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Objective:To establish a clinical laboratory diagnostic pathway for hepatitis C covering diagnosis, differential diagnosis, drug toxicity monitoring, and therapeutic and prognostic evaluation, and to explore a new teaching model for laboratory diagnostics based on the clinical laboratory diagnostic pathway.Methods:According to the clinical diagnosis and treatment guidelines for hepatitis C, laboratory testing strategies for different stages of diagnosis and treatment of the disease were formulated to establish a clinical laboratory diagnostic pathway for hepatitis C. The pathway was applied in the teaching for undergraduate medical students of the seven-year program of grade 2019 of The First Clinical College of Wuhan University, with those of grade 2018 as the control to receive traditional teaching. The teaching effect was compared through questionnaires and quizzes in class. The data were analyzed through the t test with the use of SPSS 19.0. Results:A clinical laboratory diagnostic pathway for hepatitis C recognized by clinicians was established, covering the entire process of clinical diagnosis, differential diagnosis, monitoring of drug side effects, and therapeutic and prognostic evaluation. The students of grade 2019 receiving the pathway-based teaching model had significant improvements in teaching quality evaluation indicators ( P<0.05), with the most marked improvement in "having mastered the key and difficult points of this lesson", with a score of (60.90±2.15) points for grade 2018 and (84.80±3.44) points for grade 2019. The total score for teaching evaluation was significantly higher in students of grade 2019 than in those of grade 2018 [(94.02±4.29) vs. (79.21±3.68)] points, P<0.05). Grade 2019 also had a significantly higher classroom quiz score than grade 2018 (94.60±5.63) vs. (78.10±4.92), P<0.01]. Conclusions:We established and applied a clinical laboratory diagnostic pathway of hepatitis C in the teaching model of laboratory diagnostics, which organically integrates laboratory diagnostics and clinical medicine, and significantly improves the quality of teaching.
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The gynecological tumours such as Breast cancer or female reproductive system tumors are a serious threat to female health. With the development of molecular diagnosis, the genomic changes of gynecological tumours have been revealed continuously, and the diagnosis and treatment modes of tumors have gradually changed. The detection of molecular targets which potentially participated in the transformation or progress of disease has become an important section of the management of female reproductive tumors, and accurate identification of molecular targets of tumors plays an important role in disease diagnosis, monitoring of metastasis, prediction of recurrence and treatment. This review briefly discusses the risk assessment, molecular typing, targeted therapy, toxic and side effects, and prognosis evaluation of breast and female reproductive system tumors by molecular diagnosis.
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Objective:To investigate the expression of inflammatory factors, advanced oxidation protein products (AOPP), homocysteine (Hcy), vascular cell adhesion molecule 1 (VCAM-1) and the clinical significance of pregnancy outcome in patients with gestational diabetes.Methods:From January 2019 to January 2020, 103 cases of gestational diabetes in Taizhou Hospital of Zhejiang province were selected as observation group, and 87 normal pregnant women in Taizhou Hospital of Zhejiang province from January 2019 to January 2020 were selected as control group.The fasting blood glucose (FPG) and fasting insulin (FINS) were measured by automatic biochemical analyzer.The levels of tumor necrosis factor-α(TNF-α), high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), AOPP and VCAM-1 were measured by enzyme-linked immunosorbent assay (ELISA). The Hcy level was measured by circulating enzyme method.Results:The FPG [(8.34±1.25)mmol/L] and FINS [(9.75±0.89)U/L] in the observation group were higher than those in the control group [(4.89±0.67)mmol/L and (5.93±0.45)U/L] ( t=23.088, 36.297, all P<0.05). The serum levels of TNF-α [(21.63±3.25)ng/L], hs-CRP [(6.34±1.29)mg/L], IL-6 [(147.32±28.39)ng/L] in the observation group were higher than those in the control group [(8.72±1.84)ng/L, (2.17±0.73)mg/L and (87.96±12.41)ng/L] ( t=32.861, 26.744, 18.091, all P<0.05). The serum levels of AOPP [(53.21±9.89)μg/L], Hcy [(11.23±1.36)μmol/L] and VCAM-1 [(94.27±15.46)μg/L] in the observation group were higher than those in the control group [(25.48±6.18)μg/L, (8.41±1.28)μmol/L and (62.18±6.52)μg/L] ( t=22.674, 14.627, 18.047, all P<0.05). The incidence of adverse pregnancy outcome in the observation group (29.13%) was lower than that in the control group (4.60%) (χ 2=19.313, P<0.05). Conclusion:The patients with gestational diabetes have obvious inflammatory reaction, higher serum levels of AOPP, Hcy and VCAM-1, and poor pregnancy outcome.
