Multiplex real-time quantitative polymerase chain reaction for simultaneous detection of Escherichia coli and Candida albicans genes in human whole blood / 中华临床营养杂志

Objective To establish a multiplex real-time quantitative polymerase chain reaction (MRQPCR) assay for fast and simultaneous detection of Escherichia coli (E.coli) and Candida albicans (C.albicans) genes in human whole blood,in order to facilitate differentiation of the types of microorganism and evaluation of the severity of bacterial or fungi translocation due to impaired gut barrier,hence providing help to select specific antimicrobial agents.Methods The β-D-galactosidase gene of E.coli and ITS2 gene of C.albicans were selected as the target genes for designing primers and probes.E.coli and C.albicans genomes were extracted with QIAamp(R) DNA Blood Mini Kit,and the 25 μl TaqMan MRQ-PCR amplification reaction system was established.18 simulated human whole blood samples and 10 whole blood samples from febrile surgical patients were detected for E.coli and C.albicans genes using MRQ-PCR.Results The specificity of the primers and probes were excellent.The correlation coefficients of the standard curves of E.coli and C.albicans were 0.994-0.999 and 0.994-0.998,respectively;and the efficiency of amplification were 0.894-1.022 and 0.905-1.028,respectively.In the standard samples,the lowest detection limits of E.coli and C.albicans were 13.9 copies/μl and 0.8 cfu/μl,respectively;the sensitivity was 100％ and 99.69％,the specificity was 100％ and 94.73％,respectively;the average recovery rates were (101.89 ± 5.69)％ and (103.74 ± 4.64)％ respectively;the intra-batch coefficients of variance (CV) in detecting the genes were (13.14 ± 10.27)％ and (19.18 ± 8.54)％,respectively,and the inter-batch CV were (14.35 ± 9.34)％ and (18.31 ± 10.25) ％,respectively.In human whole blood,the lowest detection limits of E.coli and C.albicans were 12 455.2 copies/ml and 800.3 cfu/ml,respectively;the average recovery rates were (111.60 ± 11.06) ％ and (99.96 ± 6.16) ％,respectively;the intra-batch CV in detecting the genes were (11.02 ± 5.65) ％ and (8.14 ± 7.29)％,respectively,and the average inter-batch CV were (12.88 ± 7.59)％ and (18.62 ± 9.14)％.Conclusions MRQ-PCR is a rapid,sensitive,specific,accurate,and reproducible method for simultaneous detection of E.coli and C.albicans genes in human whole blood,with sample-,cost-,and time-saving advantages.It is a promising technique for rapid differentiation between fungi and bacteria,which could help targeted administration and evaluation of antimicrobial agents,and help to assess the consequence of gut barrier damage and the efficacy of treatment.

Nasal cavity supernumerary tooth and maxillary sinusitis: one case reported / 临床耳鼻咽喉头颈外科杂志

Supernumerary tooth is a rare case. This report described a case of nasal cavity supernumerary tooth association with maxillary sinusitis. A 28-year-old male patient reported with the chief complaint of nasal obstruction, headache and purulent secretion for the past three months. Clinic examination and CT examination showed that there was a supernumerary tooth in the right nasal bottom, and maxillary sinus was infected in the same side. This patient was performed supernumerary tooth removing and given antibiotics for 3 days. Ten days after the operation, there was no clinical symptoms, and nasal bottom mucosa was normal. After 3 months of follow-up, reexamination of coronal CT scan appeared normal.

Detection of Aspergillus fumigatus genome load in human whole blood samples by real-time quantitative polymerase chain reaction and its clinical application / 中华临床营养杂志

