Objective:To investigate the value of targeted three-dimensional contrast-enhanced ultrasound（3D-tCEUS）in efficacy evaluation of non-surgical treatments for renal cell carcinoma（RCC）.Methods:Forty nude mice with subcutaneous xenograft tumor model of human RCC（786-O cells）were divided into four groups based on different treatment methods：control，thermal ablation（TA），sunitinib（Suni），and TA+Suni. 3D-tCEUS were performed on days 1，3，7，and 14 post-treatment using self-developed vascular endothelial growth factor receptor 2（VEGFR2）targeted microbubbles. Tumor overall volume（V T）and non-enhanced volume（V N）were measured，from which the volume of the active（enhanced）region was calculated as V A = V T - V N. The tumor total and active area volume was standardized as V T st and V A st（standardized tumor volume = tumor volume after treatment / tumor volume at the beginning of treatment）. Tumor growth curves were plotted and tumor inhibition rates calculated for V T and V A respectively. Quantitative parameters，including the area under the curve（AUC）and the difference in peak intensity before and after burst（dTE），were obtained from the viable tumor enhanced region，and the standardized targeted quantitative parameters were derived by calculating the ratios of parameters at various time points post-treatment to those pre-treatment. The differences in V T st，V A st，AUC and dTE between different treatment groups at different time points were compared. At the end of the experiment，tumor tissues were obtained for immunohistochemical staining to observe the expression of VEGFR2 and CD31 antigens. Results:During the treatment period，no statistically significant differences in weight changes were observed among groups（all P > 0.05）. When V T was taken as the research object，V T st increased across all groups during the treatment period，with the TA group showing the most significant growth，while the TA + Suni group exhibited the smoothest increase in growth curve. When analyzing the tumor enhanced region，tumor growth trend of V A st was different with V T st for all groups；the Suni group showed a slow upward trend，whereas the TA + Suni group showed a continuous decline. Significant differences in tumor inhibition rate originated from V T and V A were noted within the same experimental group（ P < 0.05 for all experimental groups）. One day post-treatment，the AUC and dTE of the TA group were higher than that of the Control group，while the dTE of the TA + Suni group was lower than that of the Control group（all P < 0.05）. By day 3，statistically significant differences in AUC and dTE were observed between each experimental group and Control groups（all P < 0.05）. At day 14，the TA group showed increased AUC and dTE compared with those before treatment，while all other groups，particularly Suni group and TA + Suni group，demonstrated significant reductions（all P < 0.05）. Immunohistochemical results revealed the highest VEGFR2 and CD31 positivity in the TA group，followed by the Control group，while the Suni and TA + Suni groups exhibited lower rates. Conclusions:The combination of TA and targeted therapy effectively induces RCC cells death，demonstrating superior efficacy compared to monotherapy. 3D-tCEUS serves as a accurate and reliable tool for early evaluating the efficacy of non-surgical RCC treatments.

Objective:To investigate the value of targeted three-dimensional contrast-enhanced ultrasound（3D-tCEUS）in efficacy evaluation of non-surgical treatments for renal cell carcinoma（RCC）.Methods:Forty nude mice with subcutaneous xenograft tumor model of human RCC（786-O cells）were divided into four groups based on different treatment methods：control，thermal ablation（TA），sunitinib（Suni），and TA+Suni. 3D-tCEUS were performed on days 1，3，7，and 14 post-treatment using self-developed vascular endothelial growth factor receptor 2（VEGFR2）targeted microbubbles. Tumor overall volume（V T）and non-enhanced volume（V N）were measured，from which the volume of the active（enhanced）region was calculated as V A = V T - V N. The tumor total and active area volume was standardized as V T st and V A st（standardized tumor volume = tumor volume after treatment / tumor volume at the beginning of treatment）. Tumor growth curves were plotted and tumor inhibition rates calculated for V T and V A respectively. Quantitative parameters，including the area under the curve（AUC）and the difference in peak intensity before and after burst（dTE），were obtained from the viable tumor enhanced region，and the standardized targeted quantitative parameters were derived by calculating the ratios of parameters at various time points post-treatment to those pre-treatment. The differences in V T st，V A st，AUC and dTE between different treatment groups at different time points were compared. At the end of the experiment，tumor tissues were obtained for immunohistochemical staining to observe the expression of VEGFR2 and CD31 antigens. Results:During the treatment period，no statistically significant differences in weight changes were observed among groups（all P > 0.05）. When V T was taken as the research object，V T st increased across all groups during the treatment period，with the TA group showing the most significant