Analysis of dynamic image features of tear film in dry eye patients / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the image features of dynamic changes of tear film break-up after corneal fluorescein staining(FL)combined with tear film lipid layer(TFLL)dynamic changes in patients with dry eye and its value of diagnosis in dry eye. METHODS: A prospective study. A total of 66 patients(132 eyes)with dry eye admitted to our hospital during September 2019 to December 2020 were divided area break-up group(17 cases, 28 eyes), circle break-up group(20 cases, 27 eyes), line break-up group(25 cases, 28 eyes), spot break-up group(21 cases, 24 eyes)and random break-up group(20 cases, 25 eyes)according to the different fluorescein tear film break-up patterns(BUPs). The image features of tear film break-up dynamic changes and the image features of TFLL dynamic changes were compared in each group, and the differences in the first non-invasive tear film break-up time(NIBUTf), average non-invasive tear film break-up time(NIBUTav), tear meniscus height(TMH)and fluorescein staining scores were compare,RESULTS:A statistically significant differences were observed in NIBUTf among the groups of patients(P<0.001). Except that there was no difference between spot break-up group and random break-up group(7.56±1.54s vs 8.02±1.86s, P=0.881), other groups had significant differences in pairwise comparisons(P<0.05). There were statistically differences among the groups for NIBUTav(P<0.001). Except that there was no difference between spot break-up group and random break-up group(9.54±2.12s vs 9.73±1.94s, P=0.997), other groups had significant differences in pairwise comparisons(P<0.05). There was statistically difference among the groups for TMH(P<0.001). Except that there was no difference between circle break-up group and line break-up group(0.16±0.03mm vs 0.17±0.03mm, P=0.986), there was no difference between spot break-up group and random break-up group(0.22±0.03mm vs 0.21±0.05mm, P=0.993), other groups had significant differences in pairwise comparisons(P<0.05). There were statistically significant differences among the groups for FL scores and TFLL grading(P<0.001).CONCLUSION:It is shown that different fluorescein BUPs intuitively reflects the tear film structure of the pathological changes according to evaluation and analysis of images feature of dynamic changes of FL tear film and TFLL combined with the results of static examination of tear film. It is helpful for clinicians to identify subtypes of dry eye, which has potential clinical value for the diagnosis and classification of dry eye.

A study of hepatitis B virus subtypes in patients chronically infected with genotype B or C of hepatitis B virus in Guizhou / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate hepatitis B virus (HBV) subtypes in patients chronically infected with genotype B or C of hepatitis B virus in Guizhou and to study the relationship between the subtypes and the progression of their liver diseases.</p><p><b>METHODS</b>Using PCR, 309 bp gene fragments in the HBV p region were amplified. The products of PCR were digested by VspI, NciI, BstEII and subjected to agarose gel electrophoresis. The subtypes of C1 and C2 were detected by restriction fragment length polymorphism (RFLP). B subtype was determined by direct sequencing of PCR product. One hundred seventy-eight patients with genotype B or C HBV infection in Guizhou, including 50 asymptomatic carriers (ASC), 100 chronic hepatitis (CHB), 14 liver cirrhosis (LC), and 14 hepatocellular carcinoma (HCC) patients were examined. The relationship between HBV C subtypes and the progression of their liver diseases was studied by analyzing the HBeAg positivity, HBV DNA loads and ALT levels of the patients.</p><p><b>RESULTS</b>Of 84 patients with HBV genotype C, 27 (32.14%) and 56 (66.67%) were subtype C1 and C2, respectively. In 94 genotype B, 93 (98.94%) were subtype Ba and only one was subtype Bj. Subtype C1 showed a trend of gradual decrease from ASC and CHB to LC/HCC groups. In contrast, subtype C2 showed a gradual increase (trend) in the same order. The HBeAg positivity was significantly lower in subtype C1 than that in subtype C2. The ALT levels and HBV DNA loads were higher in patients with subtype C2 than those in subtype C1, however no statistical significance was found in these primes (t=0.95, 0.79).</p><p><b>CONCLUSION</b>Subtype Ba is major and subtype C2 is more common in Guizhou. The distribution of subtype C1 and C2 are different in various stages of liver disease. The PCR-RFLP method is simple and accurate and can be used in a large-scale survey.</p>

