Objective To develop and evaluate a nucleic acid amplification test for spectinomycin-resistant Neisseria gonorrhoeae(N.gonorrhoeae).Methods N.gonorrhoeae-specific primers NG1/NG2 and primers specific to the N.gonorrhoeae rpsE gene mutation(80_82 delTTA)were designed.Genomic nucleic acids of spectinomycin-sensitive and resistant N.gonorrhoeae,Escherichia coli,Pseudomonas aeruginosa,and Salmonella typhi were used as templates to be amplified by PCR and quantitative real-time PCR(qPCR).The sensitivity and specificity of the method were evaluated accordingly.Results The NG1/NG2 primers could effectively amplify specific fragments of N.gonorrhoeae,yielding negative results for the nucleic acid amplification test of the other types of bacteria tested.E64/E175R and E-87/E95R could effectively differentiate the wild type and mutant(80_82 delTTA)rpsE genes.In PCR reactions,the minimum limits of NG1/NG2,E64/E175R,and E87/E95R for the target genes were 414.8 copies,414.8 copies,and 4.1 copies/μL,respectively,while those for qPCR reactions were 41.5,41.5,and 4.1×10-2 copies/μL,respectively.Conclusion A nucleic acid amplification test for spectinomycin-resistant N.gonorrhoeae with high specificity and sensitivity was successfully established in this study,which is expected to provide support for the rapid diagnosis of N.gonorrhoeae infection and treatment decision-making in clinical settings.

Objective To evaluate the current status of compliance with animal experiment reporting guidelines in medical research papers published in Chinese and to enhance the transparency of medical research reporting.Methods Using a predefined literature search strategy,we conducted searches in the China National Knowledge Infrastructure(CNKI)database for literature published in 2019 and 2022 in journals indexed in A Guide to the Core Journals of China published by Peking University Library.We focused on 22 pieces of key information required in the ARRIVE Guidelines 2.0.These pieces of information concerned 6 items of the ARRIVE Essential 10 Items,including study design,sample size,randomization,statistical methods,and experimental animals,and 3 items of the ARRIVE Recommended Set,including abstract,ethical statement,and declaration of interests.We conducted statistical analysis of the reporting rates of the key information in the research publications included in the study.Results A total of 4818 research papers were included in the analysis,and none comprehensively reported all 22 pieces of information investigated in the study.Most of the research papers published in 2019 and 2022 reported information on the control groups,with the reporting rate for the respective years being 99.8％(2461/2467)and 99.7％(2343/2351).Although the sample size reporting rates were 79.2％(1954/2467)for research papers published in 2019 and 77.2％(1815/2351)for those published in 2022,none described the method and rationale of sample size calculation.The reporting rates of randomisation and blinding methods were approximately 20％and 1％,respectively.The reporting of statistical methods increased slightly from 91.8％in 2019 to 96.0％in 2022.The reporting of information on the experimental animals showed mixed trends for 2019 and 2022,including the provenance of animals(93.8％vs.93.7％,P＞0.05),strains and substrains(99.1％vs.99.2％,P=0.514),sex(94.1％vs.92.7％,P=0.044),age(58.1％vs.70.6％,P＜0.001),weight(84.4％vs.81.9％,P=0.020),and health status(66.0％vs.75.5％,P＜0.001).Research papers published in both 2019 and 2022 had relatively low rates of reporting animal ethics review(15.8％vs.38.9％)and animal ethics principles adhered to(9.8％vs.21.3％),declaration of interests(2.3％vs.10.6％),and accurate summaries of animal-related information in the abstracts(9.16％vs.8.13％).In particular,the reporting rate of the animal ethics statement and the declaration of interests increased significantly high in 2022 compared to that in 2019(P＜0.001).Conclusions Since ARRIVE Guidelines 2.0 was released,transparency in the reporting of most of the ARRIVE checklist items of interest in this study has improved significantly.However,the reporting of randomization,blinding,ethical statement,and declaration of interests still need further improvement and should be prioritized for future efforts.