growth，while the TA + Suni group exhibited the smoothest increase in growth curve. When analyzing the tumor enhanced region，tumor growth trend of V A st was different with V T st for all groups；the Suni group showed a slow upward trend，whereas the TA + Suni group showed a continuous decline. Significant differences in tumor inhibition rate originated from V T and V A were noted within the same experimental group（ P < 0.05 for all experimental groups）. One day post-treatment，the AUC and dTE of the TA group were higher than that of the Control group，while the dTE of the TA + Suni group was lower than that of the Control group（all P < 0.05）. By day 3，statistically significant differences in AUC and dTE were observed between each experimental group and Control groups（all P < 0.05）. At day 14，the TA group showed increased AUC and dTE compared with those before treatment，while all other groups，particularly Suni group and TA + Suni group，demonstrated significant reductions（all P < 0.05）. Immunohistochemical results revealed the highest VEGFR2 and CD31 positivity in the TA group，followed by the Control group，while the Suni and TA + Suni groups exhibited lower rates. Conclusions:The combination of TA and targeted therapy effectively induces RCC cells death，demonstrating superior efficacy compared to monotherapy. 3D-tCEUS serves as a accurate and reliable tool for early evaluating the efficacy of non-surgical RCC treatments.

Objective:To explore the relationship between drug resistance occurrence and the distribution pattern of polymorphic loci in individuals with human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) treated with highly active anti-retroviral therapy (HAART).Methods:HAART-failed HIV/AIDS patients who successfully amplified the gene sequences of the pol region between June 2015 and December 2021 from 16 prefecture-level administrative regions in Yunnan Province were included.The resistant sequences were classified using the human immunodeficiency virus (HIV) basic local alignment search tool (BLAST) and validated through MEGA 6.0, and the obtained sequences were submitted to the Stanford University HIV Drug Resistance Database to identify drug resistance loci. The distribution of polymorphic loci was analyzed across patients exhibiting varying degrees of drug resistance, different treatment regimens and distinct HIV-1 subtypes.Changes of the frequencies of polymorphic loci in patients with different degrees of drug resistance were analyzed using trend chi-square test. Statistical comparisons and further paired comparisons were performed using chi-square test.Results:Gene sequences were amplified from 1 453 patients, and the resistance testing results showed 954 sensitive, 224 potentially or low resistant, 189 moderately resistant, and 86 highly resistant patients. The frequencies of mutations I15V, L19I, D60E in the HIV-1 protease region (PR region) and E36A, T39D, S48T mutations in the HIV-1 reverse transcriptase region (RT region) showed a decreasing trend as the degree of HIV-1 resistance escalated ( χ2trend=19.86, 9.16, 13.66, 37.64, 18.44 and 40.86, respectively, all P<0.01). Conversely, the mutations V77I in the PR region and K122E in the RT region showed an ascending trend ( χ2trend=12.19 and 10.03, respectively, both P<0.01). Distinct treatment groups, namely zidovudine (AZT)+ lamivudine (3TC)+ lopinavir/ritonavir (LPV/r), AZT+ 3TC+ efavirenz (EFV), AZT+ 3TC+ nevirapine (NVP), and tenofovir (TDF)+ 3TC+ EFV, were examined. Statistically significant differences in the frequencies of mutations E35D, M36I, and D60E in the PR region, as well as S48T, K122E, and R211K in the RT region, were observed among these treatment groups ( χ2=22.46, 9.32, 14.46, 26.85, 18.92 and 24.26, respectively, all P<0.05). In paired comparisons, AZT+ 3TC+ LPV/r group displayed higher frequencies of E35D, M36I, and D60E mutations, the AZT+ 3TC+ EFV group showed a higher frequency of S48T mutation, the AZT+ 3TC+ NVP group showed a higher frequency of K122E mutation, and the TDF+ 3TC+ EFV group exhibited a higher frequency of R211K mutation, all with statistically significant differences (all P<0.008). The differences in the frequencies of T12S, I15V, L19I, M36I, V77I, L89M in the PR region and E53D, I135V, S162C, R211K, K277R in the RT region among circulating recombinant form (CRF)08_BC, CRF07_BC and CRF01_AE subtype group were statistically significant ( χ2=693.60, 712.51, 798.11, 434.85, 386.91, 657.78, 932.58, 409.21, 344.39, 469.44 and 260.48, respectively, all P<0.001). In paired comparisons, the frequencies of T12S, I15V, L19I, E53D, I135V, S162C and R211K in CRF08_BC subtype, the frequencies of V77I and K277R in CRF07_BC subtype, and the frequencies of M36I and L89M in CRF01_AE subtype were higher than those in the other two groups, and the differences were all statistically significant (all P<0.017). Conclusions:The polymorphic loci resulting from HIV-1 HAART failure show different distribution patterns across various degrees of drug resistance, treatment regimens and HIV-1 subtypes.These loci demonstrate both specific and shared characteristics. It is necessary to enhance the surveillance of select polymorphic loci.

