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1.
Dig Liver Dis ; 2024 Jun 08.
Article in English | MEDLINE | ID: mdl-38853092

ABSTRACT

BACKGROUND: Anti-glycoprotein 2 (anti-GP2) IgA and antineutrophil-cytoplasmic antibodies to proteinase 3 (PR3-ANCA) have been reported as predictive markers of cholangiocarcinoma (CCA) in patients with primary sclerosing cholangitis (PSC), but their prevalence in CCA patients without PSC remains unclear. METHODS: This study involved Asian discovery (n = 118) and European validation (n = 38) cohorts of CCA patients without PSC, alongside 49 Asian and 82 European pancreatic ductal adenocarcinoma (PDAC) patients, 21 with benign pancreatic neoplasms (BPN) and 45 with hepatocellular carcinoma (HCC), and 157 healthy controls (HC) from Asia and Europe. We analyzed the prevalence of PR3-ANCA, IgA and IgG against GP21 and GP24, and the CA19-9 levels. RESULTS: Anti-GP21 IgA was the most prevalent in both CCA cohorts (discovery: 55.1 %; validation: 42.1 %) and significantly higher than in other groups except PDAC (all p < 0.05). It demonstrated the best diagnostic performance in distinguishing CCA from disease controls and HC, outperforming tumor markers. No significant correlation was found between anti-GP21 IgA levels and CA19-9 levels. CONCLUSION: Our findings show that anti-GP21 IgA revealing the loss of mucosal tolerance is a potential novel diagnostic biomarker for CCA.

2.
Clin Chim Acta ; 561: 119814, 2024 Jun 13.
Article in English | MEDLINE | ID: mdl-38879063

ABSTRACT

BACKGROUND: Hepatocellular cancer (HCC) is one of the most harmful tumors to human health. Currently, there is still a lack of highly sensitive and specific HCC biomarkers in clinical practice. In this study, we aimed to explore the diagnostic performance of prostaglandin A2 (PGA2) for the early detection of HCC. METHODS: Untargeted metabolomic analyses on normal control (NC) and HCC participants in the discovery cohort were performed, and PGA2 was identified to be dysregulated in HCC. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detecting serum PGA2 was established and applied to validate the dysregulation of PGA2 in another independent validation cohort. Receiver operating characteristic (ROC), decision curve analysis (DCA) and some other statistical analyses were performed to evaluate the diagnostic performance of PGA2 for HCC. RESULTS: At first, PGA2 was found to be dysregulated in HCC in untargeted metabolomic analyses. Then a precise quantitative LC-MS/MS method for PGA2 has been established and has passed rigorous method validation. Targeted PGA2 analyses confirmed that serum PGA2 was decreased in HCC compared to normal-risk NC and high-risk cirrhosis group. Subsequently, PGA2 was identified as a novel biomarker for the diagnosis of HCC, with an area under the ROC curve (AUC) of 0.911 for differentiating HCC from the combined NC + cirrhosis groups. In addition, PGA2 exhibited high performance for differentiating small-size (AUC = 0.924), early-stage (AUC = 0.917) and AFP (-) HCC (AUC = 0.909) from the control groups. The combination of PGA2 and AFP might be useful in the surveillance of risk population for HCC and early diagnosis of HCC. CONCLUSION: This study establishes that PGA2 might be a novel diagnostic biomarker for HCC.

