[Evaluation of fast nucleic acid detection system in severe acute respiratory syndrome coronavirus 2].

Objective: To evaluate the performance and application of a fast nucleic acid detection system for testing severe acute respiratory syndrome virus 2 (SARS-COV-2). Methods: Clinical samples were collected from February to July 2020 from Beijing Center for Diseases Prevention and Control and the Laboratory Department of China-Japan Friendship Hospital, to evaluate the sensitivity, specificity, anti-interference ability, precision and clinical sample coincidence rate of fast nucleic acid detection system for SARS-CoV-2. The analytical sensitivity was determined by a dilution series of 20 replications for each concentration. Analytical specificity study was performed by testing organisms whose infection produces symptoms similar to those observed at the onset of corona virus disease 2019 (COVID-19), and of the normal or pathogenic microflora that may be present in specimens collected. Potential interference substances were evaluated with different concentration in the interference study. Precision study was conducted by estimating intra-and inter-batch variability. Clinical evaluation was performed by testing 230 oropharyngeal swab specimens and 95 sputum specimens in fast nucleic acid detection system, comparing with conventional real-time fluorescent quantitative PCR (RT-qPCR) and clinical diagnostic results. Results: The analytical sensitivity of SARS-CoV-2 using fast nucleic acid detection system was 400 copies/ml. The result is negative for testing with the organisms that may likely in the circulating area or causing similar symptoms with SARS-CoV-2 and human nucleic acid, indicating that no cross reactivity with organisms. The results of precision test showed that the Coefficient of variation of Ct value of high, medium and low concentration samples was 1.90%-3.92%, and all of them were less than 5% in intra-and inter-batch testing. The results of the samples were still positive after adding the potential interfering substances, indicating that the possible interfering substances in the samples had no effect on the results. 98.46% and 97.85% diagnosis results of fast nucleic acid detection system were consistent with RT-qPCR and clinical diagnostic results, respectively. Conclusion: The fast nucleic acid detection system based on molecular parallel reaction can be used as a selection method for SARS-CoV-2 testing.

Evaluation of a loop-mediated isothermal amplification method for the rapid detection of Vibrio harveyi in cultured marine shellfish.

AIMS: The purpose of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the rapid, sensitive detection of Vibrio harveyi in mariculture shellfish. METHODS AND RESULTS: A set of four primers, two outer and two inner primers, were designed from the toxR gene sequence of V. harveyi. The LAMP reaction was conducted at 65 degrees C for 60 min. There were no cross-reactions with other bacterial strains indicating a high specificity of the LAMP. The detection sensitivity of the LAMP assay for V. harveyi with both of pure cultures and added shellfish cultures is about 10(-5) dilution level (equivalent to 17.2 cells per reaction). The amplification products were detected by visual inspection using SYBR Green I. The detection sensitivity using the LAMP method was 10 times higher than that of conventional PCR. CONCLUSIONS: The LAMP assay established in this study is an extremely specific, sensitive and rapid for identification of V. harveyi in mariculture shellfish. SIGNIFICANCE AND IMPACT OF THE STUDY: This LAMP technique provides an important detecting tool for the detection of V. harveyi infection both in the laboratory and field. This technique is recommended as an applied protocol for health management programme and disease surveillance of in hatcheries as well as in grow-out pond, to prevent the disease outbreak.

[Inner hair cells loss by carboplatin and the changes of cochlear compound action potential in chinchillas].

