mutation of DHPS gene,and to analyze the relationship between drug resistance and mutation.Then,through the recombinant clone which carry out mutational DHPS gene to screen effective antibiotics for mutants in vitro.Methods 1.Clinical manifestations,imaging examination,laboratory tests,therapeutical drug,prognosis and opportunistic infection type of AIDS patients suspected PCP were collected and analyzed.The lymphocyte subsets were compared in AIDS patients with/without PCP and AIDS combined with PCP patients with/without opportunistic infections respectively.2.The sputum and BALF samples from AIDS patients suspected PCP were collected,DNA after digestion was extracted,and DHPS gene was amplified by PCR.Then DHPS gene mutation was analyzed by PCR-RFLP,and was sequenced after purification of PCR products.3.The recombinant plasmid was cloned with DHPS gene and PET-28a vector,and three mutations were obtained by site-directed mutagenesis and then transferred into Escherichia coli BL21(DE3)competent cells.The recombinant protein was induced by IPTG,and the susceptibility of sulfamethoxazole,sulfadiazine and sulfamerazine were further analyzed in vitro.Results 1.AIDS patients combined with PCP mainly manifested as fever,cough,chest tightness and shortness of breath.The main imaging features of the two lungs were translucenly decreased,markings increased,and in ground-glass pattern.Drug therapy was often used with sulfamethoxazole combined with voriconazole or caspofungin.After treatment,the symptoms of dyspnea were alleviated,and the chest CT findings showed the absorption of lung shadows.2.Compared with patients of AIDS without PCP,the amount of lymphocytes,CD3+T cells,CD4+T cells,CD8+T cells and CD4+ T/CD8+T cell ratio were significantly lower in AIDS patients combined with PCP(P<0.01).AIDS combined with PCP patients have a high incidence of opportunistic infections.The CD4+T cell counts and CD4+T/CD8+T cell ratio in the patients combined with opportunistic infections were less than those of patients unconsolidated(P<0.01,P<0.05).3.The positive rate of AIDS combined with PCP was 40.82%,and the drug-related mutation rate was 3.33%.There was synonymous mutation of TCA to TCG in the 171th codon of DHPS gene.4.The protein expression of DHPS gene could be induced by IPTG.Drug sensitivity analysis in vitro found that in the same circumstance the IC50 was slightly higher at the concentration of IPTG 0.25 mM,0.5 mM than IPTG 1 mM,and the first two are closer.In addition,only sulfadiazine IC50 A to G mutant was higher than DHPS without mutation at the concentration of IPTG 0.25 mM,and the rest were lower than DHPS without mutation.Conclusion 1.The clinical symptoms and signs of PCP were not typical.The patients which were treated with sulfamethoxazole combined with voriconazole or caspofungin,have favorable results.2.With the decline of CD4+T cell number,the incidence of PCP and other opportunistic infections increased gradually.For AIDS patients combined with PCP,the CD4+T cell level is a risk factor for opportunistic infections.3.Resistant rate of DHPS related mutation was low,and clinically relevant drug resistance was not found yet.The synonymous mutation of TCA to TCG was not yet found the relevant with drug resistance,so the site may not be the active site of the enzyme,and it will not lead to sulfa resistance.4.Drug sensitivity study in vitro showed that high concentration of IPTG could inhibit the growth of Escherichia coli.Only A to G mutant of three was more resistant to sulfadiazine than DHPS without mutation,and the rest were more sensitive. |
