Objective To analyze the clinical characteristics of co-infection of Talaromyces marneffei(TM)and non-tuberculous Mycobacterium(NTM)in human immunodeficiency virus(HIV)-negative patients.Methods Clinical data of 8 HIV-negative patients with co-infection of TM and NTM in a hospital from 2019 to 2022 were co-llected.Clinical manifestations,auxiliary examination,treatment and prognosis were retrospectively analyzed.Results Among the 8 patients,5 were females and 3 were males,with an average age of(52.25±12.31)years old.All patients presented TM and NTM disseminated infection.The major involved organs were lung(100％),lymph nodes(87.5％),and skin(75.0％).Clinical symptoms mainly included cough and expectoration(87.5％),fatigue(62.5％),joint and lumbosacral pains(62.5％),fever(50.0％),as well as skin and soft tissue abscess(50.0％),etc.Anti-interferon-γ(INF-γ)autoantibodies were detected in 4 patients and the results were positive.All 8 patients(100％)had pulmonary lesions,with chest CT mainly showing spots,patches,and striped shadows in both lungs.Among them,7 cases(87.5％)had increased and enlarged mediastinal lymph nodes,4 cases(50.0％)had pleural thickening and pleural effusion,2 cases each(25.0％for each)were accompanied by pulmonary mass shadows,bronchial stenosis,as well as increased and enlarged hilar lymph nodes.One case each(12.5％for each)had pulmonary cavity formation,bronchiectasis,and pericardial effusion.Conclusion The co-infection of TM and NTM in non-HIV patients presents disseminated infection,with multiple clinical symptoms.Chest imaging shows a wide variety of pulmonary lesions.It is prone to miss diagnosis in clinic,and the effect is not ideal after treatment for single pathogen infection.

Objective:To understand the related factors of new-type drugs use and high-risk behaviors in online-dating men who have sex with men (MSM) in Shandong Province, and provide evidence for the development of targeted HIV prevention and intervention strategies.Methods:A cross-sectional study was conducted from April to July 2023 in eight sentinel surveillance sites across cities in Shandong, recruiting MSM participants. Each city enrolled a sample of 400 individuals. Face-to-face questionnaire surveys were conducted to collect data on socio-demographic characteristics, drug use and sexual behaviors, risk perception, and online-dating practices of the MSM, and χ2 test was used to compare the differences between online-dating MSM who used new-type drugs and those who didn't use. Logistic regression model was used to analyze the related factors of group sex behavior and the lack of HIV detection before sexual behavior in the online-dating MSM. Statistical analysis was performed by using software SPSS 29.0. Results:A total of 3 235 MSM were surveyed, in whom 2 787 (86.15%) used online-dating platforms to find partners in the past six months. The majority were those who were aged 25-34 years (39.18%, 1 092/2 787), unmarried/divorced/widowed (71.51%, 1 993/2 787), had an education level of college or above (69.36%, 1 933/2 787), and primarily identified as homosexual gays (77.97%, 2 173/2 787), 37.78% (1 053/2 787) believed that they were at low risk for HIV infection and 10.88% (303/2 786) believed that using new-type drugs was less harmful and 57.59% (1 605/2 787) reported new-type drugs use. Multivariate logistic regression analysis indicated that MSM who used new-type drugs had higher risk for group sex in the past six months (a OR=1.31, 95% CI: 1.06-1.63) and sexual activity without pre-HIV testing (a OR=1.57, 95% CI:1.14-2.15) compared with those who didn't use new-type drugs.MSM who believed that the risk of using new-type drugs was general, higher and very high had higher risks for group sex in the past six months (a OR=1.54, 95% CI: 1.09-2.18) and sexual activity withou pre-HIV testing (a OR=2.02, 95% CI:1.34-3.05) compared with those who believed using new-type drugs was less harmful. Conclusions:The use of new-type drugs was relatively common in the online-dating MSM in Shandong, with widespread high-risk behaviors and poor awareness of risks to health. A gap between knowledge awareness and behavior exists. It is necessary to strengthen the health education and promotion of HIV informed dating practices in MSM.