3.
Clin Exp Immunol ; 216(2): 132-145, 2024 Apr 23.
Article in English | MEDLINE | ID: mdl-38386917

ABSTRACT

Natural killer (NK) cells were reported to be involved in the pathogenesis of primary antiphospholipid syndrome (pAPS). Immunosuppressive receptor T-cell immunoreceptor with Ig and ITIM domains (TIGIT) and activating receptor cluster of differentiation 226 (CD226) are specifically expressed on NK cells with competitive functions. This study aims to investigate the expression diversities of CD226/TIGIT on NK subsets and their associations with NK subsets activation phenotypes and potential clinical significance, furthermore, to explore potential cause for CD226/TIGIT expression diversities in pAPS. We comparatively assessed the changes of CD56brightNK, CD56dimNK, and NK-like cells in 70 pAPS patients compared with control groups, including systemic lupus erythematosus, asymptomatic antiphospholipid antibodies carriers (asymp-aPLs carriers), and healthy controls and their expression diversities of CD226/TIGIT by flow cytometry. CD25, CD69, CD107α expression, and interferon gamma (IFN-γ) secretion levels of NK subsets were detected to determine the potential association of CD226/TIGIT expression with NK subsets phenotypes. CD226/TIGIT expression levels were compared among different subgroups divided by aPLs status. Moreover, in vitro cultures were conducted to explore the potential mechanisms of CD226/TIGIT expression imbalance. CD56brightNK and CD3+CD56+NK-like cells were significantly increased while CD56dimNK cells were obviously decreased in pAPS, and CD56brightNK and NK-like cells exhibited significantly higher CD226 but lower TIGIT expressions. CD226+CD56brightNK and TIGIT-CD56brightNK cells show higher CD69 expression and IFN-γ secretion capacity, and CD226+NK-like and TIGIT-NK-like cells showed higher expressions of CD25 and CD69 but lower apoptosis rate than CD226- and TIGIT+CD56brightNK/NK-like cells, respectively. The imbalanced CD226/TIGIT expressions were most significant in aPLs triple-positive group. Imbalanced expressions of CD226/TIGIT on CD56brightNK and NK-like cells were aggravated after interleukin-4 (IL-4) stimulation and recovered after tofacitinib blocking. Our data revealed significant imbalanced CD226/TIGIT expressions on NK subsets in pAPS, which closely associated with NK subsets phenotypes and more complicated autoantibody status. CD226/TIGIT imbalanced may be affected by IL-4/Janus Kinase (JAK) pathway activation.

4.
Int J Antimicrob Agents ; 62(4): 106947, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37544384

ABSTRACT

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide. However, it remains unknown whether individuals with prior SARS-CoV-1 infection are protected from SARS-CoV-2 infection. This study assessed protective antibody levels in SARS survivors with and without the COVID-19 vaccine. METHODS: We recruited 17 SARS survivors infected with SARS-CoV-1 in 2003, including 8 not vaccinated with the COVID-19 vaccine and 9 vaccinated with two doses of inactivated whole-virion COVID-19 vaccine (Sinopharm). In addition, 105 healthy adult volunteers without SARS-CoV-1 and SARS-CoV-2 infections were used as controls. The relative concentrations of three protective antibodies including anti-SARS-CoV-2 spike IgG (nCoV S-IgG), anti-SARS-CoV-2 spike receptor-binding domain IgG (nCoV RBD-IgG), and anti-SARS-CoV-2 neutralizing antibodies (nCoV NAbs) were measured to evaluate humoral immunity. RESULTS: We found that the positive rates of these antibodies in unvaccinated SARS survivors were 37.5%, 37.5%, and 62.5%, respectively. In contrast, the corresponding positive rates were all 0% in controls before vaccination. In controls, the levels of protective antibodies reached a peak ca. 28 days after the second dose of vaccine and then started to decline. Surprisingly, the levels of these antibodies were maintained at very high levels even 166 days after the second dose of vaccine in SARS survivors. CONCLUSION: Our study suggests that there are protective antibodies cross-reacting with SARS-CoV-2 in recovered SARS patients and that SARS survivors can generate a much stronger antibody response induced by the COVID-19 vaccine than can controls. These initial findings show the feasibility of developing novel pan-sarbecovirus vaccines.