Objective To establish and evaluate a microbial sensitivity test method for Neisseria gonorrhoeae based on resazurin coloration.Methods Based on the broth microdilution method,resazurin was added as a live bacteria indicator.WHO G,a WHO gonococcal reference strain,was used to optimize the incubation time for resazurin-stained bacteria and the color change was visually observed to obtain the results.Agar dilution method(the gold standard)and resazurin-based microdilution assay were used to determine the minimum inhibitory concentration(MIC)of azithromycin,ceftriaxone,and spectinomycin for 3 reference strains and 32 isolates of Neisseria gonorrhoeae.The results were analyzed based on essential agreement(EA),which reflected the consistency of the MIC values,category agreement(CA),which reflected the consistency in the determination of drug resistance,intermediary,and sensitivity,very major error(VME),which reflected false sensitivity,and major error(ME),which reflected pseudo drug resistance,to evaluate the accuracy of resazurin-based microdilution assay as a microbial sensitivity test of of Neisseria gonorrhoeae.CA and EA rates≥90%and VME and ME rates≤3%were found to be the acceptable performance rates.Results The results obtained 6 hours after resazurin was added were consistent with those of the agar dilution method and the resazurin-based microdilution assay was established accordingly based on this parameter.The EA of resazurin-based microdilution assay for measuring the MIC results of azithromycin,ceftriaxone,and spectinomycin was 97.1%,91.5%,and 94.3%,respectively,and the CA was 88.6%,94.3%,and 94.3%,respectively.The VME was 0%for all three antibiotics,while the ME was 11.4%,5.7%,and 5.7%,respectively.Conclusion The resazurin-based microdilution assay established in this study showed good agreement with agar dilution method for measuring the MIC of antibiotics against Neisseria gonorrhoeae.Moreover,the sensitivity results of this method were highly reliable and could be easily obtained through naked eye observation.Nonetheless,the results of drug resistance should be treated with caution and the optimization of parameters should be continued.

Objective To establish a viable bacteria assay for Helicobacter pylori(H.pylori)by assessing the cgt gene expression,and to develop accordingly a rapid and novel testing method for clinical precision treatment.Methods Viable bacteria count was determined in bacterial cultures.The transcriptional expression level of cgt(hp0421),the conserved gene that encodes cholesterol-α-glucosyltransferase(CGT)in H.pylori,was measured by RT-PCR.The correlation between the number of colonies and cgt gene transcription expression was analyzed and the regression model was constructed.The linear range,sensitivity,and specificity of the new method were examined accordingly.The bactericidal action of clarithromycin was assessed using this method to verify the performance of the method in determining clinical bacterial drug resistance.Results The Ct values of cgt for H.pylori colony counts of 102,104,106,and 108 CFU/mL were 29.67±0.14,23.37±0.36,17.65±0.37,and 11.38±0.39,respectively.In the range of 101-108 CFU/mL,the regression equation for cgt gene expression and viable bacterial counts determined by RT-qPCR was y=-0.350 1x+12.49,with the correlation coefficient being R2=0.9992 and the sensitivity being 101 CFU/mL,showing no cross-reaction with 13 other bacteria.The lg values of live H.pylori bacteria treated with clarithromycin at 0,5,10,20,and 40 μg/mL for 12 h were 2.57±0.02,2.45±0.01,2.19±0.02,1.91±0.07,and 1.33±0.05,respectively.The corresponding cgt gene Ct values were 27.76±0.09,28.37±0.24,29.51±0.14,30.11±0.12,and 31.66±0.11.By applying the cgt gene expression in the equation,the estimated counts of viable bacteria were found to be 2.73±0.03,2.52±0.08,2.11±0.05,1.89±0.02,and 1.33±0.04,showing no significant difference in statistical analysis(P＞0.05).Conclusion The method for assessing viable bacteria account by evaluating cgt gene expression in H.pylori was successfully established,significantly reducing the time required to determine viable bacteria count and providing a new method for clinical viable bacteria testing.