Objective To obtain tree shrew Vasorin(VASN)recombinant protein through prokaryotic expression and purification,prepare monoclonal antibody against tree shrew VASN by immunizing mice with this protein,and preliminarily evaluate its application value.Methods Reverse transcription-polymerase chain reaction(RT-PCR)was used to amplify the full-length sequence of tree shrew VASN gene in vitro.The tree shrew VASN gene fragment was inserted into pET-30a vector to construct pET-30a-VASN recombinant plasmid.The recombinant plasmid was subjected to double digestion with BamH Ⅰ and Sal Ⅰfor identification,and its correctness was further verified by sequencing.The recombinant plasmid with correct sequencing was transformed into BL21(DE3)competent cells,and isopropyl β-D-thiogalactoside(IPTG)was used to induce expression of VASN recombinant protein.Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),and the VASN recombinant protein was purified by KCI.Purified recombinant protein was used to immunize BALB/c mice for four times,and serum antibody titer was detected by enzyme-linked immunosorbent assay(ELISA).Splenocytes from mice with serum antibody titer above 1:10 000 were used for cell fusion with myeloma cells.Hypoxanthine-aminopterin-thymidine(HAT)culture medium was first used to screen hybridoma cells.ELISA was used to screen positive hybridoma cell lines that could secrete specific antibodies,and monoclonal hybridoma cell lines were obtained by limiting dilution method.VASN monoclonal antibodies were prepared in large quantities by ascites induction method,purified using rProtein G,and the affinity and in vitro reaction specificity of the monoclonal antibodies were detected by ELISA and Western blotting.Results The full-length sequence of the tree shrew VASN gene was successfully amplified and the recombinant plasmid vector of tree shrew pET-30a-VASN was constructed.The sequence obtained by sequencing of the recombinant plasmid vector was identical to the tree shrew VASN target gene sequence.Recombinant protein VASN mainly existed in the form of inclusion bodies,and the purity after purification reached 90％,meeting the requirements of subsequent immunization experiments.After four immunizations with recombinant protein VASN,mouse serum antibody titer reached 1:729 000.Monoclonal positive hybridoma cell lines were obtained through ascites induction and purification,and the constant affinity value of monoclonal antibodies measured by ELISA reached 2.59x107 L/mol.Western blotting results showed that the tree shrew VASN monoclonal antibody could bind to tree shrew VASN recombinant protein,but it showed no binding reaction with porcine retinol-binding protein 4 recombinant protein,human VASN-leucine rich repeat recombinant protein,or bovine serum albumin.Anti-tree shrew VASN monoclonal antibody could specifically recognize VASN protein in tree shrew heart,liver,spleen,lung,kidney and muscle,with clear bands and clean background.Immunohistochemical detection results showed that this monoclonal antibody could recognize VASN protein in tree shrew spleen,lung,and tree shrew immortalized fibroblasts with high VASN mRNA expression levels,and the detection results were positive.Conclusion Monoclonal antibody against tree shrew VASN is successfully prepared.This antibody can be used for immunohistochemical detection of tree shrew immortalized fibroblasts,spleen tissue,and lung tissue,providing an important tool for further research on the function of VASN in tree shrew models.