A study in the Guizhou area on pre-S region mutations of hepatitis B virus / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the relationships between hepatitis B virus (HBV ) pre-S region mutations and their genotypes and the stages of liver disease of the patients.</p><p><b>METHODS</b>The entire HBV pre-S1 and pre-S2 genes were amplified by polymerase chain reaction (PCR). The amplified products were digested by NlaIII restriction enzyme. A detecting method for pre-S2 start codon mutation was established according to the restriction fragment length polymorphism (RFLP) analysis. Pre-S region deletion was revealed by polyacrylamide gel electrophoresis (PAGE). Fourteen sera having pre-S deletions or pre-S2 start codon mutations and wild strains were directly sequenced. HBV genotypes were determined by RFLP based on S-gene PCR products. One hundred sixty serum samples were collected from patients with HBV related diseases and they were determined by the above methods. The relationships between HBV pre-S region mutations and their genotypes and the stages of liver disease of the patients were analysed.</p><p><b>RESULTS</b>Of the 160 sera, genotype B and C were identified in 81 and 79 respectively. The detected ratios of pre-S2 start codon and pre-S deletion mutations were significantly higher in genotype C than in genotype B (43.04% vs 1.23%, 36.71% vs 19.75%, P<0.05, respectively). The detection rates of pre-S2 start codon mutation were significantly different in different groups: from 50.00% (HCC), 39.47% (LC), 8.00% (CH), to ASC (0). The detection rates of pre-S deletion mutations among patients with HCC (53.13%), LC (42.11%), CH (18.00%) and ASC (7.50%) also varied significantly. The results obtained from sequencing and PCR-RFLP/PAGE were completely compatible. Multivariate analysis indicated that genotype C (OR=6.26, P<0.01) and advanced liver disease (OR=11.99, P<0.01) were significant variables for pre-S mutations development.</p><p><b>CONCLUSION</b>The pre-S2 start codon and pre-S deletion mutations are more common in genotype C than in genotype B. These mutations are closely related to the progression of liver disease.</p>

Comparative study on detection of hepatitis B virus mutants in precore region with two methods / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a mismatched polymerase chain reaction restricted fragment length polymorphism (mPCR-RFLP) method for detection of hepatitis B virus (HBV) mutation in precore A1896, and compare with direct sequencing for evaluating its applicability.</p><p><b>METHODS</b>According to the principle of mPCR, 194bp gene fragments in HBV precore region was amplified. The products of PCR were digested by Bsu36I and subjected to agarose gel electrophoresis. A method for detecting procore A1896 mutation was established by restricted fragment length polymorphism. Totally 134 sera were analyzed by both mPCR-RFLP and direct sequencing methods. Two sera which were identified having mixed infection with precore wild and mutant strains by mPCR-RFLP also were analyzed by cloning and sequencing.</p><p><b>RESULTS</b>From 134 sera, 117 could be analyzed for HBV precore 1896 situation by mPCR-RFLP method, 109 could be analyzed by sequencing. In 101 sera which could be analyzed by the two methods, 54 were mutant strains and 47 were wild strains. The results of both methods were completely compatible. There was no significant difference in detective rate of HBV precore A1896 mutation between the two methods. The sequences of five clones from one serum which was identified precore mutant by mPCR-RFLP were all A1896 mutant strains. Another serum was identified as mixed infection by mPCR-RFLP, one clone was A1896 mutant strain and four were G1896 wild strains. The results of mPCR-RFLP were verified by cloning.</p><p><b>CONCLUSION</b>Compared with sequencing, the mPCR-RFLP method is simple, accurate and can be used in large-scale surveys and clinical research.</p>

Study on the distribution of hepatitis B virus precore and basic core promoter mutations in Guizhou area / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the distribution of hepatitis B virus (HBV) precore A1896 and basic core promoter (BCP)T1762/A1764 mutations in Guizhou area.</p><p><b>METHODS</b>482 patients with chronic HBV infection, belonging to 4 nationalities, including 225 asymptomatic carriers (ASC), 158 chronic hepatitis (CH), 57 liver cirrhosis (LC), 42 hepatocellular carcinoma (HCC), from 4 areas of Guizhou province were examined. HBV A1896 and T1762/A1764 mutations were determined by direct sequencing and restriction fragment length polymorphism (RFLP). HBV genotypes were determined by PCR-RFLP based on S gene. The relationship among these mutations and genotype and the progression of liver disease were studied by multi-normal logistic regression analysis.</p><p><b>RESULTS</b>A1896 and T1762/A1764 mutations were detected 23.03% and 29.67% among 482 patients. These mutations were more prevalent in Hans than in Dong, Miao and Buyi minorities (P < 0.01, respectively). The mutations of A1896 and T1762/ A1764 were more commonly seen in HBeAg negative than in HBeAg positive patients (P < 0.01, respectively). The mutation of T1762/A1764 was significantly higher in genotype C than in genotype B (P < 0.01). There were significantly statistical differences in the detective rate of A1896 and T1762/ A1764 mutations between patients with HCC, LC and CH, ASC. The distribution of these mutations in Guiyang (31.79% and 41.06%) was higher than in Zunye (10.94%, 14.06%), Duyun (8.64%, 11.11%) or Kaili (2.86%, 2.86%). However, there was no statistical difference by multi-normal logistic regression analysis after controlling the influence of HBeAg statu, genotype and clinical types.</p><p><b>CONCLUSION</b>The distributions of A1896 and T1762/A1764 mutations were different in some nationalities of Guizhou province. The mutation of T1762/A1764 was more commonly seen in genotype C than in genotypr B. These mutations were closely related to progression of chronic liver diseases. Hepatitis B virus; Genotype; Restriction fragment length polymorphism</p>

