Performance verification of five commercial RT-qPCR diagnostic kits for SARS-CoV-2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has caused a global pandemic beginning in 2020, can be detected by reverse-transcription polymerase chain reaction (RT-PCR). However, owing to the urgent need for a large number of detection kits, the time spent researching and developing these kits has been shortened during the pandemic, and the kits that are being used commercially have not undergone full and independent evaluation. To ensure the accuracy of SARS-CoV-2 test results, performance verification of commercial Real-Time quantitative PCR (RT-qPCR) kits is required. METHODS: The performance of five commercial RT-qPCR diagnostic kits for SARS-CoV-2 used in China was evaluated using a coronavirus disease 2019 (COVID-19) RNA liquid performance verification reference product-manufactured by Guangzhou Bondson (BDS) Biotechnology Co., Ltd.,Guangzhou, China-that uses droplet digital RT-PCR technology combined with fluorescence quantitative PCR. The five kits of Novel Coronavirus 2019-nCoV nucleic acid detection kit (RT-qPCR method) evaluated were Da An (Da An Gene Co., Ltd. of Sun Yat-sen University), Liferiver (Shanghai ZJ Bio-Tech Co., Ltd.), Kinghawk (Beijing Kinghawk Pharmaceutical Co., Ltd.), eDiagnosis (Wuhan Easy Diagnosis Biomedicine Co., Ltd.), and Maccura (Maccura Biotechnology Co., Ltd.). Performance verification criteria included the coincidence rate, limit of detection (LoD), cross-reactivity, precision, and anti-interference. Finally, through the BDS performance verification reference product kit, clinical samples are used to verify its clinical diagnostic efficacy. RESULTS: The coincidence rate was 100% for all kits except for Kinghawk, which was 95%. The LoD for Da An, eDiagnosis and Maccura was 250copies/mL, and it was 1000 copies/ml for Liferiver. Kinghawk was not able to detect its advertised LoD of 500 copies/ml. The cross-reactivity test results were all negative. Moreover, all kits had a coefficient of variation less than 5%; however, Liferiver showed the best precision. Da An, Liferiver, and eDiagnosis showed higher sensitivity to the nucleocapsid (N) gene than they did to the open reading frame (ORF) 1ab genes. Anti-interference results for all five kits were positive. The results of clinical diagnostic efficacy were that the specificity of the four kits was 1.000 (0.877-1.000), the sensitivity of Da An was 1.000 (0.850-1.000), Liferiver was 0.964 (0.798-0.998), Maccura was 0.893 (0.706-0.972), and eDiagnosis was 0.857 (0.664-0.953). CONCLUSIONS: All commercial RT-qPCR diagnostic kits for SARS-CoV-2 passed the BDS performance verification, except for Kinghawk (batch No:20200608113) which failed to detect the LoD of 500 copies/mL. Da An and Liferiver have excellent clinical diagnostic specificity and sensitivity. This study can provide guidance for the selection or optimization of RT-qPCR diagnostic test kits for SARS-CoV-2.

A near-infrared light-controlled, oxygen-independent radical generating nano-system toward cancer therapy.

Anti-tumor treatment based on free radicals is often inefficient in hypoxic tumors, mainly because of the oxygen-dependent generation mechanism of reactive oxygen species (ROS). Herein, we report an NIR laser-controlled nano-system that is capable of generating alkyl radicals in situ in an oxygen-independent approach. Hollow mesoporous Prussian blue nanoparticles (HPB NPs) were developed to co-encapsulate the azo initiator (AIBI) and 1-tetradecanol as the phase change material (PCM, melting point of ∼39 °C), obtaining the AP@HPB NPs. At normal body temperature, the PCM remained in the solid state to prevent the pre-leakage of AIBI. Upon NIR laser irradiation (808 nm) at the tumor site, AP@HPB NPs generated heat upon photothermal conversion, which melted the PCM to release AIBI and decomposed AIBI to produce toxicity free alkyl radicals under both normoxic and hypoxic conditions. The alkyl free radicals efficiently killed tumor cells by causing oxidative stress and damaging DNA. Meanwhile, NIR light-induced hyperthermia cooperated with free radicals to efficiently eradicate tumors. This study therefore provides a promising strategy toward oxygen-independent free radical therapy, especially for the treatment of hypoxic tumors.

Topology-assisted, photo-strengthened DNA/siRNA delivery mediated by branched poly(ß-amino ester)s via synchronized intracellular kinetics.