Objective:To understand the related factors of new-type drugs use and high-risk behaviors in online-dating men who have sex with men (MSM) in Shandong Province, and provide evidence for the development of targeted HIV prevention and intervention strategies.Methods:A cross-sectional study was conducted from April to July 2023 in eight sentinel surveillance sites across cities in Shandong, recruiting MSM participants. Each city enrolled a sample of 400 individuals. Face-to-face questionnaire surveys were conducted to collect data on socio-demographic characteristics, drug use and sexual behaviors, risk perception, and online-dating practices of the MSM, and χ2 test was used to compare the differences between online-dating MSM who used new-type drugs and those who didn't use. Logistic regression model was used to analyze the related factors of group sex behavior and the lack of HIV detection before sexual behavior in the online-dating MSM. Statistical analysis was performed by using software SPSS 29.0. Results:A total of 3 235 MSM were surveyed, in whom 2 787 (86.15%) used online-dating platforms to find partners in the past six months. The majority were those who were aged 25-34 years (39.18%, 1 092/2 787), unmarried/divorced/widowed (71.51%, 1 993/2 787), had an education level of college or above (69.36%, 1 933/2 787), and primarily identified as homosexual gays (77.97%, 2 173/2 787), 37.78% (1 053/2 787) believed that they were at low risk for HIV infection and 10.88% (303/2 786) believed that using new-type drugs was less harmful and 57.59% (1 605/2 787) reported new-type drugs use. Multivariate logistic regression analysis indicated that MSM who used new-type drugs had higher risk for group sex in the past six months (a OR=1.31, 95% CI: 1.06-1.63) and sexual activity without pre-HIV testing (a OR=1.57, 95% CI:1.14-2.15) compared with those who didn't use new-type drugs.MSM who believed that the risk of using new-type drugs was general, higher and very high had higher risks for group sex in the past six months (a OR=1.54, 95% CI: 1.09-2.18) and sexual activity withou pre-HIV testing (a OR=2.02, 95% CI:1.34-3.05) compared with those who believed using new-type drugs was less harmful. Conclusions:The use of new-type drugs was relatively common in the online-dating MSM in Shandong, with widespread high-risk behaviors and poor awareness of risks to health. A gap between knowledge awareness and behavior exists. It is necessary to strengthen the health education and promotion of HIV informed dating practices in MSM.

Objective To obtain tree shrew Vasorin(VASN)recombinant protein through prokaryotic expression and purification,prepare monoclonal antibody against tree shrew VASN by immunizing mice with this protein,and preliminarily evaluate its application value.Methods Reverse transcription-polymerase chain reaction(RT-PCR)was used to amplify the full-length sequence of tree shrew VASN gene in vitro.The tree shrew VASN gene fragment was inserted into pET-30a vector to construct pET-30a-VASN recombinant plasmid.The recombinant plasmid was subjected to double digestion with BamH Ⅰ and Sal Ⅰfor identification,and its correctness was further verified by sequencing.The recombinant plasmid with correct sequencing was transformed into BL21(DE3)competent cells,and isopropyl β-D-thiogalactoside(IPTG)was used to induce expression of VASN recombinant protein.Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),and the VASN recombinant protein was purified by KCI.Purified recombinant protein was used to immunize BALB/c mice for four times,and serum antibody titer was detected by enzyme-linked immunosorbent assay(ELISA).Splenocytes from mice with serum antibody titer above 1:10 000 were used for cell fusion with myeloma cells.Hypoxanthine-aminopterin-thymidine(HAT)culture medium was first used to screen hybridoma cells.ELISA was used to screen positive hybridoma cell lines that could secrete specific antibodies,and monoclonal hybridoma cell lines were obtained by limiting dilution method.VASN monoclonal antibodies were prepared in large quantities by ascites induction method,purified using rProtein G,and the affinity and in vitro reaction specificity of the monoclonal antibodies were detected by ELISA and Western blotting.Results The full-length sequence of the tree shrew VASN gene was successfully amplified and the recombinant plasmid vector of tree shrew pET-30a-VASN was constructed.The sequence obtained by sequencing of the recombinant plasmid vector was identical to the tree shrew VASN target gene sequence.Recombinant protein VASN mainly existed in the form of inclusion bodies,and the purity after purification reached 90％,meeting the requirements of subsequent immunization experiments.After four immunizations with recombinant protein VASN,mouse serum antibody titer reached 1:729 000.Monoclonal positive hybridoma cell lines were obtained through ascites induction and purification,and the constant affinity value of monoclonal antibodies measured by ELISA reached 2.59x107 L/mol.Western blotting results showed that the tree shrew VASN monoclonal antibody could bind to tree shrew VASN recombinant protein,but it showed no binding reaction with porcine retinol-binding protein 4 recombinant protein,human VASN-leucine rich repeat recombinant protein,or bovine serum albumin.Anti-tree shrew VASN monoclonal antibody could specifically recognize VASN protein in tree shrew heart,liver,spleen,lung,kidney and muscle,with clear bands and clean background.Immunohistochemical detection results showed that this monoclonal antibody could recognize VASN protein in tree shrew spleen,lung,and tree shrew immortalized fibroblasts with high VASN mRNA expression levels,and the detection results were positive.Conclusion Monoclonal antibody against tree shrew VASN is successfully prepared.This antibody can be used for immunohistochemical detection of tree shrew immortalized fibroblasts,spleen tissue,and lung tissue,providing an important tool for further research on the function of VASN in tree shrew models.

