Objective To evaluate the sensitivity and specificity of a nested polymersase chain reaction (nested PCR) for detection of the Mycobacterium tuberculosis IS6110 in paraffin embedded tissues. Methods 31 samples from tuberculosis patients and 5 biopsy specimens from patients with hepatitis were subjected to detection of Mycobacterium tuberculosis IS6110 by nested PCR. PCR products of two randomly selected samples were cloned and sequenced. Results Mycobacterium tuberculosis IS6110 was positive in 28 of 31 samples of tuberculosis. The sensitivity and specificity of nested PCR for detection of IS6110 were 90.3% and 100%, respectively. The predictive value of nested PCR was 100%. The sequences of two samples were compared with known sequence of H37Rv isolate (reported by Thierry D) and with nucleotide homology of 97% and 95.3%, respectively. Conclusions Nested PCR is sensitive and specific in the diagnosis of Mycobacterium tuberculosis infection in tissues of routinely paraffin embedded. We propose the diagnostic application of nested PCR for the identification of Mycobacterium tuberculosis infection, especially in the cases which can not be distinguished with certainty from other diseases by histopathology and Ziehl Neelsen's staining.

Objective To establish an external quality assessment (EQA) scheme for cell morphological interpretation of bone marrow smears and investigate its application value.Methods The Laboratories taking part in the assessment were periodically provided with diagnosed bone marrow smears,clinical data and other examination results for cell morphological interpretation of EQA.Results The coincidence rate was improved year after year from 50% in 1988 up to 85.8% in 2005 (P

Objective To investigate the role of CD4+CD25+Foxp3 regulatory T cells in chronicity of hepatitis B and viral clearance of hepatitis B virus(HBV).Methods Nineteen patients with chronic active hepatitis B(CAH).21 HBV carriers(AsC)and 12 patients with resolved HBV infection and 1 5 healthy controls were enrolled.The frequency and phenotype of peripheral CD4+CD25+Foxp3+ T cells were detected by flow cytometry.CD4+CD25+T cells were sorted by magnetic-activated cell sorting(MACS)assay.Level of Foxp3 mRNA in CD4+CD25+T cells was examined by real time polymerase chain reaction(PCR)assay.The data were analyzed by one-way ANoVA or nonparametric statistics.Results Both frequencies of CD4+CD25+Foxp3+T cells and levels of Foxp3 mRNA in CD4+CD25+T ceils in patients with CAH or AsC were significantly higher than those in healthy controls Or resolved HBV infection(F=6.8,F=3.72,respectively;both P<0.05).Accumulation of Foxp3+T cells in liver tissue of CAH patients was higher than that of healthy controls,while that in AsC was lower than CAH.The frequency of CD4+CD25+Foxp3+T cells of hepatitis B e antigen(HBeAg)positive patients(including CAH and AsC)was significantly higher than that of HBeAg negative patients(t=2.3,P<0.05),and that of antFHBe negative patients were significantly higher than anti-HBe positive patients(t=2.4,P<0.05).Furthermore,the frequency of CD4+CD25+Foxp3 regulatory T cells was positively correlated with serum HBV DNA level of patients with chronic hepatitis B(r=0.56,P<0.01).Conclusion The findings have important implication in the understanding of the role of CD4'CD25'regulatory T cells in chronicity and viral clearance in HBV infection.