The performance of non-viral gene delivery vehicles, especially cationic polymers, is often challenged by the multiple cellular barriers that pose inconsistent requirements for material properties. A most pronounced inconsistency is exemplified by the molecular weight (MW)-related transfection efficiency and cytotoxicity. In this study, we report the development of photo-degradable, branched poly(ß-amino ester)s (BPAE-NB) to realize efficient and photo-controlled DNA and siRNA delivery. BPAE-NB possessing built-in light-responsive 2-nitrobenzene moieties in the polymer backbone was synthesized via the A2 (amine) + B3 (triacrylate) + C2 (diacrylate) polycondensation reaction from 4-amino-1-butanol (A2), trimethylolpropane triacrylate (B3), and (2-nitro-1,3-phenylene)bis(methylene) diacrylate (NPBMDA, C2). The highly branched BPAE-NB with the multivalent arrangement of cationic groups provides stronger nucleic acid binding capacity than its linear analogue LPAE-NB, and thus features stronger trans-membrane gene delivery capabilities and higher transfection efficiencies. Upon UV light irradiation, the backbone of BPAE-NB can quickly degrade into low-MW fragments as a consequence of the cleavage of the light-responsive 2-nitrobenzene, thus promoting intracellular gene release and diminishing the toxicity of materials at the post-transfection state. As such, in multiple mammalian cells, BPAE-NB exhibited remarkably higher DNA/siRNA transfection efficiency yet lower cytotoxicity than its non-responsive analogue BPAE-CC upon light irradiation, notably outperforming commercial reagents PEI 25k and Lipofectamine 2000. This study therefore provides an effective topology- and photo-controlled approach to precisely manipulate the transfection efficiency and toxicity of polycationic gene vectors, and may also provide promising additions to the existing non-viral gene delivery vectors.

Cancer-Selective Bioreductive Chemotherapy Mediated by Dual Hypoxia-Responsive Nanomedicine upon Photodynamic Therapy-Induced Hypoxia Aggravation.

Stimuli-responsive drug delivery has rendered promising utilities in cancer treatment. Nevertheless, cancer selectivity as well as sensitivity still remains critical challenges that would undermine the therapeutic efficacy of chemodrugs and cause undesired systemic toxicity. Herein, a dual hypoxia-responsive drug delivery system was developed to enable photodynamic therapy (PDT)-induced drug release and drug activation intermediated via PDT-induced hypoxia. Particularly, tumor-targeting and hypoxia-dissociable nanoparticles (NPs) were self-assembled from the amphiphilic polyethylenimine-alkyl nitroimidazole [PEI-ANI, (PA)] and hyaluronic acid-chlorin e6 (HA-Ce6) to encapsulate bioreductive chemodrug, tirapazamine (TPZ). After systemic administration, the obtained PA/HA-Ce6@TPZ NPs enabled effective tumor accumulation due to HA-mediated cancer targeting. Upon receptor-mediated endocytosis, light irradiation (660 nm, 10 mW/cm2) produced high levels of reactive oxygen species to mediate PDT and generated a severe local hypoxic environment to dissociate the NPs and selectively release TPZ, as a consequence of hypoxia-triggered hydrophobic-to-hydrophilic transformation of ANI. In the meantime, TPZ was activated under hypoxia, finally contributing to a synergistic anticancer treatment between PDT and hypoxia-strengthened bioreductive chemotherapy. This study, therefore, demonstrates a suitable strategy for cancer-selective drug delivery as well as programmed combination therapy.

[Surgical treatment of carotid stenosis].

OBJECTIVE: To summarize clinical experience of carotid endarterectomy (CEA) in treating severe carotid stenosis. METHODS: Between October 1998 and January 2010, 215 patients with carotid stenosis were treated with CEA. There were 140 males and 75 females with an average age of 66 years (range, 51-88 years). Transient ischemic attack (TIA) occurred in 127 cases, and 31 cases had history of cerebral infarction. All cases were diagnosed definitely by selective angiography and/or CT angiography, and stenosis degree was more than 80%; contralateral carotid artery was also involved in 45 cases. Ninety-six cases were found to have coronary artery stenosis by coronary angiography. CEA and coronary artery bypass grafting were performed simultaneously in 25 cases. Peripheral arterial disease was found in 43 cases and treated at the same time. RESULTS: A total of 155 patients were followed up 6-72 months. The clinical symptom significantly alleviated in 148 cases postoperatively. Two cases had complication of cerebral hemorrhage within 1 week postoperatively; one died and the other was resumed after the conservative treatment. One case had hypoglossal nerve injury. Four cases had injuring marginal mandibular branch of the facial nerve, and no special treatment was given. Restenosis was found in 25 patients, and the stenosis degree was less than 25%; moreover, the patients had no TIA. One case died of heart attack at 3 years of follow-up period. CONCLUSION: CEA is an effective and safe method for treating severe carotid stenosis.