Objective To establish a real-time quantitative polymerase chain reaction (RQ-PCR) assay for fast detection of Aspergillus fumigatus genome in human whole blood samples and explore its clinical application.Methods The primers and the TaqMan-probe were designed on the basis of the multi-copy ITS1-5. 8S region of the rDNA of Aspergillus fumigatus. The Aspergillus fumigatus genomic DNA were extracted with QIAamp(R) DNA Blood Mini Kit.A 20 μl RQ-PCR amplification system was established, and the simulated blood samples containing various given load of Aspergillus fumigatus genome and the 66 whole blood samples of the surgical febrile patients were examined. Results The detection limit of the RQ-PCR instrument is 10-1 genomes/μl DNA sample,namely 78 CFU/ml whole blood. The specificity and the sensitivity were 94. 25% and 99. 04% respectively; and the positive predictive value and negative predictive value were 97. 63% and 97. 62% respectively. The average relative error of the quantitative results was (3. 67 ±13. 19)%, and the intra- and the inter-assay average coefficients of variation were (12.38 ± 1. 53)% and (16. 27 ±2. 72)% , respectively. The average recovery rate of Aspergillus fumigatus genomic DNA in human whole blood samples was (107. 81 ±25. 92)% , and the average coefficient of variation of the average recovery rate was (26. 24 ± 5.62) % . No Aspergillus fumigatus genomic DNA was detected among the 66 blood samples of the surgical febrile patients. Conclusions The RQ-PCR assay for fast quantitative detection of Aspergillus fumigatus genome in human whole blood samples is of high sensitivity, specificity,accuracy and precision. The Aspergillus fumigatus genome was not detected in this group of surgical febrile patients.

Detection of Candida albicans in the venous blood of the surgical febrile patients by real-time quantitative PCR / 中华临床营养杂志

Objective To establish the real-time quantitative PCR (RQ-PCR) assay for detecting Candida albicans (C.albicans) in whole blood and its clinical application in the febrile surgical patients who may develop gut barrier damage and gut microorganism translocation.Methods The NAG1 gene,which is a single copy in C.albicans genome,was selected as the target gene for designing the primers and probe.The plasmid was fabricated and produced as standard samples.C.albicans genomes were extracted with QIAamp(R) DNA Blood Mini Kit,and the total 20 μl TaqMan RQ-PCR amplification reaction system was established.The 74 venous blood samples from the surgical febrile patients were detected for C.albicans load.Results The specificities of the primers and probe were excellent,the correlation coefficients of the standard curves were between 0.9918 and 0.9985,and the efficiency of amplification was 0.88-1.027 for the samples above the lowest detection limit (100 copies/μl examine fluid,or nearly 1.1 × 103 cfu/ml whole blood).The average accuracy of the RQ-PCR equipment was (99.64±2.08) %,the sensitivity was 97.46%,the specificity was 100%,and the average coefficients of variation (CV) of the intra-and inter-assay were (14.76±2.64)% and (17.85±3.53)%,respectively.The average recovery rate of C.albicans DNA in whole blood samples was (88.60±5.73) %,and the average CV of recovery rate was (11.70 ±5.36) %.The number of copies of C.albicans genes per unit blood was not significantly different among the same original blood samples stored separately under-20℃ for 3 or 6 months when compared with its freshly collected blood (P = 0.267).In the 74 whole blood samples obtained from the febrile surgical patients,the positive rate of C.albicans genes was 2.7% and the highest load was 4.42×103 cfu/ml.Conclusions RQ-PCR is a rapid,sensitive,highly specific,and reproducible method in detecting C.albicans NAG1 gene.Clinically it can be used to quantitatively evaluate the numbers of C.albicans in the whole blood.A small percentage of the febrile surgical patients may develop blood infection of C.albicans.

Diagnosis and treatment of the sphenoid sinus malignant tumor / 中国耳鼻咽喉头颈外科

OBJECTIVE To investigate the clinical characteristics,nasal endoscope and imaging findings, misdiagnosis and treatment results of sphenoid sinus malignant tumor.METHODS The clinical data of 9 cases with sphenoid sinus malignant tumor were summarized and analyzed.Headache was found in 8 patients, ophthalmic symptoms in 3 patients,nasal bleeding and obstruction in 3 patients,and cranial nerve palsy in 2 patients.They were often misdiagnosed as sphenoid sinusitis and nasopharyngeal carcinoma.RESULTS Only 1 patient with papillary carcinoma was cured for 6 years.One patient with neuroendocrine carcinoma was alive for 3 years after treatment.Four patients died at 2 to 3 years.Two patients were alive at 1 year after operation. One patient was lost to follow up.CONCLUSION Sphenoid sinus tumor had no characteristic symptoms in early stage.It is easy to misdiagnose and delay diagnosis. Patients with headache,visual symptoms and nasal bleeding should take nasal endoscopy,CT scan and MRI examination at early stage.