Objective:To understand the diagnostic value of tuberculosis (TB) pathogenic detection methods (TPDM) in pathological tissue for TB.Methods:A retrospective study was conducted with 190 pathological specimens from different tissues suspected with TB from Third People′s Hospital of Shenzhen during May 2016 and May 2019. Specimens were divided into four groups according to histomorphology: group one, necrotizing granulomatous inflammation (109 cases); group two, non-necrotic granulomatous inflammation (20 cases); group three, non-granulomatous inflammation (45 cases); group four, non-tuberculous lesions (16 cases). The positive rates of each TPDM among specimens from four groups were compared. The positive rates of all TPDM for specimens from group one were compared. Meanwhile, the influence of antituberculosis treatment course on the TPDM was analyzed. Chi-square test or Fisher′s exact test was used for statistical analysis.Results:The positive rates of Ziehl-Neelsen acid-fast staining among the four groups were 17.4%(19/109), 5.0%(1/20), 4.4%(2/45) and 0(0/16), respectively. The positive rates of Mycobacterium tuberculosis (MTB) complex culture were 32.0%(32/100), 4/19, 4.8%(2/42) and 0(0/16), respectively. The positive rates of Mycobacterium tuberculosis/rifampin resistance real-time quantitative nucleic acid amplification detection system (Xpert MTB/RIF) were 74.3%(81/109), 15.0%(3/20), 13.3%(6/45) and 0(0/16), respectively. The positive rates of fluorescent quantitative polymerase chain reaction (FQ-PCR) were 63.0%(58/92), 0(0/15), 2.6%(1/38) and 0(0/10), respectively. The positive rates of simultaneous amplification and testing (SAT) were 32.4%(24/74), 0(0/10), 0(0/15) and 0(0/10), respectively. The differences of each TPDM among four groups were all statistically significant (all P<0.05). The positive rate of Xpert MTB/RIF in group one specimens was significantly higher than those of acid-fast staining, MTB culture and SAT ( χ2=71.016, 37.162 and 35.679, respectively, all P<0.01), while the difference was not statistically significant when compared with FQ-PCR ( χ2=2.517, P=0.112). The positive rate of combined TPDM (85.3%(93/109)) was significantly higher than Xpert MTB/RIF(74.3%(81/109)) ( χ2=4.100, P=0.043). The positive rates of acid-fast staining group 1A (anti-tuberculosis treatment course was less than one month) and group 1B (anti-tuberculosis treatment course was longer than one month) were 14.3%(7/49) and 20.0% (12/60), respectively ( χ2=0.612, P=0.434); those of MTB culture were 48.9% (22/45) and 18.2% (10/55), respectively ( χ2=10.721, P=0.001); those of Xpert MTB/RIF were 69.4%(34/49) and 78.3%(47/60), respectively ( χ2=1.131, P=0.287); those of FQ-PCR were 55.0%(22/40) and 69.2%(36/52), respectively ( χ2=1.965, P=0.161); those of SAT were 43.3%(13/30) and 25.0%(11/44), respectively ( χ2=2.736, P=0.098). Conclusions:The results of TPDM correlate closely with the typical histomorphological features of tuberculosis. Xpert MTB/RIF possesses significantly higher sensitivity than any other single TPDM, and is not attenuated by early anti-tuberculosis treatment. Combined TPDM could significantly improve the sensitivity of TB pathogenic detection, which is suggested to be applied when the tissue specimen is sufficient.

Objective: To analyze the hand-foot-mouth disease (HFMD) enterovirus 71 (EV-A71) infection epidemic characteristics of Guangdong Province from 2011 to 2015. Methods: We colleted data on common cases of hand-foot-mouth disease infected with EV-A71 reported from eight sentinel hospitals in Guangdong Province from January 2011 to December 2015, through the "Guangdong Province Acute Infectious Disease Surveillance Information Platform System" , including the age and incidence of cases. Time and etiological data, etc.We also collected data on the number of reported cases of HFMD disease and the number of laboratory-confirmed cases, through the "China Disease Prevention and Control Information System" , including data on common cases of HFMD disease, data on epidemics of severe cases and deaths, and the age, onset time, and pathogens of cases. Learning data, etc.The data from two sources were used to estimate the incidence of HFMD in EV-A71 and describe its distribution characteristics.Chi-square test was used to compare the positive rate of HF-A71 infection in hand-foot-mouth disease and the difference in estimated incidence among different age groups and months. Results: Eight sentinel hospitals from 2011 to 2015 reported a total of 1 855 common cases of EV-A71 infection, of which the highest was in 2014 (31.6%, 605/1 916) and the lowest was in 2015 (6.8%, 134/1 971) (χ²=521.85, P<0.001).According to the Disease Surveillance Reporting Information System, 1 772 516 cases of HFMD disease were reported from 2011 to 2015 in Guangdong Province, and 1 902 cases of severe and fatal cases of EV-A71 infection.The composition ratio of EV-A71 infected was 72.6% (1 775/2 444) and 97.0% (127/131) of severe HFMD disease in Guangdong province during 2011-2015.The average annual incidence of HF-A71 infection in all age groups showed a decreasing trend with age (χ^2trend=990 273.20, P<0.001), and it was the highest in the 1-year-old group, which was 1 697.67/100 000, and the lowest in the 4-year-old group, which was 705.46/100 000. The difference of monthly average incidence of EV-A71 infection in HF-A71 in each month was statistically significant (χ²=401.23, P<0.001), the highest in May at 15.51 per 100 000, and the lowest in July at 9.42 per 100 000. Conclusion EV-A71 infection rate of ordinary HFMD varies in different years. The most severe and death cases of HFMD were EV-A71 infected. 1 year old children were the high-risk group of infected with EV-A71 HFMD. April was the epidemic months of EV-A71 HFMD infection.