[Open surgery for femoro-popliteal arterial occlusive disease].

OBJECTIVE: To explore the mid-term surgical results of arterial revascularization for femoro-popliteal arterial occlusive disease (lesion type C and D). METHODS: From January 2005 to February 2009, 191 arterial bypass had been performed on 170 patients (21 cases bilateral). There were 108 male and 62 female, age ranged from 45 to 85 years old with an average of 67 years old. The operative indication was claudication in 78 cases, rest pain in 62 cases, ischemic ulcer in 19 cases, and distal tissue necrosis in 11 cases. Arterial angiography were performed on all cases. According to TASC II document, type C lesions were seen in 127 limbs, type D lesions were seen in 64 limbs. Autogenous greater saphenous vein bypass in situ were done on 15 limbs, autogenous greater saphenous vein bypass reversed in 20 limbs, revascularization with artificial prosthesis in 128 limbs, composite grafts consisting of a prosthetic conduit with a distal venous segment in 28 limbs. RESULTS: There were no 30-day mortality. Follow-up periods ranged 6 to 36 months with an average of (24 + or - 6) months. Seventy-three cases were lost during follow-up periods, follow-up rate was 57% (109/191). Primary patency rate was 84.4% (92/109). The patency rate was 88.2% with artificial prosthesis, 70.8% with greater saphenous vein (in situ or reversed). Secondary patency rate was 89.9%. CONCLUSIONS: Arterial revascularization with artificial prosthesis is main treatment for diffused superficial femoral artery occlusive disease (TASC II type C and D lesion) with satisfied surgical results.

[Surgical treatment for varicose vein].

OBJECTIVE: To explore the techniques and outcomes of the surgical treatment for varicose vein. METHODS: Totally 2 200 patients with varicose vein received surgical treatment in our hospital from July 2000 to January 2006. The latest techniques for endovenous occlusion were used for most cases, among which 1 802 cases were treated with endovenous laser treatment (EVLT) combined with transilluminated powered phlebectomy (TIPP), 82 cases with radiofrequency endovenous occlusion (RFO) combined with TIPP, and 218 cases with limited invaginated vein stripping and foam sclerotherapy. The remaining 98 cases were treated with laser, radiofrequency or ligation for saphenous vein trunk, and with resection, electric coagulation, and transfixation for vein clusters as additional methods. Perforators were also cut and ligated as well. RESULTS: Satisfied surgical results were obtained in all cases. The average operative time was 40 minutes (range 20-78 minutes). CONCLUSIONS: Limited invaginated vein stripping, EVLT, and RFO can be used to treat saphenous vein reflux. Surgical resection, transfixation, electric coagulation, and sclerotherapy are reasonable options for vein cluster or tributaries. Transection and ligation of the perforators are important. TIPP is ideal for the treatment of vein clusters.

[Treating severe stenosis of internal carotid artery with carotid endarterectomy].

OBJECTIVE: To explore the effective and safety of carotid endarterectomy (CEA) in the treatment of severe stenosis of internal carotid artery. METHODS: The clinical data of 95 patients with internal carotid artery stenosis and treated by CEA in our hospital from October 1998 to October 2006 were retrospectively analyzed. Transient ischemic attack (TIA) was found in 65 patients and cerebral infarction in 21 patients before operations. Diagnoses were confirmed by selective angiography, and > 70% stenosis of internal carotid artery was found in all patients and > 95% stenosis was found in 50 patients. Stenosis or occlusion of the contralateral carotid artery was also found in 35 patients. Coronary artery stenosis was confirmed in 43 patients with preoperative coronary angiography. CEA and coronary artery bypass grafting (CABG) were simultaneously performed in 18 patients. RESULTS: After the operation, the clinical symptoms significantly improved in all patients. One case experienced hypoglossal nerve injury. One patient died of heart attack during follow-up. CONCLUSION: CEA is an effective and safe treatment for severe stenosis of internal carotid artery.