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In December, the outbreak of a novel coronavirus (2019-nCoV) in Wuhan, China, has attracted extensive global attention. On January 20, 2020, the Chinese health authorities upgraded the coronavirus to a Class B infectious disease in the Law of the People′s Republic of China on the Prevention and Treatment of Infectious Diseases, and considered it as Class A infectious diseases in disease control and prevention. On January 18, 2020, the 2019-nCoV nucleic acid detection test was listed as the diagnostic criteria in the "guidelines for diagnosis and treatment of pneumonia due to 2019-nCoV (Trial Version 2)" . Therefore, standardizing the operation process of the 2019-nCoV nucleic acid detection in clinical laboratories has become a top priority. It is of paramount importance to establish standard protocols for detection of the 2019-nCoV nucleic acids in clinical laboratories to improve the reliability of the results and ensure the biosafety of laboratory personnel.
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Objective:To investigate the positive rate for 2019-nCoV tests and co-infections in Wuhan district.Methods:A total of 8 274 cases in Wuhan were enrolled in this cross-sectional study during January 20 to February 9 in 2020, and were tested for 2019-nCoV using fluorescence quantitative PCR. Both respiratory tract samples (nasopharynx, oropharynx, sputum and alveolar lavage fluid) and non-respiratory tract samples (urine, feces, anal swabs, blood and conjunctival sac swabs) were collected. If both orf1ab and N genes are positive, they are classified as nucleic acid test positive group; if both orf1ab and N genes are negative, they are classified as negative group; if single gene target is positive, they are classified as suspicious group. Individuals were divided into male group and female group according to sex. At the same time, 316 patients were tested for 13 respiratory pathogens by multiplex PCR.Results:Among the 8 274 subjects, 2 745 (33.17%) were 2019-nCoV infected; 5 277 (63.77%) subjects showed negative results in the 2019-nCoV nucleic acid test; and 252 cases (3.05%) was not definitive (inconclusive result). The age of cases with COVID-19 patients and inconclusive cases was significantly higher than that of cases without 2019-nCoV infection (56>40, t=27.569, P<0.001; 52>40, t=6.774, P<0.001). The positive rate of 13 respiratory pathogens multiple tests was significantly lower in 104 subjects who were positive for 2019-nCoV compared with those in subjects who were negative for 2019-nCoV test (5.77% vs 18.39%, χ 2=24.105, P=0.003). Four types of respiratory tract samples and five types of non-respiratory tract sampleswere found to be positive for 2019-nCoV nucleic acid test. Conclusion:The 2019-nCoV nucleic acid positive rate inmale is higher than infemale. Co-infections should be pay close attention in COVID-19 patients. 2019-nCoV nucleic acid can be detected in non-respiratory tract samples.
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Acute respiratory tract infections ranks first in China for various infectious diseases.Lower respiratory tract infections and related diseases caused a heavy burden on China′s medical care and society. In particular, COVID has caused great losses. This article discusses the standardization of clinical pathological diagnosis of respiratory pathogen infection, in order to improve the correct diagnosis of the disease and facilitate the timely treatment of the disease.
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In December, the outbreak of a novel coronavirus (2019-nCoV) in Wuhan, China, has attracted extensive global attention. On January 20, 2020,the Chinese health authorities upgraded the coronavirus to a Class B infectious disease in the Law of the People's Republic of China on the Prevention and Treatment of Infectious Diseases, and considered it as Class A infectious diseases in disease control and prevention. On January 22, 2020, the 2019-nCoV nucleic acid detection test was listed as the diagnostic criteria in the "guidelines for diagnosis and treatment of pneumonia due to 2019-nCoV (Trial Version 2)" . Therefore, standardizing the operation process of the 2019-nCoV nucleic acid detection in clinical laboratories has become a top priority. It is of paramount importance to establish standard protocols for detection of the 2019-nCoV nucleic acids in clinical laboratories to improve the reliability of the results and ensure the biosafety of laboratory personnel.