OBJECTIVE: To detect pathological variant in two patients with Chediak-Higashi syndrome (CHS) from a consanguineous family and to explore its genotype-phenotype correlation. METHODS: Clinical data was collected for this pedigree. Genomic DNA was prepared from probands' peripheral leukocytes and their relatives' fingernail. Whole exome sequencing and Sanger sequencing were carried out to detect potential variant of the LYST gene. RESULTS: The proband presented with partial oculocutaneous albinism, immunodeficiency and acidophilic inclusion body in bone marrow and blood smears. A novel homozygous nonsense variant c.8782C>T (p.Gln2928*) was identified in exon 34 of the LYST gene in the sib pair. The same variant was found to be in heterozygous status in 6 unaffected individuals from the pedigree. CONCLUSION Above result enriched the mutational spectrum of CHS and provided a basis for genetic counseling and prenatal diagnosis for this pedigree.
Chediak-Higashi Syndrome ; genetics ; Exons ; Heterozygote ; Humans ; Mutation ; Pedigree ; Sequence Analysis, DNA ; Vesicular Transport Proteins ; genetics ; Whole Exome Sequencing

Objective:To construct the liver organoid infected with hepatitis D virus (HDV), and to investigate the role of the sodium taurocholate cotransporting polypeptide (NTCP) receptor inhibitor bulevirtide in inhibiting viral replication.Methods:Hepatocyte-like cells (HLC) differentiated from induced pluripotent stem cells (iPSC) were seeded onto inverted colloidal crystal polyethylene glycol scaffolds (ICC) to construct liver organoids. After transfecting human hepatocelluar carcinoma cells (HuH7 cells) with plasmids, HDV particles were harvested from the supernatant, while HBV particles were extracted from the HepG2.2.15 cell supernatant. The liver organoids were infected with both HBV and HDV particles, and the negative control group without HDV infection was set up. The microstructure of the liver organoid units and the expression of hepatitis D antigen (HDAg) and hepatitis B surface antigen (HBsAg) were observed under laser scanning confocal microscope by immunofluorescence method. The protein levels of NTCP and HDAg in the liver organoids were detected by Western blotting. Bulevirtide was added before HDV infection (bulevirtide pre group) and 24 hours after infection (bulevirtide post group), and interferon-alpha (IFN-α) was also added after 24 hours infection (IFN-α group), and a control group without drug treatment was set up. HDV replication was compared among the four groups after drug intervention. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to measure the relative mRNA expression levels of Nanog homeobox (NANOG), sex determining region Y-box (SOX)2, SOX17, forkhead box protein A2 (FOXA2), hepatocyte nuclear factor 4 alpha (HNF-4α), albumin (ALB), alpha-fetoprotein (AFP), NTCP during the differentiation of iPSC, and the mRNA expression of HDV after the drug intervention of the four groups. Statistical analysis was performed using two independent sample t tests. Results:Within 21 days of the differentiation of iPSC into HLC, the mRNA expression level of NANOG gradually decreased, while the expression levels of SOX17, FOXA2 initially increased then decreased, and the expression levels of the HNF-4α, ALB, AFP and NTCP progressively increased. The protein level of NTCP in iPSC (0.118±0.003) was lower than that in HLC (1.315±0.073), and the difference was statistically significant ( t=11.92, P<0.001).The protein level of HDAg in the liver organoids after HDV infection was higher than that in the negative control group without HDV infection (1.284±0.128 vs 0.157±0.040), and the difference was statistically significant ( t=23.27, P<0.001).Laser scanning confocal microscopy showed three-dimensional spheroid structures and high expressions of HDAg and HBsAg at the 14th day of infection.Compared with the control group (1.000±0.077), the HDV mRNA expressions in both IFN-α group (0.453±0.028) and bulevirtide pre group (0.136±0.012) decreased after three days of drug intervention. The differences were statistically significant ( t=19.95 and 33.15, respectively, both P<0.001). However, there was no significant difference in HDV mRNA expressions between the bulevirtide post group (0.968±0.069) and the control group ( t=0.94, P>0.05). Conclusions:The liver organoids constructed from iPSC-derived HLC and ICC can simulate human liver functions and successfully be infected by HDV particles. Early blockade with bulevirtide can effectively reduce the level of viral replication in the HDV-infected liver organoids.