Objective:To explore the clinical characteristics of tenofovir disoproxil fumarate (TDF)-related Fanconi syndrome (FS) in patients with HIV infection/AIDS (HIV/AIDS).Methods:The medical records of patients with HIV/AIDS who were hospitalized in Yunnan Provincial Hospital of Infectious Diseases from December 2017 to February 2021, treated with antiretroviral therapy (ART) containing TDF, and diagnosed as FS were collected by searching hospital information system. Information such as gender, age, body weight, body mass index (BMI), ART treatment regimen and period, time of FS diagnosis, main clinical characteristics, results of laboratory test at admission and discharge, dual energy X-ray bone mineral density (BMD) test results, and interventions and outcomes were retrospectively analyzed.Results:A total of 16 HIV/AIDS patients were diagnosed with TDF-related FS in the setting period, including 6 patients with complete FS and 10 with incomplete FS. FS were accompanied with chronic hepatitis C, hypertension, liver cancer, or depression in 7 patients. Sixteen patients received ART containing TDF for a minimum of 20 months and a maximum of 168 months with an average time of 68 months. The initial symptoms of FS were bone pain, fatigue, nausea, anorexia, polydipsia, polyuria, weight loss, etc. The time from initial symptoms to diagnosis of FS was 2 weeks at least, 24 weeks at most, with an average time of 7 weeks. Laboratory test results were as follows: all 16 patients had positive urine glucose under normoglycemic conditions and 14 patients had positive urine protein; 11, 11, 4, and 4 patients had low urine phosphorus, hypocalciuria, hypokalemia, and hyponatruria, respectively; 13, 12, 8, and 7 patients had hypophosphatemia, hypokalemia, hypocalcemia, and hyponatremia, respectively; 11 patients had serum creatinine increase; 10 patients had serum uric acid decrease; 1 patient had serum uric acid increase. Dual energy X-ray BMD detection was performed in 15 patients, of which 2, 2, and 11 patients had normal, reduced, and osteoporotic BMD, respectively. After diagnosis of TDF-related FS, 16 patients stopped using TDF immediately. After replacement of ART protocol without TDF and symptomatic treatment for an average time of 29 days, the above symptoms were alleviated, and some laboratory test indicators returned to the reference value range. The prognosis was good.Conclusions:TDF-related FS mostly occurs within 68 months of drug use. The clinical symptoms of FS are nonspecific. Laboratory tests show that urine glucose is positive under normal blood glucose. Most of the patients have low blood phosphorus, low urine phosphorus, hypocalciuria, and osteoporosis. The prognosis is better after discontinuing TDF, replacing therapy with ART regimen without TDF, and giving symptomatic treatments.

