Objective:To develop a validation method for microbial targeted next generation sequencing (tNGS) detection for respiratorypathogens, and to evaluate the performance of the pathogen-targeted high-throughput sequencing test implemented in local hospital.Methods:Cross-sectional study. A total of 14 patients with severe pulmonary infections were admitted to Huangshi Central Hospital from December 2023 to January 2024. Samples were collected as follows:Bronchoalveolar lavage fluid (BALF) samples ( n=7) subjected to culture, fluorescent PCR, and tNGS testing. Sputum samples ( n=2) analyzed via sputum culture, fluorescent PCR, and tNGS. Throat swab samples ( n=5) tested using fluorescent PCR-capillary electrophoresis and tNGS. Reference samples were prepared using representative species such as Influenza A virus, Adenovirus C, Klebsiella pneumoniae, Aspergillus fumigatus, Staphylococcus aureus, Staphylococcus epidermidis, Candida parapsilosis, and Candida albicans. Jurkat cells at different concentrations were used as a source of human cells. Traditional detection methods such as fluorescent PCR-capillary electrophoresis and culture methods were used as reference methods. The detection performance of tNGS was evaluated by assessing the detection limit, precision, human cell impact, stability, cross-reactivity, and accuracy of metagenomic next-generation sequencing for pathogen detection. Results:The detection limits for Klebsiella pneumoniae, Human Adenovirus C, and Influenza A virus were 2×10 2 copies/ml, and for Aspergillus fumigatus, Staphylococcus aureus, Staphylococcus epidermidis, Candida parapsilosis, and Candida albicans, the detection limits were 4×10 2 copies/ml. The consistency rate of repeated detection results for all pathogens in the reference samples was 100%. The impact assessment experiment of human cells showed that when the concentration of Jurkat cells reached 1×10 6 cells/ml, Influenza A virus, Adenovirus C, Klebsiella pneumoniae, and Aspergillus fumigatus could all be detected. Stability experiments showed that there was no significant change in the number of pathogen sequences after the specimens were stored at 4 ℃ and -20 ℃ for 1 day, 4 days, and 7 days, respectively. Cross-reactivity experiments showed that when the concentration ratios of Staphylococcus aureus, Staphylococcus epidermidis, Candida parapsilosis, and Candida albicans were (5∶1∶1∶5), (1∶5∶5∶1), and (1∶1∶1∶1), respectively, the detection rate of closely related microbial species was 3/3. Accuracy assessment showed that the accuracy of 19 clinical specimens was 18/19 cases. Conclusion:Compared with traditional detection methods as the reference, tNGS demonstrates high sensitivity and a high positive concordance rate, underscoring its significant clinical value in the detection of respiratory pathogens.

Objective:To develop a validation method for microbial targeted next generation sequencing (tNGS) detection for respiratorypathogens, and to evaluate the performance of the pathogen-targeted high-throughput sequencing test implemented in local hospital.Methods:Cross-sectional study. A total of 14 patients with severe pulmonary infections were admitted to Huangshi Central Hospital from December 2023 to January 2024. Samples were collected as follows:Bronchoalveolar lavage fluid (BALF) samples ( n=7) subjected to culture, fluorescent PCR, and tNGS testing. Sputum samples ( n=2) analyzed via sputum culture, fluorescent PCR, and tNGS. Throat swab samples ( n=5) tested using fluorescent PCR-capillary electrophoresis and tNGS. Reference samples were prepared using representative species such as Influenza A virus, Adenovirus C, Klebsiella pneumoniae, Aspergillus fumigatus, Staphylococcus aureus, Staphylococcus epidermidis, Candida parapsilosis, and Candida albicans. Jurkat cells at different concentrations were used as a source of human cells. Traditional detection methods such as fluorescent PCR-capillary electrophoresis and culture methods were used as reference methods. The detection performance of tNGS was evaluated by assessing the detection limit, precision, human cell impact, stability, cross-reactivity, and accuracy of metagenomic next-generation sequencing for pathogen detection. Results:The detection limits for Klebsiella pneumoniae, Human Adenovirus C, and Influenza A virus were 2×10 2 copies/ml, and for Aspergillus fumigatus, Staphylococcus aureus, Staphylococcus epidermidis, Candida parapsilosis, and Candida albicans, the detection limits were 4×10 2 copies/ml. The consistency rate of repeated detection results for all pathogens in the reference samples was 100%. The impact assessment experiment of human cells showed that when the concentration of Jurkat cells reached 1×10 6 cells/ml, Influenza A virus, Adenovirus C, Klebsiella pneumoniae, and Aspergillus fumigatus could all be detected. Stability experiments showed that there was no significant change in the number of pathogen sequences after the specimens were stored at 4 ℃ and -20 ℃ for 1 day, 4 days, and 7 days, respectively. Cross-reactivity experiments showed that when the concentration ratios of Staphylococcus aureus, Staphylococcus epidermidis, Candida parapsilosis, and Candida albicans were (5∶1∶1∶5), (1∶5∶5∶1), and (1∶1∶1∶1), respectively, the detection rate of closely related microbial species was 3/3. Accuracy assessment showed that the accuracy of 19 clinical specimens was 18/19 cases. Conclusion:Compared with traditional detection methods as the reference, tNGS demonstrates high sensitivity and a high positive concordance rate, underscoring its significant clinical value in the detection of respiratory pathogens.