Objective:To analyze the results of liver cancer screening for urban residents in Zhejiang Province from 2013 to 2018 and explore the influencing factors of the detection rate.Methods:From September 2013 to August 2019, six urban communities in Hangzhou (Jianggan District and Gongshu District), Ningbo (Haishu District, Yinzhou District and Jiangbei District), and Quzhou (Kecheng District) were selected as study sites. All permanent residents aged 40-74 (with local household registration and living in the local area for more than 3 years) were selected as the research subjects by using cluster sampling method. Patients with confirmed cancers and other serious medical and surgical diseases were excluded. A total of 166 293 research subjects were included. Basic demographic characteristics and risk factors of subjects were obtained through questionnaire surveys. The cancer risk assessment system was used to evaluate the liver cancer risk of subjects. Clinical screening participation and screening results for subjects at high risk of liver cancer were obtained from participating hospitals. The high-risk rate of liver cancer, clinical screening rate, detection rate of positive lesions, and detection rate of suspected liver cancer were analyzed. Poisson regression was used to analyze the influencing factors of detection rate.Results:The age of 166 293 subjects was (56.01±8.40) years, of which 41.36% (68 777) were males. A total of 23 765 high-risk subjects for liver cancer were screened (the high-risk rate was 14.29%). Among them, a total of 12 375 subjects participated in clinical screening for liver cancer, with a screening rate of 52.07% (12 375/23 765). A total of 297 cases of positive lesions were detected and the detection rate was 2.40% (297/12 375). A total of 8 cases of suspected liver cancer were detected, with a detection rate of 0.06% (8/12 375). The results of multivariate Poisson regression model analysis showed that compared with men, people who never smoked, never ate pickled food, had low oil content, and had no history of hepatobiliary disease, female, people who were smoking or had smoked, sometimes ate pickled food, ate higher oil content, and had a history of hepatobiliary disease had a higher detection rate of positive lesions. The incidence rate ratio (IRR) ( 95%CI) values were 1.98 (1.45-2.70), 2.23 (1.61-3.09)/2.08 (1.31-3.28), 1.82 (1.22-2.70), 1.44 (1.08-1.91), and 1.45 (1.05-2.00), respectively. Compared with those aged from 40 to 49 years old and without HBsAg test, the IRR (95% CI) of suspected liver cancer in people aged 70 to 74 years old and HBsAg positive were 16.30 (1.32-200.74) and 6.43 (1.24-33.22), respectively. Conclusion:The urban cancer early diagnosis and early treatment project in Zhejiang Province has good compliance in clinical screening of liver cancer. Abdominal ultrasound examination and serum alpha-fetoprotein detection are helpful to detect liver cancer and its precancerous lesions in the high-risk population of liver cancer.