Objective:To explore the clinical characteristics of tenofovir disoproxil fumarate (TDF)-related Fanconi syndrome (FS) in patients with HIV infection/AIDS (HIV/AIDS).Methods:The medical records of patients with HIV/AIDS who were hospitalized in Yunnan Provincial Hospital of Infectious Diseases from December 2017 to February 2021, treated with antiretroviral therapy (ART) containing TDF, and diagnosed as FS were collected by searching hospital information system. Information such as gender, age, body weight, body mass index (BMI), ART treatment regimen and period, time of FS diagnosis, main clinical characteristics, results of laboratory test at admission and discharge, dual energy X-ray bone mineral density (BMD) test results, and interventions and outcomes were retrospectively analyzed.Results:A total of 16 HIV/AIDS patients were diagnosed with TDF-related FS in the setting period, including 6 patients with complete FS and 10 with incomplete FS. FS were accompanied with chronic hepatitis C, hypertension, liver cancer, or depression in 7 patients. Sixteen patients received ART containing TDF for a minimum of 20 months and a maximum of 168 months with an average time of 68 months. The initial symptoms of FS were bone pain, fatigue, nausea, anorexia, polydipsia, polyuria, weight loss, etc. The time from initial symptoms to diagnosis of FS was 2 weeks at least, 24 weeks at most, with an average time of 7 weeks. Laboratory test results were as follows: all 16 patients had positive urine glucose under normoglycemic conditions and 14 patients had positive urine protein; 11, 11, 4, and 4 patients had low urine phosphorus, hypocalciuria, hypokalemia, and hyponatruria, respectively; 13, 12, 8, and 7 patients had hypophosphatemia, hypokalemia, hypocalcemia, and hyponatremia, respectively; 11 patients had serum creatinine increase; 10 patients had serum uric acid decrease; 1 patient had serum uric acid increase. Dual energy X-ray BMD detection was performed in 15 patients, of which 2, 2, and 11 patients had normal, reduced, and osteoporotic BMD, respectively. After diagnosis of TDF-related FS, 16 patients stopped using TDF immediately. After replacement of ART protocol without TDF and symptomatic treatment for an average time of 29 days, the above symptoms were alleviated, and some laboratory test indicators returned to the reference value range. The prognosis was good.Conclusions:TDF-related FS mostly occurs within 68 months of drug use. The clinical symptoms of FS are nonspecific. Laboratory tests show that urine glucose is positive under normal blood glucose. Most of the patients have low blood phosphorus, low urine phosphorus, hypocalciuria, and osteoporosis. The prognosis is better after discontinuing TDF, replacing therapy with ART regimen without TDF, and giving symptomatic treatments.

Objective:To explore the capability of vascular endothelial growth factor receptor 2 (VEGFR2)/integrinα vβ 3 dual-targeted microbubbles in assessing the expression level of pro-angiogenic factors during renal cell carcinoma (RCC) growth. Methods:VEGFR2/integrinα vβ 3 dual-targeted microbubbles were prepared by using biotin-avidin linkage method. Twenty subcutaneous RCC xenografts in nude mice were established by subcutaneously injecting 786-O cells and then divided into 2 groups randomly. The targeted contrast-enhanced ultrasound (t-CEUS) examination was performed for all 10 mice in the first group when xenograft tumors were metered from 5 to 10 mm and >10 to 20 mm respectively. And the quantitative parameters of RCC on t-CEUS were longitudinally evaluated during tumor growth. The second group were divided into two subgroups according to xenograft tumors′ diameter, which was 5 to 10 mm and >10 to 20 mm respectively, and underwent t-CEUS examination. Quantitative analysis was performed for all t-CEUS images to obtain the targeted quantitative parameters, which including peak intensity (PI), area under the time-intensity curve (AUC), the differential tissue enhancement (dTE, presenting the difference in PI before (P 1) and after (P 2) the process of Flash). All xenograft tumors in the second group were harvested for immunohistochemical staining to observe the expression of VEGFR2, integrinα vβ 3 and CD31, and their differences in RCC with different tumor sizes. And the correlations between quantitative parameters and VEGFR2, integrinα vβ 3 and CD31 were analyzed. Results:The longitudinal comparison showed that there were statistically significant differences between AUC and dTE of RCC with different tumor sizes (all P<0.001). The larger the tumor size, the smaller the parameters were. According to the horizontal comparison, the expression levels of VEGFR2 and integrinα vβ 3 in larger RCCs were higher than those of RCCs with smaller size (both P<0.05), but there was no significant difference in CD31 expression between the two subgroups ( P=0.754). Both the targeted quantitative parameters (AUC anddTE ) and pro-angiogenic factors (VEGFR2 and integrinα vβ 3) were negatively correlated with tumor size ( rs=-0.83, -0.81, -0.70, -0.88; all P<0.05). Further more, there were good positive correlations between AUC and VEGFR2, integrinαvβ ( rs=0.76, 0.72; all P<0.05). There were good positive correlations between dTE and VEGFR2, integrinα vβ 3 ( rs=0.81, 0.70; all P<0.05). Additionally, the parameter PI was positively correlated with the expression of CD31 ( rs=0.70, P=0.025). Conclusions:The t-CEUS, mediated by VEGFR/integrinα vβ 3 dual-targeted microbubbles, allows noninvasive assessment of the expression levels of VEGFR2 and integrinα vβ 3 in RCCs, which decrease gradually with the increase of tumor size.