Subject(s)
COVID-19 Vaccines , COVID-19 , Adult , Humans , SARS-CoV-2 , COVID-19/prevention & control , Antibody Formation , Immunoglobulin G , Survivors
5.
J Inflamm Res ; 16: 2879-2892, 2023.
Article in English | MEDLINE | ID: mdl-37456782

ABSTRACT

Background: Follicular helper T (Tfh), follicular regulatory T (Tfr), and follicular cytotoxic T (Tfc) cells play important roles in autoimmune diseases. Nevertheless, their changes of functional phenotypes in ulcerative colitis (UC), most importantly, their changes in colon tissue as the target-organ, have not been explored. Methods: DSS-colitis was induced in Balb/c mice and lymphocytes were collected from spleen, mesenteric lymph nodes, peripheral blood and colon. Tfh, Tfr, and Tfc cells were analyzed using flow cytometry based on their CD4+CXCR5+FOXP3-Tfh, CD4+CXCR5+FOXP3+Tfr and CD8+CXCR5+Tfc expressions. Various functional characterization markers including CD44, CD62L, TIGIT, CD226, PD-1, ICOS, Helios, CTLA-4 and Bcl6 were analyzed in the T cell subsets of the organs. Results: Tfh and Tfr cells in the colon were significantly increased in DSS-colitis mice. Additionally, the proportions of Tfr and Tfc cells in the peripheral blood were also increased, while Tfc cell proportions in the colon were decreased. The proportion of naïve cells in the Tfh, Tfr and Tfc cells in the colon and peripheral blood decreased, while the proportion of effector memory T cells increased. The TIGIT+CD226-Tfh and Tfc cells were upregulated in the colon of DSS-colitis mice. The PD-1+, ICOS+ and PD-1+ICOS+ Tfh cells were increased in both the colonic and peripheral blood Tfh and Tfc of DSS-colitis mice. The Bcl6+ proportions in the Tfh and Tfr were increased in the colon of DSS-colitis mice. Conclusion: The colonic and peripheral blood Tfh and Tfc cells of DSS-colitis mice have a significantly activated T cell phenotype, which may play a significant role in the pathogenesis of UC.

6.
Front Immunol ; 13: 922307, 2022.
Article in English | MEDLINE | ID: mdl-35874706

ABSTRACT

Objectives: This study aimed to elucidate the changes and associated mechanisms of circulating CD28- cytotoxic T lymphocytes (CTLs) in patients with IgG4-related disease (IgG4-RD). Methods: Fifty IgG4-RD patients and 15 healthy controls (HCs) were recruited. Peripheral blood mononuclear cells (PBMCs) were isolated, the levels of circulating CD28- CTLs were detected by flow cytometry, and the proportions of CD127lo or GZMB+CD28- CTL subsets were analyzed in the meantime. Mechanistically, PBMCs isolated from IgG4-RD patients were stimulated with IL-7 in the presence or absence of the JAK inhibitor tofacitinib. Flow cytometry was used to analyze the proliferation of CD28- CTLs and the changes in related subpopulations. Results: Circulating CD4+CD28- CTLs and CD8+CD28- CTLs were significantly increased in IgG4-RD patients compared with HCs, accompanied by an elevation of CD127lo or GZMB+ CTL subsets. The ex vivo culture of PBMCs showed that IL-7 could induce the amplification of CD4+CD28- CTLs and CD8+CD28- CTLs in IgG4-RD. Furthermore, IL-7 promotes the proliferation and functional subset changes of these CD28- CTLs in this disease. The selective JAK inhibitor tofacitinib significantly inhibited the effects of IL-7 on CD4+CD28- CTLs and CD8+CD28- CTLs. Conclusion: IL-7 can affect the immune balance of IgG4-RD patients by promoting the expansion and function of CD4+CD28- and CD8+CD28- CTLs in IgG4-RD through the JAK pathway. Blockade of the IL-7 signaling pathway may be a new therapeutic strategy for IgG4-RD.


Subject(s)
Immunoglobulin G4-Related Disease , Janus Kinase Inhibitors , CD28 Antigens , Humans , Interleukin-7/metabolism , Interleukin-7/pharmacology , Janus Kinase Inhibitors/pharmacology , Leukocytes, Mononuclear/metabolism , Signal Transduction , T-Lymphocytes, Cytotoxic
7.
J Rheumatol ; 48(5): 664-668, 2021 05.
Article in English | MEDLINE | ID: mdl-32934126