Objective: To measure the cochlear compound action potential (CAP) and the densities of hair cells (HCs) along the whole length of the basilar membrane (BM) in adult chinchillas. And to investigate the relationship between the severity of inner hair cells (IHCs) loss and the changes of CAP by using carboplatin-cochlear lesion model. Methods: Totally 18 chinchillas were recruited after ontological evaluation. They were randomly divided into three groups (with 6 subjects in each), A: control, B and C: legion groups treated with one or two shot(s) of carboplatin respectively (76 mg/kg in one shot, i.p., one-week interval between the two shots). Endpoint tests were performed 30 days after the carboplatin treatment in groups B and C, and matched time in group A. A sliver-ball electrode was placed into round window niche via hypotympanic approach in anesthetized chinchilla. CAP was measured in response to clicks and tone burst of 0.5, 1, 2, 4, 8, 16 kHz respectively under anesthesia. CAP amplitudes and thresholds were measured and compared across the groups. After the recording, the whole cochlea surface preparation was made and the HCs were stained in histochemistry against substrate of succinate dehydrogenase (SDH). Images were taken with high-resolution digital camera under light microscope and across the whole cochlea. The length of the basilar membrane (BM) and the number of both IHCs and OHCs were counted. The HC density was calculated as the number of HCs per 10% BM length. Results: The CAP thresholds were (7.1±2.6), (25.4±5.0), (24.6±5.4), (10.4±5.0), (0.4±1.4), (4.2±6.3) and (17.1±14.1) dB SPL (from 6 subjects in group A, n=12 ears) corresponding to stimuli of Click and 0.5, 1, 2, 4, 8, 16 kHz tone bursts respectively. The total number of cochlear HCs were measured as (8 936±643) (x±s) and the average length of the BMs was (17.73±1.012) mm from the six subjects in the group A (n=12 ears). The HC density was found to be varied slightly across the BM. There was no significant CAP threshold difference between the control (group A) and the group B, which received one shot of carboplatin. However, the maximal CAP amplitude was reduced by 40% in the group B and compared with group A. Correspondingly, approximately 40% loss of IHCs were seen. In contrast, a significant CAP threshold shift was seen in subjects receiving two shots of carboplatin (group C), which was accompanied by a loss of 90% IHCs. Conclusions: The CAP thresholds of adult chinchillas show typical open-V shape with the lowest values at 2, 4, and 8 kHz. IHC loss by carboplatin in certain degree is well correlated with CAP amplitude reduction, but does not change the threshold when inner hair cell loss reaches 40%, however, if inner hair cell loss exceeds 80%, the threshold shift of CAP will be inevitable.

[The clinical characteristics and treatment outcomes of 386 patients with hypopharyngeal cancer].

OBJECTIVE: To investigate the clinical characteristics, surgical treatment outcomes and prognostic factors of hypopharyngeal carcinoma. METHODS: A retrospective review of the Eye & ENT Hospital of Fudan University patients' database between January 2003 and June 2013 was conducted and a total of 386 patients were enrolled in the study. Patients' clinical and oncological information was collected and survival rates were analyzed using Kaplan-Meier curves. Prognostic factors were evaluated with multivariate Cox model survival analysis. RESULTS: Among the 386 patients 95.9% were males and 4.1% were females, with an average age of (58.4±9.4) years. The primary tumor sites were pyriform sinus (76.7%), posterior hypopharyngeal wall (17.3%) and postcricoid region (6.0%). There were 31(8.0%), 83(21.5%), 175(45.3%) and 97(25.1%) patients with stage T1 to T4, respectively, 99(25.6%), 74(19.2%), 181(46.9%) and 32(8.3%) patients with stage N0 to N3, respectively , and 3 patients with distant metastasis to lung on initial diagnosis. Second primary cancers were found in 28 cases (7.3%). The 5-year overall survival (OS), disease specific survival (DSS) and disease free survival (DFS) were 45.8%, 48.1% and 46.0% respectively according to Kaplan-Meier survival curves. Multivariate Cox regression model showed significant association between 5-year overall survival rate and T stage (P<0.001), N stage (P=0.003) and second primary tumors (P=0.017). Advanced T stage and lymphovascular invasion were associated with a higher rate of recurrence (P<0.001). CONCLUSIONS: Hypopharyngeal squamous cell carcinoma has a dismal prognosis, with high rates of submucosal infiltration, cervical lymph node metastasis and distant metastasis. Treatment choices should be made according to TNM stage and overall health conditions in order to achieve ideal oncologic and functional results. Surgery with postoperative chemoradiation therapy is the main treatment for advanced-stage hypopharyngeal cancer.