Objective:To evaluate the ability of vascular endothelial growth factor receptor 2(VEGFR2)/integrin α vβ 3 dual-targeted microubble (MBD) to target angiogenesis of renal cell carcinoma (RCC) in vivo. Methods:Non-targeted microbubble (MBN) USphere LA was employed as a template to prepare single- and dual-targeted microbubbles which could bind VEGFR2 and/or integrin α vβ 3 (MBV and MBI) by the biotin-avidin bridging method. A total of 40 RCC nude mice models were established by subcutaneously injecting 786-O cells.Twenty of the models were all injected with MBN, MBV, MBI and MBD in a random order, and the other 20 models were registered for antibody blocking assays. The results of ultrasound images were used for quantitative analyses, and the following quantitative parameters were obtained: intensity increment (a 1), peak halving speed (a 2), curve rising slope (a 3), perfusion time (t 0), time to peak (TTP), peak intensity (PI), mean transit time (MTT) and area under the curve (AUC) for the first three minutes, peak intensity at 10 s before (P 1) and after (P 2) ultrasound destruction, and the differences of tissue enhancement (dTE) between P 1 and P 2 (dTE=P 1-P 2). All the quantitative parameters of four contrast agents and the antibody blocking assays were compared.Besides, the immunohistochemical assays were performed to evaluate the expression of CD31, VEGFR2 and integrin α vβ 3 in tumor tissues. Results:The differences of parameters of a 1, a 3, t 0, TTP, PI and P 2 among four different microbubbles had no statistical significances (all P>0.05), and all parameters between the two single-targeted contrast agents were not statistically different (all P>0.05). The parameters of AUC, MTT, P 1 and dTE all showed a trend that dual-targeted bubbles > single-targeted bubbles > non-targeted bubbles (all P<0.05). On the contrary, the trend of dual-targeted bubbles < single-targeted bubbles < non-targeted bubbles (all P<0.05) was observed for a 2. In the antibody blocking experiment, a 2 was faster after the antibody injection ( P<0.001), while AUC, MTT, P 1 and dTE were all lower than those before the antibody injection ( P<0.001), and the other parameters were not statistically different before and after the antibody injection (all P>0.05). Immunohistochemical analyses confirmed the high expression of CD31, VEGFR2 and integrin β 3 in tumor tissues. Conclusions:The VEGFR2 and integrin α vβ 3 dual-targeted microbubble has a good potential to target the angiogenesis of human RCC in vivo.