ABSTRACT

OBJECTIVE: Neutrophilia is a hallmark of adult-onset Still disease (AOSD). We aimed to investigate the levels of granulocyte colony-stimulating factor (G-CSF), an essential regulator of neutrophil production and function, in the pathogenesis of AOSD. METHODS: Sera were collected from 70 patients with AOSD and 20 healthy controls (HCs). The levels of G-CSF were determined by ELISA. Low-density granulocytes (LDGs) were quantified by flow cytometry. Correlations between G-CSF levels and disease activity, laboratory variables, and LDG levels in patients with AOSD were analyzed by Spearman correlation test. RESULTS: Patients with active AOSD presented significantly higher levels of G-CSF compared to inactive AOSD patients (P < 0.001) and HCs (P < 0.0001). The G-CSF levels were significantly decreased after active AOSD patients achieved disease remission (P = 0.0015). The G-CSF levels were significantly correlated with C-reactive protein, erythrocyte sedimentation rate, ferritin, and systemic score in AOSD (P < 0.0001). Significant correlations between the levels of G-CSF and circulating neutrophils (P < 0.0001), neutrophil-to-lymphocyte ratio (P < 0.0001), percentages of LDGs in the peripheral blood mononuclear cells (P = 0.004), as well as absolute numbers of circulating LDGs (P = 0.018) were identified. Patients with fever, evanescent rash, sore throat, arthralgia, myalgia, lymphadenopathy, or hepatomegaly/elevated liver enzymes displayed significantly higher levels of G-CSF compared to patients without these manifestations (P < 0.05). CONCLUSION: Our findings indicate that G-CSF is implicated in the pathogenesis of AOSD, and targeting G-CSF may have therapeutic potential for AOSD. In addition, introducing circulating G-CSF levels into the clinical assessment system may help to monitor disease activity.


Subject(s)
Still's Disease, Adult-Onset , Granulocyte Colony-Stimulating Factor , Humans , Leukocyte Count , Leukocytes, Mononuclear , Neutrophils
8.
Dis Markers ; 2020: 3424039, 2020.
Article in English | MEDLINE | ID: mdl-32454904

ABSTRACT

OBJECTIVE: To evaluate the performance of the nuclear matrix protein 22 (NMP22) BladderChek test in urothelial carcinoma (UC). METHODS: We retrospectively analyzed 1318 patients who performed the NMP22 BladderChek tests. Of them, 103 were primary UC patients, 90 were surgical treatment UC patients, and 1125 were benign disease patients. The performance of the NMP22 BladderChek test for the diagnosis of primary and recurrent UC was evaluated. Moreover, the performance of urine cytology and the NMP22 BladderChek test for the diagnosis of primary UC was compared in 90 available subjects including 48 primary UC patients and 42 benign disease patients. RESULTS: The sensitivity and specificity of the NMP22 BladderChek test were 37.9% and 95.8%, respectively, for the diagnosis of primary UC (n = 1228). The corresponding parameters of the NMP22 BladderChek test were 31.0% and 88.5%, respectively, for the diagnosis of recurrent UC (n = 90). The sensitivity and specificity of urine cytology were 54.2% and 97.6%, respectively, for the diagnosis of primary UC (n = 90); the corresponding parameters of the NMP22 BladderChek test were 41.7% and 83.3%, respectively; the corresponding parameters of the two tests combination were 64.6% and 83.3%, respectively. There was a significant difference in the performance between the NMP22 BladderChek test and urine cytology or the combination of two tests (P = 0.017 and 0.001, respectively). CONCLUSIONS: The NMP22 BladderChek test has a low sensitivity for detecting primary and recurrent UC. Urine cytology is superior to the NMP22 BladderChek test, and combined use of the two tests improves the sensitivity in the detection of primary UC.