Objective To analyze the clinical characteristics of co-infection of Talaromyces marneffei(TM)and non-tuberculous Mycobacterium(NTM)in human immunodeficiency virus(HIV)-negative patients.Methods Clinical data of 8 HIV-negative patients with co-infection of TM and NTM in a hospital from 2019 to 2022 were co-llected.Clinical manifestations,auxiliary examination,treatment and prognosis were retrospectively analyzed.Results Among the 8 patients,5 were females and 3 were males,with an average age of(52.25±12.31)years old.All patients presented TM and NTM disseminated infection.The major involved organs were lung(100％),lymph nodes(87.5％),and skin(75.0％).Clinical symptoms mainly included cough and expectoration(87.5％),fatigue(62.5％),joint and lumbosacral pains(62.5％),fever(50.0％),as well as skin and soft tissue abscess(50.0％),etc.Anti-interferon-γ(INF-γ)autoantibodies were detected in 4 patients and the results were positive.All 8 patients(100％)had pulmonary lesions,with chest CT mainly showing spots,patches,and striped shadows in both lungs.Among them,7 cases(87.5％)had increased and enlarged mediastinal lymph nodes,4 cases(50.0％)had pleural thickening and pleural effusion,2 cases each(25.0％for each)were accompanied by pulmonary mass shadows,bronchial stenosis,as well as increased and enlarged hilar lymph nodes.One case each(12.5％for each)had pulmonary cavity formation,bronchiectasis,and pericardial effusion.Conclusion The co-infection of TM and NTM in non-HIV patients presents disseminated infection,with multiple clinical symptoms.Chest imaging shows a wide variety of pulmonary lesions.It is prone to miss diagnosis in clinic,and the effect is not ideal after treatment for single pathogen infection.

Objective:To understand the use of post-exposure prophylaxis (PEP) and analyze related factors among men who have sex with men (MSM) in Qingdao, and provide a reference for the AIDS prevention and control in this population.Methods:A cross-sectional survey conducted from April 2022 to February 2023. Relying on MSM social groups in Qingdao, a snowball sampling method was applied to recruit research subjects who met the inclusion criteria of age ≥18 years old, having had homosexual anal or oral sex in the past six months, and HIV-negative or infection status unknown. The sample size was estimated at 566. Data on demographic characteristics, sexual behavior characteristics, PEP use, and others of the research subjects was collected through on-site questionnaires. The logistic regression model was used to analyze the factors associated with using PEP.Results:A total of 811 participants were recruited, mainly aged 25-34 (53.6%, 435/811), unmarried (74.7%, 606/811), with an average monthly income of ≥5 000 yuan (52.2%, 423/811), and having lived in Qingdao for ≥10 years (75.6%, 613/811). The proportion of those who knew the HIV status of their sexual partners in the last six months was 67.1% (544/811), and those with HIV-positive partners were 3.6% (29/811). In the last six months, the proportion of participants who had group sex (86.4%, 701/811), unprotected anal sex (98.2%, 796/811), and use of club drugs (80.3%, 651/811) was high. Moreover, 28.4% (230/811) had used PEP. The multivariate logistic regression analysis showed that the factors related to the use of PEP included divorced or widowed (a OR=5.46,95% CI:1.96-15.17), average monthly income ≥5 000 yuan (a OR=2.04,95% CI:1.44-2.89), same-sex sexual orientation (a OR=0.40,95% CI:0.22-0.71), having HIV-positive sexual partners in the last six months (a OR=2.54,95% CI:1.13-5.71) and having been tested for HIV ≥3 times in the last six months (a OR=1.46,95% CI:1.04-2.06). Conclusions:The prevalence of risk behaviors among MSM in Qingdao was high, and the use of PEP was low. In the future, it is essential to increase HIV/AIDS prevention education among MSM, promote MSM to know the HIV status of their sexual partners, and reduce the prevalence of risk behaviors among this population. Additionally, explore medical insurance reimbursement plans for PEP to reduce utilization costs and promote the use of PEP by MSM after HIV exposure occurs as soon as possible.