Investigation on virus genotype in patients infected with hepatitis B virus in four cities of Guizhou / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the distribution of hepatitis B virus (HBV) genotype in Guizhou and to study the relationship between the genotype and the progression of liver disease.</p><p><b>METHODS</b>786 patients with chronic HBV infection, from 4 cities of Guizhou, including 346 asymptomatic carriers (ASC), 313 chronic hepatitis (CH), 77 liver cirrhosis (LC), 50 hepatocellular carcinoma (HCC) were examined. HBV genotype was determined by restriction fragment length polymorphism analysis and the subtypes were determined by direct sequencing of PCR product in 94 patients with HBV B genotype, the relationship between HBV genotype and the progression of liver disease was studied by multifactor analysis such as HBeAg positivity, HBV DNA load and ALT level.</p><p><b>RESULTS</b>Of the 786 patients, 7 (0.89%), 497 (63.23%), 275 (34.99%), and 7 (0.89%) belonged to genotype A, B, C, D, respectively. There was statistically significant difference in the distribution of genotype B among Kaili (96.04%), Zunyi (78.79%), Duyun (64.52%) and Guiyang (53.14%) (P< 0.01). Genotype C was more prevalent in Guiyang than in other three cities (P < 0.01, or P < 0.05). Out of 94 genotypes B, 93 (98.94%) belonged to subtype Ba, only one was subtype Bj. There were statistically significant difference in the distribution of genotype B and C among various stage of liver disease (P < 0.05 or P < 0.01). Genotype B showed a gradual decrease from ASC, CH, LC to the HCC group while in contrast, genotype C showed a gradual increase in the same order. The ALT levels and the mean age were significantly higher and older in patients with genotype C than those in genotype B (P < 0.01 or 0.05). The HBeAg positivity was significantly lower in genotype C than that in genotype B (P < 0.025).</p><p><b>CONCLUSION</b>Data showed that there were genotype A, B, C and D existing in Guizhou. Genotype B was the major one but genotype C was more commonly seen. In genotype B, subtype Ba appeared to be predominant. The geographic distribution of genotype B and C were different in some cities of Guizhou. Compared to genotype B, genotype C was associated with the development of more severe liver damage.</p>

Distribution of hepatitis B virus genotype among population of Dong, Miao minority and Han in Guizhou / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the distribution of hepatitis B virus (HBV) genotype in population of Dong, Miao minority and Han in Guizhou.</p><p><b>METHODS</b>S region nucleotides were compared in 127 strains whole sequence of HBV and three restriction enzymes which can be used for genotyping were found by DNA software analysis system. The partial gene fragment of HBV S region was amplified by nested polymerase chain reaction (nPCR). The products were digested with Mbo I, BstN I or BsmA I and subjected to electrophoresis on agarose gel, respectively. The patterns of restriction fragment length polymorphism (RFLP) were analysed. The genotypes were determined by nPCR-RFLP in 166 asymptomatic HBV carriers (ASC), including 48 Dong minority, 52 Miao minority and 66 Han subjects. Some of the ASC were also analysed by direct sequencing of PCR products.</p><p><b>RESULTS</b>The nPCR-RFLP method was simple and accurate. Of the 166 ASC, 138 (83.13%) were genotype B, and 28 (16.87%) were genotype C. Of the 48 Dong minority subjects, 47 (97.2%) were genotype B and 1 (2.08%) was type C. Of the 52 Miao minority subjects, 49 (94.23%) were genotype B and 3 (5.77%) were genotype C. Of the 66 Han subjects, 42 (63.64%) were genotype B and 24 (36.36%) were genotype C. There was a statistical significance in the distribution of genotype C between Dong, Miao minority and Han (7.85% vs 36.36% P less than 0.005).</p><p><b>CONCLUSION</b>Genotype B and C exist in Guizhou and genotype B is the major genotype. Genotype C is found more frequently in Han than in Dong and Miao minority.</p>