Subject(s)
Biomarkers, Tumor/genetics , Diagnostic Tests, Routine/methods , Histocytochemistry/methods , Neoplasms/diagnosis , Nuclear Proteins/genetics , Ureteral Neoplasms/diagnosis , Urinary Bladder Neoplasms/diagnosis , Aged , Biomarkers, Tumor/urine , Female , Humans , Kidney Pelvis/metabolism , Kidney Pelvis/pathology , Male , Middle Aged , Neoplasm Grading , Neoplasm Staging , Neoplasms/genetics , Neoplasms/pathology , Neoplasms/urine , Nuclear Proteins/urine , Recurrence , Retrospective Studies , Sensitivity and Specificity , Ureteral Neoplasms/genetics , Ureteral Neoplasms/pathology , Ureteral Neoplasms/urine , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/urine
9.
J Infect Chemother ; 24(11): 887-891, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30197093

ABSTRACT

The accuracy of the test is critical for the syphilis serology diagnosis. This study aims to evaluate the values of the Elecsys syphilis assay, the Architect syphilis assay, and the Mindray syphilis assay, as syphilis screening tests for pregnant women and patients with syphilis or other diseases. A reverse algorithm was used for the syphilis serology diagnosis. Serum samples (n = 584) were tested with three automated screening assays. All reactive sera by one, two, or three screening assays were further analyzed with the tolulized red unheated serum test (TRUST). Inconsistent results were confirmed by the Treponema pallidum particle agglutination assay (TPPA). The final patient diagnosis was made according to the results of syphilis serology, clinical evidence, and past medical history. The sensitivity, specificity, accuracy, and kappa value of each assay were as follows: for the Elecsys syphilis assay, 100.0%, 98.5%, 98.6%, and 0.927, respectively; for the Architect syphilis assay: 100.0%, 94.5%, 95.0%, and 0.770; and for the Mindray syphilis assay: 100.0%, 97.0%, 97.3%, and 0.862. The McNemar test showed that there were significant differences in the performance between the Elecsys syphilis assay and the Architect syphilis assay (P < 0.001), and between the Mindray syphilis assay and the Architect syphilis assay (P = 0.001). Our study demonstrated that three automated Treponema pallidum antibody assays generally showed high sensitivities and specificities, and so, they are suitable for use in screening for syphilis. The performances of the Elecsys syphilis assay and the Mindray syphilis assay are superior to Architect syphilis assay.


Subject(s)
Antibodies, Bacterial/isolation & purification , Pregnancy Complications, Infectious/diagnosis , Syphilis Serodiagnosis/methods , Syphilis/diagnosis , Treponema pallidum/immunology , Adult , Aged , Aged, 80 and over , Algorithms , Antibodies, Bacterial/immunology , Female , Humans , Male , Middle Aged , Pregnancy , Pregnancy Complications, Infectious/blood , Pregnancy Complications, Infectious/immunology , Pregnancy Complications, Infectious/microbiology , Prospective Studies , Sensitivity and Specificity , Syphilis/blood , Syphilis/immunology , Syphilis/microbiology , Treponema pallidum/isolation & purification , Young Adult
10.
J Clin Lab Anal ; 32(9): e22600, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30058207

ABSTRACT

BACKGROUND: Anti-hepatitis C virus (anti-HCV) antibody assays are recommended for HCV infection screening. The Mindray anti-HCV assay, based on a third-generation immunoassay, was recently launched in China. We aimed to evaluate its diagnostic performance compared with that of two other widely used assays. METHODS: Six HCV infection seroconversion panels were used to evaluate the sensitivity of the assay for early detection. A total of 1952 clinical samples were tested by the Mindray anti-HCV, Elecsys anti-HCV II, and Architect anti-HCV assays. Samples with reactive results using at least one anti-HCV assay were further tested with the recombinant immunoblot assay (RIBA). Inconsistent results were investigated by the HCV RNA assay and HCV core antigen assay. HCV infection diagnosis was made according to the results of laboratory tests and medical records. RESULTS: The Mindray anti-HCV assay and Elecsys anti-HCV II assay detected seroconversion in an average of 12.5 days and 10.5 days, respectively, and this difference was not significant (P = .818). Of the 1952 cases, 90 were categorized as "HCV infection" and 1862 were categorized as "no HCV infection." The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), and negative likelihood ratio (LR-) of each assay were as follows: the Mindray anti-HCV assay, 95.6%, 99.2%, 85.1%, 99.8%, 118.6 and 0.045, respectively; the Architect anti-HCV assay, 98.9%, 95.2%, 50.0%, 99.9%, 20.69 and 0.012, respectively; and the Elecsys anti-HCV II assay, 96.7%, 99.9%, 98.9%, 99.8%, 1799.9 and 0.033, respectively. There were significant differences in the specificity, PPV and LR+ among the three assays (P < .001). There were no significant differences in the sensitivity, NPV or LR- among the three assays (P > .05). CONCLUSIONS: The Mindray anti-HCV assay displays a similar sensitivity to the Elecsys anti-HCV II assay with respect to the early detection of HCV infection. The Mindray anti-HCV assay shows excellent diagnostic performance and is suitable for the screening of HCV infection.