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Objective@#To investigate the positive rate for 2019-nCoV tests and co-infections in Wuhan district.@*Methods@#A total of 8 274 cases in Wuhan were enrolled in this cross-sectional study during January 20 to February 9, 2020, and were tested for 2019-nCoV using fluorescence quantitative PCR. Both respiratory tract samples (nasopharynx, oropharynx, sputum and alveolar lavage fluid) and non-respiratory tract samples (urine, feces, anal swabs, blood and conjunctival sac swabs) were collected. If both orf1ab and N genes are positive, they are classified as nucleic acid test positive group; if both orf1ab and N genes are negative, they are classified as negative group; if single gene target is positive, they are classified as suspicious group. Individuals were divided into male group and female group according to sex. At the same time, 316 patients were tested for 13 respiratory pathogens by multiplex PCR.@*Results@#Among the 8 274 subjects, 2 745 (33.2%) were 2019-nCoV infected; 5 277 (63.8%) subjects showed negative results in the 2019-nCoV nucleic acid test; and 252 cases (3.05%) was not definitive (inconclusive result). The age of cases with COVID-19 patients and inconclusive cases was significantly higher than that of cases without 2019-nCoV infection (40 vs 56, t=27.569, P<0.001; 52 vs 56, t=6.774, P<0.001). The positive rate of 13 respiratory pathogens multiple tests was significantly lower in 104 subjects who were positive for 2019-nCoV compared with those in subjects who were negative for 2019-nCoV test (5.77% vs 18.39%, χ2=24.105, P=0.003). Four types of respiratory tract samples and five types of non-respiratory tract samples were found to be positive for 2019-nCoV nucleic acid test.@*Conclusion@#The 2019-nCoV nucleic acid positive rate in male is higher than in female. Co-infections should be pay close attention in COVID-19 patients. 2019-nCoV nucleic acid can be detected in non-respiratory tract samples.
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High-throughput sequencing, also known as next-generation sequencing (NGS), is a new type of genetic screening and diagnostic technology. Its continuous innovation acceleratesthe understanding of genetic markers and the molecular mechanisms of disease, especially for complex hereditary diseases. With the development of NGS technology, especially the emergence of the targeted genome sequencing, genome-wide exome sequencing and genome-wide sequencing projects, NGS has been clinically applied indetection of SNVs, structural rearrangements and CNVs, monitoring circulating tumor DNA, and analyzing the genome regions that previously challenged the management of standard bioinformatics algorithms.
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Objective To establish the system of high resolution melting for detection of aldehyde dehydro-genase 2(ALDH2) and alcohol dehydrogenase-1B (ADH1B) gene polymorphisms .Methods The short prim-ers were designed for ALDH2 and ADH1B gene .Different PCR products were analyzed using Eva Green dyes after amplification ,which were confirmed by Sanger sequencing .Results The genotypes of ALDH2 rs671 and ADH1B rs1229984 were successfully detected in the same procedure by HRM within 90min ,and the results were consistent with Sanger sequencing .Conclusion The assay of HRM is simple ,rapid ,cost-effective ,and re-liable for the detection of ALDH2 and ADH1B polymorphism and it is worthy to be popularized .