A study on detection method of lamivudine related mutations in hepatitis B virus polymerase gene / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a simple and accurate method for rapid detection of lamivudine related mutations in hepatitis B virus (HBV) polymerase gene.</p><p><b>METHODS</b>HBV polymerase gene fragments of covering B and C active region were amplified by nested polymerase chain reaction (nPCR) or nested mismatched PCR. The PCR products were digested with Nde I or Nia III and subjected to electrophoresis on agarose gel, respectively. The patterns of restriction fragment length polymorphism (RFLP) were distinguished. Using this method, thirty patients with chronic hepatitis B and treated with lamivudine for at least one year were analysed for the lamivudine related mutations in polymerase gene. Sixteen cases without lamivudine therapy were used as controls. Some of the patients were also analysed by clone sequencing.</p><p><b>RESULTS</b>The nested mismatched PCR-RFLP method was simple, accurate and rapid. The whole experiments could be finished in eleven hours. The least titers of HBV DNA which could be detected was 10.3 copies/ml. The wild or mutant strains judged by RFLP were identified by clone sequencing. Mutation in the tyrosine methionine aspartic aspartic acid (YMDD) motif of HBV polymerase gene was found in eight patients and mutations of YMDD motif associated with L526M were found in another three patients. However, there were no such mutations in the control cases.</p><p><b>CONCLUSION</b>The nested PCR-RFLP is considered as a simple and accurate method for rapid detection of lamivudine related mutations in HBV polymerase gene. It is suitable for larger number of sample detection.</p>

Establishing a new genotyping method of hepatitis B virus by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) to analysis on S region and its application / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method of genotyping HBV using Mbo I, BsTN I, BsmA I, Hpa II and investigate the relationship between genotype and clinical spectrum of hepatitis B.</p><p><b>METHODS</b>124 full-genomic HBV sequences and 13 S-genomic sequences were analyzed, genotype specific regions were identified by the restriction enzymes Mbo I, BsTN I, BsmA I, Hpa II. And 176 samples from different kinds of hepatitis B were genotyped by this method. Five samples had been randomly selected and directly sequenced their S gene, to assess the accuracy.</p><p><b>RESULTS</b>In 176 serum samples of patients with hepatitis B from Guizhou area, genotype B and C were found in 56.8% and 43.2% respectively. The proportions of genotype B and C in ASC were 40.0% and 15.7% (chi-square = 12.16, P < 0.005); and they were 31.6% and 14.0% in CHB (chi-square = 7.88, P < 0.005).</p><p><b>CONCLUSION</b>Genotyping HBV, based on S gene RFLP seems to be highly sensitive, differential and accurate and could be used in large-scale surveys. HBV genotype B and C are existed in Guizhou area.</p>

Quantitative analysis of HBV DNA amplified products with microtiter hybridization / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To establish a new assay for detecting the quantity of HBV DNA with PCR and enzyme-linked immunosorbent assay(ELISA).</p><p><b>METHODS</b>The products of PCR using primers pre-labeled with biotin were hybridized with the capture probes that were immobilized on the microtiter strips and then bond with Sav-Ap. The quantity of DNA was detected by measuring the yellow color at 405 nm wave length.</p><p><b>RESULTS</b>Totally 125 sera from patients with hepatitis B were tested for HBV DNA by this method,the sera were also tested for HBV immunological markers by solid phase radio immuno-assay (SPRIA). The HBV positive rate with PCR-ELISA was 64.9% (24/37) in samples which were positive for HBsAg, HBeAg and HBcAb; and 34.2% (13/38) in sera which were positive for HBsAg, HBeAb and HBcAb; in sera positive for HBsAg and HBcAb or only HBcAb, the positive rate was 6.7% (1/15) and 5.9% (2/34) respectively.</p><p><b>CONCLUSIONS</b>The PCR ELISA assay is simple and suitable for clinical laboratory in quantitative determination of HBV DNA.</p>

The relationship between hepatitis B virus pre-S gene mutations and progression of liver disease / 中华传染病杂志

Objective To study the relationship between hepatitis B virus(HBV)pre-S gene mutations and progression of liver disease.Methods The entire pre-S1,pre-S2 genes were amplified hy nested polymerase chain reaction(PCR)and the products were digested by NlaⅢ.The method for detecting pre-S2 start codon mutation was established based on the digested restriction fragment length polymorphism(RFLP).Pre-S gene deletion was revealed by electrophoresis on polyacrylamide gel(PAGE).Pre-C A1896 and basic core promoter T1762/A1764 mutations were identified by direct sequencing of PCR products.The 138 sera from patients with HBV-related disease,including asymp- tomatic carriers(ASC),chronic hepatitis(CH),liver cirrhosis(LC),hepatocarcinoma(HCC),were tested by these methods.Results The detection rate of pre-S deletion mutation was higher in patients with HCC(56.3%)and LC(42.9%)than those with CH(11.8%)and ASC(8.1%,P