Subject(s)
Hepatitis C Antibodies/blood , Hepatitis C/diagnosis , Hepatitis C/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Hepatitis C/blood , Humans , Male , Middle Aged , Retrospective Studies , Sensitivity and Specificity , Seroconversion/physiology , Young Adult
11.
Clin Rheumatol ; 36(12): 2769-2774, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28540606

ABSTRACT

Patients with IgG4-related disease (IgG4-RD) often have elevated serum IgG4 levels. Here, we aimed to evaluate the diagnostic performances of elevated serum IgG4 concentration and IgG4/IgG ratio for IgG4-RD. We retrospectively analyzed 1381 patients subjected to serum IgG subclass testing to differentiate IgG4-RD from other diseases at Peking University People's Hospital from 2012 to 2016. This sample included 133 IgG4-RD patients and 1248 non-IgG4-RD patients. Serum IgG subclass concentrations were measured using Siemens reagents. The median values (25th-75th percentile) for serum IgG4 concentration and IgG4/IgG ratio, respectively, were 8640 (3970-17750) mg/L and 0.339 (0.229-0.517) in IgG4-RD patients and 450 (220-920) mg/L and 0.032 (0.014-0.061) in non-IgG4-RD patients (p < 0.001). For distinguishing IgG4-RD from non-IgG4-RD, the optimal cut-off values of IgG4 and IgG4/IgG were 2100 mg/L and 0.114, respectively. The corresponding area under the curve (AUC) values were 0.964 and 0.970, respectively. Comparison of the receiver operating characteristic curves revealed a significant difference between these AUC values (p = 0.002). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), respectively, were 94.7, 91.6, 54.5, and 99.4% for the IgG4 optimal cut-off value and 96.2, 92.1, 56.4, and 99.6% for the IgG4/IgG optimal cut-off value. Our results confirmed that elevated serum IgG4 concentration and IgG4/IgG ratio were of great value for IgG4-RD diagnosis.


Subject(s)
Autoimmune Diseases/diagnosis , Immunoglobulin G/blood , Adult , Aged , Area Under Curve , Autoimmune Diseases/blood , Female , Humans , Male , Middle Aged , Retrospective Studies , Sensitivity and Specificity
12.
Intervirology ; 55(5): 342-8, 2012.
Article in English | MEDLINE | ID: mdl-22057089

ABSTRACT

OBJECTIVES: To investigate the distribution of human cytomegalovirus (HCMV) glycoprotein B (gB) genotypes and to explore the possible relationship between gB genotypes and clinical characteristics in Chinese hematopoietic stem cell transplant (HSCT) recipients. METHODS: A prospective analysis of gB genotypes was conducted on HCMV clinical isolates obtained from 102 HSCT recipients. Real-time quantitative PCR and PCR-based restriction fragment length polymorphism analysis were applied for the determination of viral loads and gB genotypes, respectively. RESULTS: The distribution of gB genotypes was as follows: gB1, 54/102 (52.9%); gB3, 21/102 (20.6%); and mixtures, 27/102 (26.5%). The rate of viral clearance at day 21 was higher in patients infected with the gB1 genotype than in those infected with the gB3 genotype (56 and 29%, respectively; p = 0.036). In contrast, the rate of HCMV reactivation/reinfection was higher in patients infected with the gB3 genotype than in those infected with the gB1 genotype (81 and 56%, respectively; p = 0.041). CONCLUSIONS: The HCMV gB1 genotype is the most prevalent among Chinese HSCT recipients; patients infected with the gB3 genotype have more difficulty eradicating the virus and have a higher risk of reactivation/reinfection than those infected with the gB1 genotype.