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Objective To observe the effect of Yunnanbaiyao combined with gelatin sponge on wound healing in patients with post-extraction hemorrhage.Methods 72 patients received dental extraction were selected,and they were divided into observation group (Yunnanbaiyao + gelatin sponge) and control group (gelatin) according to the digital table,each group in 36cases.The hemostasis after treatment and the improvement of pain after 12h and 24h after treatment were observed.The wound healing was observed in the two groups after 7 days of treatment.Results After treatment,the total effective rate was 91.67% in the observation group and 69.44% in the control group.The effective rate of hemostasis in the observation group was significantly higher than that of the control group(x2 =7.32,P =0.007).The VAS scores of the observation group at 12 h (t =23.44,P =0.000) and 24h (t =22.86,P =0.000) after pack treatment obviously decreased;The VAS scores of the control group at 12h(t =19.87,P =0.000) and 24h (t =18.47,P =0.000) after pack treatment obviously decreased;The VAS scores of the observation group at 12h and 24h after pack treatment was lower than those of the control group,the differences were statistically significant(t =7.03,5.03,all P =0.000).The wound healing of the observation group was improved after 7d treatment(t =8.12,P =0.00);The wound healing of the control group was improved after 7d treatment(t =5.39,P =0.00);The wound healing of the observation group was better than that of the control group,the differences were statistically significant (t =2.88,P =0.005).Conclusion Yunnanbaiyao combined with gelatin sponge can relieve the bleeding symptoms after tooth extraction,promote wound healing,reduce the patients' pain.
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Objective To investigate the association of rs10004195 single nucleotide polymorphisms in Toll like re-ceptor 10(TLR 10) gene with helicobacter pylori infection and associated diseases. Methods A total of 652 pa-tients who has been examined by gastroscopy were obtained, and then peripheral blood samples were collected from all the patients. Immune colloidal gold method was used to test the serological Hp antibody of all the patients. The TLR10 gene rs10004195 polymorphisms were examined by direct DNA sequencing of the PCR products. Results The frequencies of AA,TT and AT genotype on TLR10 rs10004195 were 30. 98%, 20. 71%, 48. 31%;there was significant difference between Hp antibody positive group and Hp antibody negative group in the TT frequencies of TLR10 rs10004195 ( P<0. 05 ) . No significant difference between controls and Hp associated diseases groups in Hp antibody positive group or in Hp antibody negative group were observed. Conclusion There was correlation be-tween the TLR10 rs10004195 loci genotype and the risk of Hp infection, but no correlation with Hp associated dis-eases.
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Objective To evaluated the prevalence of EGFR mutations in Hubei region,to provide reliable experimental basis for reasonable screening TKI beneficiaries in clinic.Methods EGFR mutation of 253 patients diagnosed with NSCLC in Hu-bei region during 2010 to 2015 was detected by PCR-sanger sequencing and ARMS assay,to investigate the sensitivity of dif-ferent methods,to explore the frequency and clinical significance of EGFR mutation in different gender,in different histologi-cal type and different exons.Results 93 of 253 NSCLC patients harbored EGFR mutation,mainly occurred in exon 19 and 21,accounting for 53.76% and 35.38% of the total EGFR mutation rate,respectively.ADx-ARMS method showed higher sensitivity (P=0.001).The mutation detection rate of female NSCLC was significantly higher than that of male NSCLC (P=0.001).The observed incidence of EGFR mutations in patients with adenocarcinomas (38.01%)was the highest in differ-ent histological type,followed by glandular squamous cell carcinoma (30.77%),large cell carcinoma (20%)and squamous cell carcinoma (4.55%).Nonsmoking patients had a higher EGFR mutation frequency (51.6%,81/157)than those with a history of smoking (24%,12/50).Conclusion ARMS assay was more sensitive and more convenient detection method for clinical screening for EGFR-TKI treatment subpopulation.The mutation rate of female NSCLC was significantly higher than that of male NSCLC in Hubei Province.Speculated that the sexual differences in NSCLC with EGFR mutation frequencies were related to hormone levels and smoking status.
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With the development of molecular diagnostic techniques, the study for the etiology of leukemia has been entering the era of the molecular biology.Molecular techniques for leukemia diagnosis, prognosis and individualized therapy are used widely, becoming one of the necessary routine tests for patients with leukemia. So far, molecular techniques for leukemiaincluding cytogenetic diagnosis, molecular genetics, molecular diagnostics based on PCR, mutation detection, as well as a variety of next-generation sequencing, have played an important role in the diagnosis of leukemia, minimal residual disease monitoring, prognosis and targeted therapy.
