脑瘤手术患者咽喉部吸取液中耶氏肺孢子菌的筛查

Screening pharyngolaryngeal aspirate from patients undergoing surgery for a brain tumor for Pneumocystis jirovecii

目的采用常规PCR、巢式PCR（Nest PCR,nPCR）和定量PCR（Quantitative PCR,qPCR）检测耶氏肺孢子菌（Pneumocystis jirovecii,Pj）特异性核酸片段,调查脑瘤手术患者咽喉部吸取液中的Pj。方法选取无肺炎脑瘤手术患者108例,留取手术咽喉部吸取液,分别以线粒体大亚基rRNA（Mitochondrial large subunit rRNA,mtLSUrRNA）为靶基因的常规PCR（Mt-PCR）和巢氏PCR（Mt-nPCR）,以及主要表面糖蛋白（Major surface glycoprotein,Msg）为靶基因的常规PCR（Msg-PCR）,检查咽喉部吸取液中Pj特异性核酸片段,阳性标本进行qPCR,检测分别以mtLSUrRNA和Msg为靶基因的拷贝数。结果 108例脑瘤手术患者咽部吸取液Mt-nPCR、Msg-PCR及Mt-PCR扩增阳性率率分别为41.7%（45/108）、15.7%（17/108）和4.6%（5/108）,其中3种检测方法同为阳性1例（占0.9%）,任意2种方法阳性11例（占10.2%）,Mt-nPCR和Msg-PCR任意1种方法阳性42例（占38.9%）,3种方法均阴性54例（占50.0%）。54例PCR检测阳性的标本同时进行mtLSUrRNA定量PCR（Mt-qPCR）和Msg定量PCR（Msg-qPCR）检测,其中MtqPCR检测为100-999拷贝/μl 2例（占3.7%）,1 000-9 999拷贝/μl有49例（占90.7%）,≥104拷贝/μl有3例（占5.6%）;Msg-qPCR检测为1 000-9 999拷贝/μl 14例（占25.9%）,10 000-99 999拷贝/μl 21例（占38.9%）,100 000-999 999拷贝/μl 14例（占25.9%）,≥106拷贝/μl有5例（占9.3%）。54例中Msg-qPCR拷贝数〉Mt-qPCR拷贝数51例（占94.4%）,

Objective To investigate the rates at which pharyngolaryngeal aspirate from patients undergoing surgery for a brain tumor tested positive for Pneumocystis jirovecii using conventional PCR,nested PCR（nPCR）,and quantitative PCR. Methods Subjects were 108 patients undergoing surgery for a brain tumor without any symptoms,signs,or chest X-ray findings suggesting of pneumonia（patients with no pneumocystis pneumonia,or PCP）.Pharyngolaryngeal aspirate was collected before withdrawing the intubation tube under general anesthesia.The mitochondrial large subunit rRNA（mtLSUrRNA）gene of P.jirovecii was designated as the target gene for conventional PCR（Mt-PCR）and nested PCR（Mt-nPCR）.The major surface glycoprotein（Msg）gene of P.jirovecii served as the target gene for conventional PCR（Msg-PCR）.Samples were screened with Mt-PCR,Mt-nPCR,and Msg-PCR.Samples that tested positive in screening were further tested with real-time fluorescence quantification PCR（qPCR）for mtLSUrRNA（Mt-qPCR）and Msg（Msg-qPCR）. Results Of 108 patients with no PCP,41.7%（45/108）tested positive for P.jirovecii according to Mt-nPCR,15.7%（17/108）tested positive according to Msg-PCR,and 4.6%（5/108）tested positive according to Mt-PCR.One patient（0.9%）tested positive for P.jirovecii according to all 3 methods of PCR,11（10.2%）testedpositive according to 2of the 3methods,42（38.9%）tested positive according to 1of the 3methods,and 54（50.0%）tested negative according to all 3methods.Of the 54 patients that tested positive according to Mt-qPCR,2（3.7%）were infected with P.jirovecii at a level of 100copies/l to 999copies/l,49（90.7%）were infected with P.jirovecii at a level of 1 000copies/l to 9 999copies/l,and 3（5.6%）were infected with P.jirovecii at a level of 104copies/l or more.Of the 54 patients that tested positive according to Msg-qPCR,14（25.9%）were infected with P.jirovecii at a level of1 000copies/l to 9 999copies/l,21（38.9%）were infected with P.jirovecii at a level of 10 000copies/l 99 999copies/l,14（25.9%）were infected with P.jirovecii at a level of 100 000copies/l to 999 999copies/l,and 5（9.3%）were infected with P.jirovecii at a level of 106copies/l or more.When qPCR results were compared,51patients（94.4%）had a greater copy number according to Msg-qPCR than that according to Mt-qPCR.Only 3patients（5.6%）had a smaller copy number according to Msg-qPCR than that according to Mt-qPCR. Conclusion Pharyngolaryngeal aspirate from patients with no PCP who underwent surgery for a brain tumor tested positive for P.jirovecii at a rate of50% according to a nucleic acid amplification test（NAAT）.The copy number according to Mt-qPCR was primarily 103copies/l.The copy number according to Msg-qPCR was primarily 103,104,or 105copies/l.Care is required when interpreting the clinic significance of testing positive for P.jirovecii and the copy number according to qPCR using NAAT.