Objective:To analyze the results of liver cancer screening for urban residents in Zhejiang Province from 2013 to 2018 and explore the influencing factors of the detection rate.Methods:From September 2013 to August 2019, six urban communities in Hangzhou (Jianggan District and Gongshu District), Ningbo (Haishu District, Yinzhou District and Jiangbei District), and Quzhou (Kecheng District) were selected as study sites. All permanent residents aged 40-74 (with local household registration and living in the local area for more than 3 years) were selected as the research subjects by using cluster sampling method. Patients with confirmed cancers and other serious medical and surgical diseases were excluded. A total of 166 293 research subjects were included. Basic demographic characteristics and risk factors of subjects were obtained through questionnaire surveys. The cancer risk assessment system was used to evaluate the liver cancer risk of subjects. Clinical screening participation and screening results for subjects at high risk of liver cancer were obtained from participating hospitals. The high-risk rate of liver cancer, clinical screening rate, detection rate of positive lesions, and detection rate of suspected liver cancer were analyzed. Poisson regression was used to analyze the influencing factors of detection rate.Results:The age of 166 293 subjects was (56.01±8.40) years, of which 41.36% (68 777) were males. A total of 23 765 high-risk subjects for liver cancer were screened (the high-risk rate was 14.29%). Among them, a total of 12 375 subjects participated in clinical screening for liver cancer, with a screening rate of 52.07% (12 375/23 765). A total of 297 cases of positive lesions were detected and the detection rate was 2.40% (297/12 375). A total of 8 cases of suspected liver cancer were detected, with a detection rate of 0.06% (8/12 375). The results of multivariate Poisson regression model analysis showed that compared with men, people who never smoked, never ate pickled food, had low oil content, and had no history of hepatobiliary disease, female, people who were smoking or had smoked, sometimes ate pickled food, ate higher oil content, and had a history of hepatobiliary disease had a higher detection rate of positive lesions. The incidence rate ratio (IRR) ( 95%CI) values were 1.98 (1.45-2.70), 2.23 (1.61-3.09)/2.08 (1.31-3.28), 1.82 (1.22-2.70), 1.44 (1.08-1.91), and 1.45 (1.05-2.00), respectively. Compared with those aged from 40 to 49 years old and without HBsAg test, the IRR (95% CI) of suspected liver cancer in people aged 70 to 74 years old and HBsAg positive were 16.30 (1.32-200.74) and 6.43 (1.24-33.22), respectively. Conclusion:The urban cancer early diagnosis and early treatment project in Zhejiang Province has good compliance in clinical screening of liver cancer. Abdominal ultrasound examination and serum alpha-fetoprotein detection are helpful to detect liver cancer and its precancerous lesions in the high-risk population of liver cancer.

Objective:To analyze the results and cost-effectiveness of colorectal cancer (CRC) screening program among Zhejiang urban residents so as to provide evidence for further optimization of CRC screening strategies.Methods:Based on the Cancer Screening Program in Urban China which was conducted in Zhejiang province from 2013-2018, data related to the rates on compliance and detection through the CRC screening program among the 40-74 year-old residents were analyzed. Chi-square tests were used to compare the differences among groups, and multivariate logistic regression models were applied to explore the potential risk factors. Cost-effectiveness ratio (CER) was calculated by using the cost per lesion detected as the indicator.Results:Among all the 166 285 participants who completed the risk assessment questionnaire, 21 975 (13.2%) were recognized as under the high risk of CRC and 4 389 (20.0%) of them received the colonoscopy. The detection rates of CRC, advanced adenoma, and non-advance adenoma were 0.3% (11 cases), 2.7% (119 cases), and 5.2% (229 cases), respectively. Results from the multivariate logistic regression analyses showed that factors as age, gender, education level, smoking, drinking alcohol, previous fecal occult blood test (FOBT), polyp history, and family history of CRC were significantly associated with the compliance rate of colonoscopy while age, smoking and polyp history were significantly associated with the detection rate of advanced neoplasms (CRC and advanced adenoma). The costs were ￥22 355.74 Yuan for every CER advanced neoplasm detection and ￥264 204.18 Yuan per CRC detection, respectively. The CER decreased along with ageing. Sensitivity analysis showed that CERs were expected to decrease when the compliance rate of colonoscopy was increasing.Conclusions:The current screening program seems effective in detecting the precancerous colorectal lesions, but the relatively low compliance rate of colonoscopy restricting both the diagnostic yields and economic benefits. It is necessary to improve the awareness and acceptance of colonoscopy among the high-risk CRC population.