Pain is a complex physiological and psychological activity, involving at least three dimensions, including pain sensation, pain emotion, and pain cognition. Acupuncture can clearly relieve the pain sensation of patients and improve pain emotion and pain cognition induced by pain; acupuncture participates in the multi-dimensional regulation of pain through brain regions of the limbic system such as anterior cingulate cortex (ACC), amygdala (AMY), and hippocampus. By analyzing relevant literature, it has been found that the regulation of acupuncture on pain emotion is mainly related to the activation of pertinent opioid receptors in the ACC, the decrease of the expression of extracellular signal-regulated kinase (ERK), and the promotion of the expression of glutamic acid (Glu) A1, metabotropic glutamate receptor-1 (mGluR1), and γ-aminobutyric acid aminobutyric acid (GABA) B2 protein in the AMY. The regulation of acupuncture on pain cognition is mainly related to the elevation of the expression of protein kinase A (PKA) and phospho-p38 mitogen-activated protein kinase (phospho-p38 MAPK) and the inhibition of cyclic adenosine monophosphate (cAMP)/PKA/cAMP response element-binding protein (CREB) signaling pathway in the ACC.

OBJECTIVE: To investigate the effect of intrathecal sufentanil and protein kinase C inhibitor on pain threshold and the expression of N-methyl-D-aspartate receaptors (NMDAR)/calcitonin generelated peptide (CGRP) in spinal dorsal horn in rats with neuropathic pain. METHODS: Fifty-four healthy male Sprague-Dawley rats were randomly divided into 6 groups (9 in each group). The rats in the sham group(Group S) + spared nerve injury (SNI), SP+SNI, and P+SNI were intrathecally injected sufentanil (1 μg), sufentanil (1 μg) and chelerythrine chloride (11 μg), chelerythrine chloride (11 μg) followed by 10 μL normal saline once every day for 14 days postoperatively, respectively. Similarly, rats in the control group (Group C), the sham group (Group S), and SNI model group (Group SNI) were intrathecally injected 20 μL normal saline in the uniform interval. Pain behaviours were measured on Day 1 pre-surgery and on Day 1, 2, 7, and 14 after the intrathecal injection. The expressions of NMDAR and CGRP in the spinal dorsal horn of L5 segment were determined by immunohistochemistry on Day 2, 7, and 14 after the intrathecal injection. RESULTS: Compared with Group C and Group S, mechanical allodynia threshold in group SNI was decreased after the surgery (P<0.01), and expressions of NMDAR and CGRP immunoreactive soma in the spinal dorsal horn was significantly increased (P<0.01). Mechanical stimulation pain threshold was elevated in Group S+SNI, Group P+SNI, and Group SP+SNI compared with Group SNI (P<0.01), while expressions of NMDAR and CGRP immunoreactive soma in Group S+SNI, Group P +SNI, and Group SP+SNI were significantly decreased (P<0.05 or 0.01). CONCLUSION Intrathecal administration of sulfentanil and protein kinase C inhibitor can provide significant antinociception in rats with neuropathic pain and obviously inhibit the upregulation of NMDAR and CGRP expressions in the spinal dorsal horn of SNI rat models.
Animals ; Benzophenanthridines ; administration & dosage ; Calcitonin Gene-Related Peptide ; metabolism ; Injections, Spinal ; Male ; Neuralgia ; drug therapy ; metabolism ; physiopathology ; Pain Measurement ; Posterior Horn Cells ; metabolism ; Protein Kinase C ; antagonists & inhibitors ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; metabolism ; Sufentanil ; administration & dosage