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Objective Investigating the expression of TLR3 and TLR7 mRNA in peripheral blood mononuclear cells (PBMC) of patients with chronic hepatitis C infection (CHC),to explore the effects of gender and age on Toll-like receptor expres-sion.Methods Peripheral blood was collected from 1 1 5 patients of chronic hepatitis C infection,1 1 3 healthy individuals.Ex-pression levels of TLR3 and TLR7 mRNA were detected by real-time quantitative PCR.Results The expression of TLR3 and TLR7 mRNA were significant difference between patients with CHC infection and healthy individuals (t=38.73,6.16, P0.05).The HCV-RNA load of premenopausal females was significant lower than young males and old females (t=3.49,2.51,P<0.05),the load of old males was lower than old females (t=2.35,P<0.05),however, there was no significant differences between old males and old females (t=1.20,P<0.05).Conclusion Gender and age dis-crepancies have a relationship with the expression of Toll-like receptors of patients with CHC infection,which may provide a theoretical basis and a new method for CHC.
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Objective To investigate mRNA expressions of Toll-like receptors (TLR3 and TLR7)and type Ⅰ interferons (IFN-α and IFN-β) in peripheral blood mononuclear cells (PBMCs) of patients with chronic hepatitis C (CHC).Methods Thirty patients with CHC and 30 healthy controls were collected from Renmin Hospital of Wuhan University during September 2013 and May 2014.Liver function was detected using enzyme-linked immunosorbent assay (ELISA).mRNA expressions of TLR3,TLR7,IFN-α and IFN-β were detected by real-time quantitative PCR,and their relative expression values were obtained by 2-△△Ctmethod.The t test was performed for the comparison of mean differences between CHC patients and healthy controls,and Pearson analysis was used to analyze the correlations between TLR mRNA and IFN mRNA,liver function,HCV RNA load.Results Taking mRNA expressions in healthy control group as 1,the relative mRNA expressions of TLR3,TLR7,IFN-α and IFN-β in CHC group were 0.086,0.633,0.145 and 0.423 respectively (t =4.25,2.39,2.37 and 2.80,all P < 0.01).Pearson correlation analysis showed that mRNA expressions of TLR3 and TLR7 were positively correlated with that of IFN-β (r =0.381 and 0.487,all P < 0.05),but not correlated with IFN-α mRNA expression,HCV RNA load,ALT and AST (all P > 0.05).Conclusion The mRNA expressions of TLR3,TLR7 and IFN-α,IFN-β decrease in CHC patients,and the mRNA expressions of TLR3 and TLR7 are positively correlated with that of IFN-β,indicating that increasing the expression of TLRs might be a new strategy in CHC treatment.
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Objective To investigate epidemic characteristics of HCV and the distribution of naturally occurring resistance mutations of HCVNS5B in Hubei province,and provide the basis for individualized antiviral treatment for HCV infected pa-tients.Methods Collected HCV blood samples of 253 patients from March 2011 to May 2014 in Renming Hospital of Wu-han University,and determined the subtypes of HCVNS5B by first generation of sequencing technology,then compared the result with the nucleotide sequence of HCVNS5B online,located drug resistance loci and record.Results There were 4 gen-otypes and 6 different genetic subtypes in these 253 cases,namely 1a subtype 3,1b subtype 192,2a subtype 38,3a subtype 3, 3b subtypes 11 and 6a subtypes 6.Found 27 gene locus of resistance from 234 different places.The main drug resistance lo-ciare L159F (475-477)26.42%,followed by C316Y (946-948)16.98%,V321I (961-963)15.09% and S282T (844-846) 12.58%.Conclusion The epidemic characteristics of HCV in Hubei province were consistent with those of current in Chi-na;subtype 1b was the main types,followed by 2a subtype.The number of drug-resistant of HCVNS5B was more and com-plex,and presented some popular distributions.It was complex in mutations of 2a subtype,including 13 mutations of natural resistance.There were fewer mutations in subtype 1b,including sites of L159F and C316N.These results may provide the basis for antiviral therapy for HCV patients in this region.