Subject(s)
Cytomegalovirus Infections/epidemiology , Cytomegalovirus/classification , Hematopoietic Stem Cell Transplantation/adverse effects , Phylogeny , Viral Envelope Proteins/genetics , Adult , Asian People , Cluster Analysis , Cytomegalovirus/genetics , Cytomegalovirus/isolation & purification , Cytomegalovirus Infections/virology , DNA, Viral/chemistry , DNA, Viral/genetics , Genotype , Humans , Male , Molecular Epidemiology , Molecular Sequence Data , Polymorphism, Restriction Fragment Length , Prevalence , Prospective Studies , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNA , Virus Activation
13.
Arch Virol ; 157(3): 475-81, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22205145

ABSTRACT

Human cytomegalovirus (CMV) is an opportunistic pathogen, and infections with this virus can be treated with ganciclovir (GCV). Most GCV-resistant clinical CMV isolates contain a mutation in the UL97 gene. Genotypic assays for diagnostic screening of GCV-resistant CMV have been developed. High-resolution melting analysis (HRMA) with unlabeled probe is considered a perfect tool for this purpose. In this study, we have developed an HRMA-based genotypic test for the detection of UL97 mutations. Wild type and M460V/I mutants of UL97 were constructed. HRMA with unlabeled probe was used as a genotyping method for the detection of M460V/I mutations. The melting peaks obtained directly from PCR products did not enable us to distinguish the wild type from M460 mutants. The sensitivity and accuracy of HRMA were dramatically improved by using unlabeled probe. HRMA with unlabeled probe successfully distinguished M460V from M460I and served well for the detection of M460V/I mutations in clinical samples. HRMA with unlabeled probe proves to be a sensitive and cost-effective genotyping method for the detection of M460 mutations.


Subject(s)
Cytomegalovirus/drug effects , DNA, Viral/genetics , Drug Resistance, Viral , Molecular Typing , Mutation, Missense , Phosphotransferases (Alcohol Group Acceptor)/genetics , Virology/methods , Antiviral Agents/pharmacology , Cytomegalovirus/genetics , Ganciclovir/pharmacology , Genotype , Humans , Sensitivity and Specificity , Transition Temperature
14.
Arch Virol ; 156(1): 17-23, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20878191

ABSTRACT

Human cytomegalovirus (HCMV) genomic polymorphisms have been used to investigate correlations between virus variants and clinical characteristics. We explored the distribution of HCMV glycoprotein N (gN) genotypes and their roles relative to clinical features in a population of Chinese hematopoietic stem cell transplant (HSCT) recipients. This prospective analysis involved HCMV clinical isolates obtained from 102 HSCT patients. Real-time quantitative PCR and PCR-based restriction fragment length polymorphism analysis were applied for the determination of viral loads and gN genotypes. The distribution of HCMV gN genotypes was as follows: gN1, 6/102 (5.9%); gN2, 10/102 (9.8%); gN3a, 17/102 (16.7%); gN3b, 5/102 (4.9%); gN4a, 12/102 (11.7%); gN4b, 9/102 (8.8%); gN4d, 2/102 (2.0%); and mixtures, 41/102 (40.2%). No particular HCMV gN genotype was significantly associated with specific clinical characteristics. The HCMV gN3a genotype was the most prevalent among Chinese HSCT recipients, but HCMV gN genotypes may have no correlation with clinical features in HSCT patients.


Subject(s)
Cytomegalovirus Infections/virology , Cytomegalovirus/genetics , Hematopoietic Stem Cell Transplantation/adverse effects , Viral Envelope Proteins/genetics , Antiviral Agents/therapeutic use , China , Cytomegalovirus/metabolism , Cytomegalovirus Infections/etiology , DNA, Viral/isolation & purification , Female , Foscarnet/therapeutic use , Ganciclovir/therapeutic use , Genetic Variation , Genotype , Humans , Male , Polymerase Chain Reaction , Viral Envelope Proteins/metabolism , Viral Load
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