Objective:To investigate the clinical feasibility of three-dimensional contrast-enhanced ultrasound (3D-CEUS) in the quantitative assessment of blood perfusion of hepatocellular carcinoma (HCC).Methods:Between January 2020 and August 2021, 36 HCC patients (39 lesions in total) confirmed by pathology and clinical diagnosis without any treatment from Zhongshan Hospital, Fudan University were enrolled and underwent both 2D-CEUS and 3D-CEUS examinations. Each examination last for 150 s and all images were recorded, and then the data were analyzed. A region of interest was manually drawn along the margin of the whole tumor and then the time-intensity curve (TIC) generated. The following perfusion parameters were extracted: peak intensity (PI), peak time (TTP), ascending slope (AS), mean transit time (MTT) and area under the curve (AUC). After calculating the quality of fit (QOF) of the curve, the intraobserver agreement of the 3D-CEUS quantitative parameters obtained by the same doctor between two times were assessed, and the consistency of the 3D-CEUS and 2D-CEUS quantitative parameters was evaluated when QOF>75%. The differences of the quantitative parameters between different groups (divided by depth of 8 cm and necrosis rate of 50%, respectively) in 3D-CEUS were compared.Results:There were 38 lesions (97.4%, 38/39) with QOF>75% in 3D-CEUS. The intraobserver agreement was excellent, the intraclass correlation efficient(ICC) values was 0.85-0.99. The consistency of the time quantitative parameters (TTP and MTT) were high (the ICC values of 0.87 and 0.91), and the correlation of intensity quantitative parameters were substantial, the rs values were 0.71, 0.72 and 0.71. The differences in 3D-CEUS quantitative parameters of the two groups of lesions with different depths were statistically significant (all P<0.05); but there were no significant differences in quantitative parameters between the two groups with different necrosis rate (all P>0.05). Conclusions:Quantitative 3D-CEUS is an useful and creditable tool in evaluating the blood perfusion of HCC, especially when the depth of lesion was less than 8 cm.

Objective:To investigate the value of contrast-enhanced ultrasonography (CEUS) in detecting minute renal cell carcinoma (MRCC) smaller than 15 mm (by ultrasonic measurement) and the strategy to improve its detection rate.Methods:Fifty-three pathologically confirmed MRCCs by surgery from November 2007 to October 2019 at Zhongshan Hospital of Fudan University were enrolled in this retrospectively study. All of them underwent both conventional ultrasound and CEUS examinations. The clinical and imaging data were collected and analyzed. Common features, such as tumor size, location, echogenicity, morphology, border, and blood flow signals were observed on conventional ultrasound. On CEUS, the presence of enhancement, wash in and wash out pattern, perfusion uniformity within the lesions were observed.Results:Post-operative pathology confirmed 48 clear cell carcinomas, 4 papillary carcinomas, and 1 chromophobe cell carcinoma. On conventional ultrasound, 12/53 lesions showed no protrusion out of the kidney, and 41/53 cases slightly protruded out of the kidney. There was considerable difficulty in the detection of ten lesions, which was achieved with the guidance of CT/MRI, due to their dorsal location of the kidney. On conventional ultrasound, solid, hyper-echoic, color flow signal with varying degrees were the main features of MRCC.The boundary could be well- or ill-defined, and cystic changes existed in part of cases. On CEUS, most MRCCs showed simultaneous enhancement in cortical phase, iso- to hyper-enhancement at peak, and rapid washout in parenchymal phase. The comparisons of imaging features demonstrated that the characteristics were significantly different between conventional ultrasound and CEUS with regard to boundaries, blood supply, and perfusion uniformity (χ 2=12.425, 20.247, 7.185; all P<0.01). Conclusions:CEUS can significantly improve the detection rate of MRCC, which is superior to conventional ultrasound.

Objective:To explore the value of contrast-enhanced ultrasound(CEUS) in distinguishing of renal oncocytoma(RO) and chromophobe renal cell carcinoma(chRCC).Methods:The ultrasonic image features of 49 ROs and 72 chRCCs between October 2007 and January 2020 were retrospectively analyzed, all lesions underwent ultrasonic examination (including 19 ROs and 70 chRCCs with CEUS imaging) and were pathologically approved in our institution. The statistically significant parameters from univariate analyses were then entered for further multivariable Logistic regression. The value of each ultrasonic imaging feature in differentiating RO and chRCC was evaluated.Results:According to the univariate analyses, all imaging features on conventional ultrasound were not statistically different between RO and chRCC (all P>0.05), while the characteristics of tumor wash-in/out pattern, enhancement degree and homogeneity at peak time and pseudocapsule around tumor were significantly different (all P<0.05). After multivariable analyses, tumor wash-in and wash-out pattern were excluded for tumor differentiation ( P>0.05), and the parameters of enhancement degree or homogeneity at peak time and pseudocapsule around tumor were still significantly different between tumor types (all P<0.05, odd ratio was 8.683, 6.667 and 18.774 respectively). The overall sensitivity, specificity and accuracy of these three parameters in diagnosing RO was 68.4%, 91.4% and 86.5%, respectively. Conclusions:CEUS can provide some useful information for the differentiation of RO and chRCC.