OBJECTIVE To evaluate the drug resistance of Candida isolated from patients' specimen in clinic.METHODS The minimal inhibitory concentration(MIC) test to 243 Candida strains was performed using ATB-Fungus drug susceptibity plate provided by Bio-Merieux.RESULTS The most popular species of Candida in clinic was Candida albicans(64.6%),C.glabrata(14.4%),C.tropicalis(11.1%),C.parapsilosis(5.8%)and C.krusei(4.1%).There was a certain resistance to the 4 kinds of common antifungals including flucytosine,amphotericin B,fluconazole and itraconazole.The resistance status of C.krusei was the most serious,Its resistance rate to 4 kinds of antifungals was 20.0%,50.0%,30.0% and 40.0%,respectively.The drug resistance rate of 5 species of Candida to itraconazole was higher than that to fluconazole.CONCLUSIONS The commonly encountered Candida produce particular resistance to common antifungals,and it is very necessary to detect and control them.

OBJECTIVE To explore the distribution of ?-lactamase and drug resistant gene mediated by transposon and integron in continuous isolates of Pseudomonas aeruginosa.METHODS The antibiotics susceptibility was tested by K-B method;the ?-lactamase and the outer membrane protein gene oprD2 were detected by polymerase chain reaction(PCR);the genotype of merA encoding Tn21/Tn501 type trandsposon and qacE?1-suI1 encoding Ⅰ type integron was detected by PCR and the positive genes were amplified and sequenced by PCR fluorescence spectrophotometry.RESULTS The TEM type,OXA-2,OXA-10 and CARB of ?-lactamase genes had been detected in 20 strains of P.aeruginosa,but plasmid-mediated ampC enzyme and metallo-?-lactamase had not been detected,the gene oprD2 encoding porins was not detected.The gene merA was detected in 7 strains(35.0%),and the qacE?-sul1 had been detected in 10 strains(50.0%).The gene OXA-2 in the isolate No.2 was sequenced,and translated into amino acid,and the translated amino acid sequence was compared with GenBank,the results indicated that amino acid sequence of OXA in the isolate No.2 was simlar to that in GenBank,exsited 2 different amino acid sequences,so was confirmed as a new subtype.CONCLUSIONS Resistance to ?-lactamase compounds in P.aeruginosa of our hospital is related to TEM,OXA and CARB genes,and integron and transposon contribute to the drug resistance and multidrug resistance in P.aeruginosa.

OBJECTIVE To explore the distribution of related resistance genes of chloramphenicol and tetracycline in Pseudomonas aeruqinosa isolates.METHODS The antibiotics susceptibility was tested by K-B method,catB cmlA,tetA tetB and smr-2 were detected by polymerase chain reaction(PCR),the gene cmlA was sequenced by PCR fluorescence spectrophotometry.RESULTS The drug-resistant rates of 20 strains of P.aeruginosa against 17 kinds of antibiotics ranged from 10.0% to 100.0%,and multidrug resistant strains were found.The gene of cmlA had been detected in 6 strains of 15 resistant P.aeruginosa isolates,but the genes of tetA,tetB and smr had not been detected.The cml was sequenced,and compared with GenBank,the result showed the gene fragment shared 99.0% homology in nucleotides with the GenBank sequences of cmlA7 and cmlA8.CONCLUSIONS Resistance to antibiotics in P.aeruginosa of our hospital is mainly related to cmlA.The resistance to chloramphenicol and tetracycline was not related to tetA,tetB and smr-2.

OBJECTIVE To explore the distribution of 16S rRNA methylase and aminoglycoside modifying enzymes in continuous isolates of Pseudomonas aeruginosa(PAE).METHODS The antibiotics susceptibility was tested by K-B method,the 16S rRNA methylase(rmtB) and aminoglycoside modifying enzymes were detected by polymerase chain reaction(PCR),the positive genes were amplified and sequenced by PCR fluorescence spectrophotometry.RESULTS Among 20 strains of PAE,the 16S rRNA methylase and 5 kinds of aminoglycoside modifying enzymes had been detected.The 5 kinds of aminoglycoside modifying enzymes were aac(3)-Ⅱ,aac(6′)-Ⅰb,aac(6′)-Ⅱ,ant(3″)-Ⅰ and ant(2″)-Ⅰ,respectively.And more,the rmtB in the first and third strains was sequenced,and translated into amino acid,and the translated amino acid sequence was compared with GenBank,suggesting there be different amino acid sequence.This was confirmed as two new subtypes.CONCLUSIONS Resistant to antibiotics PAE in our hospital is mainly related to 16S rRNA methylase and 5 aminoglycoside modifying enzymes,and 2 new subtypes of 16S rRNA methylase are discovered.