Objective:To compare the chemical composition of decoction and granules of Sangju Decoction; To provide a method for quality evaluation of Sangju Decoction.Methods:HPLC was used to establish fingerprints, and a comprehensive comparative study was conducted on the traditional decoction and formula granules of Sangju Decoction from four aspects: chemical composition type, fingerprint similarity, chemical pattern recognition analysis, and representative index component content.Results:The fingerprint similarity of the 10 batches of traditional decoction was >0.988. 35 peaks were identified and 12 peaks were identified as common peaks (neochlorogenic acid for peak 7, chlorogenic acid for peak 10, cryptochlorogenic acid for peak 11, 1,3-dicaffeoylquinic acid for peak 13, rutin for peak 17, lenoside A for peak 19, lignan for peak 20, isochlorogenic acid B for peak 24, ammonium glycyrrhizate for peak 25). The fingerprint similarity of the formulation pellets was >0.983, and 29 characteristic peaks were identified. Compared with the traditional decoction, some batches of the granules lacked peaks 14, 26, 27, 30, 32 and 34, and clustering analysis (CA), principal component analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA) could distinguish between the two. The contents of the 10 index components neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 1,3-dicaffeoylquinic acid, forsythia ester glycoside A, grass glycosides, isochlorogenic acid B, 3,5-O-dicaffeoylquinic acid, forsythia glycosides, monkshood glycosides in the traditional soup were higher than that in the granules, and the contents of rutin and ammonium glycyrrhizate in the granules were higher than that in traditional decoction.Conclusions:The content and composition of traditional decoction and formula granules of Sangju Decoction are significantly different. The combination of fingerprinting and chemical pattern identification effectively can effectively evaluate the difference between traditional decoction and formula granules of Sangju Decoction, which can lay a foundation for the quality control and rational clinical application of formula granules of Sangju Decoction.

Objective:To compare the characteristics of MSM with different types of primary sexual partners and to analyze the factors associated with MSM not being tested for HIV in the last six months.Methods:MSM were recruited in nine cities of Shandong Province from April to July 2021, and face-to-face questionnaires were conducted to collect information on sociodemographic characteristics, high-risk behaviors, and HIV testing of MSM. Blood samples were taken for serological tests of HIV and syphilis antibodies.Results:A total of 3 008 men who had anal sex with men in the last six months were divided into three groups according to the type of primary sexual partner in the last six months: the fixed sexual partner group (36.83%, 1 108/3 008), the commercial sexual partner group (3.06%, 92/3 008), and casual sexual partner group (60.11%, 1 808/3 008). There were statistically significant differences in the distribution of age, local residence time, education level, the primary place to find male sexual partners, use of new-type drugs in the last six months, consistent condom use every time during same-sex anal sex in the last six months, group sex in the last six months, no HIV testing in the last six months, having had a sexually transmitted disease in the last year, receiving peer education in the last year, and frequency of syphilis testing in the last year among different groups ( P<0.05). Multivariable logistic regression analysis showed that related factors of not being tested for HIV in the last six months in MSM were those aged less than 30 years old (a OR=1.39, 95% CI: 1.06-1.83), married/cohabiting (a OR=1.74, 95% CI: 1.39-2.16), high school education or less (a OR=1.39, 95% CI: 1.15-1.67), had not used new-type drugs in the last six months (a OR=2.27, 95% CI: 1.89-2.71), had not received peer education in the last year (a OR=1.59, 95% CI: 1.28-1.98), had never been tested for syphilis (a OR=11.30, 95% CI: 8.15-15.66), had not been tested in the last year but had been previously tested for syphilis (a OR=5.65, 95% CI: 4.19-7.62), the type of primary sexual partner in the last six months being a commercial sexual partner (a OR=1.80, 95% CI: 1.01-3.20), and the type of primary sexual partner in the last six months being a casual sexual partner (a OR=1.50, 95% CI: 1.26-1.80). Conclusions:The characteristics of MSM with different types of primary sexual partners are different, and the proportion of HIV testing still needs to be improved. In the future, we should make full use of the Internet and peer education to expand the coverage of HIV testing for MSM, targeting the characteristics of MSM with different types of primary sexual partners.