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Objective To explore the accuracy and clinical application of Pyrosequencing for detection of drug resistant relevant mutation in the polymerase gene of Hepatitis B virus (HBV).Methods Compared with Sanger sequencing,the accuracy and sensitivity of Pyrosequencing were assessed.Pyrosequencing was used to determine the serum of 1164 patients with chronic Hepatitis B and its results were analyzed.Results The sensitivity of Pyrosequencing was 1 × 103 KIU/L,the same as Sanger sequencing.But Pyrosequencing was more stable and specific than Sanger sequencing to detect low rate of mutation with over 10% mutation.The mutations couldn't be detected in 248 patients with chronic Hepatitis B,but positive results occurred in 919 patients,among them,61.5 % without mutation,38.5 % with mutation (including 47.5% of the single locus mutation and 52.5% of the combined mutation).Other sites had different degree of mutations except the sites of rtI169T and rtA194T; the main mutation sites in patients with chronic Hepatitis B were rtM204V/I (32.1%),rtL180M (19.8%),rtA181V/T (6.7%) and rtN236T (5.3%),in which fractional mutation had high ratio in the sites of rtA181V/T (6.7%) and rtN236T (5.3%).Conclusions Pyrosequencing has good sensitivity,specificity and accuracy and can early detect the drug resistant relevant mutation in the polymerase gene of HBV,which is suitable for monitoring patients with chronic Hepatitis B for the treatment of nucleoside analogues (acid).The different sites and frequencies of mutations may be associated with different habits of doctors for using different anti-HBV drugs.
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Objective To analyze the association of the single nucleotide polymorphism (SNP) of Toll-like receptor 7 (TLR7) and Toll-like receptor 9 (TLR9) with chronic hepatitis C virus (HCV)infection.Methods A total of 150 patients with chronic hepatitis C (CHC) admitted to Renmin Hospital of Wuhan University from January 2011 to May 2012 and 168 healthy controls were enrolled in the study.The genotypes of TLR7 IVS2-151 (rs179009) were detected by Sanger sequencing,and the genotypes of TLR9 T-1486C (rs187084) were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).SPSS 15.0 was used for statistical analysis,and goodness-of-fit test for HardyWeinberg equilibrium was also performed.Results The frequency of TLR7 IVS2-151G was higher in malepatients with CHC than that in male controls (41.4% vs.21.6%,x2 =7.250,P =0.007,OR =0.389,95% CI:0.194-0.781) ; however the female CHC patients had a higher frequency of TLR7 IVS2-151A than the female controls (76.9% vs.63.1%,x2 =7.202,P =0.007,OR =1.942,95% CI:1.192-3.164).No significant difference in the distribution of TLR9 T-1486C (rs187084) gene SNP was observed betweenCHC and control groups (P >0.05).Conclusion TLR7 IVS2-151 (rs179009) is correlated with HCV infection,which may be involved in the pathogenesis of CHC.
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Objective To evaluate the value of detection of interleukin 28B (IL28B) rs12979860 by allele-specific PCR (AS-PCR) for the prediction of antiviral treatment hepatitis C patients.Methods One hundred seventy-four blood samples were random collected from hospitalized patients with hepatitis C,who came from department of infectious diseases,Renmin Hospital of Wuhan University from May 2011 to May 2012.Two pairs of specific primers were designed for rs12979860 gene polymorphisms,and one mutated base was introduced to the second or third site of the end of 3' with the reverse primer.rs12979860 gene polymorphism of 30 cases with hepatitis C was detected by AS-PCR,and gene sequencing was further used to verify the consistency of the two methods in parallel.Then,the frequency distribution of different rs12979860 genotypes with 174 cases were analyzed by the AS-PCR method in the population.Results The genotype CC,CT or TT of rs12979860 with 30 cases could be well identified by both AS-PCR and gene sequencing,and the coincidence rate was 100% (x2 =60.0,P < 0.01).Compared to gene sequencing,both of the sensitivity and specificity of AS-PCR were 100%.Compared to the control (CC genotype),TT genotype detection sensitivity by AS-PCR was 10-5,while sequencing sensitivity was 2 × 10-1.rs12979860 polymorphism in the TT,CC and CT genotype distribution in the Chinese population frequencies were 3.45% (6/174),13.2% (23/174) and 83.3% (145/174),respectively.Conclusion AS-PCR can quickly,accurate,reliable,economic and efficiently detect IL28B rs12979860 gene polymorphism of hepatitis C in patients,which could predict the effect of antiviral therapy on patients with hepatitis C.