Helicobacter pylori(H.pylori),for a long time,has generally been considered an extracellular bacterium.However,recent findings have shown that H.pylori can gain entry into host cells,evade attacks from the host immune system and the killing ability of medication,form stable intracellular ecological niche,and achieve re-release into the extracellular environment,thus causing recurrent infections.H.pylori intracellular infection causes cellular signaling and metabolic alterations,which may be closely associated with the pathogenesis and progression of tumors,thereby presenting new challenges for clinical eradicative treatment of H.pylori.Herein,examining this issue from a clinical perspective,we reviewed reported findings on the mechanisms of how H.pylori achieved intracellular infection,including the breaching of the host cell biological barrier,immune evasion,and resistance to autophagy.In addition,we discussed our reflections and the prospects of important questions concerning H.pylori,including the clinical prevention and control strategy,intracellular derivation,and the damage to host cells.

To carry out phylogenetic analysis for drug-resistance genes from clinical isolates of Helicobacter pylori (Hp) among patients with gastric diseases from Tibet, China.

METHODSHp strains were isolated and cultured from saliva and gastric mucosal tissues derived from patients with gastric diseases. Nine strains (including 5 isolated from oral tissues, 1 isolated from gastric tissues, and 3 representative strains of SS international standard strains used for animal models) were tested for common antibiotic resistance. Together with an ACTT 11637 international standard strain, these were subjected to re-sequencing to obtain drug-resistance genes. Such genes from various sources were compared with the resistance genes of Hp strains recorded by the NCBI website. Combined with results of drug-resistance experiments, correlation between molecular evolution and drug-resistance was analyzed.

RESULTSTesting of gastric mucosal tissues and salivary samples from 217 patients has found 89 Hp strains, which yielded a total infection rate of 41.01%. The resistance rates of 9 representative Hp strains for clarithromycin, amoxicillin, metronidazole, levofloxacin and tetracycline were 77.8%, 77.8%, 44.4%, 77.8%, and 77.8%, respectively. Compared with the reference strain, the similarity between clarithromycin-resistance genes was 99%, and that between amoxicillin- and metronidazole-resistance genes was 96%-97%. A2143G mutation was also found in clarithromycin-resistant genes of three Hp strains.

CONCLUSIONThe sensitivity of Hp to metronidazole is much higher in patients from Tibet region, and the sensitivity of Hp to clarithromycin, amoxicillin, levofloxacin and tetracycline is poor. Resistance mutations are consistent with drug resistance.

OBJECTIVE: To investigate the relationship between OPRM1 118A/G gene polymorphism and oxycodone analgesic dose in patients with cancer pain. METHODS: DNA sequencing was used to detect the genotypies of OPRM1 118 A/G site in 203 patients with moderate and severe cancer pain, and to compare the relationship between the pain degree and the dose of oxycodone at 3 and 30 days after treatment in patients with different genotypes. RESULTS: The fequencies of AA, AG and GG genotypes at the OPRM1 118 A/G site were 34.78%, 52.70%, and 12.52%, respectively. The dosage of oxycodone in GG genotype was significantly higher than that in AA genotype and AG genotype (15.44±10.19 vs. 10.25±4.53, 10.49±5.26; 89.15±27.69 vs. 43.59±12.19, 48.27±18.79) on the 3 and 30 day after treatment, difference was statistically significant (P< 0.05). CONCLUSION For cancer pain patients with GG genotype of OPRM1 118A/G site, if they need to achieve the same analgesic effect as patients with AA and AG genotype, the dose of oxycodone should be increased.
Analgesics, Opioid ; administration & dosage ; Cancer Pain ; drug therapy ; Dose-Response Relationship, Drug ; Genotype ; Humans ; Oxycodone ; administration & dosage ; Polymorphism, Single Nucleotide ; Receptors, Opioid, mu ; genetics