Objective:To explore the influencing factors of AIDS-related deaths among HIV-infected patients in Shandong Province, to help reduce the risk of death and prolong survival time.Methods:The study population was HIV-infected patients in Shandong Province from 2017-2021, and Cox proportional hazards regression model was used to analyze the influencing factors of AIDS-related deaths and deaths within one year of confirmation.Results:Among 14 700 HIV- infected patients reported in Shandong Province in 2017-2021, 351 AIDS-related deaths occurred, accounting for 2.4% (351/14 700). The results of multifactorial Cox proportional hazards regression model analysis showed that the risk factors for AIDS-related deaths among HIV-infected patients included education level of junior high school, high school, and secondary school (a HR=1.37, 95% CI:1.01-1.84), sample source from healthcare institutions (a HR=1.61, 95% CI:1.22-2.12), duration of disease in AIDS stage (a HR=9.86,95% CI:6.86-14.19), baseline CD4 +T lymphocytes (CD4) undetected (a HR=3.93, 95% CI:2.69-5.75), duration of antiviral treatment (ART) <6 months (a HR=3.46, 95% CI:2.42-4.93) and no ART (a HR=1.45, 95% CI:1.02-2.07), final CD4 <200 cells/μl (a HR=3.51, 95% CI:2.18-5.65) and final CD4 undetected (a HR=10.58, 95% CI:6.15-18.19), and final viral load (VL) values of 50-999 copies/ml,≥1 000 copies/ml and undetected (a HR=2.59, 95% CI:1.07-6.26; a HR=9.50, 95% CI:5.60-16.12; a HR=15.33, 95% CI:8.91-26.36). Factors with higher risk of AIDS-related deaths within one year of confirmation of HIV-infected patients included samples originating from healthcare facilities (a HR=1.68, 95% CI: 1.19-2.36), AIDS stage of disease (a HR=10.60, 95% CI:7.13-15.75), baseline CD4 undetected (a HR=3.71, 95% CI:2.34-5.90), duration of ART <6 months (a HR=4.30, 95% CI:2.85-6.49) and no ART (a HR=2.05, 95% CI:1.35-3.13), final CD4 <200 cells/μl (a HR=5.45,95% CI:2.04-14.60) and final CD4 undetected (a HR=20.95, 95% CI: 7.69- 57.04), and final VL values of 50-999 copies/ml, ≥1 000 copies/ml and undetected (a HR=15.21, 95% CI: 2.54-91.21; a HR=42.93, 95% CI:9.64-191.20; a HR=61.35, 95% CI:13.85-271.77). Conclusions:Expanding the coverage of testing, promoting early detection and treatment, strengthening regular follow-up and the test of HIV-infected patients, grasping the progress of the disease to provide accurate management and treatment are important for reducing the disease mortality rate and prolonging the survival time of HIV-infected patients.